DELIVERY OF A PANCREATIC BETA CELL - DERIVED HORMONE TO ERYTHROCYTES BY ALBUMIN AND DOWNSTREAM CELLULAR EFFECTS By Yueli Liu A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of Chemistry - Doctor of Philosophy 2015 ABSTRACT DELIVERY OF A PANCREATIC BETA CELL - DERIVED HORMONE TO ERYTHROCYTES BY ALBUMIN AND DOWNSTREAM CELLULAR EFFECTS By Yueli Liu Erythrocytes (ERYs) deliver oxygen to orga ns. However, another important role of ERYs is to regulate blood flow by resp onding to va rious stimuli . In blood vessels, flowing ERYs deform and release adenosine triphosphate (ATP). ATP is able to stimulate the production of a well - kno wn vasodilator, nitric oxide (NO) in the endothelium, thereby serving as an indirect determinant of blood flow. NO relaxes vascular smooth muscle cells enabling the dilation of blood vessels to maintain normal blood flow. In addition to deformation, ERYs a lso respond to other stimuli in the bloodstream such as C - peptide and zinc to release ATP. C - peptide is a 31 amino acid peptide co - - cells. Insulin is known for controlling blood glucose levels , and insulin replacement is approved therapy for type 1 diabetic patients, whose pancreas fails to produce insulin and C - peptide. In the 1990s, beneficial effects of C - peptide in reducing diabetes complications were discovered, and were thought to be related to improved blood flow . However, the mechanism of C - peptide remains elusive, which beca me the major obstacle for C - peptide administration to b e used along with insulin . Previously, it has been shown that C - peptide, when combined with zinc, can enhance ATP rele a se from ERYs by 50%. In turn, a n increase in NO production from the endothelium was also measured using an in vitro platform. However, the interaction of C - peptide and zinc with ERYs is unclear. En zyme - linked immunosorbent assay (ELISA) and scintillation counting results showed that both C - peptide and zinc could bind to the ERY, although no binding between C - peptide and zinc could be detected. T he results in this dissertation provide evidence that albumin is indispensable for uptake of both species, and that C - peptide uptake by the ERY is requisite for zinc uptake. Isotheral titration calorimetry experiments revealed specific binding of C - peptide and zinc to albumin, suggesting that albumin transports C - peptide and zinc to ERYs for biological effects. Uptake of C - peptide and zinc led to an improvement in ERY membrane deformability, which could explain the subsequent increase of A TP release when subjected to flow - induced shear stress. To demonstrate the broader impact of these findings, t he response of stored ERYs to C - peptide and zinc is found compromised in the current FDA approved AS - 1 solution . Decreased re sponse was reversed up to 12 - 15 days of storage . Excess glucose in the AS - 1 solution was found responsible for the loss of response. C - pepti de and zinc are components of pancrea - cell secretion, therefore suggesting that transfusion of ERYs stored in AS - 1 for longer than 15 days can cause more hea l th issues than post transfusion complications by not properly responding to healthy organs su ch as the pancreas, whereas a normoglycemic storage conditions may be a solution to the issues. The ability of C - peptide and zinc to have combined effects with molecul e s secreted from other tissues was also investigated. Specifically, leptin, a hormone se creted from adipose cells, acts on receptors in the arcuate nucleus of the hypothalamus to control appetite, and achieve energy balance. Results of ATP release experiments showed that, in the presence of C - peptide and zinc, leptin further enhanced ATP rele ase from ERYs by an additional 30% . This may suggest another mechanism of leptin for glucose clearance that involves a site of action on the ERY also gover ned by the efficacy of C - peptide and zinc. iv ACKNOWLEDGMENTS First, I would like to thank my advisor, Dr. Dana Spence, for all the help and support I could possibly get during my PhD program. His patience and willingness to guide me has always been the cou rage and confidence for me to pursue an advanced degree. He is always supportive and open to discussions and new ideas which have been the valuable source of inspiration for my project, and have deepened my interest in pharmaceutical research and modern he althcare, which contribute to the shaping of my future career path. As a nice person, all the group parties Dr. Spence has hosted gave me the opportunity to experience American culture which filled my life in the US with relaxation and joy. I am also thank ful for the guidance provided by my committee members, Dr, Gary Blanchard, Dr. Thomas Hamann, and Dr. Jenifer Fenton. Because of their help, my understanding for the importance of my research was strengthened. The Spence group members have been very suppor tive in work and nice friends at other times as well, without whom my project would not have gone smoothly. Steve, Kari, Suzanne and Paul, I want to thank you for all of your valuable advices and suggestions which have been so helpful in improving my prese ntation skills and problem solving skills. Adam and Wathsala, both of you have been nice teachers of mine who taught me lab techniques and shared ideas on the C - peptide project. Yimeng, I appreciated the great time we went to watch college football games t ogether for three seasons, and the unforgettable trip to Mexico. Jayda, Sarah and Bethany, cheers for the ups and downs we have had in our own PhD life, and I am very glad we have made it or will make it, and I will always cherish the suggestions and help I have received from you and the thoughts we have shared. Kristen, thanks for being such a warm and enthusiastic person, my life would have been a lot harder v without you in the lab. Ruipeng and Tiffany, thanks for the free rides you have offered me. For al l new people in the lab, you have brought more fun to this lab and I hope you the best in your projects. In addition, I am thankful for all group activities we have had, especially the baking times we enjoyed together let me feel warm as in a big family. T hese memories are precious to me. My research could not have been done without close collaborations with other research laboratories. Dr. offered great support in the purification and identification of C - peptide and mutants. Dr. i n the Department of Biochemistry and Molecular Biology provided me with free access to the isothermal titration calorimeter, which I used to discover C - pepti de binding. I want to express my gratitude for Dr.Hoogstraten for the time he spent on training me in the use of the instrument and discussions on sample preparation. I also want to thank Chengpeng Chen in the Spence lab for our collaborations in the proje going personality made our collaboration very fruitful and pleasant. Finally, I want to thank family for encouraging me and supporting me to obtain an advanced degree in chemistry. motivation fo r me to c ome to this new height in academia, and it will continue to be my motivation to achieve a successful career in this field . PhD program usually require s extensive r esearch work w hich makes the summer vacation to C hina every year a luxury. I want to expre ss my great appreciation that my parents not only understand my situation, but also have tried to plan a few trips to the US to visit me, stay with me and help me . I am very fortunate to have their love and care, and will always appreciate and cherish it. vi TABLE OF CONTENTS LIST OF TABLES . . . i x LIST OF FIGURE .. x KEY TO ABBREVIATIONS x vi i Chapter 1 - Introduction 1.1 Brief Introduction to Diabete 1 1. 2 Diagnosis, Causes and Classification of Diabet 2 1.3 Current Treatments for Diabe 7 1.4.1 Acute Compli 7 1.4.2 Chronic Complicati 8 1.5 Biosynthesis of Insulin an d C - 1.6 Biological Effects of Insu 22 1.7 C - 24 1.7.1 Discovery of C - 24 1.7.2 Improvement of Ner ve Function in Diabetes via C - peptide Replacement 27 1.7.3 Improvement of Kidney Function in Diabetes via C - peptide Replacement ..33 1.7.4 Cytoprotective Effects of C - peptide on the Endothe 1.8 Theory of a C - 3 8 1.9 Molecular Mechanism of Zn 2+ Activated C - peptide on Improving . 43 R EFERENCES . . . 5 2 Chapter 2 - Serum Albumin as a Carrier for C - peptide and Zinc t o the ERY 2.1 Introduction 7 2 2.1.1 Structure of Huma n C - .72 2.1.2 Serum A 2.1.2.1 Transp ..73 2.1.2.2 Albumin as a Drug Carrier... . 74 2.2 Expe rimental Proced ..77 2.2.1 Collection and Purification o ..77 2.2.2 Purification and Characterization of C - peptide and Mutant E27A u sing High Performance Liquid Chromatography ( HPLC) a nd Mass Spectrometry (MS ...77 2.2.3 ELISA - based Determination of C - ...81 2.2.4 Radiometric Determination of 65 Zn 2+ 2.2.5 Isothermal Titration Calorimetry (ITC) Determination of Interaction of Albumin, C - peptide (or Mutant E27A) and Z . 85 2.2.6 Measurement of Static AT 87 2.2.7 Measurement of Flow - induced ATP Release from ERYs on a 3D Printed Fluidic 88 92 vii 2.3.1 Purification of . 92 2.3.2 C - peptide and Zinc Uptake by ERY 92 2.3.3 Interaction between C - peptide and Zinc and Their Binding to Serum A 92 2.3.4 Interaction between E27A a 9 6 2.3.5 Enhanced Static ATP Release from ERYs by Albumin/ C - 101 2.3.6 Enhanced Flow - induced ATP Release from ERYs by A lbumin/C - peptide . 101 106 R EFERENCES ... ..11 5 Chapter 3 - Stored ERYs Response to Insulinoma Cell Line (INS - 1) .. 1 22 3.1.1 Overall Introduction to Blood Transfusion and Blood 1 23 3.1.2 Procedure of Bloo d Collecting an 1 23 3.1.3 Post Transfusion Com ... 1 26 3.1.3.1 Transfusion - transmit 1 26 3.1.3.2 Acute Transf . 1 27 3.1.3.3 Insufficient Nitric Oxide B ioavailability .. 12 8 3.1.3.4 Modified Storage Solut . 12 8 3.2 Experimental Proce 3.2.1 Preparation of Solutions f 129 3.2.2 Collectio . 130 3.2.3 ERY Sample Pr ep ... 132 3.2.4 Determination of ATP Release from Stored ERYs on a 3D P rinted Fluidic Devic ..133 3.2.5 Rat INS - 1 Cell Culture in Inserts a nd Integration on Device . ... 134 .138 3.3.1 Extracellular Glucose Environm 138 3.3.2 Reversibility of Defect of ATP Re .. 138 3.3.3 C - peptide Uptake by ... 142 3.3.4 Increase of ATP Release by Stored ERYs in Response to Zn 2+ - C - pept 142 3.3.5 Increase of ATP Release by Stored ERYs in Response to Rat INS - 1 C 146 147 R EFERENCES ... .. 15 3 Chapter 4 - Mechanistic Studies of Albumi n/ Zn / C - peptide Effects on ERY - derivedATP Release .. 158 4.1.1 Shear Stress - induced Deformabilit y of ERY and ATP Releas e . 158 . 159 4.2 Experimenta l Proce 161 4.2.1 ERY Sample Pre .. 161 4.2.2 Characterization of Deformability of ERYs using 3D Printed 162 viii 4.2.3 Measurement of Static ATP Release ..163 4.3 Res . 163 4.3.1 Increase of Membrane Deformability of Fresh ERYs by Albumin/ Zn/C - pe 163 4.3.2 Increase of Membrane Deformability of Stored ERYs by Albumin/ Zn/C - pe ... ... 167 4.3.3 Further I ncrease of ERY - derive 168 168 R EFERENCES . .. . 17 5 Chapter 5 - Conclusions and Future Directions . 180 5.1.1 C - peptide Replace ment Therapy Potentially Complements In sulin Therapy fo . 1 80 5.1.2 Normolgycemic Storage Condition Reduces ERY Storage L .. 184 5.1.3 Leptin May Affect Energy Balance through Interaction with E 188 5.2 Future Direction . 189 R EFERENCES . .. .. 19 4 ix LIST OF TABLES Table 2.1: Amino acid sequences and of human C - peptide and mutant E27A. Their multi - segment gradients and conditions for HP LC purification are also .... ...80 Table 3.1: Constituents and their concentrations in anticoagulant solutions for blood collection and storage solutions for ERY storage. These solutions are currently used for blood banking in the US. CPD and CP2D are two types of anticoagulant solutions for blood collection. AS - 1, AS - 3 and AS - 5 are three types of storage solutions for ERY storage. Note that the glucose concentrations in these solutions all exceed normal phy siological glucose level (4 - 6 mM). 1 25 x LIST OF FIGURES Fig 1.1: Increased polyol pathway flux in hyperglycemia leading to diabetic complications. Aldose reductase reduces aldehydes generated by reactive oxygen species (ROS) to inactive alco hols. When intracellular glucose levels become too high, aldose reductase reduces glucose to sorbitol, consuming NADPH in the process. Sorbitol dehydrogenase (SDH) oxidizes sorbitol to fructose using NAD + as a co - factor. When the activity of aldose reducta se is sufficient to deplete reduced 10 Fig 1.2: Hypothesis of increased formation of AGEs and diabetic complications. Intracellular proteins, including those involved in the regulati on of gene transcription, are irreversibly modified with glucose. AGE precursors can diffuse out of cells and modify extracellular matrix molecules, and thus cause cellular dysfunction. AGE precursors are also capable of modifying circulating plasma protei ns. These modified proteins can then bind to AGE receptors, leading to production of inflammatory cytokines and growth factors, which in turn causes abnormal vascular 11 Fig 1.3: The hypothesis of increased hexosamine pathway flux and diabetic co mplications. Excess intracellular glucose is metabolized through glycolysis and converted to glucose - 6 phosphate (Gluc - 6 - P) and then fructose - 6 phosphate (Fruc - 6 - P). Next, some Fruc - 6 - P is converted by an enzyme called GFAT (glutamine:fructose - 6 phosphate amidotransferase) to form glucosamine - 6 phosphate which is finally converted to uridine diphosphate N - acetyl glucosamine, resulting in pathologic changes in gene expression. As a result, expression of transforming growth factor - - activator inhibitor - 1 (PA - 1) are both increased, giving rise to adverse effects i 1 4 Fig 1.4: Amino acid sequence of human proinsulin. Human proinsulin is comprised of A chain, B chain, and a connecting peptide, known as C - pept ide, that connects the two chains. A chain contains a disulfide bond, and A chain and B chain are linked together by two more disulfide bonds, which together form a specific conformation of proinsulin. C - peptide also stabilizes the structure of proinsulin. When cleaved by certain enzymes, segments of A chain and B chain in blue form a single molecule of insuliln, and C - peptide is freed. As shown, C - peptide and insulin are produced in 1 9 F ig 1.5: Maturation and exocytosis of insulin, C - peptide and zinc from beta cell granules. The biosynthesis of insulin and C - - cells granules. Proinsulin is produced as a precursor and is able to fold quickly into its native and is then transported to the Golgi apparatus via vesicular transfer and packaged into immature secretory vesicles as hexamers. The synthesis is highly controlled by pH condition and regulated by zinc. As the vesicles become mature, proinsulin hexamers are c onverted into insulin hexamers by endopeptidases and carboxypeptidase, freeing the connecting peptide as C - peptide and yielding insulin hexamers. Insulin and C - peptide are stored together in the secretion granules along with small amounts of intact proinsu lin and other cleavage products, as well as zinc, until released via exocytosis, primarily in response to elevated blood glucose levels. Insulin and C - peptide are released in equimolar amounts with zinc simultaneously 20 xi Fig 1.6: The pathway of insulin signaling and GLUT4 translocation. The uptake of glucose into cells is achieved by facilitated diffusion. The receptor for insulin (IR) is located on the cell membrane of muscle and fat cells. IR is consisted of two i nsulin - - - subunits). - subunits, - autophosphory lation is activated, which in turn phosphorylates other protein substrates such as insulin - receptor substrate (IRS). Then, the lipid kinase activity of phosphatidylinositol 3 - kinase (PI3K) is stimulated, which activates several kinases including Akt, prote in kinase B (PKB), and protein kinase C (PKC). Finally, translocation of an intracellular pool of GLUT4 to the plasma membrane occurs and glucose uptake is stimulated. Glucose is then stored in muscle cells in the form of glycogen. Insulin also acts on the liver to force storage of . 26 Fig 1.7: Multiple pathways of C - peptide in improving nerve function in diabetes. C - peptide has been shown to increase eNOS activity, Na + , K + - ATPase activity and transcriptional factors, which will lead to improved nerve function in diabetes, alleviating pains in patients caused by diabetic peripheral neuropathy. Improved nerve function can be indicated by an increase in nerve conduction velocity and vibration perception, and decrease in axonal atrophy and demyelin Fig 1.8: Multiple pathways of C - peptide in improving renal function in diabetes. C - peptide is able to reduce glomerular permeability, leading to decreased proteinuria. C - peptide has also been shown to inhibit tubular sodium reabsorpt ion, reducing afferent arteriolar diameter and GFR. A decrease in GFR further leads to decrease in proteinuria and prevention of the progression of glomerular hypertrophy. Both of reduction of proteinuria and prevention of glomerular hypertrophy result in attenuation of microalbuminuria, glomerulosclerosis, and tubulointerstitial fibrosis, .34 Fig 1.9: Proposed mechanism of Zn 2+ - C - peptide improving vascular health via ATP regulation. C - peptide, i n the presence of Zn 2+ , increases ATP release from ERYs under flowing condition, which then increases NO production from the endothelium. NO is a well - known blood vasodilator. An increase in NO levels in the bloodstream leads to smooth muscle cells relaxa tion and vasodilaton. As a result, blood flow is increase. According to result from the Spence group, C - peptide does not have its efficacy without the presence of zinc and serum albu . 45 Fig 2.1: Reaction scheme for the detection of C - peptide using human C - peptide ELISA. Sample containing C - peptide is added into a well of the ELISA plate, where C - peptide binds to the coated primary antibody on the bottom of the well. The secondary antibody conjugated with the enzyme converts the added substrat e into a colored product. The absorbance intensity of the colored product is proportional to the amount of C - ...83 Fig 2.2: Schematic diagram of an ITC instrument. Solution filled in the syringe is titrated into the solution filled in the sample cell in the adiabatic shield. Power was continuously supplied to the sample cell during a titration experiment, to keep the sample and reference cell at the same temperature. Therefore, each injection of the titrant result s in a peak corresponding to the power being supplied to the sample cell xii to maintain constant temperature as a function of time. The negative peaks shown indicate an exothermic binding event. The value of enthalpy change can be directly obtained from integ ration of the peaks, and the binding stoichiometry and affinity can be obtained by the molar ratio at the inflection point and the slope, respectivel ..84 Fig 2.3: Schematic diagram showing measurement of flow - induced ATP release from ERYs using a 3D - prin ted fluidic device. Six channels were fabricated in the device, with three wells above each channel. A transwell insert (6 mm in diameter) whose bottom is a piece of porous (0.4 micron) polyester membrane is placed in each of these wells. Inlets and outlet s of each channel are connected by finger tight fittings and tygon tubing to form a closed loop system. Once samples are loaded into the channels by a peristaltic pump and the loop is closed, a circulation mimic is formed. All samples were allowed to flow collected for 20 minutes at room temperature and measured in the middle insert of each channel. Four ATP standards (concentrations of 0, 100, 200 and 400 nM) were measured first to obtain a standard curve, .91 Fig 2.4: ELISA determination of C - peptide binding to ERYs. C - peptide binding to ERYs in the presence of Zn 2+ (open circles) was not significantly different from the uptake in the absence of Zn 2+ (filled circle s). In both cases, C - peptide binding to the ERY displays a specific binding curve which saturates at approximately 2 .93 Fig 2.5: Measurement of C - peptide and Zn binding to ERY samples prepared in album in - free PSS. Neither C - peptide nor Zn was able to bind to EYRs in the absence of albumin. This indicates that albumin is necessary for C - peptide and Zn binding to the ERY, and that albumin is probably a plasma transporter for C - peptide and Zn as well. Erro .94 Fig 2.6: Radiometric determination of 65 Zn 2+ uptake by ERYs. 65 Zn 2+ uptake by ERYs in the presence of C - peptide (filled circles) displayed a specific binding curve which also saturated at around 2 pmoles an d was not significantly different from that of C - peptide. However, 65 Zn 2+ uptake did not occur in the absenc e of C - peptide (open circles ) ...95 Fig 2.7: ITC analysis of C - peptide and zinc interaction at pH ~5.8. A 3 mM zinc solu - peptide solution and no specific binding was detected. Electrostatic attraction was the only interaction observed. All solutions Fig 2.8: ITC analysis of interactions betwe en C - peptide and zinc at pH ~5.8. A 250 - attraction, without specific binding, was the only interaction detected. All solutions . 9 8 Fig 2.9: ITC analysis of C - - peptide solution and specific binding was detected. Solutions were prepared in metal - free Tris - HCl buffer. Average binding affinity K a = (1.75 ± 0.64) × 10 5 M - 1 and binding xiii stoichiometry N = 0.53 ± 0.03. Error bars = S.E.M., n = 9 9 Fig 2.10: ITC analysis of C - peptide/zinc mixture binding to albumin at pH = 7.40 ± n (HSA) solution was titrated into a C - - phase binding event was detected. All solutions were prepared in metal - free Tris - HCl buffer. Phase 1 indicates zinc - albumin binding, with averaged binding affinity K a1 = (5.08 ± 0.98) ×10 7 M - 1 . Phase 2 indicates C - peptide - albumin binding, with averaged binding affinity K a2 = (2.66 ± 0.25) ×10 5 M - 1 . ..100 Fig 2.11: ITC analysis of interactions of E27A and HSA at pH = 7.40 ± 0.01 . A 200 without specific binding was detected. All solutions were prepared in Tris - HCl 102 Fig 2.12: The effect of albumin, C - peptid e and zinc on static ATP release from ERYs. Values of ATP release are normalized to the first black bar, the untreated 7% ERYs in PSS. ERYs suspended in PSS (black bars) had significantly increased ATP release only when C - peptide and zinc were both present , compared to untreated ERY control sample. The previously seen increase of ATP release was abandoned when C - peptide was mutated to E27A. C - peptide and zinc did not increase ATP release from ERYs in BSA - free PSS (grey bars). Error bars = S.E.M., n = 103 Fig 2.13: The effect of albumin, C - peptide and zinc on flow - induced ATP release from ERYs. A significant increase in ATP release was only measured from the ERY sample prepared in an albumin - containing PSS buffer that had been incubated with both C - pept ide and zinc (p < 0.005). The absence of any one of these 3 components (C - peptide, zinc, or albumin) resulted in no significant increase in ERY - derived ATP. Error bars = S.E.M., n = .104 Fig 2.14: Comparison of ATP released from E RYs treated with combination of C - peptide/zinc and E27A/zinc. In albumin - containing PSS, C - peptide/zinc increased the ATP release significantly (p < 0.005), whereas E27A/zinc did not show such an effect. In albumin free PSS, however, the role that C - peptid e/zinc has on increasing the ATP release from ERYs was notobserved. Error bars = S.E.M., . .105 Fig 2.15: ITC analysis of C - peptide and Ca 2+ interaction at pH ~5.8. A 3 mM CaCl 2 - peptide solution and electrostatic attraction without specific binding was detected. Solutions w Fig 2.16: ITC analysis of C - peptide and Na + interaction at pH ~ 5.8. A 3 mM NaCl solution - peptide solution and electrostatic attraction without specific binding was detected. Solutions were .111 Fig 3.1 Illustration of the process of blood collection and ERY sample preparation. Whole blood is draw n into blood tubes from healthy donors, centrifuged, and the plasma and white cell parts discarded by aspiration. About 0.5 volumes of either AS - 1 or AS - 1N solution is added to ERYs in the tube and gently mixed well to form a evenly distributed ERY suspens ion. Then, the ERY suspension is aliquoted and xiv transferred into pre - sterilized PVC bags, which are then heat sealed and stored at 4 ºC in a refrigerator for 5 weeks. On the day of experiments, PVC bags are opened and some stored ERYs are placed in microcen trifuge tubes, followed by addition of freshly prepared PSS or PSSH buffer to a final hematocrit of 5% for ATP release experiment, or a final hematocrit of 7% for C - 131 Fig 3.2: Top view of the experimental setup of the 3D pri nted fluidic device for ATP release experiment using cultured INS - 1 cells. Inserts that are cultured with INS - 1 cells are tightly placed in wells in row B of channels 7, 9 and 11. Two sets of the three samples (AS - 1N - PSS, AS - 1 - PSSH, AS - 1 - PSS) are prepared at 5% hematocrit and loaded into all six channels of the device simultaneously using a peristaltic pump (channels 1, 3 and 5 will not contain INS - 1 cells secretion whereas channels 7, 9 and 11 will contain INS - 1 cells secretion). After sample introduction, the device is incubated at 37 ºC for 2 hours for the best effect of Zn 2+ - C - luciferin/luciferase assay solution is simultaneously added to each insert in row E, ............ 136 Fig 3.3: Extracellular glucose environments in AS - 1N and AS - 1 storage bags. Extracellular glucose concentration in AS - 1N storage bags (open circles) was maintained at a healthy level around 4 - 6 mM over 36 days of storage with periodic feeding, whereas glucose concentration in AS - 1 bags (closed circles) stayed exce ssively high around 40 - 50 mM. Error bars are ±SEM, n = 137 Fig 3.4: Flow - induced ATP release from stored ERYs without the addition of Zn 2+ - C - peptide. After transfused to PSS, AS - 1N stored ERYs (AS - 1N - PSS samples, black bars) always released the mo st ATP around 220 nM. AS - 1 stored ERYs after transfused to PSSH (AS - 1 - PSSH samples, light grey bars) released least ATP which also declined with time. Same AS - 1 stored ERYs but transfused to PSS (AS - 1 - PSS samples, dark grey bars) completely reversed ATP r elease to 200 nM until day 5, and partially reversed until day 12, and lost reversibility after day 15. Error bars . ...140 Fig 3.5: ELISA determination of C - peptide uptake by stored ERYs. A constant 2 pmol of C - peptide uptake by ERYs of AS - 1N - PSS samples was measured (black bars). C - peptide uptake by AS - 1 - PSSH samples (light grey bars) continued to decrease over time. C - peptide uptake by ERYs of AS - 1 - PSS samples (dark grey bars) was completely reversed to 2 pmol in the first 5 days and partially reversed until day 12, but not reversed after day 15. Error bars are ±SEM, n = Fig 3.6: Increase of flow - induced ATP release from stored ERYs by Zn 2+ - C - peptide. After incubated with Zn 2+ - C - peptide, ATP rel ease from AS - 1N - PSS samples (black bars) went up to 320 nM. AS - 1 - PSSH samples (light grey bars) did not respond to Zn 2+ - C - peptide very well as there was not much significant increase in ATP release observed. ATP release from AS - 1 - PSS samples (dark grey bar s) was increased to 300 nM until day 5; the increase became less obvious in the following week and ...143 Fig 3.7: Flow - induced ATP release from stored ERYs without access to INS - 1 cells. This d ata serves as a control experiment for flow - induced ATP release from stored xv ERYs that had been in contact with INS - 1 cell secretion. Here, results of ATP release were comparable to data in Fig 3.4. Briefly, AS - 1N - PSS samples (black bars) released the most ATP around 220 nM throughout 36 days of storage. AS - 1 - PSSH samples (light grey bars) released least ATP which also declined with time. AS - 1 - PSS samples (dark grey bars) were able to completely reverse ATP release on day 1, and then lost reversibility on an 144 Fig 3.8: Response of stored ERYs to INS - 1 cells determined by increase of ATP release. AS - 1N - - cells very well as an increase in ATP release was observed throughout the p eriod of storage. AS - 1 - PSSH - cells normally as no significant increase in ATP release was observed. Response of AS - 1 - PSSN samples (dark grey - cells was observed before day 8, and was reduced after day 8 and lost 145 Fig 4.1: Schematic diagram of 3D printed cell filter for characterization of ERY cell membrane deformability. a) Design of the cell filter. Two pieces of 3D - printed slab s with a hole in the center are clamped together with a piece of filter. Tubing for sample introduction was connected to the hole of the top slab, and each ERY sample was dri ven through the cell filter by a peristaltic pump with a pressure fixed at 5 cmH 2 O (~0.075 psi). 10 minute filtration was allowed for collection of each sample into a cuvette. b) Picture showing each part of the cell filter. c) Picture showing the actual s etup of the system, including the cell filter, cuvette and pump. An ERY sample is flowing in the tubing and being filtered through Fig 4.2: The change of deformability of fresh ERYs in the pre sence of Zn 2+ - C - peptide characterized by 3D printed cell filter. No cell lysis was detected during the deformability tests. The number of cells counted for the untreated ERY sample in PSS (left - most black bar) was set to deformability of 100%, to which the cell numbers of all other samples were normalized. Incubation of fresh ERYs with 10 nM Zn 2+ - C - peptide in PSS led t o 50% increase of membrane deformability compared to untreated ERYs. Incubation with 10 nM of either C - peptide or Zn 2+ alone did not lead to any significant change in membrane deformability. No change in deformability was observed either when ERYs were suspended in albumin - free PSS. Error bars are . ..165 Fig 4.3: Deformability of stored ERYs. Cell count s of day 1 AS - 1N - PSS samples were set to 100%, to which cell counts of all other samples were normalized. Regardless of the length of storage, AS - 1N - PSS samples (black bars) displayed consistent cell membrane deformability (cell count ~20 million) to a lev el that was not different than that of fresh ERYs. As comparison, AS - 1 - PSSH samples (light grey bars) gradually lost their ability to deform while remained in hyperglycemic buffer, which decreased from 80% on day 1 to 60% on day 36. After transferred to ph ysiological PSS, AS - 1 - PSS samples (dark grey bars) displayed 100% reversibility in deformability up until day 5, and partial reversibility between day 8 and 12. Beyond day 15, deformability of AS - 1 - PSS samples was statistically the same as that of AS - 1 - PSS H samples and was no longer reversible. Error bars are ±SEM, n = 166 xvi Fig 4.4: Change of deformability of stored ERYs in the presence of Zn 2+ - C - peptide. Cell counts in this figure were all normalized to the cell counts of day 1 AS - 1N - PSS samples in Fig 4.3. In the presence of Zn 2+ - C - peptide, deformability of AS - 1N - PSS samples consistently increased by 30 - 40% over 5 weeks without showing a significant decrease with the length of storage. Zn 2+ - C - peptide was able to improve the deformability of AS - 1 - PSSH s amples by 25% on day 1 and 20% on day 5, followed by a minor 10% in week 2 after which the efficacy was totally abolished. AS - 1 - PSS samples demonstrated 30 - 40% increase of deformability with Zn 2+ - C - peptide that was statistically the same as the increase of AS - 1N - PSS samples on day 1 and day 5, which gradually weakened afterwards. Error bars are ±SEM, n = 4 .. Fig 4.5: Characterization of static ATP release from fresh and healthy ERYs in the presence of a series of concentrations of leptin. Leptin (0, 0.95, 1.9, 3.8 and 7.6 nM) was added to ERYs. After 2 hour incubation, levels of static ATP release from ERY samples were characterized by chemiluminescence intensity in the luciferin/luciferase enzymatic reaction. No change of level of static ATP release was observed after ERYs were incubated with any of the chosen concentration of leptin. Fig 4.6: Further increase of static ATP release from ERYs in the presence of both leptin and Zn 2+ - C - peptide. Incuba tion with 3.8 nM leptin in the presence of 20 nM Zn 2+ - C - peptide led to an additional 30% increase in the chemiluminescence intensity, which resulted from an additional increase of ATP release. However, leptin with either C - peptide or Zn 2+ alone did not lea d to a signi ficant increase of ATP release. Error bars are ±SEM, n = xvii KEY TO ABBREVIATIONS ATP: Adenosine triphosphate AS - 1: Additive solution 1 AS - 1N: Additive solution 1 (normoglycemic) CPD: Citrate - phosphate - dextrose solution CPD - N: Citrate - phosphate - dextrose (normoglycemic) CFTR: Cystic fibrosis transmembrane conductance regulator ERY: Erythrocyte solution i.v.: Intravenous INS - 1: Rat insulinoma beta cells ITC: Isothermal Titration Calorimetry NO: N itric oxide PBS: Phosphate buffered salt solution PSS: Physiological salt solution PSSH: P hysiological salt solution ( hyperglycemic ) 1 C hapter 1 - Introduction 1.1 Brief Introduction to Diabetes Mellitus Diabetes mellitus, commonly referred to as diabetes, is a metabolic disease characterized by chronic hyperglycemia due to defects in insulin biosynthesis, insulin efficacy, or both. 1 Insulin is a polypeptide hormone (MW of 5800 Da) produced by - cells that can promote the storage of blood glucose in skeletal muscle cells and fat cells. 2 Due to the inability of insulin to help clear glucose from the bloodstream, diabetic patients have a buildup of glucose in their bloodstream leading to hyperglycemic conditions, while also suffering from starvation cells that rely on insulin for glucose uptake. Hyperglycemia can lead to a variety of complications. For example, in the short term, patients can experience symptoms of increased thirst, hunger, and weight loss. However, in the lo ng term, more severe and damaging complications can develop, such as cardiovascular disease (including high blood pressure and stroke), retinopathy (which can lead to blindness), neuropathy (which can lead to amputation), and nephropathy (which can result in renal failure). These disorders and complications are contributing factors to diabetes as the seventh leading cause of death in the United States. 3 According to the National Diabetes Statistics Report in 2014, 21 million people in the United States are currently diagnosed with diabetes, and it is estimated that there are another 8.1 million undiagnosed diabetic patients, or about 9.3% of the US population. 4 The annual report by the International Diabetes Federation in 2009 showed that 285 million people worldwide have diabetes, and it was predicted that this 2 number will exceed 435 million in 2030. Among all risk factors, the correlation between obesity and the development of diabetes is expected to have a profound influence on the number of people diagnos ed with the disease in the future. There have been studies that describe the possible correlation between racial/ethnic differences and the diagnosis of diabetes. During 2010 - 2012 in the US, compared to non - hispanic white adults, the percent of people diag nosed with diabetes was 18% higher among Asian Americans, 66% higher among Hispanics, and 77% higher among non - Hispanic blacks. However, this correlation cannot be elucidated until data collection from a global survey is achieved. 3 There is a large economi c burden of diabetes on society. The National Diabetes Statistics Report estimated the cost of diabetes in the US in 2012 to be around $245 billion, with direct medical costs of $176 billion, and indirect costs (disability, work loss, premature death, etc. ) of $69 billion. 5 With the increasing incidence of diabetes, especially among younger people, the economic burden of diabetes will continue to increase unless necessary actions are taken to effectively control the onset of diabetes and its consequences, a therapies. 6 1.2 Diagnosis, Causes and Classification of Diabetes Mellitus A hallmark feature of diabetes is elevated blood glucose level; therefore, blood glucose testing is used for screening. Ac cording to the American Diabetes Association (ADA) 7 and the World Health Organization (WHO), 8 the diagnostic cutoff point for diabetes is a fasting plasma glucose level of 126 mg/dL (7.0 mM), the level at which a unique microvascular complication of diabet es, retinopathy, becomes detectable; or a 3 plasma glucose level of 200 mg/dL (11.1 mM) after an oral glucose tolerance test (OGTT). Though the OGTT has been reported to identify more cases of diabetes than the fasting plasma glucose test, due to greater exp enses, complexity, and reduced precision associated with the OGTT, the fasting plasma glucose test has been the preferred test in the US. 9 A diagnosis of diabetes is confirmed by an additional fasting plasma glucose test on a different day, or by other tes ts, such as an OGTT or glycated hemoglobin (HbA1c) level, which measures the percentage of glycated hemoglobin. HbA1c is typically elevated in diabetes, while a healthy individual has a HbA1c level of 6.5%. Pathologically, there are four major categories o f diabetes: type 1 diabetes, type 2 diabetes, gestational diabetes and other specific types of diabetes. 11 Determining the cause and type of diabetes of a patient can be difficult, depending on the circumstances present at the time of diagnosis, but it is important for the disease to be treated effectively. 11 Type 1 diabetes (T1D, also known as insulin - dependent or juvenile diabetes) is characterized by progressive immune - mediated destruction of insulin producing - cells, resulting in absolute insulin deficiency in the bloodstream. 11 Typ e 1 diabetes accounts for 5 10% of people with diabetes, and it is usually diagnosed in children and young adults, though it can occur in adults as well. 12 T1D is known to have - cells is determined by a combination of genetic, environmental and immune factors some of which are not very clear. 13 For example, although rare, obese T1D diabetic patients are prone to other 4 autoimmune disorders as well, such as Graves' disease and autoimmune hepatitis. 11 Viruses and other microbes seem to be associated with the pathogenesis of T1D as well, among which congenital rubella and childhood enterovirus (EV) infections have the strongest association. 14 - 16 Type 2 diabetes (T2D), the most common form of diabetes, ac counts for 90 95% of cases. T2D has been previously referred to as non - insulin - dependent diabetes. 17 The mechanisms unde rlying insulin resistance are not yet fully understood, however, studies have shown impaired insulin action in the skeletal muscle of T2D humans 18 Accumulation of specific lipid metabolites (diacylglycerols and/or ceramides) in liver and skeletal muscle h as also been reported to be related to impaired insulin signaling and insulin resistance. 19 Genetic susceptibility and poor lifestyle habits can both cause insulin resistance and lead to T2D. In fact, most patients with T2D have poor lifestyle habits such as consumption of unhealthy meals and lack of exercise. W eight loss and exercise can slow the progression from impaired glucose tolerance to T2D. 20 - cell dysfunction exists with insulin resistance in subjects with T2D when hyperglycemia develops. 21 There has been debate over the past few decades regarding the relative - cell dysfunction and insulin resistance. Many researchers have - cell dysfunction arises from the prolonged, increased secre tory demand - cells by insulin resistance. 22 - 23 In contrast, others have indicated 5 otherwise. 24 - 26 - cell and the insulin sensitive tissues as a regulated and communicating feedback system in order for glucose homeos tasis, - cell function are both found in the course of the development of T2D. 27 Gestational diabetes mellitus (GDM) is the third type of diabetes and develops during pregnancy. It is defined as any degree of glucose i ntolerance with onset or first recognition during pregnancy. 28 GDM affects between 2% and 5% of pregnant women; 29 and it represents nearly 90% of all pregnancies complicated by diabetes. 28 Most cases of GDM resolve with delivery, however, women diagnosed w ith GDM during pregnancy may continue to be hyperglycemic after delivery for different extents of time. Furthermore, GDM has a 10% risk of converting to T2D, with the greatest risk within the first 5 years following the pregnancy. 29 - 30 In addition to the three major types of diabetes described above, the American Diabetes Association (ADA) recognizes more than 56 other specific types of diabetes. 31 Some specific types are associated with insulin deficiency caused by non - immune - mediated injury to the pancre - cells. The forms of injury include diseases such as cystic fibrosis, pancreatitis, pancreatic resection, and hemochromatosis as well as injury by trauma. 31 Excess secretion of several hormones that oppose the action of insulin can also lead to diabe tes, such as cortisol, growth hormone, glucagon, and epinephrine. These hormones are capable of increasing hepatic glucose production and decreasing insulin sensitivity, thus leading to glucose intolerance. 31 - 32 Other than these types, 6 diabetes can also be - cell or insulin action, and by drugs or chemicals. 11 1.3 Current Treatments for Diabetes Mellitus Effective treatment for diabetes is decided according to the disease pathogenesis. T1D patients must monitor blood glucose and receive exogenous insulin. 33 In order to improve pharmacokinetics, the injected insulin molecule has been altered and insulin analogues are now available that can be absorbed more rapidly with decreased variability of insulin absorption (e.g., Humalogue and Aspart insulin). 34 - 36 Rapid - acting insulin analogues are usually administered with, or after, meals, either using pens or pumps. It is worth noting that insulin, since its discovery in 1921, has been thought to represent a cure for type 1 di abetes, it is not, in fact, a complete solution for the disease when considering the occurrences of severe diabetes associated complications that greatly increase morbidity and mortality of diabetic patients. 37 - 38 The Diabetes Control and Complications Trial (DCCT) research group has indicated the importance of strict metabolic control for the delay and prevention of severe complications. 39 There are other treatments for type 1 that can replace the need for insulin injections, including pancreas transplantation 40 and islet transplantation. 41 However, such limitations as lack of organ donors and the need for continuous immunosuppression following transplationation still remain. 33 In addition, use of stem - cell regeneration thera py of T1D is also being studied with greath interest in recent years. 42 In contrast, insulin therapy, is not usually the first choice for treating 7 T2D. It is suggested that newly diagnosed T2D patients be initially treated with diet and exercise, along wit h common oral T2D medications such as metformin. 43 When such initial treatment cannot meet the target blood glucose level, additional medications from different classes will be r ecommended. These medications fro m different classes have different working me chanisms to control blood glucose levels. For example, - cells to release more insulin; while thiazolidinediones can help muscle and fat tissue cells use insulin more effectively and may also reduce the blood glucose released by the l iver. Unfortunately, T2D patients may eventually need to take external insulin therapy due to the progressive nature of T2D of decreasing islet functions. 44 1.4 Diabetes Associated Complications 1.4.1 Acute Complications Both acute and chronic complication s can be developed in patients with diabetes. Common acute metabolic complications of diabetes include diabetic ketoacidosis (DKA), hyperosmolar non - ketotic coma (HNC), lactic acidosis (LA), and hypoglycemia. DKA is clinically defined by absolute insulin d eficiency causing hyperglycemia, and fat being released from fat cells and converted to ketoacids in the liver. DKA most commonly occurs in patients with T1D, although it can also occur in patients with T2D during a transition to insulin deficiency. HNC is clinically defined by the presence of relative insulin deficiency and hyperglycemia, with elevated serum osmolality, dehydration, and stupor, without the presence of ketosis or acidosis. HNC occurs in people with T2D, who have sufficient circulating insul in to prevent lipolysis and ketosis. It is a life threatening situation that can result in coma and even death. LA is a relatively 8 rare complication seen in diabetic patients. It is characterized by elevated lactic acid with acidosis, but without ketoacido sis. Hypoglycemia can occur in patients using insulin therapy or insulin releasing medications such as sulfonylurea. Hypoglycemia is very unlikely to occur in T2D patients who are only treated with lifestyle changes or blood sugar normalizing medications. 4 5 1.4.2 Chronic Complications Chronic diabetic complications often include vascular diseases, retinopathy, neuropathy, and nephropathy. Currently there are four main hypotheses linking hyperglycemia to diabetic complications: (1) increased polyol pathway f lux; (2) increased formation of advanced glycation end products (AGEs); (3) activation of protein kinase C (PKC) isoforms; and (4) increased hexosamine pathway flux. Thus far there has been no unifying hypothesis linking these four mechanisms. 46 Increased polyol pathway flux was the first hypothesized mechanism (1966) relating hypgerglycemia to diabetic complications. 47 As shown in Fig 1.1, this mechanism hypothesizes that when intracellular glucose levels become too high, aldose reductase reduces glucose t o sorbitol, consuming NADPH in the process. 48 NADPH is the essential cofactor for regenerating intracellular antioxidants, which are crucial for cellular health. By consuming NADPH, the polyol pathway increases susceptibility to intracellular oxidative str ess, and leads to osmotic vascular damage. Positive effects of aldose reductase inhibition on diabetic neuropathy has been shown in humans. 49 Increased polyol pathway flux was the first hypothesized mechanism (1966) relating hypgerglycemia to diabetic comp lications. 47 As shown in Fig 1.1, this mechanism 9 hypothesizes that when intracellular glucose levels become too high, aldose reductase reduces glucose to sorbitol, consuming NADPH in the process. 48 NADPH is the essential cofactor for regenerating intracell ular antioxidants, which are crucial for cellular health. By consuming NADPH, the polyol pathway increases susceptibility to intracellular oxidative stress, and leads to osmotic vascular damage. Positive effects of aldose reductase inhibition on diabetic n europathy has been shown in humans. 49 The second hypothesis, increased formation of AGEs was proposed in the late 1970s. According to this theory, intracellular proteins, including those involved in the regulation of gene transcription, are irreversibly mo dified with glucose. 50 - 51 AGE precursors can diffuse out of cells and modify extracellular matrix molecules, and thus cause cellular dysfunction. 52 - 53 In addition, these AGE precursors are also capable of modifying circulating proteins in the bloodstream, such as albumin. These modified proteins can then bind to AGE receptors, leading to production of inflammatory cytokines and growth factors, which in turn causes abnormal vascular pathology. 54 - 55 The mechanism of increased formation of AGEs is shown in Fig 1.2. Animal models have shown that pharmacologic inhibition of AGEs prevents late structural changes of experimental diabetic retinopathy. 56 10 Fig 1.1: Increased polyol pathway flux in hyperglycemia leading to diabetic complications. Ald ose reductase reduces aldehydes generated by reactive oxygen species (ROS) to inactive alcohols. When intracellular glucose levels become too high, aldose reductase reduces glucose to sorbitol, consuming NADPH in the process. Sorbitol dehydrogenase (SDH) o xidizes sorbitol to fructose using NAD + as a co - factor. When the activity of aldose reductase is sufficient to deplete reduced glutathione (GSH), intracellular oxidative stress is increased. 11 Fig 1.2: Hypothesis of increased formation of AGEs and di abetic complications. Intracellular proteins, including those involved in the regulation of gene transcription, are irreversibly modified with glucose. AGE precursors can diffuse out of cells and modify extracellular matrix molecules, and thus cause cellul ar dysfunction. AGE precursors are also capable of modifying circulating plasma proteins. These modified proteins can then bind to AGE receptors, leading to production of inflammatory cytokines and growth factors, which in turn causes abnormal vascular pat hology. 12 In the late 1980s and early 1990s, the third theory, hyperglycemia - induced activation of PKC isoforms, wa s proposed. In this theory, intracellular hyperglycemia increases the synthesis of diacylglycerol in the cell, activating isoforms of protein kinase - C, - - - 57 - 59 Activated PKC isoforms can impact gene expression in adverse ways, for ex ample, decreasing endothelial nitric oxide synthase (eNOS) while increasing the vasoconstrictor endothelin - 1. 60 - 61 Results from animal studies have shown that inhibition of PKC prevented early changes in the diabetic retina and kidney. 62 - 63 The fourth h ypothesis, increased hexosamine pathway flux was proposed in the late 1990s. 64 According to this hypothesis, as shown in Fig 1.3, excess intracellular glucose is metabolized through glycolysis and converted to glucose - 6 phosphate and then fructose - 6 phosphate. Next, some fructose - 6 - phosphate is converted by an enzyme called GFAT (glutamine:fructose - 6 phosphate amidotransferase) to form glucosamine - 6 phosphate which is finally converted to uridine diphosphate N - acetyl glucosamine, resulting in patho logic changes in gene expression. 65 - 67 As a result, expression of transforming g rowth factor - - 1 are both increased, giving rise to adverse effects in diabetic blood vessels. 68 The increased hexosamine pathway flux has been reported to play a role in hyperglycemia - induced abnormalities of glomerula r cel l gene expression, 65 and in hyperglycemia - induced cardiomyocyte dysfunction in cell culture, 69 both of which are important factors in the pathogenesis of diabetic complications. 64 13 Chronic diabetic complications can affect the whole body through stroke , blindness, heart attack, kidney failure, or even the need for amputation. These complications contribute to most of the cost of diabetes care, and greatly increase the morbidity rate. Chronic diabetic complications can be divided into microvasc ular complications (diabetic nephropathy, neuropathy, and retinopathy) and macrovascular complications (coronary artery disease, peripheral arterial disease, and stroke). 70 14 Fig 1.3: The hypothesis of increased hexosamine pathway flux and diabetic complications. Excess intracellular glucose is metabolized through glycolysis and converted to glucose - 6 phosphate (Gluc - 6 - P) and then fructose - 6 phosphate (Fruc - 6 - P). N ext, some Fruc - 6 - P is converted by an enzyme called GFAT (glutamine:fructose - 6 phosphate amidotransferase) to form glucosamine - 6 phosphate which is finally converted to uridine diphosphate N - acetyl glucosamine, resulting in pathologic changes in gene expre ssion. As a result, expression of transforming growth factor - - - 1 (PA - 1) are both increased, giving rise to adverse effects in diabetic blood vessels. 15 Diabetic retinopathy is responsible for approximately 10,000 new cases of blindness every year in the United States. 71 Most T1D patients develop retinopathy within 20 years of diagnosis, whereas for T2D patients, retinopathy may begin to develop as early as 7 years after diagnosis. 72 - 73 Formation of microaneurysms, thickenin g of basement membranes, and the loss of pericytes cause loss of vision through increased polyol pathway flux, where conversion of glucose into sorbitol causes sorbitol accumulation in cells, resulting in osmotic stress. Unfortunately, aldose reductase inh ibition treatment has been disappointing. 71,74 - 75 Cells are also thought to be injured by glycoproteins according to the AGE theory. 71 In addition, oxidative stress caused by high glucose levels can lead to cellular injury. Animal studies suggested that tr eatment with antioxidants, such as vitamin E, attenuated some vascular dysfunction by correcting retinal blood flow, but it did not alter the progression of retinopathy. 71,76 Growth factors including vascular endothelial growth factor (VEGF) have been foun d in increased levels in diabetic retinopathy, and suppressing VEGF production has been associated with less progression of retinopathy. 77 Diabetic retinopathy is generally classified as either background or proliferative. Background retinopathy includes small hemorrhages in the middle layers of the retina. Proliferative retinopathy is characterized by the formation of new blood vessels on the surface of the retina. If proliferation continues, blindness can occur through vitreous hemorrhage and tr action retinal detachment . 78 Diabetic nephropathy is the leading cause of renal failure in the United States, 71 16 and is associated with the greatest mortality of diabetic patients. 79 Nephropathy develops in 35 - 45% of T1D patients, and less than 20% of T2D p atients. 80 - 81 Within the first five years after the onset of diabetes, nephropathy begins with the development of microalbuminuria in patients with glomerular hyperfiltration. 82 - 83 In the next 5 - 10 years of diseases progression, the glomerular filtration r ate falls and overt proteinuria develops in patients who have end - stage nephropathy, which will and that will lead to renal failure. 84 It has been noticed that, with the progress of nephropathy, there is an increase in the glomerular basement membrane thi ckness, microaneurysm formation, mesangial nodule formation, and other changes, which involve some or all of the same mechanisms as diabetic retinopathy, including an increase in vascular endothelial growth factors (VEGF). 70,85 It has been reported that in addition to aggressive treatment of elevated blood glucose levels, patients with diabetic nephropathy also benefit from treatment with antihypertensive drugs. 70 Diabetic neuropathy is a type of nerve damage that occurs in patients with diabetes. It most o ften damages nerves in the legs and feet. As with other microvascular complications, risk of developing diabetic neuropathy is proportional to both the magnitude and duration of hyperglycemia, and is sometimes also affected by genetic attributes. 70 The nat ure of injury to the peripheral nerves from hyperglycemia is likely related to mechanisms such as polyol accumulation, injury from AGEs, and oxidative stress. 86 Diabetic neuropathy can be divided into several forms. The most common form is known as the chr onic sensorimotor distal symmetric polyneuropathy. Typically, 17 pain, but some patients only experience simple numbness. 87 Pure sensory neuropathy is a relatively rare form of diabetic neuropathy, which is characterized by isolated sensory findings without signs of motor neuropathy. 88 Mononeuropathies can involve any nerve, but most commonly affect the median, ulnar, and radial nerves. One major manifestation of diabetic mononeuropathy is diabetic amyotrophy that causes severe pain and muscle weakness, usually in large thigh muscles. 88 Diabetic autonomic neuropathy is another form of diabetic neuropathy that can cause significant morbidity and mortality in diabetic patien ts due to the neurological dysfunction in most organ systems. 88 Peripheral neuropathy can lead to loss of sensation and is the major cause of non - traumatic amputations. 89 Importantly, since neuronal ischemia is a well - established characteristic of diabetic neuropathy, vasodilatory agents (e.g., ACE - antagonists) that are able to improve blood supply to nerves have shown beneficial effects in increasing neuronal blood flow and in nerve conduction. 90 1.5 Biosynthesis of Insulin and C - peptide The pancreas is a multi - functional glandular organ in the human body. As an exocrine gland of the digestive system, the pancreas secretes pancreatic fluid that contains digestive enzymes that pass to the small intestine to help further metabolize carbohydrate s, proteins, and lipids. The pancreas is also an endocrine gland in which - - cells (secrete insulin, 18 which decreases b - - - - cells that secrete pancreatic polypeptide). 91 The biosynthesis of insulin and C - - cells. Insulin is initially manufactured at the r ibosome as a single chain polypeptide of 110 amino acid residues known as preproinsulin. The signal sequence is then cleaved to generate proinsulin across the membrane of the rough endoplasmic reticulum (RER). Proinsulin consists of chain A and chain B of insulin, which are connected by C - peptide, as shown in Fig 1.4. 92 Proinsulin is able to fold quickly into its native structure with the help of three disulfide bonds and is then transported to the Golgi apparatus via vesicular transfer and packaged into im mature secretory vesicles as hexamers, as shown in Fig 1.5. 93 As the vesicles become mature, proinsulin hexamers are converted into insulin hexamers by endopeptidases and carboxypeptidase, freeing the connecting peptide asC - peptide and yielding insulin hex amers. 94 Insulin and C - peptide are stored together in the secretion granules along with small amounts of intact proinsulin and other cleavage products, as well as zinc, until released, primarily in response to elevated blood glucose levels. C - peptide and i nsulin are released at equimolar concentrations into the bloodstream. 95 19 Fig 1.4: Amino acid sequence of human proinsulin. Huma n proinsulin is comprised of A chain, B chain, and a connecting peptide, known as C - peptide, that connects the two chains. A chain contains a disulfide bond, and A chain and B chain are linked together by two more disulfide bonds, which together form a spe cific conformation of proinsulin. C - peptide also stabilizes the structure of proinsulin. When cleaved by certain enzymes, segments of A chain and B chain in blue form a single molecule of insuliln, and C - peptide is freed. As shown, C - peptide and insulin ar e produced in equalmolar concentrations during the cleavage of proinsulin. 20 Fig 1.5: Maturation and exocytosis of insulin, C - peptide and zinc from beta cell granules. The biosynthesis of insulin and C - - cells granules. Pr oinsulin is produced as a precursor and is able to fold quickly into its native and is then transported to the Golgi apparatus via vesicular transfer and packaged into immature secretory vesicles as hexamers. The synthesis is highly controlled by pH condit ion and regulated by zinc. As the vesicles become mature, proinsulin hexamers are converted into insulin hexamers by endopeptidases and carboxypeptidase, freeing the connecting peptide as C - peptide and yielding insulin hexamers. Insulin and C - peptide are s tored together in the secretion granules along with small amounts of intact proinsulin and other cleavage products, as well as zinc, until released via exocytosis, primarily in response to elevated blood glucose levels. Insulin and C - peptide are released i n equimolar amounts with zinc simultaneously released. 21 Zinc and pH in the granules both play an indispensable role in the biosynthesis of insulin and C - peptide. 96 - cells, after proinsulin is produced, it is stored in its hexameric form. It has been reported that the Golgi has an aqueous environment containing high concentrations of zinc and calcium. 97 A specific zinc transporter, ZnT - 8 is localized in insulin secretory granules and transports zinc into th e granules to provide zinc for insulin processing, storage and secretion. One proinsulin hexamer is assembled with two zinc ions at its center that are coordinated by a histidine residue from each monomer. 98 The hexamer form is very stable and soluble, but inactive. The sign of the maturation of the secretory granules is characterized by a drop in pH value. 99 - 100 The membrane of these granules contains an ATP - dependent proton pump that regulates pH inside the vesicle. When the proinsulin hexamer is cleaved by endopeptidases and carboxypeptidases to form the insulin hexamer, the pH condition drops to around 5.5, which is the optimal pH of the enzymes. 96 Insulin hexamers can bind more zinc and this facilitates the crystallization of zinc - insulin so lid hexamers. This precipitation further increases the stability of the mature insulin, making it inaccessible to proteases. 101 Upon glucose stimulation, the interior of the granule undergoes a rapid pH change from 5.5 to 7.4. This pH change is necessary f or the dissolution and dissociation of the zinc - insulin hexamer when released from the granules into the bloodstream by exocytosis. 102 The monomeric insulin is the biologically active form of insulin, and when insulin and C - peptide are released from the - cells, a large amount of zinc is released as well. 95 22 - cell granules serves several functions during insulin biosynthesis: assembly of the proinsulin hexamers, increasing solubility of the proinsulin hexamer, and crystallization of insulin hexamers. Formation of Zn - insulin crystals is believed to protect the insulin molecule from proteolytic enzymes. In fact, the - cell is among the highest in the body (~20 mM). Zinc deficiency has been repor ted to lead to a lower production of insulin in some ZnT - 8 knockout mice studies, and it has also been linked to the progress of both type 1 and type 2 diabetes. 103 - 105 1.6 Biological Effects of Insulin Insulin was discovered in 1921 by Frederick Banting a nd J.J.R. Macleod, who were awarded the Nobel Prize in 1923 for the discovery and shared the prize with Charles Best and James Collip. 106 The primary sequence of insulin was determined in 1954 by Sanger and coworkers, 107 who were awarded the Nobel Prize in Chemistry in 1958. The tertiary structure of insulin was determined by X - ray crystallography in 1969. 108 Human insulin consists of two chains, the A - chain (21 amino acid residues) and the B - chain (30 amino acid residues), which are linked together by dis ulfide bonds as shown in Fig 1.1. It has a molecular weight of 5,808 Da. The amino acid sequence of insulin is strongly conserved between species. For example, bovine insulin differs from human insulin by only three amino acid residues, and porcine insulin by a single residue. - cell granules and released into the bloodstream primarily in response to increased blood glucose levels. Briefly, upon - cells throu gh 23 the glucose transporters, GLUT2 on the cell membrane. Glucose is then phosphorylated and subjected to glycolysis, producing adenosine triphosphate (ATP), and leading to a rise in the ratio of ATP/ADP (adenosine diphosphate). The ATP - sensi tive K + channels (KATP channels) close, which causes a buildup of intracellular potassium ions and thus membrane depolarization. The membrane depolarization opens voltage - gated Ca 2+ channels that allow Ca 2+ ions to enter into the cells by facilitated diffu sion. 109 - 113 Elevated Ca 2+ levels rapidly lead to insulin exocytosis from the storage secretory vesicles. It is important to note that in vivo , insulin secretion is finely tuned by a variety of stimulatory and inhibitory factors. 114 Insulin plays an indisp ensable role in regulating glucose homeostasis at many sites by reducing hepatic glucose output via decreasing gluconeogenesis and breakdown of glycogen , and by promoting glucose uptake, primarily into muscle and adipose tissue. 115 The uptake of glucose in to cells is achieved by facilitated diffusion. Briefly, the receptor for insulin is a heterotetrameric transmembrane protein located on the cell membrane of muscle and fat cells. This receptor belongs to the class of glycoproteins consisting of two insulin - - subunits) and two signal transduction - subunits). Binding of insulin to its receptor causes a conformational change - - Receptor autophosphorylation is activated, 116 - 117 which in turn phosphorylates other protein substrates beginning with insulin - receptor substrate (IRS) 1 and 2. 115,118 - 120 The phosphorylation network stimulates the lipid kinase activity of phosphatidylinositol 24 3 - kinase (PI3K), which activates several kinases, such as PI - dependent protein kinase - 1 and - 2, 121 Akt, 122 protein kinase C (PKC), 123 and wortmannin - sensitive and insulin - stimulated serine kinases. 124 Finally, translocation of an intracellular pool of GLUT4 to the plasma membran e occurs and glucose uptake is stimulated. 125 - 126 Glucose is then stored in muscle cells in the form of glycogen. Insulin also acts on the liver to force storage of glucose in there. The pathway of insulin signaling and GLUT4 translocation is shown in Fig 1.6. Insulin also has an active role in lipid metabolism. For example, insulin lowers the plasma fatty acid level by decreasing the rate of lipolysis in adipose tissue, 127 it increases fatty acid and triacylglycerol synthesis in tissues, 128 - 129 and increas es the uptake of triglycerides from the blood into adipose and muscle cells. Insulin also decreases the rate of fatty acid oxidation in muscle and liver. 130 In addition, insulin effect s protein metabolism by increasing protein synthesis in muscle, adipose tissue and the liver, and by decreasing the rate of protein degradation in muscle. 131 1.7 C - peptide 1.7.1 Discovery of C - peptide C - peptide was first discovered in 1967, 132 however, its bioactivity was unknown until the 1990s. C - peptide is a 31 amino acid p eptide that connects the A - and B - chains of insulin in its precursor proinsulin. After cleavage of proinsulin, C - peptide is freed and is co - - cells upon glucose stimulation. 25 For decades after the discovery of C - peptide, it was regarded as a biologically inert peptide, serving merely as a link that stabilized the two chains of insulin to facilitate the correct folding of proinsulin through disulfide bond formation during its biosynthesis. 133 C - peptide is released with insulin at equimolar concentrations into the circulati on and therefore was found to be a successful indicator for the secretion and hepatic extraction of insulin in a variety of clinical situations (in short, it is a biomarker for endogenous insulin production). 134 Direct measurement of insulin levels in the bloodstream can be very difficult due to its short half - life and the presence of circulating anti - insulin antibodies in insulin - treated diabetic patients. 135 - 136 26 Fig 1.6: The pathway of insulin signaling and GLUT4 translocation. The uptake of glucose into cells is achieved by facilitated diffusion. The receptor for insulin (IR) is located on the cell memb rane of muscle and fat cells. IR is consisted of two insulin - - - subunits). - subunits, - s autophosphorylation is activated, which in turn phosphorylates other protein substrates such as insulin - receptor substrate (IRS). Then, the lipid kinase activity of phosphatidylinositol 3 - kinase (PI3K) is stimulated, which activates several kinases including Akt, protein kinase B (PKB), and protein kinase C (PKC). Finally, translocation of an intracellular pool of GLUT4 to the plasma membrane occurs and glucose uptake is stimulated. Glucose is then stored in muscle ce lls in the form of glycogen. Insulin also acts on the liver to force storage of glucose in there. 27 The investigation into the possible insulin - like biological effects of C - peptide began soon after the discovery of the peptide, but unfortunately there was little success. Ever though the use of C - peptide was temporarily focused as a marker for endogenous insulin secretion, interest in the physiological role of C - p eptide persisted. It was reported - cell activity had a lower chance of developing chronic diabetic complications than those with total C - peptide deficiency. 137 - 138 Additionally, amelioration of diabetic neurop athy and nephropathy was also noticed in T1D patients whose insulin and C - peptide levels were both restored after receiving islet or pancreas transplantation. 139 - 140 These findings have served as motivation for researchers to continue their studies on the bioactivity of C - peptide and make it a treatment for diabetes. 1.7.2 Improvement of Nerve Function in Diabetes via C - peptide Replacement Experiments in both animals and humans have proven the potential of C - peptide supplementation in conjunction wit h insulin therapy in reducing neural and vascular complications caused by T1D. Thus it has been proposed that the lack of C - peptide may play a role in the development of diabetic complications. Both animal models and patients with T1D have de monstrated improvements in peripheral and autonomic nerve function, both after short term and long term treatment with C - peptide. Diabetic BB/Wor rats (a model of spontaneous T1D) were administered C - peptide at low physiological concentration (0.5 - 0.7 nM) from the onset of diabetes for 2 months and 8 months, and between 5 - 8 months. According to their results, 2 months of C - peptide replacement therapy upon the onset of diabetes resulted in a 59% partial 28 prevention of acute nerve conduction velocity (NCV) def ect in T1D rats compared to rats treated with placebo. In agreement with the improvement of NCV, the decreased neural Na + , K + - ATPase activity was also partially corrected by 55%, leading to a 66% improvement in the acute paranodal swelling as a result. 141 Since impaired blood flow and increased polyol - pathway activity 142 are believed to be leading causes of impaired NCV, 143 and can both have a profound adverse effect on Na + , K + - ATPase activity, 144 combined with C - blood flow, 145 the residual defects of NCV and Na + , K + - ATPase activity are probably due to the uncorrected elevated polyol - pathway activity. 142,146 C - peptide successfully prevented chronic diabetic polyneuropathy as well. After 8 months of C - peptide replac ement therapy, the chronic nerve conduction defects including axonal atrophy and degeneration were prevented by 71%. The axoglial dysjunction and paranodal demyelination were corrected completely. The loss of nerve function due to T1D can be irreversible. When the BB/Wor rats were treated after 5 months of onset of T1D, C - peptide treatment through month 8 showed a 13% improvement in NCV, and 33% improvement in the axoglial dysjunction. The neuroprotective role of C - peptide was also reflected by the correcti on of axonal degeneration and nerve fiber regeneration. C - peptide treatment from month 5 of onset of T1D through month 8 led to complete correction of paranodal dysjunction and axonal degeneration in rats. Nerve fiber regeneration was also increased 4 - fold , reported as fiber number, as a result of C - peptide treatment. 29 Similar C - peptide administration therapy was performed in streptozotocinon (STZ) - induced diabetic rats (a second model of T1D); 2 weeks of C - peptide replacement at week 6 after the onset of di abetes led to 62% and 78% corrections of the loss of sciatic motor NCV and saphenous sensory NCV, respectively. 147 Impaired sciatic blood flow and vascular conductance by diabetes were partially corrected after C - peptide treatment by 57 - 66%. It is worth no ting that the nitric oxide synthase inhibitor NG - nitro - Larginine (L - NNA) was given with C - peptide to another group of diabetic rats, and results showed that C - peptide effects on nerve conduction improvements were abolished, and corrections of sciatic blood flow and vascular conductance markedly attenuated, confirming the hypothesis that C - peptide effects are achieved through subsequent stimulation of nitric oxide (NO) - mediated vasodilation. It was indicated by the author that C - peptide could possibly influe nce the NO system through two mechanisms, with the first mechanism being direct stimulation of the activity and expression of nitric oxide synthase (NOS), 148 - 150 and the second mechanism being indirectly increasing blood flow via an increase in e ndothelial flow - induced NO production. 151 In this study, scrambled C - peptide was also tested in another group of diabetic rats and was shown to have no effect on nerve conduction or perfusion, suggesting the amino acid sequence of C - peptide plays an import ant role in its efficacy. Clinical studies in humans have also confirmed the beneficial effects of C - peptide in improving nerve functions. For example, a 3 - month C - peptide replacement therapy was shown to correct the initial reduction in sensory NCV in T1D patients with early - stage neuropathy by 80%. 152 In a larger clinical study, a 6 - month C - peptide replacement 30 therapy in T1D patients with neuropathy resulted in significant improvements in sensory NCV. It was also noticed that the positive effects of C - pep tide were most marked in the patients who had the least amount of neuropathy at baseline in the beginning of the study, which emphasizes the importance of early intervention in diabetic complications. Additionally, clinical neurological impairment and vibr ation perception were both improved in T1D patients treated with C - peptide. 153 C - peptide infusion in patients with T1D also demonstrated improvements in cardiac autonomic nerve function shown by increased heart rate variability (HRV), which is capable of d ecreasing the chances for cardiac arrhythmias and sudden death. 154 - 155 Fig 1.7 connects multiple factors that are believed to be involved in the beneficial effects of C - peptide on nerve dysfunction. 156 Nerve blood flow has been found to be substantially re duced in diabetic rats. 147,157 C - peptide replacement in these animals greatly improved endoneurial blood flow to a level comparable to normal, which is assumed to be due to the stimulation of endothelial nitric oxide synthase (eNOS), and increased NO bioav ailability. It was reported that C - peptide increased endothelial NO production in a concentration and time - dependent manner, and at physiological concentrations of C - peptide, endothelial NO production was more than doubled. Increased Ca 2+ influx into endot helial cells was found important to the stimulation of eNOS. 158 Blocking eNOS led to abolishment of C - peptide effects on both endoneurial blood flow and nerve conduction velocity. T1D also impairs the activity of Na + , K + - ATPase in peripheral nerve 31 tissue b y diminishing activity of Na + channels, causing intra - axonal sodium accumulation and swelling of the paranodal region. 156,159 It was reported that C - peptide replacement therapy in physiological concentrations was able to activate Na + channels and diminish paranodal swelling, therefore correcting nerve Na +, K + - ATPase activity and improving nerve function in diabetes. 159,141 Other researchers have observed that C - peptide replacement therapy also increased gene expression of several neurotropic factor s, including NGF, NT3, and IGF - I through increasing transcription factors such as c - fos and c - jun. As a result, thermal hyperalgesia as well as degeneration of unmyelinated fibers due to diabetes could be prevented, alleviating pains in patients caused by diabetic peripheral neuropathy. 160 - 161 3 2 Fig 1.7: Multiple pathways of C - peptide in improving nerve function in diabetes. C - peptide has been shown to increase eNOS activity, Na + ,K + - ATPas e activity and transcriptional factors, which will lead to improved nerve function in diabetes, alleviating pains in patients caused by diabetic peripheral neuropathy. Improved nerve function can be indicated by an increase in nerve conduction velocity and vibration perception, and decrease in axonal atrophy and demyelination. 33 1.7.3 Improvement of Kidney Function in Diabetes via C - peptide Replacement Diabetic nephropathy is the single leading cause of end - stage renal disease (ESRD), which leads to high morbidity and mortality. 162 Cur rent treatments for diabetic nephropathy include glycemic and blood pressurecontrol. However, the increasing prevalence of diabetes and obesity will continue to add to the cost and burden of diabetic nephropathy, suggesting an urgent need for development o f novel treatments. As mentioned previously, the early stage of diabetic nephropathy usually indicates glomerular hyperfiltration, characterized by increased glomerular filtration rate (GFR), glomerular hypertrophy, and microalbuminuria. 162 - 163 A bout 70% of human diabetic nephropathy is associated with elevated mean arterial pressure (MAP). As diabetic nephropathy further progresses, symptoms including proteinuria, nodular glomerulosclerosis, tubulointerstitial fibrosis, and a decline in GFR will appear, and eventually beco me an ESRD. 162 In recent years, the potential of C - peptide to be a novel therapeutic treatment for diabetic nephropathy has been uncovered. C - peptide prevents early nephropathy symptoms such as glomerular hyperfiltration, glomer ular hypertrophy and microlabuminuria. In an animal study to evaluate the acute effect of C - peptide on kidney function, STZ - induced diabetic rats were infused with C - peptide for over 2 weeks. As a result, C - peptide lowered GFR and urinary protein excretion (UPE) to a maximum amount level of 40 - 50%. The effect of C - peptide was dose - dependent and was not related to alterations of blood glucose levels. 164 Unresponsiveness of non - diabetic control rats to infused C - peptide was observed. In another group of anima l tests, the size of the glomeruli in the T1D rats treated with a 34 continuous 2 - week infusion of physiological doses of C - peptide was not significantly different than that of the normal control rats, and was smaller than that of non - C - peptide treated T1D r ats, proving the effects of C - peptide in preventing the development of glomerular hypertrophy. C - peptide replacement also significantly reduced glomerular hyperfiltration and albuminuria in T1D rats. 165 Fig 1.8: Multiple pathways of C - peptide in improving renal function in diabetes. C - peptide is able to reduce glomerular permeability, leading to decreased proteinuria. C - peptide h as also been shown to inhibit tubular sodium reabsorption, reducing afferent arteriolar diameter and GFR. A decrease in GFR further leads to decrease in proteinuria and prevention of the progression of glomerular hypertrophy. Both of reduction of proteinur ia and prevention of glomerular hypertrophy result in attenuation of microalbuminuria, glomerulosclerosis, and tubulointerstitial fibrosis, which are all improvements for renal function. 35 In human clinical trials, 21 patients with microal buminuria received either C - peptide injection or placebo for 3 months along with insulin replacement treatment. A time - dependent reduction in urine albumin excretion was observed in patients receiving C - peptide. However, C - peptide had no effect on GFR, whi ch was probably due to an already normal GFR in patients. 155 Other studies indicated that treatment of C - peptide in rats with elevated GFR attenuated further progression of glomerular hyperfiltration. 166 Interestingly, the C - terminal pentapeptide of rat C - peptide, EVARQ, showed similar beneficial effects on GFR and blood pressure to the full - length C - peptide, suggesting at least one active site within this region of C - peptide. 164,167 - 169 C - peptide is known to exert beneficial effects in diabetic nephropath y through several different mechanisms. The first mechanism states that C - peptide normalizes GFR via inhibiting tubular sodium reabsorption and constricting efferent arteries. 170 - 171 Other mechanisms of C - sing renal Na + , K + - ATPase activity, 170 enhancing eNOS expression and NO production, 158 inhibiting tumor necrosis factor (TNF) - - induced apoptosis, 172 and diminishing transforming growth factor (TGF) - - induced epithelial - to - mesenchymal transition. 173 Even though the aforementioned studies performed in T1D (insulin dependent) animal models o r humans were conducted in the presence of insulin , the renoprotective role of C - peptide was in fact, insulin independent. Experimental evidence shows that 2 weeks of C - peptide treatment in STZ - induced T1D rats that were not receiving insulin also attenua ted glomerular hyperfiltration and microalbuminuria in a similar way to the 36 rats receiving insulin; 166 and that C - peptide also had acute beneficial effects on the kidney without the presence of insulin. 165,171 The renoprotective role of C - peptide in T2D or nondiabetic renal disease was also studied by some groups, and the results were positive. For example, in a clinical study, T2D patients with higher levels of C - peptide demonstrated reduced chances of microvascular complications. 174 In another animal stud y, rats with chronic, nondiabetic renal disease were treated with C - peptide, and the rats showed reduced albuminuria, glomerular permeability, and renal inflammation, with unchanged levels of blood glucose or GFR, indicating that C - peptide is renoprotectiv e independent of blood glucose regulation. 175 Therefore, C - peptide may provide a potential benefit in the treatment of both diabetic and non - diabetic renal disease. 156 1.7.4 Cytoprotective Effects of C - peptide on the Endothelium Vascular diseases are commo n, chronic diabetic complications. They form by the combination of hyperglycemia and inflammatory responses generating oxidative stress and endothelial dysfunction. 176 - 177 T1D patients are known to have increased monocyte activity, a biomarker for inflamma tion and oxidative stress due to their ability to release pro - inflammatory cytokines, including interleukins (IL) IL - - 6 and IL - 8. The direct consequences of monocyte activation are unknown, but are known to be deleterious to the endothelium, which ca n involve activation of endothelial cells, altered endothelial function and monocyte adherence to vessel walls, eventually leading to vascular damage. In recent years, it has been reported by many researchers that C - 37 peptide is capable of modulating or elim inating inflammatory responses in diabetes. 156 C - peptide has been shown to lower circulating levels of the proinflammatory cytokines by inhibiting the activation of monocytes. 177 - 178 Among the proinflammatory cytokines, IL - 8 is important for the adhesion o f leukocytes to the endothelial wall. Another molecule, monocyte chemoattractant protein (MCP) - 1 is also required by leukocytes for adhesion. Using C - peptide to inhibit the secretion of both molecules during hyperglycemia, atherosclerosis plaque formation and endothelial dysfunction can be prevented. 179 - 181 Additionally, increasing evidence demonstrates that expression of several other adhesion proteins required by leukocyte attachment to endothelial cells, such as intercellular adhesion molecule (ICAM) - 1, vascular cellular adhesion molecule (VCAM) - 1, and P - selectin, can also be reduced through exposure to physiological levels of C - peptide. 156 In addition to reducing leukocyte attachment to endothelial cells, C - peptide is also able to promote re - endotheliali zation by significantly increasing endothelial cell numbe r , thereby limiting neointima formation in human saphenous vein. 182 Moreover, C - peptide has also been reported to affect the proliferation of vascular smooth muscle cells, another factor leading to v ascular disease and atherosclerotic plaque formation. However, there has been a debate upon C - peptide action. 156 While many groups claim that C - peptide inhibits hyperglycemia - induced proliferation of vascular smooth muscle cells, 182 - 184 another reports th e opposite. 185 Revisiting these experiments with strict control on glucose and C - peptide concentrations and the source of mooth muscle cells 38 would help uncover the exact role of C - peptide. 156 Other mechanisms of how C - peptide protects endothelial cells inc lude inhibition of the proapoptotic enzyme transglutaminase - 2 and activation of the major energy - sensing 186 restoring mitochondrial function and physiological reactive oxygen species (ROS) pr oduction in endothelial cells. 187 Although physiological levels of ROS play an important signaling role in the maintenance of cellular functions, excessive ROS production and accumulation in endothelial cells can cause cellular injury. 187 M any studies have shown that reduction of excessive intracellular ROS production by C - peptide is the key link to its anti - inflammatory effects. 188 - 190 For example, when human aortic endothelial cells were exposed to high glucose to induce intracellular NAD(P)H oxidase - dep endent ROS generation, cells that were also incubated with C - peptide showed quenched ROS production via decreased NAD(P)H oxidase activity and translocation of RAC - 1 from the cytoplasm to the membrane. 189 The function of C - peptide as an endogenous antioxid ant protected endothelial cells from high - glucose - induced apoptosis. Collectively, all of the aforementioned experimental results, both in animal models and in humans, have made C - peptide a compelling, novel therapeutic candidate for treating diabetes. Unf ortunately, the unclear molecular mechanism of C - peptide, including receptors involved in its anti - inflammatory effects, has become the major obstacle of C - 1.8 Theory of a C - Peptide Receptor 39 Despite C - treating diabetic complications, a receptor for C - peptide still remains undiscovered, which has become the major obstacle in the approval of C - peptide as a therapy for diabetes. However, the efforts of searching for a C - peptide receptor continue and provi de several ongoing theories. The evidence of the existence of a C - peptide receptor was first realized by the demonstration that C - peptide could bind to human cell membranes. Human C - peptide was labeled with rhodamine and used to study binding of C - peptide to human skin fibroblasts. Experiments displacing rhodamine - labeled C - peptide with unlabeled C - peptide demonstrated specific binding of the peptide to the cell membrane of fibroblasts, with a binding affinity of 3 x 10 9 M - 1 , suggesting the exi stence of a cell membrane receptor. C - peptide was shown to bind in the nanomolar range with saturation of approximately 0.9 nM, close to the physiological plasma concentration of C - peptide. 191 C - peptide has also been reported to specifically bind to renal tubular cells with a high affinity of 3.3 x 10 9 M - 1 , and to saphenous vein endothelial cells with a similar affinity of 2.0 x 10 9 M - 1 . The observation of specific binding of C - peptide to saphenous vein endothelial cells, but not to umbilical cord vein endo thelial cells, agrees with the finding that C - peptide stimulates NOS activity in aortic endothelial cells, but not in umbilical vein cells. Again, saturation of specific binding of C - peptide occurred in the nanomolar concentration range. 192 In an additiona l experiment, failure of scrambled C - peptide and D - enantio C - peptide to displace bound Rh - labeled C - peptide further provided evidence for C - peptide binding specificity with a stereospecific nature. 192 40 The C - terminal fragments of C - peptide are essential for C - activity, and have been shown to be replaceable for the full - length C - peptide in some binding processes and for biological effects. For example, the unlabeled C - terminal pentapeptide (EGSLQ) was able to displace bound Rh - labeled C - p eptide in the same manner as full - length C - peptide, suggesting the C - terminal pentapeptide is involved in the binding process. However, proinsulin containing the same pentapeptide segment fails to displace bound Rh - labeled C - peptide, indicating the C - term inus end of the segment needs to be free. 192 The C - terminal tetra - and penta - peptides were found to elicit 92 - 103% of the full - length C - + , K + - ATPase in renal tubular segments. Interestingly, the mid - segment of C - peptide als o exerted 36 - 80% of the full - length C - - (27 - 31) C - peptide showed no effect, indicating the length of the peptide segment may be a factor. 193 In another study where each amino acid in the pentapeptide (EGSLQ) was indivi dually replaced with alanine to compare the change in binding affinity to human renal tubular cell membranes, replacement of Glu27 with Ala abolished membrane binding, while replacement of Gly28 with Ala demonstrated little effect, and replacement of any o f the other three residues had intermediate effects. Surprisingly, free Glu amino acid displaced about 50% of membrane bound C - peptide. However, not all Glu - containing peptides compete with C - peptide binding. The author also noted the possibility of more than one receptor being involved in the mediation of C - peptide binding, and perhaps, function, which could explain why C - peptide as a specific ligand is difficult to establish. 194 41 Insulin, IGF - I, IGF - II, and proinsulin failed to displace specifically bound Rh - labeled C - peptide, and Rh - labeled insulin bound to cell membranes was not displaced by added unlabeled C - peptide. These experiments indicated that C - peptide does not seem to interact with the insulin receptor. 192 However, a conflict to this finding wa s found in another report, where C - peptide was shown to specifically bind to a proinsulin receptor with low affinity. 195 The most popular hypothesis on the existence of a C - peptide receptor thus far is that C - peptide probably directly interacts with a G protein coupled receptor (GPCR) or family of GPCRs. GPCRs are important integral membrane proteins found in the cell membranes of eukaryotes. GPCRs function by sensing molecules outside the cell and activating signal transduction pathways and cellula r responses as a result. GPCRs are involved in a wide variety of physiological processes, including regulation of the immune system and inflammation, autonomic nervous system transmission, the visual sense, the gustatory sense, etc. The activity of GPCRs c an be abolished by pertussis toxin, and many of the cellular actions of C - peptide are pertussis toxin sensitive as well. 192,172 In one study, an orphan GPCR, GPR146 was hypothesized to be the C - peptide receptor. 196 In this study, GPR146 was expressed in th e gastric tumor cell line KATOIII, and was essential for C - peptide - induced cFos expression in these KATOIII cel ls. The author reported that C - peptide was able to stimulate internalization of GPR146 in KATOIII cells, and facilitate colocalization between C - peptide and GPR146 on KATOIII cell membranes, strongly suggesting GPR146 is a candidate receptor for C - peptide. 42 Even though GPR146 appears to be the C - peptide receptor, other possibilities exist. It cannot be ruled out that GPR146 may be a co - receptor of the C - peptide receptor/receptors or part of a C - peptide signaling complex or signalosome. Fortunately, the specificity of GPR146 for C - peptide was confirmed by no alterations in the responsiveness of KATOIII cells to neuronostatin upon knockdown of GPR146. However, this study did not demonstrate any direct physical interaction between C - peptide and GPR146, which would be further confirmatory evidence for the functional interaction between the two molecules. Binding experiments need to be completed. 198 Invol vement of GPCR in C - specific binding of C - peptide to cell membranes was inhibited by pertussis toxin. 192 The initiation of multiple intracellular signaling cascades by C - peptide is usually associated with the activation of a GPCR. 197 For example, in a clinical study, C - peptide was infused in patients with T1D at a rate of 3 pmol/min/kg for 60 minutes, followed by a rate of 10 pmol/min/kg for another 60 minutes. Venous blood was collected from patients at 0, 60 and 120 min of infusion for determination of erythrocyte membrane Na + , K + - ATPase activity. Results demonstrated a linear relationship between plasma C - peptide levels and erythrocyte Na + , K + - ATPase activity during the infusion. 148 C - peptide activat ion of Na + , K + - ATPase has been shown to lead to enhanced erythrocyte deformability and ATP release from the cells. C - peptide also activates eNOS and is calcium dependent. 198 These mechanisms are all closely related to the observed microvascular effects of C - peptide in patients with T1D. C - peptide is also found able to 43 activate the family of mitogen - activated protein kinases (MAPKs). 197 MAPKs are serine threonine - specific kinases that respond to extracellular signals, and link cell - surface receptors, or chem ical and physical stresses to fundamental regulatory targets within cells. The MAPK family includes the ERKs 1 and 2, c - Jun N - terminal kinases, p38s, and ERK5. 199 Phosphorylation activation of ERK 1 and 2 in Swiss 3T3 cells by C - peptide was detected and was time and dose dependent, with the maximum activation at 1 min and at 1 nM C - peptide. Similar to the activation of Na + , K + - ATPase, the activation of ERKs by C - peptide in this study was also abolished by pertussis toxin, suggesting the involvement of GPC R(s). 200 There have been other theories existing in the literature that point against the notion that C - peptide interacts with the cells through a specific receptor. 156 For example, the internalization and localization of C - peptide to the cytosol of HEK 293 and Swiss 3T3 cells, 201 the interaction of C - peptide with intracellular protein tyrosine phosphatase 1B, 202 - enolase that can localize to the cell membrane. 203 Additionally, another group reported that C - peptide could act via nonchiral interactions with the cell membrane instead of stereospecific receptors or binding sites based on the observation that synthetic reverse sequence (retro) and all - D - amino acid (enantio) C - peptides were equally bioactive in preventing vascular dysfunction compared to native C - peptide. 159 1.9 Molecular Mechanism of Zn 2+ Activated C - peptide on Improving Blood Flow 44 In support of the literature concerning C - related complications by improving blood flow in patients via increasing NO bioavailability in the bloodstream, the Spence group has proposed a pathway in which C - peptide is able to increase NO production from vascular endothelial cells, which can potentially lead to improved blood flow and reduced diabe tic complications. As shown in Fig 1.9, the Spence group has found that C - peptide, in the presence of a transition metal, significantly increases ATP release from erythrocytes (ERYs). The ATP molecules released by ERYs play an important role in regulating blood flow. ATP diffuses to endothelial cells and stimulates endothelial NO production. The Spence group hypothesizes that the increased level of NO bioavailability by metal - bound C - peptide in the bloodstream is an important mechanism of C - tive role in maintaining blood vessel health and correcting microvascular diseases in diabetes. 204 - 205 45 Fig 1.9: Proposed mechanism of Zn 2+ - C - peptide improving vascul ar health via ATP regulation. C - peptide, in the presence of Zn 2+ , increases ATP release from ERYs under flowing condition, which then increases NO production from the endothelium. NO is a well - known blood vasodilator. An increase in NO levels in the bloods tream leads to smooth muscle cells relaxation and vasodilaton. As a result, blood flow is increase. According to result from the Spence group, C - peptide does not have its efficacy without the presence of zinc and serum albumin. 46 However, in contrast to other reports, the Spence group has not seen any biological effects of pure C - peptide unless it is co - administered with a metal ion, such as Cr 3+ , Zn 2+, or Fe 2+.204 - 205 Meyer et. al . has reported that C - peptide in the presence of Fe 2+ as an impurity in the commercially available C - peptide product, significantly increased ATP release from rabbit ERYs. This increase of ATP rel ease reached approximately 2.9 - fold over a period of 8 hours of incubation. The interaction of C - peptide and Fe 2+ was verified by mass spectrometry. Fe 2+ bound to C - peptide (Fe 2+ - C - peptide) was then tested on ERYs obtained from T2D patients, whose ERY - deri ved ATP release may be less than that of healthy controls due to increased oxidative stress within the ERYs leading to decreased cell deformability. The results showed that after 6 hours of incubation, Fe 2+ - C - peptide restored ATP release from the ERYs of T 2D patients to a level that was statistically equivalent to that of healthy controls. At the same time, Fe 2+ - C - peptide was found to double the ATP release from healthy ERYs. C - peptide in the presence of Cr 3+ (Cr 3+ - C - peptide) increased ERY - derived ATP releas e similar to Fe 2+ , except that the activity was maintained for 72 hours, which did not occur with Fe 2+ . This discrepancy could be explained by the fact that Fe 2+ usually has a faster exchange rate than Cr 3+ for ligand binding, which means that Fe 2+ will bi nd to C - peptide for a shorter period of time. Additionally, ERY in the presence of phloretin, a GLUT1 (glucose transporter on the ERY membrane) inhibitor, abolished the increase of ERY - derived ATP release by metal - C - peptide. Furthermore, metal - C - peptide si gnificantly increased 47 glucose transport into the ERYs. Together, these results suggested that C - peptide increases ERY - derived ATP release due to increased glycolysis within the ERY via increased cellular glucose transport through GLUT1. 204 Zn 2+ was another metal ion that was found to be able to activate C - peptide and increase ERY - derived ATP release. Zn 2+ - C - peptide had similar beneficial effects on ERYs from healthy rats by increasing ATP release by about 80%. However, ERYs from T2D rats demonstrated an app arent resistance to Zn 2+ - C - peptide, exerting only 31% increase in ATP release, less than half of the increase seen in healthy ERYs. This suggested that T2D patients can have both insulin resistance and C - peptide resistance. In addition to ATP release, simi lar resistance was also observed with glucose uptake by ERYs. Zn 2+ - C - peptide only increased glucose accumulation by about 36% in T2D ERYs, compared to 65% increase in healthy ERYs. The resistance of T2D ERYs to C - peptide can be corrected by metformin. Metf ormin is one of the most widely prescribed drugs to treat T2D. It has been reported in the literature that metformin can increase glucose transport into ERYs in diabetic patients and improve blood flow as a result. 206 - 207 This has been thought to be associ ated with increased fluidity of the cell membranes. 208 - 209 Results by Meyer et al. showed that 2 hour pretreatment of ERYs from T2D rats with metformin prior to 4 hour incubation with Zn 2+ - C - peptide resulted in an increase of ATP release that was not signi ficantly different than that of healthy ERYs. Additionally, metformin also reversed the defect of increase of glucose uptake by Zn 2+ - 48 C - peptide in T2D ERYs. It was worth noting that metformin by itself, or metformin with C - peptide alone or Zn 2+ alone did not significantly increase ATP release and glucose uptake by either healthy or diabetic ERYs. 205 It has been demonstrated that hyperglycemia causes externalization of phosphatidylserine (PS) from the inner leaflet to the outer leaflet of the ERY cell membrane, which can lead to cell apoptosis. 210 - 211 PS and C - peptide are both negatively charged, and the increased appearance of PS in ERY cell membrane is thought to further repulse C - peptide binding. Even though the mechanism of how metformin reesta blished the biological effects of C - peptide on diabetic ERYs is not fully understood, it may be associated with a change in the amount of negatively charged PS in cell membrane, thereby facilitating C - with the diabetic ERY membrane. 20 5 Importantly, these biological effects of C - peptide were not achieved in the presence of insulin, which seem to create a conflict against most of previous reports about the necessity of insulin for the beneficial effects of C - peptide in vivo . However, sin ce pure C - peptide does not have any biological effects without the presence of a metal according to this data, and the sources of insulin used in previous C - peptide studies by other groups were not clearly demonstrated to be free of metal contamination, th e co - administration of insulin in those reports may have accidentally introduced Zn 2+ to the ERY samples leading to C - pure C - peptide is necessary. Among these transition metals, Zn 2+ has the h ighest potential of activating C - peptide in vivo , because of its high abundance and co - localization with C - peptid e and insulin in 49 - cells. Even though how Zn 2+ is involved in the stimulation of ERY - derived ATP release is not understood, there are other reports that support the idea that Zn 2+ plays an important role in the treatment and prevention of di abetes. The concentration of Zn 2+ - cell granules can reach millimolar levels. Insulin is produced in a solid hexamer around Zn 2+ , and two Zn 2+ ions are required by every insulin hexamer. 212 - 214 Interestingly, a relationship between Zn 2+ and in sulin was established even before the role of Zn 2+ - cells for insulin production was well understood. It was reported that the effect of insulin would be extended by the addition of zinc. 215 Zn 2+ has also been reported to be involved in the proper function for some important antioxidant enzymes, and thus affects the cellular oxidant status of people - cells in T1D patients can cause in less bioavailability of zinc for such antioxid ant enzymes and eventually lead to free radical production and tissue damage. 215 - 216 Zinc bioavailability may also play a role in the progression of T2D. Since in the early stage of T2D, there is increased secretion of insulin and therefore zinc as well. T his accelerated depletion of zinc from the pancreas can possibly lead to islet dysfunction and destruction. 215,217 Unfortunately, a solid mechanism for the role of zinc remains unclear. Due to the colocation of Zn 2+ and C - - cells, it is hypothesized that Zn 2+ activates C - - cells granules before being released into the bloodstream and having physiological effects. C - peptide has five 50 acidic residues in its sequence at p osition 1, 3, 4, 11 and 27 which can possess electrostatic interactions with Zn 2+ ions. The importance of these five residues was evaluated by the Spence group, using C - peptide mutants where each of the residues was replaced by alanine, referred to as the E1A, E3A, D4A, E11A and E27A mutants. C - peptide and zinc interaction was first studied by mass spectrometry, and it was found that substitution of any of the five acidic residues led to a decrease in overall Zn 2+ ion interaction. Biological experimental da ta indicated that substitution of any one of the acidic residues resulted in a reduction in ATP release by nearly 50%, with the E27A mutant yielding the greatest reduction. Furthermore, full activity for ATP stimulation was observed with incubation of ERYs with the C - terminal pentapeptide segment EGSLQ, whereas no activity was observed with mutant AGSLQ. 218 These results not only suggested that all of the five acidic residues of C - peptide are important for Zn 2+ - al activity of the C - terminal pentapeptide containing Glu27, which is consistent with previous findings. In the effort to understand how C - peptide and Zn 2+ change the metabolism of ERYs and if this biological effect is due to a specific membrane receptor, cellular uptake of C - peptide and Zn2+ was studied in the Spence group. Results suggest C - peptide and Zn2+ uptake by ERYs are both required for the subsequent increase of ATP release. In addition, cellular uptake of C - peptide seems to be a prerequisite for the uptake of Zn 2+ ions, where the opposite is not the case. In this dissertation, the interaction of C - peptide and Zn 2+ with 51 the ERYs and subsequent biological effects will be discussed as assistance in further understanding the mechanism of C - peptide a ctivity in order to support C - peptide as a therapeutic agent in diabetes treatment. Results suggest both C - peptide and Zn 2+ can interact with serum albumin through specific binding under physiological condition. Albumin seems to function as a transporter f or both C - peptide and Zn 2+ , and deliver them to the ERY to be utilized by the cell. C - been evaluated, which again points out the importance of a normoglycemic environment for maintaining the health of ERYs. Additionally, the effect of leptin on ERY - derived ATP release was also investigated, showing enhanced C - peptide activity. Furthermore, when measuring ATP release from ERY samples, instead of applying traditional ERY sample preparation in test tubes, ERY samples were flowed on a novel 3D - printed fluidic device in a way of mimicking real blood flow. 52 REFERENCES 53 REFERENCES 1. Gepts, W., Pathologic anatomy of the pancreas in juvenile diabetes mellitus. Diabetes 19 65, 14 (10), 619 - 33; 2. Pedersen, J. S.; Hansen, S.; Bauer, R., The aggregation behavior of zinc - free insulin studied by small - angle neutron scattering. European biophysics journal: EBJ 1994, 22 (6), 379 - 89. 3. Directors, N. A. o. C. D., Diabetes Prevent ion and Control Program FY 2015 Appropriations Fact Sheet. 2015 . 4. Promotion, N. C. f. C. D. P. a. H ., National Diabetes Statistics Report. 2014 . 5. American Diabetes, A., Economic costs of diabetes in the U.S. in 2012. Diabetes care 2013, 36 (4), 10 33 - 46. 6. Herman, W. H., The economic costs of diabete s: is it time for a new treatment paradigm? Diabetes care 2013, 36 (4), 775 - 6. 7. Report of the Expert Committee on the Diagnosis and Classification of Diabetes Mellitus. Diabetes care 1997, 20 (7), 118 3 - 97. 8. Alberti, K. G.; Zimmet, P. Z., Definition, diagnosis and classification of diabetes mellitus and its complications. Part 1: diagnosis and classification of diabetes mellitus provisional report of a WHO consultation. Diabetic medicine : a journal o f the British Diabetic Association 1998, 15 (7), 539 - 53. 9. Inzucchi, S. E., Diagnosis of diabetes. The New Eng land journal of medicine 2013, 368 (2), 193. 10. Nathan, D. M.; Balkau, B.; NBonora, E., International Expert Committee report on the role of the A1C assay in the diagnosis of diabetes. Diabetes care 2009, 32 (7), 1327 - 34. 11. American Diabetes, A., Diagnosis and classification of diabetes mellitus. Diabetes care 2013, 36 Suppl 1 , S67 - 74. 12. C orvilain, J.; Gepts, W.; Beghin, P.; Vis, H.; Ver banck, M.; Verniory, A., [Renal functional and histoenzymologic exploration in a case of Toni - Debre - Fanconi syndrome]. Journal d'urologie et de nephrologie 1965, 71 (4), 354 - 70. 13. Li, M.; Song, L. J.; Qin, X. Y., Advances in the cellular immunological pathogenesis of type 1 diabetes. Journal of cellular and molecular medicine 2014, 18 (5), 749 - 58. 14. Hober, D.; Sauter, P., Pathogenesis of type 1 diabetes mellitus: interplay between 54 enterovirus and host. Nature reviews. Endocrinology 2010, 6 (5), 279 - 8 9. 15. Viskari, H.; Paronen, J.; Keskinen, P.; Simell, S.; Zawilinska, B.; Zgorniak - Nowosielska, I.; Korhonen, S.; Ilonen, J.; Simell, O.; Haapala, A. M.; Knip, M.; Hyoty, H., Humoral beta - cell autoimmunity is rare in patients with the congenital rubella s yndrome. Clinical and experimental immunology 2003, 133 (3), 378 - 83. 16. Kondrashova, A.; Hyoty, H., Role of viruses and other microbes in the pathogenesis of type 1 diabetes. International reviews of immunology 2014, 33 (4), 284 - 95. 17. Fowler, G. C.; Vas udevan, D. A., Type 2 diabetes mellitus: managing hemoglobin A(1c) and beyond. Southern medical journal 2010, 103 (9), 911 - 6. 18. Choi, K.; Kim, Y. - B., Molecular Mechanism of Insulin Resistance in Obesity and Type 2 Diabetes. The Korean Journal of Internal Medicine 2010, 25 (2), 119 - 29. 19. Samuel, V. T.; Shulman, G. I., Integrating Mechanisms for Insulin Resistance: Common Threads and Missing Links. Cell 2012, 148 (5), 852 - 71. 20. Knowler, W. C.; Barrett - Connor, E.; Fowler, S. E.; Hamman, R. F.; Lachin, J. M.; Walker, E. A.; Nathan, D. M.; Diabetes Prevention Program Research, G., Reduction in the incidence of type 2 diabetes with lifestyle intervention or metformin. The New England journal of medicine 2002, 346 (6), 393 - 403. 21. Kahn, S. E.; Porte, D., Jr. , Pathophysiology of type II diabetes mellitus. In: Porte D Jr, Sherwin RS (eds) Diabetes mellitus. . Appleton and Lange, Stamford 1996 , 487 - 512. 22. DeFronzo, R. A.; Ferrannini, E., Insulin resistance. A multifaceted syndrome responsible for NIDDM, obesit y, hypertension, dyslipidemia, and atherosclerotic cardiovascular disease. Diabetes care 1991, 14 (3), 173 - 94. 23. Kruszynska, Y. T.; Olefsky, J. M., Cellular and molecular mechanisms of non - insulin dependent diabetes mellitus. Journal of investigative med icine : the official publication of the American Federation for Clinical Research 1996, 44 (8), 413 - 28. 24. Porte, D., Jr., Banting lecture 1990. Beta - cells in type II diabetes mellitus. Diabetes 1991, 40 (2), 166 - 80. 25. Mitrakou, A.; Kelley, D.; Mokan, M .; Veneman, T.; Pangburn, T.; Reilly, J.; Gerich, J., Role of reduced suppression of glucose production and diminished early insulin release in impaired glucose tolerance. The New England journal of medicine 1992, 326 (1), 22 - 9. 26. Kahn, S. E., Clinical r eview 135: The importance of beta - cell failure in the development and progression of type 2 diabetes. The Journal of clinical endocrinology and metabolism 2001, 86 (9), 4047 - 58. 55 27. Kahn, S. E., The relative contributions of insulin resistance and beta - cel l dysfunction to the pathophysiology of Type 2 diabetes. Diabetologia 2003, 46 (1), 3 - 19. 28. American Diabetes, A., Diagnosis and classification of diabetes mellitus. Diabetes care 2006, 29 Suppl 1 , S43 - 8. 29. Gilmartin, A. B.; Ural, S. H.; Repke, J. T., Gestational diabetes mellitus. Reviews in obstetrics & gynecology 2008, 1 (3), 129 - 34. 30. Association, A. D., Diagnosis and classification of diabetes mellitus. Diabetes care 2013, 36 Suppl 1 , S67 - 74. 31. Fowler, M. J., Classification of Diabetes: No t All Hyperglycemia is the Same. Clinical Diabetes 2007, 25 (2), 74 - 6. 32. Leibowitz, G.; Tsur, A.; Chayen, S. D.; Salameh, M.; Raz, I.; Cerasi, E.; Gross, D. J., Pre - clinical Cushing's syndrome: an unexpected frequent cause of poor glycaemic control in o bese diabetic patients. Clinical endocrinology 1996, 44 (6), 717 - 22. 33. Atkinson, M. A.; Eisenbarth, G. S., Type 1 diabetes: new perspectives on disease pathogenesis and treatment. Lancet 2001, 358 (9277), 221 - 9. 34. Vajo, Z.; Duckworth, W. C., Geneticall y engineered insulin analogs: diabetes in the new millenium. Pharmacological reviews 2000, 52 (1), 1 - 9. 35. Garg, S. K.; Anderson, J. H.; Perry, S. V.; Mackenzie, T.; Keith, P.; Jennings, M. K.; Hansen, M. M.; Chase, H. P., Long - term efficacy of humalog in subjects with Type 1 diabetes mellitus. Diabetic medicine : a journal of the British Diabetic Association 1999, 16 (5), 384 - 7. 36. Mortensen, H. B.; Lindholm, A.; Olsen, B. S.; Hylleberg, B., Rapid appearance and onset of action of insulin aspart in paedi atric subjects with type 1 diabetes. European journal of pediatrics 2000, 159 (7), 483 - 8. 37. Matsushima, M.; LaPorte, R. E.; Maruyama, M.; Shimizu, K.; Nishimura, R.; Tajima, N., Geographic variation in mortality among individuals with youth - onset diabete s mellitus across the world. DERI Mortality Study Group. Diabetes Epidemiology Research International. Diabetologia 1997, 40 (2), 212 - 6. 38. Podar, T.; Solntsev, A.; Reunanen, A.; Urbonaite, B.; Zalinkevicius, R.; Karvonen, M.; LaPorte, R. E.; Tuomilehto, J., Mortality in patients with childhood - onset type 1 diabetes in Finland, Estonia, and Lithuania: follow - up of nationwide cohorts. Diabetes care 2000, 23 (3), 290 - 4. 39. Retinopathy and nephropathy in patients with type 1 diabetes four years after a trial of intensive therapy. The Diabetes Control and Complications Trial/Epidemiology of Diabetes Interventions and Complications Research Group. The New England journal of medicine 2000, 342 (6), 381 - 9. 56 40. Robertson, R. P., Prevention of recurrent hypoglycemi a in type 1 diabetes by pancreas transplantation. Acta diabetologica 1999, 36 (1 - 2), 3 - 9. 41. Shapiro, A. M.; Lakey, J. R.; Ryan, E. A.; Korbutt, G. S.; Toth, E.; Warnock, G. L.; Kneteman, N. M.; Rajotte, R. V., Islet transplantation in seven patients with type 1 diabetes mellitus using a glucocorticoid - free immunosuppressive regimen. The New England journal of medicine 2000, 343 (4), 230 - 8. 42. Calafiore, R.; Basta, G., Stem cells for the cell and molecular therapy of type 1 diabetes mellitus (T1D): the ga p between dream and reality. American journal of stem cells 2015, 4 (1), 22 - 31. 43. Gerich, J.; Raskin, P.; Jean - Louis, L.; Purkayastha, D.; Baron, M. A., PRESERVE - beta: two - year efficacy and safety of initial combination therapy with nateglinide or glybur ide plus metformin. Diabetes care 2005, 28 (9), 2093 - 9. 44. American Diabetes, A., (7) Approaches to glycemic treatment. Diabetes care 2015, 38 Suppl , S41 - 8. 45. Fishbein, H.; Palumbo, P. J., Chapter 13 Acute Metabolic Complications in Diabetes. 1995 , 283 - 92. 46. Brownlee, M., Biochemistry and molecular cell biology of diabetic complications. Nature 2001, 414 (6865), 813 - 20. 47. Gabbay, K. H.; Merola, L. O.; Field, R. A., Sorbitol pathway: presence in nerve and cord with substrate accumulation in diabetes. Science 1966, 151 (3707), 209 - 10. 48. Lee, A. Y.; Chung, S. S., Contributions of polyol pathway to oxidative stress in diabetic cataract. FASEB journal : official publication of the Federation of American Societies for Experimental Biology 1999, 13 (1), 23 - 30. 49 . Greene, D. A.; Arezzo, J. C.; Brown, M. B., Effect of aldose reductase inhibition on nerve conduction and morphometry in diabetic neuropathy. Zenarestat Study Group. Neurology 1999, 53 (3), 580 - 91. 50. Giardino, I.; Edelstein, D.; Brownlee, M., No nenzymatic glycosylation in vitro and in bovine endothelial cells alters basic fibroblast growth factor activity. A model for intracellular glycosylation in diabetes. The Journal of clinical investigation 1994, 94 (1), 110 - 7. 51 . Shinohara, M.; Thornalley, P. J.; Giardino, I.; Beisswenger, P.; Thorpe, S. R.; Onorato, J.; Brownlee, M., Overexpression of glyoxalase - I in bovine endothelial cells inhibits intracellular advanced glycation endproduct formation and prevents hyperglycemia - induced increases in macro molecular endocytosis. The Journal of clinical investigation 1998, 101 (5), 1142 - 7. 52. McLellan, A. C.; Thornalley, P. J.; Benn, J.; Sonksen, P. H., Glyoxalase system 57 in clinical diabetes mellitus and correlation with diabetic complications. Clinical scie nce 1994, 87 (1), 21 - 9. 53. Charonis, A. S.; Reger, L. A.; Dege, J. E.; Kouzi - Koliakos, K.; Furcht, L. T.; Wohlhueter, R. M.; Tsilibary, E. C., Laminin alterations after in vitro nonenzymatic glycosylation. Diabetes 1990, 39 (7), 807 - 14. 54. Li, Y. M.; Mit suhashi, T.; Wojciechowicz, D.; Shimizu, N.; Li, J.; Stitt, A.; He, C.; Banerjee, D.; Vlassara, H., Molecular identity and cellular distribution of advanced glycation endproduct receptors: relationship of p60 to OST - 48 and p90 to 80K - H membrane proteins. P roceedings of the National Academy of Sciences of the United States of America 1996, 93 (20), 11047 - 52. 55. Vlassara, H.; Brownlee, M.; Manogue, K. R.; Dinarello, C. A.; Pasagian, A., Cachectin/TNF and IL - 1 induced by glucose - modified proteins: role in nor mal tissue remodeling. Science 1988, 240 (4858), 1546 - 8. 56. Hammes, H. P.; Martin, S.; Federlin, K.; Geisen, K.; Brownlee, M., Aminoguanidine treatment inhibits the development of experimental diabetic retinopathy. Proceedings of the National Academy of S ciences of the United States of America 1991, 88 (24), 11555 - 8. 57 . Koya, D.; King, G. L., Protein kinase C activation and the development of diabetic complications. Diabetes 1998, 47 (6), 859 - 66. 58. Derubertis, F. R.; Craven, P. A., Activation of protein kinase C in glomerular cells in diabetes. Mechanisms and potential links to the pathogenesis of diabetic glomerulopathy. Diabetes 1994, 43 (1), 1 - 8. 59. Xia, P.; Inoguchi, T.; Kern, T. S.; Engerman, R. L.; Oates, P. J.; King, G. L., Characterization of th e mechanism for the chronic activation of diacylglycerol - protein kinase C pathway in diabetes and hypergalactosemia. Diabetes 1994, 43 (9), 1122 - 9. 60. Koya, D.; Jirousek, M. R.; Lin, Y. W.; Ishii, H.; Kuboki, K.; King, G. L., Characterization of protein k inase C beta isoform activation on the gene expression of transforming growth factor - beta, extracellular matrix components, and prostanoids in the glomeruli of diabetic rats. The Journal of clinical investigation 1997, 100 (1), 115 - 26. 61. Kuboki, K.; Jia ng, Z. Y.; Takahara, N.; Ha, S. W.; Igarashi, M.; Yamauchi, T.; Feener, E. P.; Herbert, T. P.; Rhodes, C. J.; King, G. L., Regulation of endothelial constitutive nitric oxide synthase gene expression in endothelial cells and in vivo : a specific vascular a ction of insulin. Circulation 2000, 101 (6), 676 - 81. 62. Ishii, H.; Jirousek, M. R.; Koya, D.; Takagi, C.; Xia, P.; Clermont, A.; Bursell, S. E.; Kern, T. S.; Ballas, L. M.; Heath, W. F.; Stramm, L. E.; Feener, E. P.; King, G. L., Amelioration of vascular dysfunctions in diabetic rats by an oral PKC beta inhibitor. Science 1996, 272 (5262), 728 - 31. 58 63. Koya, D.; Haneda, M.; Nakagawa, H.; Isshiki, K.; Sato, H.; Maeda, S.; Sugimoto, T.; Yasuda, H.; Kashiwagi, A.; Ways, D. K.; King, G. L.; Kikkawa, R., Amelior ation of accelerated diabetic mesangial expansion by treatment with a PKC beta inhibitor in diabetic db/db mice, a rodent model for type 2 diabetes. FASEB journal : official publication of the Federation of American Societies for Experimental Biology 2000, 14 (3), 439 - 47. 64. Brownlee, M., The pathobiology of diabetic complications: a unifying mechanism. Diabetes 2005, 54 (6), 1615 - 25. 65. Kolm - Litty, V.; Sauer, U.; Nerlich, A.; Lehmann, R.; Schleicher, E. D., High glucose - induced transforming growth fact or beta1 production is mediated by the hexosamine pathway in porcine glomerular mesangial cells. The Journal of clinical investigation 1998, 101 (1), 160 - 9. 66. Sayeski, P. P.; Kudlow, J. E., Glucose metabolism to glucosamine is necessary for glucose stimu lation of transforming growth factor - alpha gene transcription. The Journal of biological chemistry 1996, 271 (25), 15237 - 43. 67. Wells, L.; Hart, G. W., O - GlcNAc turns twenty: functional implications for post - translational modification of nuclear and cyto solic proteins with a sugar. FEBS letters 2003, 546 (1), 154 - 8. 68. Du, X. L.; Edelstein, D.; Rossetti, L.; Fantus, I. G.; Goldberg, H.; Ziyadeh, F.; Wu, J.; Brownlee, M., Hyperglycemia - induced mitochondrial superoxide overproduction activates the hexosami ne pathway and induces plasminogen activator inhibitor - 1 expression by increasing Sp1 glycosylation. Proceedings of the National Academy of Sciences of the United States of America 2000, 97 (22), 12222 - 6. 69. Clark, R. J.; McDonough, P. M.; Swanson, E.; Tr ost, S. U.; Suzuki, M.; Fukuda, M.; Dillmann, W. H., Diabetes and the accompanying hyperglycemia impairs cardiomyocyte calcium cycling through increased nuclear O - GlcNAcylation. The Journal of biological chemistry 2003, 278 (45), 44230 - 7. 70. Fowler, M. J . , Microvascular and Macrovascular Complications of Diabetes. Clinical Diabetes 2008, 26 (2), 277 - 82. 71. Fong, D. S.; Aiello, L. P.; Ferris, F. L., 3rd; Klein, R., Diabetic retinopathy. Diabetes care 2004, 27 (10), 2540 - 53. 72. Intensive blood - glucose control with sulphonylureas or insulin compared with conventional treatment and risk of complications in patients with type 2 diabetes (UKPDS 33). UK Prospective Diabetes Study (UKPDS) Group. Lancet 1998, 352 (9131), 837 - 53. 73. Keenan, H. A.; Costacou, T.; Sun, J. K.; Doria, A.; Cavellerano, J.; Coney, J.; Orchard, T. J.; Aiello, L. P.; King, G. L., Clinical factors associated with resistance to microvascular complications in diabetic patients of extreme disease duration: the 50 - 59 year medalist study. Diabetes care 2007, 30 (8), 1995 - 7. 74. Gabbay, K. H., Hyperglycemia, polyol metabolism, and complications of diabetes mellitus. Annual review of medicine 1975, 26 , 521 - 36. 75. Gabbay, K. H., Aldose reductase inhibition in the treatment of diabetic neuropathy: where are we in 2004? Current diabetes reports 2004, 4 (6), 405 - 8. 76. Kunisaki, M.; Bursell, S. E.; Clermont, A. C.; Ishii, H.; Ballas, L. M.; Jirousek, M. R.; Umeda, F.; Nawata, H.; King, G. L., Vitamin E prevents diabetes - induced abnormal re tinal blood flow via the diacylglycerol - protein kinase C pathway. The American journal of physiology 1995, 269 (2 Pt 1), E239 - 46. 77. Aiello, L. P.; Pierce, E. A.; Foley, E. D.; Takagi, H.; Chen, H.; Riddle, L.; Ferrara, N.; King, G. L.; Smith, L. E., Supp ression of retinal neovascularization in vivo by inhibition of vascular endothelial growth factor (VEGF) using soluble VEGF - receptor chimeric proteins. Proceedings of the National Academy of Sciences of the United States of America 1995, 92 (23), 10457 - 61. 78. Watkins, P. J., Retinopathy. Bmj 2003, 326 (7395), 924 - 6. 79. Nathan, D. M., Long - term complications of diabetes mellitus. The New England journal of medicine 1993, 328 (23), 1676 - 85. 80. Anderson, A. R.; Christiansen, J. S.; Anderson, J. K.; Kreiner, S.; Deckert, T., Diabetic Nephropathy iin Type 1 (inslulin - dependent) Diabetes: An Epidemiological Study. Diabetologia 1983, 25 , 496 - 501. 81. Ballard, D. J.; Humphrey, L. L.; Melton, L. J., 3rd; Frohnert, P. P.; Chu, P. C.; O'Fallon, W. M.; Palumbo, P. J., Epidemiology of persistent proteinuria in type II diabetes mellitus. Population - based study in Rochester, Minnesota. Diabetes 1988, 37 (4), 405 - 12. 82. Selby, J. V.; FitzSimmons, S. C.; Newman, J. M.; Katz, P. P.; Sepe, S.; Showstack, J., The natural history and epidemiology of diabetic nephropathy. Implications for prevention and control. Jama 1990, 263 (14), 1954 - 60. 83. Mogensen, C. E., Early glomerular hyperfiltration in insulin - dependent diabetics and late nephropathy. Scandinavian journa l of clinical and laboratory investigation 1986, 46 (3), 201 - 6. 84. Kussman, M. J.; Goldstein, H.; Gleason, R. E., The clinical course of diabetic nephropathy. Jama 1976, 236 (16), 1861 - 3. 85. Cooper, M. E.; Vranes, D.; Youssef, S.; Stacker, S. A.; Cox, A. J.; Rizkalla, B.; Casley, D. J.; Bach, L. A.; Kelly, D. J.; Gilbert, R. E., Increased renal expression of vascular endothelial growth factor (VEGF) and its receptor VEGFR - 2 in experimental diabetes. Diabetes 1999, 48 (11), 2229 - 39. 60 86. F owler, M. J., Micr ovascular and Macrovascular Complications of Diabetes. Clinical Diabetes 2008, 26 (2), 77 - 82. 87. Abbott, C. A.; Carrington, A. L.; Ashe, H.; Bath, S.; Every, L. C.; Griffiths, J.; Hann, A. W.; Hussein, A.; Jackson, N.; Johnson, K. E.; Ryder, C. H.; Torki ngton, R.; Van Ross, E. R.; Whalley, A. M.; Widdows, P.; Williamson, S.; Boulton, A. J.; North - West Diabetes Foot Care, S., The North - West Diabetes Foot Care Study: incidence of, and risk factors for, new diabetic foot ulceration in a community - based patie nt cohort. Diabetic medicine : a journal of the British Diabetic Association 2002, 19 (5), 377 - 84. 88. Boulton, A. J.; Vinik, A. I.; Arezzo, J. C.; Bril, V.; Feldman, E. L.; Freeman, R.; Malik, R. A.; Maser, R. E.; Sosenko, J. M.; Ziegler, D.; American Dia betes, A., Diabetic neuropathies: a statement by the American Diabetes Association. Diabetes care 2005, 28 (4), 956 - 62. 89. Boulton, A., Foot Problems in Patients with Diabetes Mellitus. Textbook of Diabetes 1997 , 1 - 58. 90. Cameron, N. E.; Cotter, M. A., Effects of a nonpeptide endothelin - 1 ETA antagonist on neurovascular function in diabetic rats: interaction with the renin - angiotensin system. The Journal of pharmacology and experimental therapeutics 1996, 278 (3), 1262 - 8. 91. Poretsky, L., Principles of Diabetes Mellitus. Kluwer Academic Publisher Group: Norwell 2002 , p 57. 92. Steiner, D. F.; Kemmler, W.; Tager, H. S.; Peterson, J. D., Proteolytic processing in the biosynthesis of insulin and other proteins. Federation proceedings 1974, 33 (10), 2105 - 15. 93. Orci, L., Patterns of cellular and subcellular organization in the endocrine pancreas. The Sir Henry Dale lecture for 1983. The Journal of endocrinology 1984, 102 (1), 3 - 11. 94. Hutton, J. C., Insulin secretory granule biogenesis and the proinsulin - pr ocessing endopeptidases. Diabetologia 1994, 37 Suppl 2 , S48 - 56. 95. Chimienti, F.; Favier, A.; Seve, M., ZnT - 8, a pancreatic beta - cell - specific zinc transporter. Biometals : an international journal on the role of metal ions in biology, biochemistry, and m edicine 2005, 18 (4), 313 - 7. 96. Hutton, J. C., The insulin secretory granule. Diabetologia 1989, 32 (5), 271 - 81. 97 . Wollheim, C. B.; Sharp, G. W., Regulation of insulin release by calcium. Physiological reviews 1981, 61 (4), 914 - 73. 98. Creemers, J. W.; Jackson, R. S.; Hutton, J. C., Molecular and cellular regulation of prohormone processing. Seminars in cell & developmental biology 1998, 9 (1), 3 - 10. 61 99. Hutton, J. C.; Peshavaria, M., Proton - translocating Mg2+ - dependent ATPase activity in insulin - secreto ry granules. The Biochemical journal 1982, 204 (1), 161 - 70. 100. Hutton, J. C., The internal pH and membrane potential of the insulin - secretory granule. The Biochemical journal 1982, 204 (1), 171 - 8. 101. Emdin, S. O.; Dodson, G. G.; Cutfield, J. M.; Cutfie ld, S. M., Role of zinc in insulin biosynthesis. Some possible zinc - insulin interactions in the pancreatic B - cell. Diabetologia 1980, 19 (3), 174 - 82. 102. Van Schaftingen, E., Short - term regulation of glucokinase. Diabetologia 1994, 37 Suppl 2 , S43 - 7. 103. Kinlaw, W. B.; Levine, A. S.; Morley, J. E.; Silvis, S. E.; McClain, C. J., Abnormal zinc metabolism in type II diabetes mellitus. The American journal of medicine 1983, 75 (2), 273 - 7. 104. Chausmer, A. B., Zinc, insulin and diabetes. Journal of the Ameri can College of Nutrition 1998, 17 (2), 109 - 15. 105. Kim, B. J.; Kim, Y. H.; Kim, S.; Kim, J. W.; Koh, J. Y.; Oh, S. H.; Lee, M. K.; Kim, K. W.; Lee, M. S., Zinc as a paracrine effector in pancreatic islet cell death. Diabetes 2000, 49 (3), 367 - 72. 106. Ros enfeld, L., Insulin: discovery and controversy. Clinical chemistry 2002, 48 (12), 2270 - 88. 107. Ryle, A. P.; Sanger, F.; Smith, L. F.; Kitai, R., The disulphide bonds of insulin. The Biochemical journal 1955, 60 (4), 541 - 56. 108. Blundell, T. L.; Cutfield, J. F.; Cutfield, S. M.; Dodson, E. J.; Dodson, G. G.; Hodgkin, D. C.; Mercola, D. A.; Vijayan, M., Atomic positions in rhombohedral 2 - zinc insulin crystals. Nature 1971, 231 (5304), 506 - 11. 109. Dean, P. M.; Matthews, E. K., Electrical activity in pancrea tic islet cells. Nature 1968, 219 (5152), 389 - 90. 110. Ashcroft, F. M.; Harrison, D. E.; Ashcroft, S. J., Glucose induces closure of single potassium channels in isolated rat pancreatic beta - cells. Nature 1984, 312 (5993), 446 - 8. 111. Cook, D. L.; Hales, C . N., Intracellular ATP directly blocks K+ channels in pancreatic B - cells. Nature 1984, 311 (5983), 271 - 3. 112. Inagaki, N.; Gonoi, T.; Clement, J. P. t.; Namba, N.; Inazawa, J.; Gonzalez, G.; Aguilar - Bryan, L.; Seino, S.; Bryan, J., Reconstitution of IKA TP: an inward rectifier subunit plus the sulfonylurea receptor. Science 1995, 270 (5239), 1166 - 70. 62 113. Inagaki, N.; Gonoi, T.; Seino, S., Subunit stoichiometry of the pancreatic beta - cell ATP - sensitive K+ channel. FEBS letters 1997, 409 (2), 232 - 6. 114. K omatsu, M.; Takei, M.; Ishii, H.; Sato, Y., Glucose - stimulated insulin secretion: A newer perspective. Journal of diabetes investigation 2013, 4 (6), 511 - 6. 115. Pessin, J. E.; Saltiel, A. R., Signaling pathways in insulin action: molecular targets of insu lin resistance. The Journal of clinical investigation 2000, 106 (2), 165 - 9. 116. Hubbard, S. R.; Wei, L.; Ellis, L.; Hendrickson, W. A., Crystal structure of the tyrosine kinase domain of the human insulin receptor. Nature 1994, 372 (6508), 746 - 54. 117. Hu bbard, S. R., Crystal structure of the activated insulin receptor tyrosine kinase in complex with peptide substrate and ATP analog. The EMBO journal 1997, 16 (18), 5572 - 81. 118. Previs, S. F.; Withers, D. J.; Ren, J. M.; White, M. F.; Shulman, G. I., Contr asting effects of IRS - 1 versus IRS - 2 gene disruption on carbohydrate and lipid metabolism in vivo. The Journal of biological chemistry 2000, 275 (50), 38990 - 4. 119. Samuel, V. T.; Petersen, K. F.; Shulman, G. I., Lipid - induced insulin resistance: unrave lling the mechanism. Lancet 2010, 375 (9733), 2267 - 77. 120. Kido, Y.; Nakae, J.; Accili, D., Clinical review 125: The insulin receptor and its cellular targets. The Journal of clinical endocrinology and metabolism 2001, 86 (3), 972 - 9. 121. Alessi, D. R.; D eak, M.; Casamayor, A.; Caudwell, F. B.; Morrice, N.; Norman, D. G.; Gaffney, P.; Reese, C. B.; MacDougall, C. N.; Harbison, D.; Ashworth, A.; Bownes, M., 3 - Phosphoinositide - dependent protein kinase - 1 (PDK1): structural and functional homology with the Dr osophila DSTPK61 kinase. Current biology : CB 1997, 7 (10), 776 - 89. 122. Bellacosa, A.; Testa, J. R.; Staal, S. P.; Tsichlis, P. N., A retroviral oncogene, akt, encoding a serine - threonine kinase containing an SH2 - like region. Science 1991, 254 (502 9), 274 - 7. 123. Le Good, J. A.; Ziegler, W. H.; Parekh, D. B.; Alessi, D. R.; Cohen, P.; Parker, P. J., Protein kinase C isotypes controlled by phosphoinositide 3 - kinase through the protein kinase PDK1. Science 1998, 281 (5385), 2042 - 5. 124. Deprez, J.; Be rtrand, L.; Alessi, D. R.; Krause, U.; Hue, L.; Rider, M. H. , Partial purification and characterization of a wortmannin - sensitive and insulin - stimulated protein kinase that activates heart 6 - phosphofructo - 2 - kinase. The Biochemical journal 2000, 347 Pt 1 , 3 05 - 12. 125. Suzuki, K.; Kono, T., Evidence that insulin causes translocation of glucose transport activity to the plasma membrane from an intracellular storage site. 63 Proceedings of the National Academy of Sciences of the United States of America 1980, 77 ( 5), 2542 - 5. 126. Cushman, S. W.; Wardzala, L. J., Potential mechanism of insulin action on glucose transport in the isolated rat adipose cell. Apparent translocation of intracellular transport systems to the plasma membrane. The Journal of biolo gical chemistry 1980, 255 (10), 4758 - 62. 127. Randle, P. J., Regulatory interactions between lipids and carbohydrates: the glucose fatty acid cycle after 35 years. Diabetes/metabolism reviews 1998, 14 (4), 263 - 83. 128. Frayn, K. N., Adipose tissue as a buf fer for daily lipid flux. Diabetologia 2002, 45 (9), 1201 - 10. 129. Coppack, S. W.; Patel, J. N.; Lawrence, V. J., Nutritional regulation of lipid metabolism in human adipose tissue. Experimental and cl inical endocrinology & diabetes : official journal, Germ an Society of Endocrinology [and] German Diabetes Association 2001, 109 Suppl 2 , S202 - 14. 130. Dimitriadis, G.; Mitrou, P.; Lambadiari, V.; Maratou, E.; Raptis, S. A., Insulin effects in muscle and adipose tissue. Diabetes research and clinical practice 20 11, 93 Suppl 1 , S52 - 9. 131. Liu, Z.; Barrett, E. J., Human protein metabolism: its measurement and regulation. American journal of physiology. Endocrinology and metabolism 2002, 283 (6), E1105 - 12. 132. Steiner, D. F.; Cunningham, D.; Spigelman, L.; Aten, B ., Insulin biosynthesis: evidence for a precursor. Science 1967, 157 (3789), 697 - 700. 133. Clark, J. L.; Cho, S.; Rubenstein, A. H.; Steiner, D. F., Isolation of a proinsulin connecting peptide fragment (C - peptide) from bovine and human pancreas. Biochemic al and biophysical research communications 1969, 35 (4), 456 - 61. 134. Polonsky, K. S.; Rubenstein, A. H., C - peptide as a measure of the secretion and hepatic extraction of insulin. Pitfalls and limitations. Diabetes 1984, 33 (5), 486 - 94. 135. Kuzuya, H.; B lix, P. M.; Horwitz, D. L.; Steiner, D. F.; Rubenstein, A. H., Determination of free and total insulin and C - peptide in insulin - treated diabetics. Diabetes 1977, 26 (1), 22 - 9. 136. Scarlett, J. A.; Mako, M. E.; Rubenstein, A. H.; Blix, P. M.; Goldman, J.; Horwitz, D. L.; Tager, H.; Jaspan, J. B.; Stjernholm, M. R.; Olefsky, J. M., Factitious hypoglycemia. Diagnosis by measurement of serum C - peptide immunoreactivity and insulin - binding antibodies. The New England journal of medicine 1977, 297 (19), 1029 - 32. 64 137. Sjoberg, S.; Gunnarsson, R.; Gjotterberg, M.; Lefvert, A. K.; Persson, A.; Ostman, J., Residual insulin production, glycaemic control and prevalence of microvascular lesions and polyneuropathy in long - term type 1 (insulin - dependent) diabetes mellitus. Diabetologia 1987, 30 (4), 208 - 13. 138. Zerbini, G.; Mangili, R.; Luzi, L., Higher post - absorptive C - peptide levels in Type 1 diabetic patients without renal complications. Diabetic medicine : a journal of the British Diabetic Association 1999, 16 (12), 1 048. 139. Navarro, X.; Sutherland, D. E.; Kennedy, W. R., Long - term effects of pancreatic transplantation on diabetic neuropathy. Annals of neurology 1997, 42 (5), 727 - 36. 140. Fiorina, P.; Folli, F.; Zerbini, G.; Maffi, P.; Gremizzi, C.; Di Carlo, V.; Soc ci, C.; Bertuzzi, F.; Kashgarian, M.; Secchi, A., Islet transplantation is associated with improvement of renal function among uremic patients with type I diabetes mellitus and kidney transplants. Journal of the American Society of Nephrology : JASN 2003, 14 (8), 2150 - 8. 141. Sima, A. A.; Zhang, W.; Sugimoto, K.; Henry, D.; Li, Z.; Wahren, J.; Grunberger, G., C - peptide prevents and improves chronic Type I diabetic polyneuropathy in the BB/Wor rat. Diabetologia 2001, 44 (7), 889 - 97. 142. Greene, D. A.; Sima , A. A.; Stevens, M. J.; Feldman, E. L.; Killen, P. D.; Henry, D. N.; Thomas, T.; Dananberg, J.; Lattimer, S. A., Aldose reductase inhibitors: an approach to the treatment of diabetic nerve damage. Diabetes/metabolism reviews 1993, 9 (3), 189 - 217. 143. Ste vens, E. J.; Carrington, A. L.; Tomlinson, D. R., Nerve ischaemia in diabetic rats: time - course of development, effect of insulin treatment plus comparison of streptozotocin and BB models. Diabetologia 1994, 37 (1), 43 - 8. 144. Stevens, M. J.; Obrosova, I.; Cao, X.; Van Huysen, C.; Greene, D. A., Effects of DL - alpha - lipoic acid on peripheral nerve conduction, blood flow, energy metabolism, and oxidative stress in experimental diabetic neuropathy. Diabetes 2000, 49 (6), 1006 - 15. 145. Jensen, M. E.; Messina, E. J., C - peptide induces a concentration - dependent dilation of skeletal muscle arterioles only in presence of insulin. The American journal of physiology 1999, 276 (4 Pt 2), H1223 - 8. 146. Sima, A. A.; Prashar, A.; Zhang, W. X.; Chakrabarti, S.; Greene, D. A., Preventive effect of long - term aldose reductase inhibition (ponalrestat) on nerve conduction and sural nerve structure in the spontaneously diabetic Bio - Breeding rat. The Journal of clinical investigation 1990, 85 (5), 1410 - 20. 147. Cotter, M. A.; Ekbe rg, K.; Wahren, J.; Cameron, N. E., Effects of proinsulin C - peptide in experimental diabetic neuropathy: vascular actions and modulation by nitric oxide synthase inhibition. Diabetes 2003, 52 (7), 1812 - 7. 65 148. Forst, T.; De La Tour, D. D.; Kunt, T.; Pfutzn er, A.; Goitom, K.; Pohlmann, T.; Schneider, S.; Johansson, B. L.; Wahren, J.; Lobig, M.; Engelbach, M.; Beyer, J.; Vague, P., Effects of proinsulin C - peptide on nitric oxide, microvascular blood flow and erythrocyte Na+,K+ - ATPase activity in diabetes mell itus type I. Clinical science 2000, 98 (3), 283 - 90. 149. Johansson, B. - L.; Pernow, J.; Wahren, J., Muscle vasodilatation by C - peptide is NO - mediated (Abstract). Diabetologia 1999, 42 (Suppl. 1) , A342. 150. Kunt, T.; Forst, T.; Closs, E.; Wallerath, U.; For stermann, R.; Lehamnn, R.; Pfutzner, A.; Harzer, O.; Engelback, M.; Beyer, J., Activation of endothelial nitric oxide synthase (eNOS) by C - peptide (Abstract). Diabetologia 1998, 41 (Suppl. 1) , A176. 151. Cotter, M. A.; Cameron, N. E., Correction of neurova scular deficits in diabetic rats by beta2 - adrenoceptor agonist and alpha1 - adrenoceptor antagonist treatment: interactions with the nitric oxide system. European journal of pharmacology 1998, 343 (2 - 3), 217 - 23. 152. Ekberg, K.; Brismar, T.; Johansson, B. L. ; Jonsson, B.; Lindstrom, P.; Wahren, J., Amelioration of sensory nerve dysfunction by C - Peptide in patients with type 1 diabetes. Diabetes 2003, 52 (2), 536 - 41. 153. Ekberg, K.; Brismar, T.; Johansson, B. L.; Lindstrom, P.; Juntti - Berggren, L. ; Norrby, A.; Berne, C.; Arnqvist, H. J.; Bolinder, J.; Wahren, J., C - Peptide replacement therapy and sensory nerve function in type 1 diabetic neuropathy. Diabetes care 2007, 30 (1), 71 - 6. 154. Johansson, B. L.; Borg, K.; Fernqvist - Forbes, E.; Odergren, T .; Remahl, S.; Wahren, J., C - peptide improves autonomic nerve function in IDDM patients. Diabetologia 1996, 39 (6), 687 - 95. 155. Johansson, B. L.; Borg, K.; Fernqvist - Forbes, E.; Kernell, A.; Odergren, T.; Wahren, J., Beneficial effects of C - peptide on inc ipient nephropathy and neuropathy in patients with Type 1 diabetes mellitus. Diabetic medicine : a journal of the British Diabetic Association 2000, 17 (3), 181 - 9. 156. Yosten, G. L.; Maric - Bilkan, C.; Luppi, P.; Wahren, J., Physiological effects and therap eutic potential of proinsulin C - peptide. American journal of physiology. Endocrinology and metabolism 2014, 307 (11), E955 - 68. 157. Stevens, M. J.; Zhang, W.; Li, F.; Sima, A. A., C - peptide corrects endoneurial blood flow but not oxidative stress in type 1 BB/Wor rats. American journal of physiology. Endocrinology and metabolism 2004, 287 (3), E497 - 505. 158. Wallerath, T.; Kunt, T.; Forst, T.; Closs, E. I.; Lehmann, R.; Flohr, T.; Gabriel, M.; Schafer, D.; Gopfert, A.; Pfutzner, A.; Beyer, J.; Forstermann, U., Stimulation of endothelial nitric oxide synthase by proinsulin C - peptide. Nitric oxide : biology and chemistry / official journal of the Nitric Oxide Society 2003, 9 (2), 95 - 102. 66 159. Ido, Y.; Vindigni, A.; Chang, K.; Stramm, L.; Chance, R.; Heath, W. F.; DiMarchi, R. D.; Di Cera, E.; Williamson, J. R., Prevention of vascular and neural dysfunction in diabetic rats by C - peptide. Science 1997, 277 (5325), 563 - 6. 160. Kamiya, H.; Zhang, W.; Sima, A. A., C - peptide prevents nociceptive sensory neuropathy i n type 1 diabetes. Annals of neurology 2004, 56 (6), 827 - 35. 161. Dyck, P. J.; Lambert, E. H.; O'Brien, P. C., Pain in peripheral neuropathy related to rate and kind of fiber degeneration. Neurology 1976, 26 (5), 466 - 71. 162. Remuzzi, G.; Macia, M.; Ruggen enti, P., Prevention and treatment of diabetic renal disease in type 2 diabetes: the BENEDICT study. Journal of the American Society of Nephrology : JASN 2006, 17 (4 Suppl 2), S90 - 7. 163. Alpers, C. E.; Hudkins, K. L., Mouse models of diabetic nephropathy. Current opinion in nephrology and hypertension 2011, 20 (3), 278 - 84. 164. Huang, D. Y.; Richter, K.; Breidenbach, A.; Vallon, V., Human C - peptide acutely lowers glomerular hyperfiltration and proteinuria in diabetic rats: a dose - response stud y. Naunyn - Schmiedeberg's archives of pharmacology 2002, 365 (1), 67 - 73. 165. Samnegard, B.; Jacobson, S. H.; Jaremko, G.; Johansson, B. L.; Sjoquist, M., Effects of C - peptide on glomerular and renal size and renal function in diabetic rats. Kidney interna tional 2001, 60 (4), 1258 - 65. 166. Flynn, E. R.; Lee, J.; Hutchens, Z. M., Jr.; Chade, A. R.; Maric - Bilkan, C., C - peptide preserves the renal microvascular architecture in the streptozotocin - induced diabetic rat. Journal of diabetes and its compli cations 2013, 27 (6), 538 - 47. 167. Nordquist, L.; Moe, E.; Sjoquist, M., The C - peptide fragment EVARQ redu ces glomerular hyperfiltration in streptoz otocin - induced diabetic rats. Diabetes/metabolism research and reviews 2007, 23 (5), 400 - 5. 168. Samnegard, B.; Jacobson, S. H.; Johansson, B. L.; Ekberg, K.; Isaksson, B.; Wahren, J.; Sjoquist, M., C - peptide and captopril are equally effective in lowering glomerular hyper filtration in diabetic rats. Nephrolo gy, dialysis, transplantation: official publication of the European Dialysis and Transplant Association - European Renal Association 2004, 19 (6), 1385 - 91. 169. Sjoquist, M.; Huang, W.; Johansson, B. L., Effects of C - peptide on renal function at the early stage of experimental diabetes. Kidney international 1 998, 54 (3), 758 - 64. 170. Nordquist, L.; Brown, R.; Fasching, A.; Persson, P.; Palm, F., Proinsulin C - peptide reduces diabetes - induced glomerular hyperfiltration via efferent arteriole dilation and inhibition of tubular sodium reabsorption. Amer ican journal of physiology. Renal physiology 2009, 297 (5), F1265 - 72. 171. Nordquist, L.; Lai, E. Y.; Sjoquist, M.; Patzak, A.; Persson, A. E., Proinsulin C - 67 peptide constricts glomerular afferent arterioles in diabetic mice. A potential renoprotective mech anism. American journal of physiology. Regulatory, integrative and comparative physiology 2008, 294 (3), R836 - 41. 172. Al - Rasheed, N. M.; Willars, G. B.; Brunskill, N. J., C - peptide signals via Galpha i to protect against TNF - alpha - mediated apoptosis o f opossum kidney proximal tubular cells. Journal of the American Society of Nephrology : JASN 2006, 17 (4), 986 - 95. 173. Hills, C. E.; Al - Rasheed, N.; Al - Rasheed, N.; Willars, G. B.; Brunskill, N. J., C - peptide reverses TGF - beta1 - induced changes in ren al proximal tubular cells: implications for treatment of diabetic nephropathy. American journal of physiology. Renal physiology 2009, 296 (3), F614 - 21. 174. Bo, S.; Gentile, L.; Castiglione, A.; Prandi, V.; Canil, S.; Ghigo, E.; Ciccone, G., C - peptide and the risk for incident complications and mortality in type 2 diabetic patients: a retrospective cohort study after a 14 - year follow - up. European journal of endocrinology / European Federation of Endocrine Societies 2012, 167 (2), 173 - 80. 175. Sawyer, R. T. ; Flynn, E. R.; Hutchens, Z. M., Jr.; Williams, J. M.; Garrett, M. R.; Maric - Bilkan, C., Renoprotective effects of C - peptide in the Dahl salt - sensitive rat. American journal of physiology. Renal physiology 2012, 303 (6), F893 - 9. 176. Devaraj, S.; Dasu, M. R.; Jialal, I., Diabetes is a proinflammatory state: a translational perspective. Expert review of endocrinology & metabolism 2010, 5 (1), 19 - 28. 177. Haidet, J.; Cifarelli, V.; Trucco, M.; Luppi, P., C - peptide reduces pro - inflammatory cytokine secretion in LPS - stimulated U937 monocytes in condition of hyperglycemia. Inflammation research : official journal of the European Histamine Research Society ... [et al.] 2012, 61 (1), 27 - 35. 178. Chima, R. S.; Maltese, G.; Lamontagne, T.; Piraino, G.; Dene nberg, A.; O'Connor, M.; Zingarelli, B., C - peptide ameliorates kidney injury following hemorrhagic shock. Shock 2011, 35 (5), 524 - 9. 179. Luppi, P.; Cifarelli, V.; Tse, H.; Piganelli, J.; Trucco, M., Human C - peptide antagonises high glucose - induced endothe lial dysfunction through the nuclear factor - kappaB pathway. Diabetologia 2008, 51 (8), 1534 - 43. 180. Scalia, R.; Coyle, K. M.; Levine, B. J.; Booth, G.; Lefer, A. M., C - peptide inhibits leukocyte - endothelium interaction in the microcirculation during acute endothelial dysfunction. FASEB journal : official publication of the Federation of American Societies for Experimental Biology 2000, 14 (14), 2357 - 64. 181. Young, L. H.; Ikeda, Y.; Scalia, R.; Lefer, A. M., C - peptide exerts cardioprotective effect s in myo c ardial ischemia - reperfusion. American journal of physiology. Heart and circulatory physiology 2000, 279 (4), H1453 - 9. 68 182. Mughal, R. S.; Scragg, J. L.; Lister, P.; Warburton, P.; Riches, K.; O'Regan, D. J.; Ball, S. G.; Turner, N. A.; Porter, K. E., Cel lular mechanisms by which proinsulin C - peptide prevents insulin - induced neointima formation in human saphenous vein. Diabetologia 2010, 53 (8), 1761 - 71. 183. Cifarelli, V.; Luppi, P.; Tse, H. M.; He, J.; Piganelli, J.; Trucco, M ., Human proinsulin C - pepti de reduces high glucose - induced proliferation and NF - kappaB activation in vascular smooth muscle cells. Atherosclerosis 2008, 201 (2), 248 - 57. 184. Kobayashi, Y.; Naruse, K.; Hamada, Y.; Nakashima, E.; Kato, K.; Akiyama, N.; Kamiya, H.; Watarai, A.; Nak ae, M.; Oiso, Y.; Nakamura, J., Human proinsulin C - peptide prevents proliferation of rat aortic smooth muscle cells cultured in high - glucose conditions. Diabetologia 2005, 48 (11), 2396 - 401. 185. Walcher, D.; Babiak, C.; Poletek, P.; Rosenkranz, S.; Bach, H.; Betz, S.; Durst, R.; Grub, M.; Hombach, V.; Strong, J.; Marx, N., C - Peptide induces vascular smooth muscle cell proliferation: involvement of SRC - kinase, phosphatidylinositol 3 - kinase, and extracellular signal - regulated kinase 1/2. Circulation researc h 2006, 99 (11), 1181 - 7. 186. Bhatt, M. P.; Lim, Y. C.; Kim, Y. M.; Ha, K. S., C - peptide activates AMPKalpha and prevents ROS - mediated mitochondrial fission and endothelial apoptosis in diabetes. Diabetes 2013, 62 (11), 3851 - 62. 187. Dugan, L. L.; You, Y. H.; Ali, S. S.; Diamond - Stanic, M.; Miyamoto, S.; DeCleves, A. E.; Andreyev, A.; Quach, T.; Ly, S.; Shekhtman, G.; Nguyen, W.; Chepetan, A.; Le, T. P.; Wang, L.; Xu, M.; Paik, K. P.; Fogo, A.; Viollet, B.; Murphy, A.; Brosius, F.; Naviaux, R. K.; Sharma, K ., AMPK dysregulation promotes diabetes - related reduction of superoxide and mitochondrial function. The Journal of clinical investigation 2013, 123 (11), 4888 - 99. 188. Bhatt, M. P.; Lim, Y. C.; Hwang, J.; Na, S.; Kim, Y. M.; Ha, K. S., C - peptide prevents h yperglycemia - induced endothelial apoptosis through inhibition of reactive oxygen species - mediated transglutaminase 2 activation. Diabetes 2013, 62 (1), 243 - 53. 189. Cifarelli, V.; Geng, X.; Styche, A.; Lakomy, R.; Trucco, M.; Luppi, P., C - peptide reduces high - glucose - induced apoptosis of endothelial cells and decreases NAD(P)H - oxidase reactive oxygen species generation in human aortic endothelial cells. Diabetologia 2011, 54 (10), 2702 - 12. 190. Vejandla, H.; Hollander, J. M.; Kothur, A.; Brock, R. W., C - Pe ptide reduces mitochondrial superoxide generation by restoring complex I activity in high glucose - exposed renal microvascular endothelial cells. ISRN endocrinology 2012, 2012 , 162802. 191. Henriksson, M.; Pramanik, A.; Shafqat, J.; Zhong, Z.; Tally, M.; Ek berg, K.; Wahren, J.; Rigler, R.; Johansson, J.; Jornvall, H., Specific binding of proinsulin C - peptide to intact and to detergent - solubilized human skin fibroblasts. Biochemical and biophysical research communications 2001, 280 (2), 423 - 7. 69 192. Rigler, R. ; Pramanik, A.; Jonasson, P.; Kratz, G.; Jansson, O. T.; Nygren, P.; Stahl, S.; Ekberg, K.; Johansson, B.; Uhlen, S.; Uhlen, M.; Jornvall, H.; Wahren, J., Specific binding of proinsulin C - peptide to human cell membranes. Proceedings of the National Academy of Sciences of the United States of America 1999, 96 (23), 13318 - 23. 193. Ohtomo, Y.; Bergman, T.; Johansson, B. L.; Jornvall, H.; Wahren, J., Differential effects of proinsulin C - peptide fragments on Na+, K+ - ATPase activity of renal tubule segments. Diab etologia 1998, 41 (3), 287 - 291. 194. Pramanik, A.; Ekberg, K.; Zhong, Z.; Shafqat, J.; Henriksson, M.; Jansson, O.; Tibell, A.; Tally, M.; Wahren, J.; Jornvall, H.; Rigler, R.; Johansson, J., C - peptide binding to human cell membranes: importance of Glu27. Biochemical and biophysical research communications 2001, 284 (1), 94 - 8. 195 . Jehle, P. M.; Fussgaenger, R. D.; Angelus, N. K.; Jungwirth, R. J.; Saile, B.; Lutz, M. P., Proinsulin stimulates growth of small intestinal crypt - like cells acting via specific receptors. The American journal of physiology 1999, 276 (2 Pt 1), E262 - 8. 196. Yosten, G. L.; Kolar, G. R.; Redlinger, L. J.; Samson, W. K., Evidence for an interaction between proinsulin C - peptide and GPR146. The Journal of endocrinology 2013, 218 (2), B1 - 8. 197. Hills, C. E.; Brunskill, N. J., C - Peptide and its intracellular signaling. The review of diabetic studies : RDS 2009, 6 (3), 138 - 47. 198. Forst, T.; Kunt, T., Effects of C - peptide on microv ascular blood flow and blood hemorheology. Exp erimental diabesity research 2004, 5 (1), 51 - 64. 199. Chang, L.; Karin, M., Mammalian MAP kinase signalling cascades. Nature 2001, 410 (6824), 37 - 40. 200. Kitamura, T.; Kimura, K.; Jung, B. D.; Makondo, K.; Okamoto, S.; Canas, X.; Sakane, N.; Yoshida, T.; Saito, M., Proinsulin C - peptide rapidly stimulates mitogen - activated protein kinases in Swiss 3T3 fibroblasts: requirement of protein kinase C, phosphoinositide 3 - kinase and pertussis toxin - sensitive G - protein. The Biochemical journal 2001, 355 (Pt 1), 123 - 9. 201. Lindahl, E.; Nyman, U.; Melles, E.; Sigmundsson, K.; Stahlberg, M.; Wahren, J.; Obrink, B.; Shafqat, J.; Joseph, B.; Jornvall, H., Cellular internalization of proinsulin C - peptide. Cellular and molecular life sciences : CMLS 2007, 64 (4), 479 - 86. 202. Jagerbrink, T.; Lindahl, E.; Shafqat, J.; Jornvall, H., Proinsulin C - peptide interaction with protein tyrosine phosphatase 1B demonstrated with a labeling reaction. Biochemical and biophysical research communications 2009, 387 (1), 31 - 5. 203. Ishii, T.; Fukano, K.; Shimada, K.; Kamikawa, A. ; Okamatsu - Ogura, Y.; Terao, A.; Yoshida, T.; Saito, M.; Kimura, K., Proinsulin C - peptide activates alpha - enolase: implications for C - peptide -- cell membrane interaction. Journal of biochemistry 70 2012, 152 (1 ), 53 - 62. 204. Meyer, J. A.; Froelich, J. M.; Reid, G. E.; Karunarathne, W. K.; Spence, D. M., Metal - activated C - peptide facilitates glucose clearance and the release of a nitric oxide stimulus via the GLUT1 transporter. Diabetologia 2008, 51 (1), 175 - 82. 205. eyer, J. A.; Subasinghe, W.; Sima, A. A.; Keltner, Z.; Reid, G. E.; Daleke, D.; Spence, D. M., Zinc - activated C - peptide resistance to the type 2 diabetic erythrocyte is associated with hyperglycemia - induced phosphatidylserine externalization and reversed by metformin. Molecular bioSystems 2009, 5 (10), 1157 - 62. 206. Magalhaes, F. O.; Gouveia, L. M.; Torquato, M. T.; Paccola, G. M.; Piccinato, C. E.; Foss, M. C., Metformin increases blood flow and forearm glucose uptake in a group of non - obese t ype 2 diabetes patients. Hormone and metabolic research = Hormon - und Stoffwechselforschung = Hormones et metabolisme 2006, 38 (8), 513 - 7. 207. Signore, A.; Fiore, V.; Chianelli, M.; Ronga, G.; Pozzilli, P., Effect of metformin on liver insulin metabolism and regional blood flow. Diabetes/metabolism reviews 1995, 11 Suppl 1 , S13 - 21. 208. Muller, S.; Denet, S.; Candiloros, H.; Barrois, R.; Wiernsperger, N.; Donner, M.; Drouin, P., Action of metformin on erythrocyte membrane fluidity in vitro and in vivo. Eur opean journal of pharmacology 1997, 337 (1), 103 - 10. 209. Wiernsperger, N. F., Membrane physiology a s a basis for the cellular effects of metformin in insulin resistance and diabetes. Diabetes & metabolism 1999, 25 (2), 110 - 27. 210. Daleke, D. L., Regulati on of phospholipid asymmetry in the erythrocyte membrane. Current opinion in hematology 2008, 15 (3), 191 - 5. 211. Dumaswala, U. J.; Wilson, M. J.; Wu, Y. L.; Wykle, J.; Zhuo, L.; Douglass, L. M.; Daleke, D. L., Glutathione loading prevents free radical inj ury in red blood cells after storage. Free radical research 2000, 33 (5), 517 - 29. 212. Zalewski, P. D.; Millard, S. H.; Forbes, I. J.; Kapaniris, O.; Slavotinek, A.; Betts, W. H.; Ward, A. D.; Lincoln, S. F.; Mahadevan, I., Video image analysis of labile zinc in viable pancreatic islet cells using a specific fluorescent probe for zinc. The journal of h istochemistry and cytochemistry : official journal of the Histochemistry Society 1994, 42 (7), 877 - 84. 213. Derewenda, U.; Derewenda, Z.; Dodson , G. G.; Hubbard, R. E.; Korber, F., Molecular structure of insulin: the insulin monomer and its assembly. British medical bulletin 1989, 45 (1), 4 - 18. 214. Baker, E. N.; Blundell, T. L.; Cutfield, J. F.; Cutfield, S. M.; Dodson, E. J.; Dodson, G. G.; Hodg kin, D. M.; Hubbard, R. E.; Isaacs, N. W.; Reynolds, C. D.; et al., The structure of 2Zn pig insulin crystals at 1.5 A resolution. Philosophical transactions of 71 the Royal Society of London. Series B, Biological sciences 1988, 319 (1195), 369 - 456. 215. Meye r, J. A.; Spence, D. M., A perspective on the role of metals in diabetes: past findings and possible future directions. Metallomics 2009, 1 , 32 - 41. 216. Rabinovitch, A.; Suarez, W. L.; Thomas, P. D.; Strynadka, K.; Simpson, I., Cytotoxic effects of cytokin es on rat islets: evidence for involvement of free radicals and lipid peroxidation. Diabetologia 1992, 35 (5), 409 - 13. 217. Sprietsma, J. E.; Schuitemaker, G. E., Diabetes can be prevented by reducing insulin production. Medical hypotheses 1994, 42 (1), 15 - 23. 218. Keltner, Z.; Meyer, J. A.; Johnson, E. M.; Palumbo, A. M.; Spence, D. M.; Reid, G. E., Mass spectrometric characterization and activity of zinc - activated proinsulin C - peptide and C - peptide mutants. The Analyst 2010, 135 (2), 278 - 88. 72 Chapter 2 Serum Albumin as a Carrier for C - peptide and Zinc to the ERY 2.1 Introduction 2.1.1 Structure of Human C - peptide Human proinsulin C - peptide is a 31 amino acid peptide, containing 5 acidic residues and no basic residues, causing it to be negatively charge d overall. 1 The attempt to obtain an ordered structure of C - peptide has not been successful. Many researchers have thus claimed that this peptide is largely unstructured, or referred to as a random coil. 2,3,4 However, more recently there have been studies showing partial ordered secondary structure of C - peptide in aqueous solutions. 5 For example, using 2D NMR spectroscopy, in a solution of 50% H2O / 50% 2,2,2 - trifluoroethanol (TFE, added to strengthen and stabilize secondary - structure formation), 6 C - peptide demonstrated substructures with defined local conformations. 6 The C - terminal region (Glu27 - Gln31), which is the most active site for C - peptide activity, presented the most highly structured part of the molecule. Hydrogen bonds formed between Glu27 and r esidues Leu30 and - turn formed by Gly28 and Ser29 that is stabilized by hydrogen bonding are included in this part of the molecule. 5 Interestingly, Gly28 and Ser29 are not absolutely conserved across species, and are absent in rat C - peptides, which could explain reports claiming no binding of rat C - peptide to human cells. 7,8 The second most highly structured part of the molecule appears to be the N - terminal Ala2 - Leu5 region, including a hydrogen bond formed by Ala2 and Leu5, and a ty - turn formed by residues 3 and 4. The N - terminal tetrapeptide has been reported to be highly 73 conserved among species, which has been proposed to have functional activity in the junction site of the B chain of insulin and C - peptide in proinsulin, and to be part of the type I endopeptidase recognition site during cleavage of proinsulin. 9,10 In addition, the three central regions of the molecule (Gln9 - Leu12, Gly15 - Ala18 and Gln22 - Ala25) did not present any well - defined structural element, however, sl ight - bends were actually detected in the peptide main chain. 5 2.1.2 Serum Albumin 2.1.2.1 Transporter role Human serum albumin (HSA) is the most abundant protein in human plasma and constitutes approximately 50% of the protein p resent in the plasma, with serum concentration of around 40 g/L, or 600 mM. It is produced in the liver at a rate of 9 to 12 g/day, and immediately released into the bloodstream. HSA is a monomeric, multidomain macromolecule, which has a single polypeptide chain of 585 amino acids with a molecular weight around 66 kDa. HSA has a strong negative charge under physiological conditions, allowing it to have excellent solubility and function in the maintenance of plasma oncotic pressure and modulation of fluid di stribution throughout the body. 11 - 13 HSA displays extraordinary ligand - binding capacity, and is a known carrier for many endogenous and exogenous ions, low molecular weight compound s, peptides, and proteins. 14 - 16 For example, HSA bind s up to nine equivalen ts of long chain fatty acids (FAs), with the nine binding sites, referred to as FA1 - FA9 all showing different binding affinities. 17 - 18 This is important for FA based drug design because binding to HSA 74 improves plasma solubility and the half - life of drugs, but at the same time reduces their free active concentration. 19 HSA binds to a wide variety of metal ions, including Mg(II), Al(III), Ca(II), Mn(II), Co(II/III), Ni(II), Cu(I/II), Zn(II), Cd(II), Pt(II), Au(I/II), Hg(II), and Tb(III), with the binding site s of many of these metals being previously determined. 20 - 23 For example, the nitrogen donor atoms of the N - terminal Asp1, Ala2, and His3 are involved in the binding of Cu(II), Co(II), and Ni(II) ions. 24 Au(I), Hg(II), and Pt(II) ions are observed to intera ct with the free Cys34 thiol; 25 whereas Cd site A (MBS - A site), which involves His67, Asn99, His247, and Asp249 residues, are the primary binding site for Zn(II) and Cd(II) ions, and the secondary binding site for Cu(II) and Ni(II). 26 Binding sites for oth er metal ions are either undefined or defined to be unspecific. 27 HSA is the major transporter for Zn 2+ in plasma, binding more than 98% of Zn 2+ ions in plasma. 28 The primary binding site for Zn 2+ is the MBS - A site having a dissociation constant in the mic romolar range. 29,30 A secondary binding site for Zn 2+ has also been reported. 27 Albumin transport of Zn 2+ ions is critical for cell functions. Examples include modulating zinc uptake into endothelial cells, 31 and a receptor - mediated endocytosis pathway tha t has been suggested for zinc - albumin complexes. 32 Additionally, albumin facilitates uptake of zinc by erythrocytes, 33 where it binds to glutathione 34 and hemoglobin, and increasing its oxygen affinity. 35 2.1.2.2 Albumin as a Drug Carrier 75 Due to the excell ent solubility, stability, availability and biodegradability of albumin, as well as its lack of toxicity, immunogenicity and preferential uptake in tumor and inflamed tissue, albumin is emerging as an ideal candidate for drug delivery. 36 Interestingly, alb umin improve s the pharmacokinetic profile of peptide or protein - based drugs. 36 Therapeutically relevant peptide and protein drugs are playing an increasing role in the treatment of viral, malignant and autoimmune diseases such as diabetes. Unfortunately, major difficulties in the application of such treatments currently include short shelf - life, costly production, immunogenic and allergic potential, and sensitivity towards peptidases. Utilizing albumin as a drug carrier has been proposed to be a solution t o such difficulties. The Albumin Fusion Technology is the first technology that has been developed for this purpose. 37 By genetically fusing HSA and albinterferon alpha - 2b (alb - IFN), a recombinant polypeptide composed of IFN - alpha2b to treat chronic hepati tis C, the half - life of the peptide is extended in vivo , and dosing frequency in individuals was able to be reduced. Another example of Albumin Fusion Technology is the development of Albulin, a long - acting insulin analog obtained by direct gene fusion of a single - chain human insulin to HSA, which achieved sustained glucose normalization in diabetic mice.38 This albumin - fusion technology is currently being investigated for other therapeutically relevant peptides with short half - lives. 37,39 A secon d technology using albumin as a drug carrier is The Drug Affinity Complex - Drug Affinity Complex (PC - platforms. 40 Each DAC contains the peptide/protein drug, a connector, and a reactive 76 group that is responsib le for the permanent bonding of the DAC to target proteins. Through biding to albumin, DAC peptides are successfully protected from rapid degradation and excretion, and distributed throughout tissues and organs in the body due to the pharmacokinetic proper ties of albumin. An albumin conjugate of Exendin - 4, a glucagonlike peptide - 1 (GLP - 1) homolog for treating type 2 diabetes, has been created by ConjuChem, Inc. This peptide was bound selectively to the cysteine - 34 position of circulating albumin. 41 Phase I/ II clinical trials demonstrated a half - life of 9 - 15 days in humans after subcutaneous administration, and no signs of immunogenicity in multiple - dose regimens. 40 ConjuChem also has invented an albumin conjugate with insulin (PC - n - binding derivative of a C34 peptide that targets gp41 of 36 A third technology using albumin as a drug carrier is based on the development of peptide fatty acid derivatives that bind to circulating albumin. 42 An example of this t echnology is the development of a long - acting insulin analogue acylated with a C14 - fatty acid chain. Through reversibly binding to albumin, the slow - release of the active species was realized. 43 In this chapter, specific binding of albumin to both C - peptid e and zinc has been detected, and the idea of albumin being a transporter of the two is proposed. Upon delivery, C - peptide and zinc bind to ERYs and result in increase of ATP release from the cells. The increased is more than 50%. Result s suggest that horm one replacement therapy in diabetes may be improved by exogenous administration of a C - peptide ensemble that includes zinc and albumin, rather than insulin alone. 77 2.2 Experimental Procedure 2.2.1 Collection and Purification of Erythrocytes (ERYs) Whole blo od was obtained from consenting and healthy human donors by venipuncture and collected into heparinized tubes. ERYs were obtained on the day of use. Blood was centrifuged at 500 g at 4 °C for 10 min and the plasma and buffy coat were discarded. ERYs were w ashed three times by re - suspending in a physiological salt solution (PSS) [PSS; in mM, 4.7 KCl, 2.0 CaCl 2 , 140.5 NaCl, 12 MgSO 4 , 21.0 tris(hydroxymethyl)aminomethane, 5.5 glucose with 5% bovine serum albumin (final pH 7.40)]. The hematocrit was determined using a hematocrit measurement device (CritSpin®, Iris sample processing, Westwood, MA). The final hematocrit was then adjusted to 70% in PSS. BSA - free PSS was also prepared for comparison studies involving the functions of ERYs in an albumin - absent enviro nment. For these studies, ERYs were washed and re - suspended in the same manner in BSA free PSS. 2.2.2 Purification and Characterization of C - peptide and Mutant E27A using High Performance Liquid Chromatography (HPLC) and Mass Spectrometry (MS) Sy nthesized human C - peptide and its single amino acid mutant E27A (~80 % pure) were purchased from Peptide 2.0 (Chantilly, VA), and purified using reverse phase high performance liquid chromatography (RP - HPLC) with a Shimadzu (Columbia, MD) LC - 20AB solvent s ystem and a SPD - 20AV UV - Vis absorbance detection system. To purify C - peptide, 10 mg of crude C - peptide were first dissolved into 1 mM HPLC - grade water (Sigma - Aldrich, St. Louis, MO) and centrifuged at 22,000 g for 10 min. The supernatant with dissolved cr ude C - peptide was carefully collected into another tube with precipitate completely excluded. Subsequently, 1 mL 10% HPLC - grade acetonitrile (ACN,EMD Biosciences, Gibbstown, NJ) in deionized distilled water (DDW, Easypure®II ultrapure 78 water system, Barnste ad) was added to the undissolved precipitate and vortexed to facilitate solubility. The tube was centrifuged again, and the supernatant was carefully collected and combined into the previous portion of crude C - peptide solution. The rest of the precipitate was discarded. The mutant E27A was treated in the same manner prior to HPLC separation. The HPLC separation system contains a Rheodyne injection valve (model 9215, Oak Harbor, WA), a 2 mL stainless steel sample loop, and a C18 preparative HPLC column (Grac e, Deerfield, IL), (25 cm column length, 5 µm particle size and 5 mm i.d.). C - peptide and E27A were purified separately by gradient elution. All parameters for the HPLC are listed in Table 2.1. Collected C - peptide and E27A peaks were lyophilized overnight. Lyophilized C - peptide was then aliquoted at 0.25 mg in 1.5 mL centrifuge tubes and stored at - 20 ºC. Frozen C - peptide and E27A were thawed and dissolved in DDW to the desired C - peptide in 10 mL DDW. Exact concentration and purity of this C - peptide solution were determined by human C - peptide ELISA and MS/MS respectively. The analysis of purity was performed by direct infusion using a Thermo Scientific LTQ Orbitrap (Thermo, SanJ ose, CA) equipped with an NanoMate electrospray source (Advion, Ithaca, NY). The C - peptide and E27A stock solutions were diluted in MS buffer (1% acetic acid in 96 - well plate and set in the MS auto sampler. The ion transfer tube was set at 100 ºC and the spray voltage was maintained at 1.4 kV. The spectra were recorded for 1 min and MS/MS spectra were also collected for sequence confirmation purposes. The activation time for MS/MS was maintained at 79 30 ms usin g an activation q value of 0.25. The isolation window was maintained at 2.0 m/z while the collision energy of HCD was set at 30. The collision peaks of each peptide from the MS/MS spectrum were compared with the theoretical collision peaks generated using showed up as a pure parent ion on the MS spectrum, which showed same collision peaks on the corresponding MS/MS spectrum as given by the Protein Prospector. 80 Table 2.1: Amino acid sequences and of human C - peptide and mutant E27A. Their multi - segment gradients and conditions for HPLC purification are also described. - - - - - 81 2.2.3 ELISA - based Determination of C - peptide Binding to ERYs A C - at 4 - peptide in DDW was prepared from the stock by dilution, as was the zinc chloride working solution. To prepa re 7% ERY samples in volumes of 1 mL, first, in two sets of six 1.5 mL centrifuge tubes, 0, 1, 2, 4, 10 and 20 uL of C - peptide working solution were added to each set. In one set, same volumes of zinc working solution were also added to C - peptide to obtain a mixture of equal molar zinc and C - peptide. Zinc solution was not added to the other set for the control purpose so that the effect of zinc on C - peptide binding to ERYs could be investigated. Corresponding amounts of DDW were added to each of the 12 tub es to a total of 40 uL so that they had equal amounts of volume. Then 860 uL of PSS buffer were added to each tube, quickly followed by 100 uL of PSS - purified 70% ERYs. For albumin - free samples, BSA - free PSS was used in place of PSS in the 12 tubes. All sa mples were mixed well and incubated at 37 °C for 2 hours. After incubation, samples were centrifuged at 500 g for 5 min and the supernatant collected into separate tubes, diluted in DDW 20 - fold, and used as the samples in the ELISA - based determination of C - peptide without further treatment. C - peptide standards with concentrations of 0, 0.2, 0.4, 0.8, 1.0 nM were also prepared in DDW by diluting the 1 uM C - peptide working solution immediately before analysis involving ELISA. The reaction mechanism of the ELI SA assay is illustrated in Fig 2.1. The assay antibodies specifically capture C - peptide from samples added in wells, and the enzymatically - tagged secondary antibody subsequently binds to the captured C - peptide and catalyzes a colorimetric reaction. The a bsorbance of this reaction is monitored at 450 nm, and is proportional to the amount of 82 C - peptide present in the sample. A standard plate reader (Molecular Devices LLC, Sunnyvale, CA) was used to measure all absorbance values. To determine the C - peptide bi nding to the ERYs, the moles of C - peptide remaining in the supernatant were subtracted from the original number of moles added to the ERYs. 2.2.4 Radiometric Determination of 65Zn2+ Binding to ERYs To determine zinc uptake by ERYs, a series of 7% ERY sampl es were prepared in a similar manner as in the C - peptide uptake studies described in the previous section, except a radioisotopic form of zinc, 65 Zn 2+ , was used. Briefly, 7% ERY samples were prepared in PSS or BSA - free PSS, containing varying concentration s (0, 1, 2, 4, 10, 20 nM) of combined C - peptide and 65 Zn 2+ , or 65 Zn 2+ alone to study the effect of C - peptide on zinc uptake. All samples were incubated at 37 °C for 2 hours after the addition of the ERYs, followed by 5 min centrifugation at 500 g to collec t the supernatant. The remaining amount of 65 Zn 2+ counter. Standard solutions containing 0, 2, 4, 10, 20, 40 nM 65 Zn 2+ in DDW were also prepared for quantitati ve determination and measured in the same manner. The amount of 65 Zn 2+ taken up by the cells at each concentration point was determined by subtracting the 65 Zn 2+ concentration in the supernatant from the amount originally added. 83 Fig 2.1: Reac tion scheme for the detection of C - peptide using human C - peptide ELISA. Sample containing C - peptide is added into a well of the ELISA plate, where C - peptide binds to the coated primary antibody on the bottom of the well. The secondary antibody conjugated w ith the enzyme converts the added substrate into a colored product. The absorbance intensity of the colored product is proportional to the amount of C - peptide present in the sample. 84 Fig 2.2: Schematic di agram of an ITC instrument. Solution filled in the syringe is titrated into the solution filled in the sample cell in the adiabatic shield. Power was continuously supplied to the sample cell during a titration experiment, to keep the sample and reference c ell at the same temperature. Therefore, each injection of the titrant results in a peak corresponding to the power being supplied to the sample cell to maintain constant temperature as a function of time. The negative peaks shown indicate an exothermic bin ding event. The value of enthalpy change can be directly obtained from integration of the peaks, and the binding stoichiometry and affinity can be obtained by the molar ratio at the inflection point and the slope, respectively. 85 2.2.5 Isothermal Titration Calorimetry (ITC) Determination of Int eraction of Albumin, C - peptide (or Mutant E27A) and Zinc To mimic the pH condition in insulin secreting granules (pH=5.50), DDW (pH around 5.8) was used to prepare C - peptide and zinc solutions. The zinc solution was prepared by dissolving 4.09 mg of zinc c hloride in 10 mL DDW in a volumetric flask to obtain a zinc solution in DDW. The C - peptide solution was prepared by dissolving approximately 5 mg of the frozen pure C - pepti de powder in 5 mL DDW. The concentration of C - peptide was measured by a human C - peptide ELISA, and then diluted in DDW to a final To mimic the pH condition in the human bl oodstream (pH=7.40), a 0.5 mM Tris - HCl buffer (pH=7.40) was prepared to dissolve albumin, C - peptide and zinc solution separately in order to study the interactions of these three molecules by ITC. Tris - HCl (0.5 mM) buffer solution was prepared by dissolvin g 0.0151 g ultrapure Tris (2 - Amino - 2 - (hydroxymethyl) - 1,3 - propanediol) (Invitrogen, Carlsbad, CA) in 250 mL DDW, with pH adjusted to 7.40. The albumin solution was prepared by dissolving 20 mg of pure HSA powder (Sigma - Aldrich, St. Louis, MO) in 1 mL of the Tris - HCl buffer, and adjusting to - peptide solution was prepared by dissolving 0.5 mg of purified C - peptide in 5 mL Tris - HCl buffer, and adjusting the concentration to the desired value by human C - peptide ELISA. A mixed C - peptide/zinc - peptide 2 Tris - HCl buffer. - 86 HCl buffer, and the adjusting the concentration to the desired value using the Pierce BCA Protein Assay Kit. The determination of all thermodynamic par ameters, including binding heat, binding number, and binding stoichiometry, were obtained using a microisothermal titration calorimeter (MicroCal, Piscataway, NJ) at 25 °C. Solutions were degased for 10 minutes under vacuum before being filled into the sample cell and the syringe of the calorimeter. The schematic diagram and brief introduction of ITC ins trument is shown in adiabatic sample cell with 120 s spacing between injections, and 310 rpm stirring speed. ITC measurements of C - peptide interactions with zinc were performed by - peptide solution, or by titrating the - solutions and was filled into the ITC refe rence cell). Measurements of C - peptide binding - peptide solution (Tris - HCl buffer was the solvent for both solutions and was filled into the ITC reference cell, hereinafter). Measurements of combined C - p eptide/zinc mixture - - peptide/zinc solution. Measurements of E27A binding to HSA was The entire instrument was thermally equilibrated at 25 °C prior to all experiments. A 310 rpm independent titration experiment, with a 2 minute spacing between injections to e nsure the signal was returned to baseline before the next inj ection was made. The background 87 dilution heats were determined in separate experiments by titration of HSA solution into buffer, and buffer into C - peptide, C - peptide/zinc, and E27A solution. All measurements were performed in triplicate to confirm reproducibility. The background dilution heats were subtracted for binding experiment, and the net heat analyzed for binding parameters by Origin software using the one - site independent binding model wit h the excep tion of the HSA titration to C - peptide/zinc mixture, where a two - site binding model was used. 2.2.6 Measurement of Static ATP Release from ERYs For quantification of ATP release from ERYs, an ATP specific luciferin/luciferase chemiluminescence a ssay was utilized. Luciferin/luciferase solution was prepared by dissolving 2.0 mg of D - luciferin (Sigma Aldrich, St. Louis, MO) in 5 mL of water, and adding the resultant solution into one 50 mg vial of firefly extract (Sigma Aldrich, St. Louis, MO). DDW was used in all steps of sample preparation to ensure strict control of the presence of zinc and other metal ions. Briefly, four 1.5 mL centrifuge tubes were C - - peptide and zinc chl oride solutions and no extra DDW. Then, - purified 70% ERY suspension. For experiments in the absence of albumin, ERY samples were prepared in a similar manner as described ab ove, except that BSA - free PSS was used in place of 88 and used in place of C - peptide to verify the importance of Glu27. All samples were incubated for 2 hours at 37 ° C. After i ncubation, samples were centrifuged at 500 g for 5 minutes and the supernatant collected into separate tubes. Two sets of fresh ATP standards were prepared in corresponding buffers by dissolving 0.0101 g of ATP (Sigma - Aldrich, St. Louis, MO) in DDW in a 1 0 mL volumetric flask to - or BSA - free PSS. Finally, two sets of standards were generated by ATP working solution to 0, 100, 200, and 400 nM in each buffer. luciferin/luciferase so lution were then added into each well, and the chemiluminesence intensity was measured using a plate reader exactly 20 seconds after the addition of the enzyme. Static ATP release from ERY samples was quantified against the static ATP standard curve. 2.2.7 Measurement of Flow - induced ATP Release from ERYs on a 3D Printed Fluidic Device This part of work was performed through collaboration with another student in the Spence lab, Chengpeng Chen. The 3D printed fluidic device was first designed and characteriz ed by our group. 44 Briefly, the device design was modeled to the size of a 96 - well plate for subsequent measurement on a standard plate reader. Six channels were fabricated in the device, with three wells above each channel that align with the internal det ectors of the plate reader for convenient measurement. A transwell insert (6 89 mm in diameter) whose bottom is porous (0.4 micron) polyester is placed in each of these wells. Inlets and outlets of each channel are connected by finger tight fittings and Tygon tubing to form a closed loop system. Once samples are loaded into the channels by a peristaltic pump (IDEX Health & Science LLC, Oak Harbor, WA) and the loop is closed, a circulation mimic is formed. Instrument setup and schematic diagram showing collecti on and measurement of flow - induced ATP release from ERYs is shown in Fig 2.3. ERY samples and ATP standard solutions were prepared in the same manner as described above. Before use, the fluidic device, tubing and fittings were cleaned thoroughly by sonicat ing in 1:50 bleach:DI water for 30 seconds, followed by rinsing with a large quantity of DI water, and one subsequent rinsing with PSS. Before loading samples, the inserts were carefully dried with a Kimwipe without damaging the membrane. Prior to measurin g the ERY samples, four ATP standards (concentrations of 0, 100, 200 and 400 nM) were loaded into four channels of the fluidic device using a peristaltic pump and then the loops were closed. Inserts in row E were chosen for ATP collection and quantificatio upon diffusion into the inserts, ATP can be collected in buffer. All ATP standards were allowed to flow through the device and loop for 20 minutes at room temperature at a flow rate of fter flowing for 20 min, the fluidic device was detached from the tubing and placed in a plate reader (Molecular Devices LLC, Sunnyvale, CA), each insert of row E. At exactly 18 s, chemiluminescence intensity was measured in row E by the plate reader, and a standard curve was generated by plotting the 90 chemiluminescence intensity values against the known ATP concentrations. After measuring the ATP standards, t he fluidic device, tubing and fittings were cleaned as described above. ERY samples were then loaded into the channels and the loops closed. To better mimic blood circulation temperature, the pump, fluidic device and tubing were carefully placed in an incu bator at 37 °C. All ERY samples were device and tubing were removed from the incubator to the bench at room temperature. To ensure a contamination - free measurement, the inserts of row E were quickly rinsed - derived ATP via diffusion through the membrane pores into the inserts. ATP was collected and measured as described above for the ATP standards, and concentrations determined from the standard curve. 91 Fig 2.3: Schematic diag ram showing measurement of flow - induced ATP release from ERYs using a 3D - printed fluidic device. Six channels were fabricated in the device, with three wells above each channel. A transwell insert (6 mm in diameter) whose bottom is a piece of porous (0.4 m icron) polyester membrane is placed in each of these wells. Inlets and outlets of each channel are connected by finger tight fittings and tygon tubing to form a closed loop system. Once samples are loaded into the channels by a peristaltic pump and the loo p is closed, a circulation ATP from the flow is collected for 20 minutes at room temperature and measured in the middle insert of each channel. Four ATP standards (concentratio ns of 0, 100, 200 and 400 nM) were measured first to obtain a standard curve, followed by ERY samples. 92 2.3 Results 2.3.1 Purification of Peptides Crude C - peptide and mutant E27A both had approximately 80% purity before purification. C - peptide has a s lightly higher solubility than E27A in aqueous solution, and therefore has a smaller amount of precipitate upon dissolving, both requring 10% was then verified by MS a nd MS/MS. Purified C - peptide and E27A were then frozen at - 20 °C for later experiments. 2.3.2 C - peptide and Zinc Uptake by ERYs An ELISA for human C - peptide was used to determine the amount of C - peptide bound to the ERYs. The data displayed in Fig 2.4 show s that C - peptide binds to the ERY in both the presence and absence of zinc. C - peptide binding to the ERY is specific and saturates at approximately 2 pmoles, or 1800 molecules per cell in both cases. However, Fig 2.5 shows that C - peptide uptake does not oc cur when albumin is absent from the buffer. While C - peptide binds to the ERY in the presence and absence of zinc, Fig 2.6 shows that zinc uptake by ERYs occurs only in the presence of C - peptide; and it requires albumin as well, as shown in Fig 2.5. Additio nally, the saturation value of zinc uptake is statistically equal to that of C - peptide (p>0.95). 2.3.3 Interaction between C - peptide and Zinc and Their Binding to Serum Albumin Fig 2.7 and Fig 2.8 show the interaction between C - peptide and zinc as determin ed by ITC - cel l granules. 45 Regardless of the titration order, ITC data showed weak heat signals from both experiments, which is a result from non - specific electrostatic attraction between the 93 zinc ions and the C - - containing groups. Specific binding was not detected between C - peptide and zinc. Fig 2.4: ELISA determination of C - peptide binding to ERYs. C - peptide binding to ERYs in the presence of Zn 2+ (open circles) was not significantly different from the uptake in the absence of Zn 2+ (filled circles). In both cases, C - peptide binding to the ERY displays a specific binding curve which saturates at approximately 2 pmoles, or 1800 molecules per ERY. 94 Fig 2.5: Measurement of C - peptide and Zn binding to ERY samples prepared in albumin - free PSS. Neither C - peptide nor Zn was able to bind to EYRs in the absence of albumin. This indicates that albumin is necessary for C - peptide and Zn binding to the ERY, and that albumin is probably a plasm a transporter for C - peptide and Zn as well. Error bars = S.E.M., n = 4. 95 Fig 2.6: Radiometric determination of 65 Zn 2+ uptake by ERYs. 65 Zn 2+ uptake by ERYs in the presence of C - peptide (filled circles) displayed a specific binding curve which also sa turated at around 2 pmoles and was not significantly different from that of C - peptide. However, 65 Zn 2+ uptake did not occur in the absence of C - peptide (open circles). 96 Fig 2.9 shows the specific binding event between C - peptide and HSA detected by ITC at pH 7.40 ± 0.01, a pH condition that mimics the pH environment in the serum. The binding isotherm was fit using a one - site independent binding model where an affinity of (1.75 ± 0.64) × 10 5 M - 1 and a binding stoichiometry of 0.53 ± 0.03 were determi ned, which indicates two C - peptide molecules bind to a single HSA molecule. The ability of albumin to bind to both C - peptide and zinc was also evaluated by ITC. Fig 2.10 shows a two - phase binding event when HSA was titrated into a mixture containing both C - peptide and zinc. This two - phase binding event was fit into a two - site independent binding model. The first phase of the binding event indicates the specific binding between albumin and zinc with a higher affinity K a1 = (5.08 ± 0.98) × 10 7 M - 1 , and bind ing stoichiometry of 0.33 ± 0.01, suggesting that three zinc ions are bound to one albumin molecule. The second phase of binding indicates C - peptide and albumin binding, with a binding affinity K a2 = (2.66 ± 0.25) × 10 5 M - 1 , and binding stoichiometry of 1. 15 ± 0.01, suggesting one albumin molecule binds to one C - peptide. 2.3.4 Interaction between E27A and Serum Albumin Fig 2.11 shows the interactions between C - peptide mutant E27A and HSA at pH 7.40 ± 0.01 measured by ITC. In contrast to the C - peptide - albumi n binding curve, after the glutamic acid at position 27 of C - peptide was mutated, the specific binding curve of the C - peptide and albumin also disappeared. Much weaker signals of heat release indicated non - specific electrostatic attractions only, but no sp ecific binding. 97 Fig 2.7: ITC analysis of C - peptide and zinc interaction at pH ~5.8. A 3 mM zinc so - peptide solution and no specific binding was detected. Electrostatic attraction was the only interaction observed. All solutions were prepared in DDW. 98 Fig 2.8: ITC analysis of interactions between C - peptide and zin c at pH - solution, and electrostatic attraction, without specific binding, was the only interaction detected. All solutions were prepared in DDW. 99 Fig 2.9: ITC analysis of C - peptide bindin g to albumin at pH = 7.40 ± - peptide solution and specific binding was detected. Solutions were prepared in metal - free Tris - HCl buffer. Average binding affinity K a = (1.75 ± 0.64) x 10 5 M - 1 and binding stoichiometry N = 0.53 ± 0.03. Error bars = S.E.M., n = 3. 100 Fig 2.10: ITC analysis of C - peptide/zinc mixture binding to albumin at pH = 7.40 ± into a C - peptide/zinc mixture, each at - phase binding event was detected. All solutions were prepared in metal - free Tris - HCl buffer. Phase 1 indicates zinc - albumin binding, with averaged binding affinity K a1 = (5.08 ± 0.98) × 10 7 M - 1 . Phase 2 indicates C - peptid e - albumin binding, with averaged binding affinity K a2 = (2.66 ± 0.25) × 10 5 M - 1 . Error bars = S.E.M., n = 3. 101 2.3.5 Enhanced Static ATP Release from ERYs by Albumin/C - peptide/Zn ERYs (7% in hematocrit) were suspended in either PSS or BSA - free PSS, and incubated with 20 nM C - peptide or zinc al one, or both. Changes in ATP release after the treatments were compared and are shown in Fig 2.12. All measured values of ATP release were normalized to that of the untreated ERY control sample in PSS, the leftmost black bar shown in the figure. For ERY sa mples prepared in PSS, shown in the black bars, incubation with both C - peptide and zinc resulted in a significant increase in ATP release, by approximately 50% (n = 5, p < 0.01), whereas incubation with C - peptide or zinc alone did not significantly change the amount of ATP released. However, an increase of ATP release following addition of C - peptide and zinc was not observed when the ERY samples were prepared in an albumin - free PSS buffer (grey bars), indicating the indispensable role of albumin in this bio logical pathway. In the same figure, when ERYs were incubated with E27A and zinc instead of C - peptide and zinc, no increase of ATP release was observed, even in albumin - containing PSS. 2.3.6 Enhanced Flow - induced ATP Release from ERYs by Albumin/C - peptide/ Zn In order to better characterize ERY - derived ATP release, a previously designed 3D - printed fluidic device was employed. 44 ERY samples were prepared in the same manner as static ATP samples for flow - induced ATP release quantification using the fluidic dev obtained using the fluidic device rather than static measurements. As shown in Fig 2.13, ERYs incubated with both C - peptide and zinc in PSS had the highest ATP release (319.8 ± 15.2 nM), which was significantly higher than the ERYs in the absence of any of the three components from albumin, C - peptide and zinc (p < 0.005). ERYs incubated 102 with E27A and zinc did not show significant increase in ATP release (p > 0.95), even in albumin - co ntaining PSS, as shown in Fig 2.14. Fig 2.11: ITC analysis of interactions of E27A and HSA at pH = 7.40 ± 0.01. A and electrostatic attraction without specific binding was detected. All solutions were prepared in Tris - HCl buffer. 103 Fig 2.12: The effect of albumin, C - peptide and zinc on static ATP release from ERYs. Values of ATP release are normalized to the firs t black bar, the untreated 7% ERYs in PSS. ERYs suspended in PSS (black bars) had significantly increased ATP release only when C - peptide and zinc were both present, compared to untreated ERY control sample. The previously seen increase of ATP release was abandoned when C - peptide was mutated to E27A. C - peptide and zinc did not increase ATP release from ERYs in BSA - free PSS (grey bars). Error bars = S.E.M., n = 5. 104 Fig 2.13: The effect of albumin, C - peptide and zinc on flow - induced ATP release from ER Ys. A significant increase in ATP release was only measured from the ERY sample prepared in an albumin - containing PSS buffer that had been incubated with both C - peptide and zinc (p < 0.005). The absence of any one of these 3 components (C - peptide, zinc, or albumin) resulted in no significant increase in ERY - derived ATP. Error bars = S.E.M., n = 5. 105 Fig 2.14: Comparison of ATP released from ERYs treated with combination of C - peptide/zinc and E27A/zinc. In albumin - containing PSS, C - peptide/zinc increase d the ATP release significantly (p < 0.005), whereas E27A/zinc did not show such an effect. In albumin free PSS, however, the role that C - peptide/zinc has on increasing the ATP release from ERYs was not observed. Error bars = S.E.M., n = 5. 106 2.4 Discussion Over the past ten years, proinsulin C - peptide has shown its ability to ameliorate diabetes complications in animals and humans. 46 For example, complications such as neuropathy and n ephropathy were decreased; 47 - 49 and blood flow in skin, muscle tissue and the microvascular system were improved in type 1 diabetic patients. 50 - 51 Unfortunately, C - peptide has not been officially approved to be administrated along with insu lin in the current hormone replacemen t therapy for type 1 diabetes. This is mainly due to the lack of knowledge of the mechanisms of action of C - peptide, including the identification of a receptor, and then systematically designed experiments that may include animal experiments to test the results of C - peptide administration. 52 In human blood vessels, especially small capillaries, blood flow is highly influenced by the funct ions of flowing erythrocytes. 53 - 54 Blood flow creates shear st ress on ERYs, and in response to that shear stress, erythrocytes deform and release ATP. 55 - 56 ATP is able to affect blood flow by stimulating a well - known vasodilator, nitric oxide (NO) from the blood vessel endothelium. 57 Once the blood vessel is dilated, blood flow is improved. 58 In support of the theories from many researchers showing that C - peptide can improve blood circulation and reduce diabetes complications, our group has shown that C - peptide can enhance the ability of erythrocytes to release more A TP, which will lead to increased NO production in the bloodstream and therefore increased blood flow. However, our group has never observed any beneficial bioactivity from C - peptide unless the peptide is co - administered to the erythrocytes with certain tra nsition metal ions, 107 such as Fe(II), Cr(III) or Zn(II). 59 - 60 Meyer et. al. first found that C - peptide, in the presence of Fe 2+ , Cr 3+ , or Zn 2+ , has the ability to increase ATP release from ERYs, whereas some of the common metal ions in the bloodstream, such as Na + and K + , do not. 59 This increase of ATP release may be due to an increase of glucose transport into the ERYs through the glucose transporter 1 (GLUT1) on the cell membrane. 59 Fe 2+ was then determined by mass spectrometry to be an impurity in the co mmercially obtained C - peptide, and removal of Fe 2+ from the commercially obtained C - p eptide lost its ability to increase ATP release and glucose clearance by ERYs. Therefore, in all subsequent experiments, an HPLC - purified high purity C - peptide containing no transition metal ions was used in sample preparation, rather than the commercially obtained C - peptide. In a healthy human body, Zn 2+ - cell granules, 61 - 62 where C - peptide and insulin are stored prior to secretion. 63 - 64 Due to the availability of Zn 2+ for C - peptide in respect of both concentration and location, the Spence l ab proposed that zinc may be the metal ion responsible for C - in vivo . 60 Similar results have shown that zinc and C - peptide together can enhance ATP rele ase from ERYs, while zinc or C - peptide alone failed to enhance ATP release. 65 The effect of C - peptide and zinc on enhancing ATP release from ERYs was not completely understood, beyond the fact that both were required for cellular activity. The Spence lab initially proposed a mechanism for C - peptide, which included zinc facilitating 108 the delivery of C - peptide to the ERY. However, this hypothesis was then replaced by a new one, since data in Fig 2.4 showed that C - peptide uptake by the ERY is zinc - independent; on the contrary, zinc uptake by the ERY is dependent upon the successful uptake o f C - peptide. Additionally, both C - peptide and zinc binding curves are specific with similar saturation values around 1800 molecules per ERY cell. This 1:1 binding ratio of C - peptide and zinc to the ERY provided us with guidance in ERY sample preparation th at best efficacy for ATP increase was achieved when C - peptide and zinc were added to the ERY at equimolar concentrations. Calculation was applied to the two binding curves, and affinity of ERYs for C - peptide and zinc was found to be both around 10 9 M - 1 . Ot her groups have also reported specific binding of C - peptide to other cell types such as venous endothelial cells, human renal tubular cells, and fibroblasts, with affinities on the same order of magnitude of 10 9 . 66 Unfortunately, no cell membrane receptor for C - peptide has been identified. Since C - peptide appears to be able to interact with multiple cell types, it is reasonable to expect that these cell types share a common element of the mechanism for C - peptide interaction. A modified hypothesis stated tha t C - peptide was zinc bound when secreted from the - cell granules, and delivered zinc to the ERY. ITC experiments were designed to identify the binding between C - peptide and zinc, although ITC results did not support this hypothesis. The weak heat signals from the interaction of C - peptide/zinc even though at high concentrations in Fig 2.7, could not be successfully fit into any theoretical binding model with reasonable calculated binding constant and binding stoichiometry, suggesting the heat signals were a ctually derived from non - specific electrostatic attraction rather than specific binding. To confirm the conclusion, zinc and C - peptide 109 solutions were inversely titrated by ITC, with the supposed ligand, zinc, filled in the sample cell, and the suppos ed receptor C - peptide filled in the syringe. A similar conclusion was drawn from the ITC data shown in Fig 2.8. To further confirm this non - specific interaction between C - peptide and zinc, more negative control experiments were designed and performe d. Titrations of other types of cations that are not known for activating C - peptide, including calcium (CaCl 2 , Sigma - Aldrich) and sodium (NaCl, Sigma - Aldrich) against C - peptide were carried out in the same manner as with zinc. Both the values of the heat s ignals and the shape of isotherm shown in Fig 2.15 and Fig 2.16 highly resembled the results with zinc in Fig 2.7. It is not unusual for a peptide like C - peptide, which carries six negative charges, to interact with multiple types of metal ions, and this f urther confirmed that C - peptide and zinc interact through non - specific electrostatic attraction rather than specific binding. It is interesting that according to previous circular dichroism spectroscopy data, 67 the interaction between C - peptide and zinc le d to a decrease of randomness in the secondary structure of C - peptide, as shown by a reduction of the peak at 198 nm with the addition of zinc. 68 Due to the absence of binding between C - peptide and zinc, it was anticipated that another molecule was partici pating in the delivery of both C - peptide and zinc to the ERY. All ERY samples prepared for C - peptide/zinc uptake quantification and ATP release measurement were prepared in PSS buffer that contained 0.05% BSA. However, when albumin was not present in the b uffer, even though C - peptide and zinc were added at equimolar concentrations to the ERY samples, neither cellular uptake of C - peptide/zinc nor increase of ATP release from static ERYs could be observed, as 110 shown in Fig 2.5 and 2.13, respectively. Albumin i n the circulating serum is a well - known transporter for fatty acids, hormones, metal ions, and drug molecules. 29 The transporter role of albumin for zinc ions via high - affinity binding has been well - established by many groups, however, with the primary bin ding site for zinc well discovered, the secondary or more binding sites are only suggested. 27,30 Fig 2.15: ITC analysis of C - peptide and Ca 2+ interaction at pH ~5.8. A 3 mM CaCl 2 - peptide solution and electrostatic attraction without specific binding was detected. Solutions were prepared in DDW. 111 Fig 2.16: ITC analysis of C - peptide and Na + interaction at pH ~5.8. A 3 - peptide solution and electrostatic attraction without specific binding was detected. Solutions were prepared in DDW. 112 ITC was also utilized to measure possible binding between HSA and C - peptide. The specific binding discovered between them suggest that album in is also a transporter for C - peptide in the human bloodstream, and perhaps delivers C - peptide to its site of action, including to the ERY. Additionally, the solubility of C - peptide in aqueous solutions was greatly enhanced with albumin present, due to the binding of the two molecules. Fig 2.10 suggests the possibility of simultaneous binding of C - peptide and zinc to albumin, indicating that albumin might bind both C - peptide and zinc upon their - cells and deliver them to the ER Y. Albumin has a higher affinity for zinc (K a ~10 7 M - 1 ) therefore their binding appears in the earlier stage in ITC binding curve in Fig 2.10, and the medium affinity for C - peptide (K a ~10 5 M - 1 ) appears in the later stage of binding curve. It is worth noti ng that the affinities of albumin for C - peptide and zinc are both lower than the affinities of those molecules to ERYs, which makes possible for albumin to release the molecules to ERYs. However, it is not known if an interaction exists between C - peptide a nd zinc when both are bound to albumin, which can potentially be useful for understanding the process of cellular uptake of C - peptide and zinc solely based on this ITC data. It is obvious that albumin is required for C - peptide and zinc to increase ATP rele ase from ERYs, which can be crucial in understanding the mechanism of how C - peptide and zinc interact with the ERY cell membrane, and it is also an important bridge connecting active molecules and sites of action through the circulating bloodstream. Intere stingly, albumin was found present in sample preparation in most of the previously published C - peptide studies when revisiting the literature. A three - component system of albumin/C - peptide/zinc was therefore proposed due to 113 their binding and potential to b e used as a molecular ensemble with therapeutic efficacy. To expand our knowledge and understanding of the formation and function of this three - component system, obtaining the structure of this ensemble and identifying the binding sites for C - peptide and z inc on albumin would provide more solid evidence. Preliminary studies were preformed to identity amino acids important for C - peptide and albumin binding. A wealth of literature explains the importance of C - acid residue at position 27 (E2 7). To test the importance of E27, a single residue mutant, E27A, was used in place of C - peptide, and it was determined that binding to albumin was abolished (Fig 2.11). Zinc uptake and increase of ATP release form ERYs were also reduced in the E27A mutant samples. These data suggested that E27A was not able to form the three - component ensemble with albumin and zinc, which was the direct cause for the loss of effects on ERYs, including increase in ATP release. This means the carboxylate group of E27 may pla y a key role in the interaction of C - peptide with albumin and the delivery of zinc. Once again, data have supported the literature by showing the importance of the glutamic acid residue at position 27 of C - peptide for its bioactivity. There is still more s tructural information to be discovered to further confirm the model of this three - component ensemble. The ability of albumin/C - peptide/zinc to increase ATP release from ERYs was investigated for both static and flow conditions. Fig 2.12 shows that when ERY s were not subjected to flow, incubation wit h albumin/C - peptide/zinc resulted in approximately a 50% increase in ATP release. When these ERYs were loaded into a 3D - printed fluidic device channel, samples that contained albumin/C - peptide/zinc showed roughly 60% increase of ATP release. Even though both static and flow conditions led to similar 114 increase of ATP release from ERYs, the 3D - printed fluidic device served as a good in vitro mimic of the actual bloodstream. It was noticed that the observation of the increase of ATP release by albumin/C - peptide/zinc was more reproducible when the ERYs were subject to flow, which strongly suggested a flow - related mechanism and will be discussed in the next chapter. The fluidic device was originally designed by a collabo rator in the group, Chengpeng Chen, 44 who also measured downstream NO production from the endothelial cells using the same device, and found that significantly more NO was produced when ATP release was increased by C - peptide/zinc (data not shown here). This further confirmed the potential of albumin/C - peptide/zinc in improving blood flow rate, and reducing diabetes complications when given to type 1 diabetic patients. 115 REFERENCES 116 REFERENCES 1. Steiner, D. F., The pr oinsulin C - peptide -- a multirole model. Experimental diabesity research 2004, 5 (1), 7 - 14. 2. Weiss, M. A.; Frank, B. H.; Khait, I.; Pekar, A.; Heiney, R.; Shoelson, S. E.; Neuringer, L. J., NMR and photo - CIDNP studies of human proinsulin and prohormone pro cessing intermediates with application to endopeptidase recognition. Biochemistry 1990, 29 (36), 8389 - 401. 3. Henriksson, M.; Shafqat, J.; Liepinsh, E.; Tally, M.; Wahren, J.; Jornvall, H.; Johansson, J., Unordered structured of proinsulin C - peptide in aqu eous solution and in the presence of lipid vesicles. Cellular and molecular life sciences : CMLS 2000, 57 (2), 337 - 42. 4. Snell, C. R.; Smyth, D. G., Proinsulin: a proposed three - dimensional structure. The Journal of biological chemistry 1975, 250 (16), 62 91 - 5. 5. Munte, C. E.; Vilela, L.; Kalbitzer, H. R.; Garratt, R. C., Solution structure of human proinsulin C - peptide. The FEBS journal 2005, 272 (16), 4284 - 93. 6. Rajan, R.; Balaram, P., A model for the interaction of trifluoroethanol with peptides and pr oteins. International journal of peptide and protein research 1996, 48 (4), 328 - 36. 7. Wahren, J.; Ekberg, K.; Johansson, J.; Henriksson, M.; Pramanik, A.; Johansson, B. L.; Rigler, R.; Jornvall, H., Role of C - peptide in human physiology. American journal of physiology. Endocrinology and metabolism 2000, 278 (5), E759 - 68. 8. Pramanik, A.; Ekberg, K.; Zhong, Z.; Shafqat, J.; Henriksson, M.; Jansson, O.; Tibell, A.; Tally, M.; Wahren, J.; Jornvall, H.; Rigler, R.; Johansson, J., C - peptide binding to human cel l membranes: importance of Glu27. Biochemical and biophysical research communications 2001, 284 (1), 94 - 8. 9. Chen, L. M.; Yang, X. W.; Tang, J. G., Acidic residues on the N - terminus of proinsulin C - Peptide are important for the folding of insulin precurso r. Journal of biochemistry 2002, 131 (6), 855 - 9. 10. Gross, D. J.; Villa - Komaroff, L.; Kahn, C. R.; Weir, G. C.; Halban, P. A., Deletion of a highly conserved tetrapeptide sequence of the proinsulin connecting peptide (C - peptide) inhibits proinsulin to ins ulin conversion by transfected pituitary corticotroph (AtT20) cells. The Journal of biological chemistry 1989, 264 (36), 21486 - 90. 11 . Petersen, C. E.; Ha, C. E.; Jameson, D. M.; Bhagavan, N. V., Mutations in a specific human serum albumin thyroxine bindin g site define the structural basis of familial dysalbuminemic hyperthyroxinemia. The Journal of biological chemistry 1996, 271 (32), 19110 - 7. 117 12. Evans, T. W., Review article: albumin as a drug -- biological effects of albumin unrelated to oncotic pressure. Alimentary pharmacology & therapeutics 2002, 16 Suppl 5 , 6 - 11. 13. Mendez, C. M.; McClain, C. J.; Marsano, L. S., Albumin therapy in clinical practice. Nutrition in clinical practice : official publication of the American Society for Parenteral and Enteral Nutrition 2005, 20 (3), 314 - 20. 14. Fasano, M.; Curry, S.; Terreno, E.; Galliano, M.; Fanali, G.; Narciso, P.; Notari, S.; Ascenzi, P., The extraordinary ligand binding properties of human serum albumin. IUBMB life 2005, 57 (12), 787 - 96. 15. A hmed - Ouameur , A.; Diamantoglou, S.; Sedaghat - Herati, M. R.; Nafisi, S.; Carpentier, R.; Tajmir - Riahi, H. A., The effects of drug complexation on the stability and conformation of human serum albumin: protein unfolding. Cell biochemistry and biophysics 2006, 45 (2), 20 3 - 13. 16. Varshney, A.; Sen, P.; Ahmad, E.; Rehan, M.; Subbarao, N.; Khan, R. H., Ligand binding strategies of human serum albumin: how can the cargo be utilized? Chirality 2010, 22 (1), 77 - 87. 17. Bhattacharya, A. A.; Curry, S.; Franks, N. P., Binding of the general anesthetics propofol and halothane to human serum albumin. High resolution crystal structures. The Journal of biological chemistry 2000, 275 (49), 38731 - 8. 18. Simard, J. R.; Zunszain, P. A.; Hamilton, J. A.; Curry, S., Location of high and low affinity fatty acid binding sites on human serum albumin revealed by NMR drug - competition analysis. Journal of molecular biology 2006, 361 (2), 336 - 51. 19. Ascenzi, P.; Bocedi, A.; Notari, S.; Fanali, G.; Fesce, R.; Fasano, M., Allosteric modu lation of drug binding to human serum albumin. Mini reviews in medicinal chemistry 2006, 6 (4), 483 - 9. 20. Sokolowska, M.; Wszelaka - Rylik, M.; Poznanski, J.; Bal, W., Spectroscopic and thermodynamic determination of three distinct binding sites for Co(II) ions in human serum albumin. Journal of inorganic biochemistry 2009, 103 (7), 1005 - 13. 21. Sokolowska, M.; Pawlas, K.; Bal, W., Effect of common buffers and heterocyclic ligands on the binding of Cu(II) at the multimetal binding site in human serum albumin . Bioinorganic chemistry and applications 2010 , 725153. 22. Deng, B.; Wang, Y.; Zhu, P.; Xu, X.; Ning, X., Study of the binding equilibrium between Zn(II) and HSA by capillary electrophoresis - inductively coupled plasma optical emission spectrometry. Analyt ica chimica acta 2010, 683 (1), 58 - 62. 23. Duff, M. R., Jr.; Kumar, C. V., The metallomics approach: use of Fe(II) and Cu(II) footprinting to examine metal binding sites on serum albumins. Metallomics : integrated biometal science 2009, 1 (6), 518 - 23. 118 24. Sadler, P. J.; Tucker, A.; Viles, J. H., Involvement of a lysine residue in the N - terminal Ni 2+ and Cu 2+ binding site of serum albumins. Comparison with Co 2+ , Cd 2+ and Al 3+ . The FEBS journal 2005, 220 , 193 - 200. 25. Shaw, C. F., The protein chem istry of antiarthritic gold(I) thiolates and related complexes. Comments on Inorganic Chemistry 1989, 8 , 233 - 267. 26. Blindauer, C. A.; Harvey, I.; Bunyan, K. E.; Stewart, A. J.; Sleep, D.; Harrison, D. J.; Berezenko, S.; Sadler, P. J., Structure, properti es, and engineering of the major zinc binding site on human albumin. The Journal of biological chemistry 2009, 284 (34), 23116 - 24. 27. Andre, C.; Guillaume, Y. C., Zinc - human serum albumin association: testimony of two binding sites. Talanta 2004, 63 (2), 503 - 8. 28. Giroux, E. L.; Henkin, R. I., Macromolecular ligands of exchangeable copper, zinc, and cadmium in human serum. Bioinorg. Chem. 1973, 2 (125 - 133). 29. Fanali, G.; di Masi, A.; Trezza, V.; Marino, M.; Fasano, M.; Ascenzi, P., Human serum albumin: from bench to bedside. Molecular aspects of medicine 2012, 33 (3), 209 - 90. 30 . Bal, W.; Christodoulou, J.; Sadler, P. J.; Tucker, A., Multi - metal binding site of serum albumin. Journal of inorganic biochemistry 1998, 70 (1), 33 - 9. 31. Bobilya, D. J.; Brisk e - Anderson, M.; Reeves, P. G., Ligands influence Zn transport into cultured endothelial cells. Proceedings of the Society for Experimental Biology and Medicine. Society for Experimental Biology and Medicine 1993, 202 (2), 159 - 66. 32. Tibaduiza, E. C.; Bobi lya, D. J., Zinc transport across an endothelium includes vesicular cotransport with albumin. Journal of cellular physiology 1996, 167 (3), 539 - 47. 33. Galvez, M.; Moreno, J. A.; Elosegui, L. M.; Escanero, J. F., Zinc uptake by human erythrocytes with and without serum albumins in the medium. Biological trace element research 2001, 84 (1 - 3), 45 - 56. 34. Rabenstein, D. L.; Isab, A. A., The complexation of zinc in intact human erythrocytes studied by 1H spin - echo NMR. FEBS letters 1980, 121 (1), 61 - 4. 35. Oels hlegel, F. J., Jr.; Brewer, G. J.; Knutsen, C.; Prasad, A. S.; Schoomaker, E. B., Studies on the interaction of zinc with human hemoglobin. Archives of biochemistry and biophysics 1974, 163 (2), 742 - 8. 36. Kratz, F., Albumin as a drug carrier: desig n of prodrugs, drug conjugates and nanoparticles. Journal of controlled release : official journal of the Controlled Release Society 2008, 132 (3), 171 - 83. 119 37. Subramanian, G. M.; Fiscella, M.; Lamouse - Smith, A.; Zeuzem, S.; McHutchison, J. G., Albint erferon alpha - 2b: a genetic fusion protein for the treatment of chronic hepatitis C. Nature biotechnology 2007, 25 (12), 1411 - 9. 38. Duttaroy, A.; Kanakaraj, P.; Osborn, B. L.; Schneider, H.; Pickeral, O. K.; Chen, C.; Zhang, G.; Kaithamana, S.; Singh, M.; Schulingkamp, R.; Crossan, D.; Bock, J.; Kaufman, T. E.; Reavey, P.; Carey - Barber, M.; Krishnan, S. R.; Garcia, A.; Murphy, K.; Siskind, J. K.; McLean, M. A.; Cheng, S.; Ruben, S.; Birse, C. E.; Blondel, O., Development of a long - acting insulin analog usi ng albumin fusion technology. Diabetes 2005, 54 (1), 251 - 8. 39. Haag, R.; Kratz, F., Polymer therapeutics: concepts and applications. Angewandte Chemie 2006, 45 (8), 1198 - 215. 40. Giannoukakis, N., CJC - 1131. ConjuChem. Current opinion in investigational d rugs 2003, 4 (10), 1245 - 9. 41. Kim, J. G.; Baggio, L. L.; Bridon, D. P.; Castaigne, J. P.; Robitaille, M. F.; Jette, L.; Benquet, C.; Drucker, D. J., Development and characterization of a glucagon - like peptide 1 - albumin conjugate: the ability to activate the glucagon - like peptide 1 receptor in vivo. Diabetes 2003, 52 (3), 751 - 9. 42. Kurtzhals, P., Pharmacology of insulin detemir. Endocrinology and metabolism clinics of North America 2007, 36 Suppl 1 , 14 - 20. 43. Kurtzhals, P.; Havelund, S.; Jonassen, I .; Markussen, J., Effect of fatty acids and selected drugs on the albumin binding of a long - acting, acylated insulin analogue. Journal of pharmaceutical sciences 1997, 86 (12), 1365 - 8. 44. Chen, C.; Wang, Y.; Lockwood, S. Y.; Spence, D. M., 3D - print ed fluidic devices enable quantitative evaluation of blood components in modified storage solutions for use in transfusion medicine. The Analyst 2014, 139 (13), 3219 - 26. 45. Hutton, J. C., The internal pH and membrane potential of the insulin - secretory gra nule. The Biochemical journal 1982, 204 (1), 171 - 8. 46. Wahren, J.; Ekberg, K.; Jornvall, H., C - peptide is a bioactive peptide. Diabetologia 2007, 50 (3), 503 - 9. 47. Johansson, B. L.; Borg, K.; Fernqvist - Forbes, E.; Kernell, A.; Odergren, T.; Wahren, J., B eneficial effects of C - peptide on incipient nephropathy and neuropathy in patients with Type 1 diabetes mellitus. Diabetic medicine : a journal of the British Diabetic Association 2000, 17 (3), 181 - 9. 48. Ekberg, K.; Brismar, T.; Johansson, B. L.; Lindstro m, P.; Juntti - Berggren, L.; Norrby, A.; Berne, C.; Arnqvist, H. J.; Bolinder, J.; Wahren, J., C - Peptide replacement therapy and sensory nerve function in type 1 diabetic neuropathy. Diabetes care 2007, 30 (1), 71 - 6. 120 49. Samnegard, B.; Jacobson, S. H.; Jare mko, G.; Johansson, B. L.; Sjoquist, M., Effects of C - peptide on glomerular and renal size and renal function in diabetic rats. Kidney international 2001, 60 (4), 1258 - 65. 50. Stevens, M. J.; Zhang, W.; Li, F.; Sima, A. A., C - peptide corrects endoneurial b lood flow but not oxidative stress in type 1 BB/Wor rats. American journal of physiology. Endocrinology and metabolism 2004, 287 (3), E497 - 505. 51. Forst, T.; Kunt, T.; Pohlmann, T.; Goitom, K.; Engelbach, M.; Beyer, J.; Pfutzner, A., Biological activity o f C - peptide on the skin microcirculation in patients with insulin - dependent diabetes mellitus. The Journal of clinical investigation 1998, 101 (10), 2036 - 41. 52. Luzi, L.; Zerbini, G.; Caumo, A., C - peptide: a redundant relative of insulin? Diabetologia 200 7, 50 (3), 500 - 2. 53. Secomb, T. W., Red blood cell mechanics and capillary blood rheology. Cell biophysics 1991, 18 (3), 231 - 51. 54. Ellsworth, M. L.; Forrester, T.; Ellis, C. G.; Dietrich, H. H., The erythrocyte as a regulator of vascular tone. The Ameri can journal of physiology 1995, 269 (6 Pt 2), H2155 - 61. 55. Sprague, R. S.; Ellsworth, M. L.; Stephenson, A. H.; Kleinhenz, M. E.; Lonigro, A. J., Deformation - induced ATP release from red blood cells requires CFTR activity. The American journal of physiolo gy 1998, 275 (5 Pt 2), H1726 - 32. 56. Sprague, R. S.; Ellsworth, M. L.; Stephenson, A. H.; Lonigro, A. J., ATP: the red blood cell link to NO and local control of the pulmonary circulation. The American journal of physiology 1996, 271 (6 Pt 2), H2717 - 22. 57 . Furchgott, R. F.; Zawadzki, J. V., The obligatory role of endothelial cells in the relaxation of arterial smooth muscle by acetylcholine. Nature 1980, 288 (5789), 373 - 6. 58. Dietrich, H. H.; Ellsworth, M. L.; Sprague, R. S.; Dacey, R. G., Jr., Red blood cell regulation of microvascular tone through adenosine triphosphate. American journal of physiology. Heart and circulatory physiology 2000, 278 (4), H1294 - 8. 59. Meyer, J. A.; Froelich, J. M.; Reid, G. E.; Karunarathne, W. K.; Spence, D. M., Meta l - activated C - peptide facilitates glucose clearance and the release of a nitric oxide stimulus via the GLUT1 transporter. Diabetologia 2008, 51 (1), 175 - 82. 60. Medawala, W.; McCahill, P.; Giebink, A.; Meyer, J.; Ku, C. J.; Spence, D. M., A Molecular Leve l Understanding of Zinc Activation of C - peptide and its Effects on Cellular Communication in the Bloodstream. The review of diabetic studies : RDS 2009, 6 (3), 148 - 58. 61. Hutton, J. C., The insulin secretory granule. Diabetologia 1989, 32 (5), 271 - 81. 121 62. Lemaire, K.; Ravier, M. A.; Schraenen, A.; Creemers, J. W.; Van de Plas, R.; Granvik, M.; Van Lommel, L.; Waelkens, E.; Chimienti, F.; Rutter, G. A.; Gilon, P.; in't Veld, P. A.; Schuit, F. C., Insulin crystallization depends on zinc transporter ZnT8 expr ession, but is not required for normal glucose homeostasis in mice. Proceedings of the National Academy of Sciences of the United States of America 2009, 106 (35), 14872 - 7. 63. Blundell, T. L.; Cutfield, J. F.; Cutfield, S. M.; Dodson, E. J.; Dodson, G. G. ; Hodgkin, D. C.; Mercola, D. A., Three - dimensional atomic structure of insulin and its relationship to activity. Diabetes 1972, 21 (2 Suppl), 492 - 505. 64. Emdin, S. O.; Dodson, G. G.; Cutfield, J. M.; Cutfield, S. M., Role of zinc in insulin biosynthesis. Some possible zinc - insulin interactions in the pancreatic B - cell. Diabetologia 1980, 19 (3), 174 - 82. 65. Meyer, J. A.; Subasinghe, W.; Sima, A. A.; Keltner, Z.; Reid, G. E.; Daleke, D.; Spence, D. M., Zinc - activated C - peptide resistance to the type 2 diab etic erythrocyte is associated with hyperglycemia - induced phosphatidylserine externalization and reversed by metformin. Molecular bioSystems 2009, 5 (10), 1157 - 62. 66. Rigler, R.; Pramanik, A.; Jonasson, P.; Kratz, G.; Jansson, O. T.; Nygren, P.; Sta hl, S.; Ekberg, K.; Johansson, B.; Uhlen, S.; Uhlen, M.; Jornvall, H.; Wahren, J., Specific binding of proinsulin C - peptide to human cell membranes. Proceedings of the National Academy of Sciences of the United States of America 1999, 96 (23), 13318 - 23. 67 . Medawala, W., Mechanistic and Funtional Studies of Zinc (II) Activation of C - peptide and Its Effect on Red Blood Cell Metabolism. Ph.D. Dissertation. Michigan State University. 2010 . 68. Sakai - Kato, K.; Ishiguro, A.; Mikoshiba, K.; Aruga, J.; Utsunomiya - Tate, N., CD spectra show the relational style between Zic - , Gli - , Glis - zinc finger protein and DNA. Biochimica et biophysica acta 2008, 1784 (7 - 8), 1011 - 9. 122 Chapter 3 Stored ERYs Response to Insulinoma Cell Line (INS - 1) 3.1 Introduction In chapter 2, al bumin as a transporter for C - peptide and zinc has been discussed. C - peptide and zinc are delivered to ERYs by albumin, leading to an increase of ERY - derived ATP release, and subsequent increase of NO production from endothelial cells. 1 NO is a well - known vasodilator which relaxes blood vessels and maintains adequate blood flow and vascular health. 2 The correlation of reduced blood flow and cause of diabetic complications has been reported. 3 ERYs obtained from diabetic rabbits have shown a decrea sed level of ATP release. 4 And according to previous results by the Spence group, ERYs obtained from diabetic rats have also shown reduced interaction with C - peptide and zinc fo r subsequent increase of ATP r elease. 5 Recently, the Spence group has reported hyperglycemic environment in the current FCA approved ERY collection and storage solutions (129 mM and 111 mM, respectively). 6 Similar to diabetic ERYs (blood glucose 7 - 9 mM in diabetic patients), the Spence group has noticed a significant reduction in ATP release from stored ERYs, compared to ERYs stored in our modified normoglycemic solution. 6 In this chapter, other possible - peptide and zinc from the p ancreatic secretions are investigated, which may shed light on another major health problem involving stored ERYs. 123 3.1.1 Overall Introduction to Blood Transfusion and Blood Banking A blood transfusion is the transfer of whole blood (WB) or blood component s (e.g., erythrocytes, platelets, plasma and cryoprecipitate) from a donor into the bloodstream of a recipient. Usually an adequate and reliable supply of safe blood can be assured by voluntary unpaid donors, family members and paid donors. Blood transfusi on is a lifesaving measure. 7 Blood and blood components are collected and stored under standard procedures to be available for situations of blood transfusion. The process of collecting, separating, and storing whole blood and blood components is referred to as blood banking. Blood may be collected and stored for the purpose of lab research and medical use. Most blood collected for medical use is transfused into patients who need blood because of trauma, for surgery or as therapeutic treatment of diseases, such as sickle cell disease, anemia and as a result of chemotherapy. 8 Blood transfusion has become an important part in modern clinical healthcare, and the need for blood transfusion is large. According to American Red Cross, there are approximately 15.7 m illion donations collected in the US each year from 9.2 million donors. And more than 41,000 donations are needed each day. A total of 30 million blood components are transfused each year in the U.S. with the average erythrocyte (ERY) transfusion requiring approximately 3 pints of ERYs. 3.1.2 Procedure of Blood Collecting and Storage of Components Generally, blood from donors undergoes collection, processing, testing, storage and distribution before it is transfused to a patient. 9 Standard protocols recomme nded by well - established organizations must be used for blood collection. During donation, about 124 1 pint of blood and several small test tubes are collected from each donor, which will be transported to a Red Cross center while stored in iced coolers. Antic oagulants are used during the collectio n of anticoagulated blood. 10 - 12 Heparin is a common type of anticoagulant for blood collection that binds to antithrombin III and accelerates the inactivation of thrombin and other clotting factors. 9 EDTA is another t ype of anticoagulant that functions by chelating metals, such as calcium and magnesium. However, as an anticoagulant, EDTA has been reported to be well suited for DNA - based assays, but problematic for cytogenetic analyses. 13 A nother type of anticoagulant is acid citrate dextrose. It also chelates calcium to prevent blood from clotting and citrate - stabilized blood was found to provide better quality RNA and DNA than other anticoagulants and yields more lymphocytes for culture. 13 Therefore the currently appr oved anticoagulant solution for blood collection is a citrate - based solution known as Citrate Phosphate Dextrose Solution (CPD). There are currently two types of CPD solutions being used in the US, CPD and CP2D, the components of which are listed in Table 3.1. 14 125 Table 3.1: Constituents and their concentrations in anticoagulant solutions for blood collection and storage solutions for ERY storage. These solutions are currently used for blood banking in the U S. CPD and CP2D are two types of anticoagulant solutions for blood collection. AS - 1, AS - 3 and AS - 5 are three types of storage solutions for ERY storage. Note that the glucose concentrations in these solutions all exceed normal physiological glucose level ( 4 - 6 mM). - - - - - - - - - - - - - - - - - - - 126 After collection, blood is centrifuged as soon as possible and the transfusable components separated to reduce contamination. Transfusable components include ERYs, platelets, and plasma. Plasma can be further processed into cryoprecipitate ( a s ource of fibrinogen, blood clotting agent ). ERYs and single donor platelets are then leuko - reduced. On each unit of donated blood, tests for blood type and infectious diseases are established. Units are discarded if test results are not qualified, and the donor is notified. Whereas units that are suitable for transfusion are labeled and stored. To provide prolonged preservation, plasma and cryoprecipitate are stored in freezers at - 80 ºC for up to one year. 15 ERYs are stored in refrigerators at 1 - 6 ºC for u p to 42 days. Platelets are stored at room temperature in agitators for up to five days.16 Specifically for the storage of ERYs, there are several types of storage solutions, also known as additive solutions (AS) that have been licensed by the Food and Dru g Administration (FDA) and commercialized in the US, including AS - 1, AS - 3, AS - 5, the components of which a re listed in Table 3.1. 14,17 - 19 3.1.3 Post Transfusion Complications 3.1.3.1 Transfusion - transmitted Infections (TTIs) TTI refers to a virus, parasit e, or other potential pathogen in donated blood that can be passed on to a recipient through transfusion. Due to the active involvement of a variety of techniques, including donor history screening and infectious disease screening tests, the risk of TTIs h as decreased dramatically during the past few decades. 20 For example, in 2005, the reported estimated risk of transfusion - associated HIV was about 1:2.3 million. 21 And in 2007, the risks of transfusion - associated hepatitis 127 B and C were reduced to 1:0.28 mi llion and 1:1.9 million, respectively. 22 However, in spite of success in reducing the risk of TTI, emerging infections are continuingly being recognized and form new threats to transfusion safety. 23 Blood components can also be contaminated by bacteria vi a donor bacteremia, contamination of blood collection bags at the time of manufacture, and contamination of the venipuncture site, with the last mechanism being the most common case. ERY component contamination is caused most commonly by Yersinia enterocol itica; and platelet product by normal skin flora, e.g., Staphylococcus aureus. 24 The rate of bacterial contamination of ERYs has been reported to be approximately 1:38,000 units, and platelets 1:2000 to 1:3000. 25 Bacterial screening of apheresis platelets has been suggested to reduce the risk of septic transfusion reactions. 26 3.1.3.2 Acute Transfusion Reactions Acute transfusion reactions include simple allergic transfusion reactions, anaphylactic transfusion reactions, 27 hemolytic transfusion reactions, 28 hypotensive reactions, 29 febrile, nonhemolytic transfusion reactions, 30 - 31 transfusion - related acute lung injury (TRALI), 32 and transfusion - associated circulator overload (TACO). 33 It has been reported that, with the decreasing risks of TTI during the las t 2 to 3 decades, noninfectious risks such as acute transfusion reactions have changed little in incidence and are now much more frequently associated with adverse patient outcomes. 20 Among the acute transfusion reactions, TRALI is the most common cause of transfusion - related 128 fatality reported to the Food and Drug Administration, with 127 fatalities reported between 2005 and 2009 (48% of all reported fatalities). 34 3.1.3.3 Insufficient Nitric Oxide Bioavailability (INOBA) Recently, it has been reported that people who receive a transfusion can also suffer from insufficient nitric oxide bioavailability (INOBA), 29 which can be briefly stated as a reduction of local NO bioavailability after ERY transfusions that is insufficient to meet metabolic demands leading to morbidity and mortality in the recipient. INOBA can further lead to insufficient vasodilation and therefore inadequate blood flow and O 2 delivery to organs and tissues. As a result, transfusion recipients can have increased risks for multiorgan failure , and death in in severe cases. 35 Importantly, some studies have indicated patients transfused with ERYs stored for more than 14 days have had statistically higher rates of in - hospital mortality tha units. 36 - 37 The INOBA hypo thesis indicates that altered RBC storage and/or processing conditions may be necessary. 35 3.1.3.4 Modified Storage Solution for ERYs The CPD solution used during whole blood collection from donors contains a glucose concentration of ~129 mM prior to colle ction, and >20 mM after collection. The AS - 1 solution for ERY storage also has a glucose concentration of ~111 mM, and after ERYs are separated from whole blood and stored in AS - 1 solution, the final glucose concentration in the storage bag is still ~40 mM . 6 Given that a heathy individual has a bloodstream glucose concentration of 4 - 6 mM, and a diabetic patient of 7 - 9 mM, 38 the 129 glucose concentration in CPD and AS - 1 may adversely affect ERY functions during storage. Interestingly, many of the adverse ERYprop erties found in people with diabetes (e.g., oxidative stress, 39 advanced glycation endproducts 40 ) also occur in stored ERYs (referred to as ERY storage lesions). 41 - 42 To test the hypothesis that hyperglycemic processing and storage conditions may cause adv erse effects on the function of ERYs, normoglycemic versions of CPD and AS - 1 (referred to as CPDN and AS - 1N, respectively) were created and used for collection of whole blood from donors and storage of ERYs, as described in a publication by Y. Wang et al . 3 2 ERYs stored in AS - 1N were fed periodically with glucose to maintain glucose concentrations in the range of normoglycemic levels. Results showed that ATP release levels, over four weeks of storage, from normoglycemia - stored ERYs were always statistically higher than that of hyperglycemia - stored ERYs. In addition, the ERY - derived ATP had a significant impact on endothelium - derived NO. Results strongly suggest that maintenance of normoglycemic levels in collection and storage of ERYs may help increase NO bio availability during and after transfusion. 6 3.2 Experimental Procedure 3.2.1 Preparation of Solutions for Storage of ERYs All reagents were purchased from Sigma - Aldrich (St. Louis, MO) unless specified otherwise. 100 mL of anticoagulant citrate phosphate d extrose solution (CPD; in mM, 101.9 sodium citrate, 15.6 citric acid, 128.8 glucose, and 16.1 monobasic sodium phosphate, pH = 5.6) were prepared. 100 mL of additive solution (AS - 1; in mM, 154.0 sodium chloride, 41.2 mannitol, 1.8 adenine, 111.1 glucose, p H of 5.8) were also 130 prepared. Normoglycemic versions of CPD and AS - 1 solutions (CPDN and AS - 1N, respectively) were prepared similarly, except that the glucose concentration was 5.5 mM. A saline solution containing 200 mM glucose was also prepared for futu re feeding purpose for ERYs stored in AS - 1N. All solutions were autoclaved at 10 bar and 121 °C and stored at 4 °C. 3.2.2 Collection and Storage of ERYs Whole blood collection from healthy donors was approved by the Biomedical and Health Institutional Revi ew Board (IRB) at Michigan State University. Prior to blood collection, six 10 mL untreated blood collection glass tubes (BD, Franklin Lakes NJ) were prepared in the sterile environment. Using syringes, four of the six tubes were injected with 1 mL CPD sol ution as the anticoagulant; and two tubes were injected with 1 mL CPDN solution. About 7 mL of whole blood from a healthy donor were collected into each of the six blood collection tubes. Tubes with blood were gently inverted several times every 5 min for 30 min at room temperature to ensure even mixing of CPD (or CPDN) solution and blood. All tubes were then centrifuged at 2000 g for 10 min and sprayed with 70% ethanol and placed in the sterile environment. Serum and buffy coat were carefully removed by as piration. Note, an additional top 2 mm layer of the packed ERYs were also aspirated and discarded to minimize the presence of leukocytes. Without further purification, four tubes of ERYs collected in CPD were combined in a separate sterilized test tube, an d re - suspended in 0.5 volumes (1:1 dilution) of AS - 1 solution and gently mixed so that the hematocrit would be around 50%. Similarly, the other two tubes of ERYs collected in CPDN were combined into another sterilized test tube, and re - suspended in 0.5 vol umes of AS - 1N solution and gently mixed. The ERY 131 suspension was then divided into aliquots of 1 mL and transferred into miniaturized, pre - sterilized PVC bags, which were then heat sealed and stored at 4 oC in a refrigerator for 5 weeks. The process of bloo d collection and ERY storage is illustrated in Fig 3.1. Fig 3.1 : Illustration of the process of blood collection and ERY sample preparation. Whole blood is drawn into blood tubes from healthy donors, centrifuged, and the plasma and white cell parts discarded by aspiration. About 0.5 volumes of either AS - 1 or AS - 1N solution is added to ERYs in the tube and gently mixed well to form a n evenly di stributed ERY suspension. Then, the ERY suspension is aliquoted and transferred into pre - sterilized PVC bags, which are then heat sealed and stored at 4 ºC in a refrigerator for 5 weeks. On the day of experiments, PVC bags are opened and some stored ERYs a re placed in microcentrifuge tubes, followed by addition of freshly prepared PSS or PSSH buffer to a final hematocrit of 5% for ATP release experiment, or a final hematocrit of 7% for C - peptide uptake experiment. 132 Extracellular glucose levels in storage bags of both storage conditions (AS - 1 and AS - 1N) were monitored using a portable Accu - Chek Aviva glucose meter (Indianapolis, IN). When glucose was measured , bags storing ERYs were taken out of the refrigerator and transferred into the sterile environment. Bags were carefully cut open, and a 10 uL suspension from each bag was used for the determination of glucose concentration. Due to consumption of glucose, ERYs stored in AS - 1N were fed weekly by adding 10 uL of a 200 mM glucose saline solution to maintain a normal glucose range of 4 - 6 mM. When glucose concentration measurement and feeding were complete, the bags were re - sealed and placed back in the refriger ator. 3.2.3 ERY Sample Preparation The sample preparation procedure is briefly illustrated in Fig 3.1. On each day of an experiment, 1 mL of AS - 1 and AS - 1N stored ERYs was removed from storage bags and placed in microcentrifuge tubes. Hematocrit was immedi ately determined by a microhematocrit centrifugation method with a microhematocrit centrifuge (CritSpin M960 - 22, Statspin, Westwood, MA ) and a microcapillary reader (Statspin, Westwood, MA). Fresh physiological salt solution (PSS) was prepared as described in the previous chapter, and a hyperglycemic version of PSS, PSSH, containing excess glucose with a concentration equal to the extracellular glucose concentration in the AS - 1 storage bag measured on the day of experiment, was also prepared. To mimic the p rocess of ERY transfusion, stored ERYs (both AS - 1 and AS - 1N storage conditions) were transferred to PSS, labeled as AS - 1 - PSS and AS - 1N - PSS, respectively. As a control, some AS - 1 stored ERYs were transferred to PSSH to remain in a hyperglycemic environment in order to investigate any defect of cellular function 133 due to hyperglycemic storage conditions, referred to as AS - 1 - PSSH. Samples of 1 mL in volume were prepared for all testing, including determination of saturating amount of C - peptide uptake by ERYs usi ng ELISA, and ATP release in the presence and absence of Zn 2+ - C - peptide or INS - 1 cells using a 3D printed fluidic device. To study the saturating amount of C - peptide uptake by ERYs, all three samples were prepared at a final hematocrit of 7%, containing 20 nM of C - peptide. Samples were incubated at 37 ° C for 2 hours and then centrifuged at 500 g for 5 min. Supernatant was collected for ELISA quantification of C - peptide, the result of which was subtracted from 20 pmole originally added to calculate the amoun t of C - peptide uptake by the ERYs in each sample. To measure ATP release, samples were prepared to a 5% final hematocrit, containing none, or 10 nM of exogenous human C - peptide and zinc. Samples were then immediately loaded onto the 3D - pritned fluidic devi ce prior to incubation, which will be described in the following section. 3.2.4 Determination of ATP Release from Stored ERYs on a 3D - printed Fluidic Device The 3D - printed fluidic device as previously described in Chapter 2 was used again to measure ATP r elease from the six ERY samples. Briefly, the fluidic device has six Once samples are introduced to the loop by a pump, flowing ERY samples in the closed loop serves a s an in vitro mimic of blood circulation. Three wells are printed above each channel for placing transwell inserts whose porous membrane at the bottom separates the channel from the well, but enables maintaining communication via diffusion of small 134 molecul es through the porous membrane. Inserts from row E are chosen as the ATP collection and detection insert, where ATP molecules released by the flowing ERYs diffuse through the porous membrane into the insert, and are quantitatively determined by the lucifer in/luerferase assay. ATP standards prepared in PSS (concentrations of 0, 100, 200 and 400 nM) were measured to obtain a linear standard curve, followed by the six ERY samples. Samples were loaded into the channels using a pump and then loops w ere closed to f orm a circulation mimic. Standards were allowed to flow for 30 minutes at room collect ATP. ERYs samples were allowed to flow for 1.5 hours, first at 37 °C in an incuba tor, and then for another 30 minutes at room temperature to collect ATP. During the 30 minutes of ATP collection, inserts of row E were covered by a piece of wet Kimwipe to prevent loss of solvent in the inserts from evaporation. After ATP collection, the device was detached and placed i n a plate reader, and ATP measured by assay solution into each inserts of row E, simultaneously. ATP release from the ERY sample was calculated according to the ATP standard curve. 3.2.5 Rat INS - 1 Cell Cultu re in Inserts and Integration on Device of a exposure to UV light. Rat INS - 1 cells were cultured in cell culture flasks with RPMI - 1640 medium (Life Technologies, Carlsbad, CA) containing 1 mM sodium pyruvate, 100 U/mL 13 5 mM L glutamine and 10 mM HEPES. Upon reaching con fluence, INS - 1 cells were detached from the flask using 1 mL of trypsin/EDTA and transferred to a microtube, followed by centrifugation at 1000 g for 4 min. With the supernatant removed, the pellet was re - - he new suspension of INS - 1 cells were loaded onto a hemacytometer (Reichert, Buffalo, NY) and cell density determined, based on which, the INS - 1 cell suspension was further diluted to a final cell l cell suspension was added into each coated insert, which was then placed in an incubator at 37 °C with 5% CO2. Two - 1640 medium were added. Inserts were then placed back in the incub ator to allow the INS - 1 cells to grow for another 24 hours before use. Prior to being placed on the 3D printed fluidic device, RPMI - 1640 medium was containing 12 mM glucose and added to each insert. These inserts were then placed back in the incubator for another 1 hour to induce rat INS - 1 cell secretion. After incubation, these inserts were tightly placed in wells in row B of channels 7, 9, 11. Two sets of the three samples were prepared and introduced into all six channels of the device simultaneously (channels 1, 3, 5 did not contain INS - 1 cells secretion; channels 7, 9, 11 contained INS - 1 cells secretion). Fig 3.2 illustrat es the experimental setup of the device. ATP release from ERYs was then measured in the same manner as described in the previous chapter. 136 Fig 3.2: Top view of the experiment al setup of the 3D printed fluidic device for ATP release experiment using cultured INS - 1 cells. Inserts that are cultured with INS - 1 cells are tightly placed in wells in row B of channels 7, 9 and 11. Two sets of the three samples (AS - 1N - PSS, AS - 1 - PSSH, A S - 1 - PSS) are prepared at 5% hematocrit and loaded into all six channels of the device simultaneously using a peristaltic pump (channels 1, 3 and 5 will not contain INS - 1 cells secretion whereas channels 7, 9 and 11 will contain INS - 1 cells secretion). Afte r sample introduction, the device is incubated at 37 ºC for 2 hours for the best effect of Zn 2+ - C - simultaneously added to each insert in row E, and the amount of ATP release from each ERY sample is determined. 137 Fig 3.3: E xtracellular glucose environments in AS - 1N and AS - 1 storage b ags. Extracellular glucose concentration in AS - 1N storage bags (open circles) was maintained at a healthy level around 4 - 6 mM over 36 days of storage with periodic feeding, whereas glucose concentration in AS - 1 bags (closed circles) stayed excessively high around 40 - 50 mM. Error bars are ±SEM, n=5. 138 3.3 Results 3.3.1 Extracellular Glucose Environment in ERY Storage Bags It was previously found by our group t hat stored ERYs consume glucose in storage bags as their source of food, for this reason ERYs stored in AS - 1N must be periodically and carefully fed with glucose so that the extracellular glucose environment in the storage bags can be maintained in a healt hy range of 4 - 6 mM throughout the period of storage. On the other hand, ERYs stored in the current hyperglycemic solution (AS - 1) do not require further feeding since the glucose level is already extremely excessive. As shown in Fig 3.3, for a 36 - day storag e, the glucose level in AS - 1N bags was maintained at 4 - 6 mM with a weekly feeding regimen, whereas the extracellular glucose level in AS - 1 bags was always above 40 mM, which was close to 10 times higher than the healthy glucose range, despite a minor decre ase due to cellular consumption during the period of storage. 3.3.2 Reversibility of Defect of ATP Release from Stored ERYs Previously, Wang et al . reported that ERYs that had been stored in AS - 1 released significantly less ATP than ERYs stored in AS - 1N wi th periodic feeding. The reduction in ATP release was easily determined, even on the first day of storage. Furthermore, the ability of AS - 1 stored ERYs to stimulate NO production from endothelial cells was also diminished significantly compared to AS - 1N st ored ERYs, as a result of the defect of ATP release. However, it was not known if the reduced ATP release could actually be entirely, or partially, reversed when AS - 1 stored ERYs are returned to a normoglycemic condition as in the real case of transfusion. PSS is a widely used in vitro physiological buffer, therefore transfusion of stored 139 ERYs into PSS would be a reasonable solvent condition when attempting to mimic an actual transfusion. Accordingly, samples in AS - 1N - PSS and AS - 1 - PSS were both considered p ost - transfusion mimics, where AS - 1N and AS - 1 stored ERYs were transfused to normoglycemic PSS. However, sample AS - 1 - PSSH was considered pre - transfusion control sample for the hyperglycemic storage condition, since stored ERYs were transfused to and remaine d in a hyperglycemic buffer, PSSH rather than the physiological condition as the other two samples. Therefore, sample AS - 1 - PSSH was a necessary and important control sample, which would indicate any loss of cellular function arising from hyperglycemic stor age condition. In Fig 3.4, black bars represent flow - induced ATP release from AS - 1N - PSS samples. Regardless of the length of storage, AS - 1N stored ERYs displayed ATP release level of around 220 nM when transfused to physiological PSS, and this ATP release level was maintained over 5 weeks of storage without showing a significant decrease (p > 0.99). In contrast, ERYs stored in hyperglycemic AS - 1 gradually lost their ability to release ATP when measured in hyperglycemic PSSH, shown by the light grey bars, wh ich was significantly suppressed by 30% (144 nM) on the first day of storage; over 55% of their ability to release ATP (90 nM) was lost by day 36 of storage. However, when AS - 1 stored ERYs were transfused to physiological PSS buffer, AS - 1 - PSS samples, rep resented by dark grey bars, ATP release levels were not significantly different than AS - 1N - PSS samples until day 5 of storage. On day 8, even though there was a 22% decrease in ATP release by the AS - 1 - PSS sample compared to AS - 1N - PSS sample, there was abou t 20% more ATP release when compared to AS - 1 - PSSH sample. Unfortunately, this reversibility did not last beyond 15 days of storage, during 140 which time the ATP release from AS - 1 - PSS samples was not significantly different than that of AS - 1 - PSSH samples. Fig 3.4: Flow - induced ATP release from stored ERYs without the addition of Zn2= - C - peptide. After transfused to PSS, AS - 1N stored ERYs (AS - 1N - PSS samples, black bars) always released the most ATP around 22 0 nM. AS - 1 stored ERYs after transfused to PSSH (AS - 1 - PSSH samples, light grey bars) released least ATP which also declined with time. Same AS - 1 stored ERYs but transfused to PSS (AS - 1 - PSS samples, dark grey bars) completely reversed ATP release to 200 nM until day 5, and partially reversed until day 12, and lost reversibility after day 15. Error bars are ±SEM, n = 6. 141 Fig 3.5: ELISA determination of C - peptide uptake by stored ERYs. A constant 2 pmol of C - peptide uptake by ERYs of AS - 1N - PSS samples was measured (black bars). C - peptide uptake by AS - 1 - PSSH samples (light grey bars) continued to decrease over time. C - peptide uptake by ERYs of AS - 1 - PSS samples (dark grey bars) was completely reversed to 2 pmol in the first 5 days and partially reversed unti l day 12, but not reversed after day 15. Error bars are ±SEM, n = 7. 142 3.3.3 C - peptide Uptake by Stored ERYs Shown in Fig 3.5, there was about 2.1 picomoles (pmol) of C - peptide bound by ERYs of the AS - 1N - PSS sample (7% hematocrit) on day 1 of storage, which stayed stable throughout 5 weeks of storage. On day 36, C - p eptide uptake by AS - 1N - PSS sample was approximately 1.9 pmol, which was not statistically different than day 1. AS - 1 - PSSH samples showed only about 1.6 pmol of C - peptide uptake, a significant 27% decrease on day 1, when compared to AS - 1N - PSS samples, and this binding capacity continued to decrease to less than 0.8 pmol on day 36. However, for those AS - 1 - PSS samples where AS - 1 stored ERYs were transferred to physiological PSS as transfusion mimic, C - peptide uptake completely reversed to 2.1 pmol through day 1 - 5, which was not statistically different than AS - 1N - PSS samples. On day 8 and 12, C - peptide uptake by ERYs of AS - 1 - PSS samples was significantly less than that of AS - 1N - PSS samples, but significantly higher than AS - 1 - PSSH samples, suggesting a partial r eversibility upon being transfused to a normoglycemic PSS buffer. However, after day 15, no reversibility of the loss of C - peptide binding to AS - 1 stored ERYs could be observed, and C - peptide uptake by ERYs of AS - 1 - PSSH and AS - 1 - PSS samples were statistic ally the same, a trend very similar to that shown for ERY - derived ATP release in 3.3.2 3.3.4 Increase of ATP Release by Stored ERYs in Response to Zn 2+ - C - peptide As described in the previous chapter, over 50% increase of ATP release has been observed from fresh and healthy ERYs in response to C - peptide and Zn uptake. Storage of ERYs in normoglycemic AS - 1N resulted in normal C - peptide binding capacity to cells, and potentially Zn binding capacity as well, which led to a 50% increase in ATP release (as shown by the black bars in Fig 3.6). This increase of ATP 143 release was well preserved throughout the entire period of storage. Storage of ERYs in hyperglycemic solutions lowered C - significant extent over time; the subs equent increase in ATP release was diminished as well as shown by the light grey bars in Fig 3.6. Fig 3.6: Increase of flow - induced ATP release from stored ERYs by Zn2= - C - peptide. After incubated with Zn 2+ - C - peptide, ATP release from AS - 1N - PSS samples (black bars) went up to 320 nM. AS - 1 - PSSH samples (light grey bars) did not respond to Zn 2+ - C - peptide very well as there was not much significant increase in ATP release observed. ATP release from AS - 1 - PSS sam ples (dark grey bars) was increased to 300 nM until day 5; the increase became less obvious in the following week and disappeared after day 15. Error bars are ±SEM, n = 6. 144 Fig 3.7: Flow - induced ATP release from stored ERYs without access to INS - 1 cel ls. This data serves as a control experiment for flow - induced ATP release from stored ERYs that had been in contact with INS - 1 cell secretion. Here, results of ATP release were comparable to data in Fig 3.4. Briefly, AS - 1 N - PSS samples (black bars) released the most ATP around 220 nM throughout 36 days of storage. AS - 1 - PSSH samples (light grey bars) released least ATP which also declined with time. AS - 1 - PSS samples (dark grey bars) were able to completely reverse ATP release on day 1, and then lost reversibi lity on and after day 8. Error bars are ±SEM, n = 3. 145 Fig 3.8: Response of stored ERYs to INS - 1 cells determined by increase of ATP release. AS - 1N - - cells very well as an increase in ATP release was observed throu gho ut the period of storage. AS - 1 - - cells normally as no significant increase in ATP release was observed. Response of AS - 1 - - cells was observed before day 8, and was reduce d after day 8 and lost after day 15. Error bars are ±SEM, n = 3. 146 Zn 2+ - C - peptide was able to induce more ATP release from ERYs stored in AS - 1 - PSSH samples from 144 nM (without Zn 2+ - C - peptide) to 163 nM on day 1, but this minor efficacy was not significant by statistical analysis and became even less noticeable with length of storage and was no longer observed beyond day 12 of storage. When AS - 1 stored ERYs were transfused to PSS buffer, the AS - 1 - PSS samples displayed a 50% increase in ATP release in the presence of Zn 2+ - C - peptide on day 1 and 5 of storage, which was statistically the same to that of AS - 1N - PSS samples. Whereas on day 8 and 12, increase of ATP release from ERYs of AS - 1 - PSS sample s in response to the addition of Zn 2+ - C - peptide was reduced to less than that of AS - 1N - PSS samples. For example, ATP release from ERYs of AS - 1 - PSS samples increased in the presence of Zn 2+ - C - peptide by only 39% (166 nM to 232 nM) on day 8 and 22% (142 nM t o 174 nM) on day 12. After day 15, Zn 2+ - C - peptide was no longer able to increase ATP release from ERYs of AS - 1 - PSS samples, and ATP release from ERYs of AS - 1 - PSS and AS - 1 - PSSH samples were statistically the same. 3.3.5 Increase of ATP Release by Stored ERY s in Response to Rat INS - 1 Cells To validate the results in the previous section, where exogenous sources of C - peptide and Zn 2+ were used to induce ATP release, rat INS - 1 cells were cultured and stimulated in inserts that were placed in the fluidic device. This construct would allow direct interaction of endogenous C - peptide and Zn 2+ secreted from INS - 1 cells with ERYs flowing through device channels below the inserts. In spite of other components 147 in INS - 1 cell secretions, including insulin, it was hypothes ized that C - peptide and Zn 2+ are the only two components that can induce ATP release from ERYs. Rat INS - 1 cells, instead of human, were used due to resource convenience and substitutability among species. In fact, our own group has used human C - peptide on rat ERYs with success in the past. Here, we attempted rat C - peptide on human ERYs. Results of ATP release from ERY samples that were not affected by INS - 1 cell secretions are shown in Figs 3.7 and Fig 3.8, respectively. Data in Fig 3.8 is comparable to th e data in Fig 3.6. Briefly, AS - 1N - PSS samples always responded to INS - 1 cells, with a consistent 50% increase in ATP release over the entire storage period. AS - 1 - PSSH samples did not respond to INS - 1 cells as well as AS - 1 - PSSN samples, and a significantly less increase in ATP release was observed since the beginning of storage, which diminished rapidly with length of storage. Whereas AS - 1 - PSSN samples were able to respond to INS - 1 cells prior to day 5, this response did not occur past 2 weeks of storage. I t is hypothesized that this phenomena is, once again, due to the ERYs ability to shed any adhered glucose for the ~ first 14 days of storage in high glucose media; however, beyond that point the effect of the hyperglycemic conditions is permanent. 3.4 Disc ussion Blood transfusion plays an important role in modern critical healthcare. According to the National Blood Collection and Utilization Survey Report of 2011, approximately 13.8 million units of WB/ERYs were transfused to patientsannually. The American Red Cross also reported that blood transfusions are received by nearly 5 million people each year in the US. Unfortunately, the existing post - transfusion complications such as infections 148 and multi - organ failure (e.g., renal failure and heart failure) can s ometimes put patients at increased risks of mortality, and have long been a major concern of the safety of the blood transfusion industry. In relation to increased adverse health risks, it is worth noting that patients who received transfusions of ERYs tha t were stored for more than two weeks had higher chances for in - hospital mortality, complications occurrence, and even a higher mortality rate one year after transfusion. 36,43 The causes of post - transfusion complications and two - week time mark for storage of blood components with fewer risks remain unclear. It was recently reported that patients who receive transfusions could suffer from insufficient nitric oxide bioavailability (INOBA) that could impair blood flow. Nitric oxide (NO) is a well - known vasodi lator that maintains blood flow. ERY - derived ATP is capable of inducing NO production from vascular endothelium and platelets. Therefore the regular function of stored ERYs regarding the ability to release ATP can have an impact on the health of blood circ ulation of patients who receive stored ERYs. Even though it is not proven if INOBA is caused by a decrease in ERY - stimulated NO, researchers have noticed some physiological damage of ERYs (e.g., oxidative stress 39 and advanced glycation endproducts 40 ) duri ng storage, known as ERY storage lesions. 41 Importantly, such lesions are also found in ERYs obtained from diabetic patients. 42 The hallmark feature of diabetic patients is high blood glucose level (fasting blood sugar level higher than 7 mM), whereas a n ormal fasting sugar level for human beings is currently considered to be less than 5.5 mM. 38 Surprisingly, as calculations show, the extracellular glucose environment in current blood storage bags that contain ERYs and 149 an additive solution (AS - 1) is over 4 0 mM, which is 5 times higher than that of the diabetic condition. 6 The Spence group has hypothesized that the hyperglycemic storage condition i s a potential cause of ERY storage lesions, although the molecular mechanism is not known. AS - 1N storage solutio n, a normoglycemic version of AS - 1 solution was first proposed by the Spence group and tested for ERY storage. In comparison to ERYs stored in the current FDA approved AS - 1 solution, ERYs stored in AS - 1N storage solution displayed significantly higher ATP release, which also induced subsequent N O production by the endothelium . 6 The decrease in ATP release from AS - 1 stored ERYs was not due to less intracellular ATP production, since the intracellular ATP levels in AS - 1 stored ERYs were actually higher than t hose of AS - 1N stored ERYs, and it did not significantly decrease over 5 weeks of storage, suggesting some disorders in the ATP release process is responsible. An important feature of these prior experiments was that the measurements were performed as the c ells remained in storage solutions. Therefore, more insight could be gained by investigating whether or not the loss of ATP release due to hyperglycemic storage is reversible when stored ERYs are transfused back to the physiological condition (e.g., during a real in vivo also an important aspect of evaluating the health and safety of stored ERYs, and of knowing whether hyperglycemic storage conditions influences t he response fro m the ERY. Due to the ability of C - peptide to stimulate ERY - derived ATP (an NO - cell secretion and reversibility upon being transfused to physiological condition would be a reaso nable starting point. 150 Collaborating with Chengpeng Chen in the Spence group, a 3D printed fluidic device was applied to improve simultaneous measurements of flow - induced ATP release from ERY samples while mimicking certain aspects of the circulation at. Da ta shown in Fig 3.4 confirms our previous findings that over the entire 5 week period of storage, our modified normoglycemic storage condition can preserve ATP release from stored ERYs on a level that is not different than fresh ERYs. ATP release from stor ed ERYs is significantly reduced under hyperglycemic storage conditions. Even after the AS - 1 stored ERYs are transfused to PSS, the reduction of ATP release can be fully reversed only if the cells are stored for less than 5 days, and partially reversed if stored for no more than 15 days. Beyond this 15 day time mark, no reversibility can be observed at any time, suggesting some permanent change has occurred to the stored ERYs that ATP. Fig 3.6 shows that normoglycemia s tored ERYs are able to respond to Zn 2+ - C - peptide as fresh ERYs can do, resulting in a consistent 50% increase in ATP release throughout the storage period. However, hyperglycemic stored ERYs fail to respond normally when the cells remain in PSSH, with a mi nor 14% increase even on day 1, and no significant increase after day 8. When hyperglycemia stored ERYs are transfused to PSS, the stored ERYs show a normal response to Zn 2+ - C - peptide with approximately 50% increase in ATP release on day 1 and day 5 indica ting full reversibility. Unfortunately the reversibility is gradually weakened with the length of storage and completely not observable on and after day 15. This 15 day time mark can again indicate permanent damage to ATP releasing ability of ERYs due to h yperglycemic storage, but it can also indicate permanent damage of stored ERYs response to Zn 2+ - 151 C - peptide. And processing and storing ERYs in normoglycemic solutions is essential to maintaining the ATP releasing ability of stored ERYs and their response to Zn 2+ - C - peptide at the same time. As discussed in chapter 2, C - peptide uptake by ERYs is an indispensable step for subsequent increase of ATP release. Data in Fig 3.5 indicates a saturation uptake of C - peptide of approximately 2.1 pmol by normoglycemia sto red ERYs on day 1. This value is not different than that of fresh ERYs, and remains consistent throughout the storage period, which explains the increase in ATP release from AS - 1N - PSS samples. C - peptide uptake by hyperglycemia stored ERYs is significantly reduced on day 1 and decreased with length of storage. AS - 1 - PSS samples also show full or partial reversibility in C - peptide uptake if the storage is less than 15 days, again consistent with the increase and ATP release in Fig 3.6. It is noticed that beyon d the time mark of 15 days, even though there was close to 1 pmol of C - peptide uptake, it did not lead to a significant increase in ATP release. This may suggest that there is a threshold amount of C - peptide uptake by ERYs in vivo in order for subsequent increase of ATP release, but it may also not be reflecting real in vivo situations because of reduced response to C - peptide during hyperglycemic storage. Even though C - peptide and Zn 2+ - cell secretion that can affect ATP release fr om ERYs, they were exogenously added to the ERYs, and it would - cells were directly endogenously and the effect on stored ERYs measured. In Fig 3.8, rat INS - 1 cells are stimulated to release C - peptide and Zn 2+ to interact with stored ERY samples. In can be seen that the trend of ATP increase 152 stimulated by INS - 1 cells in Fig 3.8 matched the trend in Fig 3.6 where C - peptide and Zn 2+ are exogenously added as the stimulus. Therefore results in Fig 3.6 are validated and proved another key advantage of using normoglycemic storage solutions which is - cell secretions after transfusion, and perhaps other organs and tissues as well. This can be important to ensuring sufficient ATP level in the circulation, nitr ic oxide bioavailability and blood flow, and potentially lowering the chances of patients developing post transfusion complications. It is also implied that ERYs that have been stored in hyperglycemic condition for more than 15 days have permanent defects in cellular function which are not reversible after transfusion. This is consistent with some reported clinical observations which also show longer than two weeks. Accor ding to our data, the hyperglycemic processing and storage condition for ERYs may explain this weak link in current transfusion medicine, though more evidence and detailed molecular mechanism will be required. As mentioned in chapter 2, the mechanism of ho w Zn 2+ - C - peptide increases ATP release from ERYs is still unclear. Sprague reported that sheer stress induced deformation of ERYs leads to ATP release through a CFTR involved mechanism, indicating a causal link between deformability of ERYs and the subsequ ent ATP release. Studying the change of deformability of both fresh and stored ERYs in the presence of Zn 2+ - C - peptide may provide a clue to understand the change of ATP release, which will be discussed in the next chapter. 153 RE FERENCES 154 REFERENCES 1. Liu, Y.; Chen, C.; Summers, S.; Medawala, W.; Spence, D. M., C - peptide and zinc delivery to erythrocytes requires the presence of albumin: implications in diabetes explored with a 3D - printed fluidic device. Integrative biology : qua ntitative biosciences from nano to macro 2015, 7 (5), 534 - 43. 2. Furchgott, R. F.; Zawadzki, J. V., The obligatory role of endothelial cells in the relaxation of arterial smooth muscle by acetylcholine. Nature 1980, 288 (5789), 373 - 6. 3. Stevens, M. J.; Zh ang, W.; Li, F.; Sima, A. A., C - peptide corrects endoneurial blood flow but not oxidative stress in type 1 BB/Wor rats. American journal of physiology. Endocrinology and metabolism 2004, 287 (3), E497 - 505. 4. Carroll, J.; Raththagala, M.; Subasinghe, W.; B aguzis, S.; D'Amico Oblak, T.; Root, P.; Spence, D., An altered oxidant defense system in red blood cells affects their ability to release nitric oxide - stimulating ATP. Molecular bioSystems 2006, 2 (6 - 7), 305 - 11. 5. Meyer, J. A.; Subasinghe, W.; Sima, A. A .; Keltner, Z.; Reid, G. E.; Daleke, D.; Spence, D. M., Zinc - activated C - peptide resistance to the type 2 diabetic erythrocyte is associated with hyperglycemia - induced phosphatidylserine externalization and reversed by metformin. Molecular bioSystems 2009, 5 (10), 1157 - 62. 6. W ang, Y.; Giebink, A.; Spence, D. M., Microfluidic evaluation of red cells collected and stored in modified processing solutions used in blood banking. Integrative biology : quantitative biosciences from nano to macro 2014, 6 (1 ), 65 - 75. 7. S harma, S.; Sharma, P.; Tyler, L. N., Transfusion of blood and blood products: indications and complications. American family physician 2011, 83 (6), 719 - 24. 8. Liumbruno, G.; Bennardello, F.; Lattanzio, A.; Piccoli, P.; Rossetti, G., Recommen dations for the transfusion of red blood cells. Blood transfusion = Trasfusione del sangue 2009, 7 (1), 49 - 64. 9. Vaught, J. B., Blood collection, shipment, processing, and storage. Cancer epidemiology, biomarkers & prevention : a publication of the Americ an Association for Cancer Research, cosponsored by the American Society of Preventive Oncology 2006, 15 (9), 1582 - 4. 10. Campbell, L. D.; Betsou, F.; Garcia, D. L.; Giri, J. G.; Pitt, K. E.; Pugh, R. S.; Sexton, K. C.; Skubitz, A. P.; Somiari, S. B., Devel opment of the ISBER Best Practices for Repositories: Collection, Storage, Retrieval and Distribution of Biological Materials for Research. Biopreservation and biobanking 2012, 10 (2), 232 - 3. 11. Caporaso, N.; Vaught, J., Collection, processing, and analysis of preneoplastic specimens. In: Franco EL, Rohan TE, editors. Cancer precursors: 155 epidemiology, detection, and prevention. New York: Springer - Verlag. 2002 , p. 33 - 45. 12. Landi, M. T.; Caporaso, N., Sample collection, processing, and storage. In: Applications of biomarkers in cancer epidemiology, IARC Scientific Pub. No. 142. Lyon, France: IARC. 1997 , p. 223 - 36. 13. Holland, N. T.; Smith, M. T.; Eskenazi, B.; Bastaki, M., Biological sample collection and processing for molecular epidemiological stu dies. Mutation research 2003, 543 (3), 217 - 34. 14. Heaton, A.; Miripol, J.; Aster, R.; Hartman, P.; Dehart, D.; Rzad, L.; Grapka, B.; Davisson, W.; Buchholz, D. H., Use of Adsol preservation solution for prolonged storage of low viscosity AS - 1 red blood cells. British journal of haematology 1984, 57 (3), 467 - 78. 15. O'Shaughnessy, D. F.; Atterbury, C.; Bolton Maggs, P.; Murphy, M.; Thomas, D.; Yates, S.; Williamson, L. M.; British Committee for Standards in Haematology, B. T. T. F., Guidelines for the use of fresh - frozen plasma, cryoprecipitate and cryosupernatant. British journal of haematology 2004, 126 (1), 11 - 28. 16. Jones, A.; Heyes, J., Processing, testing and selecting blood components. Nursing times 2014, 110 (37), 20 - 2. 17. Simon, T. L. ; Marcus, C. S.; Myhre, B. A.; Nelson, E. J., Effects of AS - 3 nutrient - additive solution on 42 and 49 days of storage of red cells. Transfusion 1987, 27 (2), 178 - 82. 18. Cicha, I.; Suzuki, Y.; Tateishi, N.; Shiba, M.; Muraoka, M.; Tadokoro, K.; Maeda, N., Gamma - ray - irradiated red blood c ells stored in mannitol - adenine - phosphate medium: rheological evaluation and susceptibility to oxidative stress. Vox sanguinis 2000, 79 (2), 75 - 82. 19. Walker, W. H.; Netz, M.; Ganshirt, K. H., [49 day storage of erythrocyte concentrates in blood bags with the PAGGS - mannitol solution]. Beitrage zur Infusionstherapie = Contributions to infusion therapy 1990, 26 , 55 - 9. 20. Squires, J. E., Risks of transfusion. Southern medical journal 2011, 104 (11), 762 - 9. 21. Busch, M. P.; Gl ynn, S. A.; Stramer, S. L.; Strong, D. M.; Caglioti, S.; Wright, D. J.; Pappalardo, B.; Kleinman, S. H.; Group, N. - R. N. S., A new strategy for estimating risks of transfusion - transmitted viral infections based on rates of detection of recently infected do nors. Transfusion 2005, 45 (2), 254 - 64. 22. Bihl, F.; Castelli, D.; Marincola, F.; Dodd, R. Y.; Brander, C., Transfusion - transmitted infections. Journal of translational medicine 2007, 5 , 25. 23. Stramer, S. L.; Hollinger, F. B.; Katz, L. M.; Kleinman, S.; Metzel, P. S.; Gregory, 156 K. R.; Dodd, R. Y., Emerging infectious disease agents and their potential threat to transfusion safety. Transfusion 2009, 49 Suppl 2 , 1S - 29S. 24. Brecher, M. E.; Hay, S. N., Bacterial contamination of blood components. Clinical mi crobiology reviews 2005, 18 (1), 195 - 204. 25. Hillyer, C. D.; Josephson, C. D.; Blajchman, M. A.; Vostal, J. G.; Epstein, J. S.; Goodman, J. L., Bacterial contamination of blood components: risks, strategies, and regulation: joint ASH and AABB educational session in transfusion medicine. Hematology / the Education Program of the American Society of Hematology. American Society of Hematology. Education Program 2003 , 575 - 89. 26. Eder, A. F.; Kennedy, J. M.; Dy, B. A.; Notari, E. P.; Weiss, J. W.; Fang, C. T.; Wagner, S.; Dodd, R. Y.; Benjamin, R. J.; American Red Cross Regional Blood, C., Bacterial screening of apheresis platelets and the residual risk of septic transfusion reactions: the American Red Cross experience (2004 - 2006). Transfusion 2007, 47 (7), 113 4 - 42. 27. Greenberger, P., Plasma anaphylaxis and immediate type reactions. in Rossi EC, Simon TL, Moss GS (ed). Principles of Transfusion Medicine. Baltimore, MD, Williams and Wilkins. 1991 , 635 - 39. 28. Sazama, K., Reports of 355 transfusion - associated de aths: 1976 through 1985. Transfusion 1990, 30 (7), 583 - 90. 29. Bruno, D. S.; Herman, J. H., Acute hypotensive transfusion reactions. Lab Med 2006, 37 , 542 - 45. 30. de Rie, M. A.; van der Plas - van Dalen, C. M.; Engelfriet, C. P.; von dem Borne, A. E., The se rology of febrile transfusion reactions. Vox sanguinis 1985, 49 (2), 126 - 34. 31. Heddle, N. M.; Klama, L.; Singer, J.; Richards, C.; Fedak, P.; Walker, I.; Kelton, J. G., The role of the plasma from platelet concentrates in transfusion reactions. The New E ngland journal of medicine 1994, 331 (10), 625 - 8. 32. Kleinman, S.; Caulfield, T.; Chan, P., Toward an understanding of transfusion - related acute lung injury: statement of a consensus panel. Transfusion 2004, 44 , 774 - 89. 33. Marriott, H. L.; Kekwick, A., V olume and Rate in Blood Transfusion for the Relief of Anaemia. British medical journal 1940, 1 (4147), 1043 - 6. 34. Fatalities reported to FDA following blood collection and transfusion: annual summary for fiscal year 2009. March 22, 2010. 2010 . 35. Roback, J. D.; Neuman, R. B.; Quyyumi, A.; Sutliff, R., Insufficient nitric oxide bioavailability: a hypothesis to explain adverse effects of red blood cell transfusion. Transfusion 2011, 51 (4), 859 - 66. 157 36. Koch, C. G.; Li, L.; Sessler, D. I.; Figueroa, P.; Hoel tge, G. A.; Mihaljevic, T.; Blackstone, E. H., Duration of red - cell storage and complications after cardiac surgery. The New England journal of medicine 2008, 358 (12), 1229 - 39. 37. Hebert, P. C.; Wells, G.; Blajchman, M. A.; Marshall, J.; Martin, C.; Pagl iarello, G.; Tweeddale, M.; Schweitzer, I.; Yetisir, E., A multicenter, randomized, controlled clinical trial of transfusion requirements in critical care. Transfusion Requirements in Critical Care Investigators, Canadian Critical Care Trials Group. The Ne w England journal of medicine 1999, 340 (6), 409 - 17. 38. Engelgau, M. M.; Narayan, K. M.; Herman, W. H., Screening for type 2 diabetes. Diabetes care 2000, 23 (10), 1563 - 80. 39. Sharifi, S.; Dzik, W. H.; Sadrzadeh, S. M., Human plasma and tirilazad mesylat e protect stored human erythrocytes against the oxidative damage of gamma - irradiation. Transfusion medicine 2000, 10 (2), 125 - 30. 40. Mangalmurti, N. S.; Chatterjee, S.; Cheng, G.; Andersen, E.; Mohammed, A.; Siegel, D. L.; Schmidt, A. M.; Albelda, S. M.; Lee, J. S., Advanced glycation end products on stored red blood cells increase endothelial reactive oxygen species generation through interaction with receptor for advanced glycation end products. Transfusion 2010, 50 (11), 2353 - 61. 41. Kor, D. J.; Van Bus kirk, C. M.; Gajic, O., Red blood cell storage lesion. Bosnian journal of basic medical sciences / Udruzenje basicnih mediciniskih znanosti = Association of Basic Medical Sciences 2009, 9 Suppl 1 , 21 - 7. 42 . Wautier, J. L.; Wautier, M. P.; Chappey, O.; Zouk ourian, C.; Guillausseau, P. J.; Capron, L., Diabetic erythrocytes bearing advanced glycation end products induce vascular dysfunctions. Clin. Hemorheol 1996, 16 , 611 - 67. 43. Tinmouth, A.; Fergusson, D.; Yee, I. C.; Hebert, P. C.; Investigators, A.; Canadi an Critical Care Trials, G., Clinical consequences of red cell storage in the critically ill. Transfusion 2006, 46 (11), 2014 - 27. 158 Chapter 4 Mechanistic Studies of Albumin - Zn 2+ - C - peptide Effects on ERY - derived ATP Release 4.1 Introduction It still remai ns unclear how the conjugate of albumin - Zn 2+ - C - peptide increases ATP release from ERYs. ERYs release ATP in response to specific stimuli, including mechanical deformation, 1,2 lowering of pH, 3 hypoxia, 4 and osmotic pressure. 5 The possibility of mechanical d eformation being the mechanism for albumin - Zn 2+ - C - peptide is studied in this chapter. Leptin, a hormone that is secreted by adipocytes, 6 is able to inhibit food intake and increase energy expenditures. 6 - 7 Recently, leptin has been reported to have relaxing effect on blood vessels mediated by NO released from endothelium.8 Whether leptin can signal to the ERY and induce glucose clearance and subsequent increased ATP release which leads to endothelium - induced NO production is also investigated in this study. 4.1.1 Shear Stress - induced Deformability of ERY and ATP Release Endothelium - derived NO is known to relax vascular smooth muscle cells and lead to increased blood flow. 9 An increased shear stress in the blood vessel has been shown to be a major stimulus for NO release from the endothelium . 10,11 Sprague et al. reported that, in isolated perfused rabbit lungs, alterations in shear stress resulted in no change in NO release in the absence of ERYs, suggesting there is another link between ERYs and endothelium - in duced NO production. 12 Followed by this study, Sprague et al . proposed that when subjected to mechanical deformation in the circulation, ERYs 159 release ATP, which then stimulate the synthesis and release of NO from the endothelium. This mechanism is supporte d by evidence from rabbit and human ERYs. 13 Further investigations with more controlled shear stress conditions not only confirmed the idea of shear stress as a stimulus of ATP release from ERYs, but also indicated that the amount of ATP released is correl ated to the magnitude and the duration of shear stress. 14 - 18 In addition, stiffened ERY membranes (e.g., by adding diamide a known cell stiffening agent) decreased the amount of ATP released, confirming that deformation is a trigger for ATP release. 18 4.1. 2 Leptin and Energy Balance Leptin is a hormone that is secreted by adipocytes (fat cells). 6 It is thought to signal to the brain to inhibit food intake and increase energy expenditures. 6,7 Higher levels of leptin have been found in obese rodents and human s. Leptin increases in humans are shown to be linear with body mass index (BMI, a measure of body fat based on weight and height). 19 Leptin injection treatment was successful in inhibiting food intake and decreasing body weight in obese animals lacking lep tin. 20,21 However, since humans with obesity are usually associated with elevated levels of leptin in their circulation, the idea of leptin treatment in obese human remains in question. 21,22 This can be partially explained by the fact that humans and roden ts lacking a functional leptin or leptin receptor can result in voracious feeding and obesity. 6 In addition, humans and rodents who become obese on a high - fat diet do not respond to leptin well. 21,22 However, 160 studies have revealed that leptin replacement a ugmented the effect of a weight loss drug in obese subjects by further decreasing food intake and stimulating fatty acid oxidation. 23 It is hoped that leptin treatment in obese patients can sustain the effects of dieting and drug treatment. 24 Currently, an other interesting area of study involving leptin is leptin therapy in insulin - deficient T1D. This area of research was driven by the fact that insulin monotherapy for T1D patients cannot duplicate the metabolic homeostasis provided by endogenous insulin pr oduced by pancreatic islets. 25 For example, peripherally injected insulin may contribute to glycemic lability due to the inherent differences in insulin requirements of its target tissues; 25 whereas chronic hyperinsulinemia can enhance cholesterologenesis 26 causing high risks for coronary artery disease. 27 - 28 In mice, the therapeutic actions of insulin and leptin were compared using a nonobese diabetic mouse model. Results indicated both hormones are able to successfully prevent ketoacidosis, cachexia, and death and that both restore HbA1c to normal levels. Because the two hormones have very distinct effects on lipid metabolism (leptin suppresses lipogenesis; insulin enhances lipogenesis and factors involved in cholesterologenesis), the authors suggested th at leptin, either alone or combined with insulin therapy, provides equivalent or superior glycemic stability without the increase in body fat and up - regulation of cholesterologenic and lipogenic transcription factors and enzymes observed with insulin monot herapy. 25 Another study reported that insulin therapy in T1D mice promotes ectopic fat deposition in liver and muscle and causes 161 insulin resistance that is prevented by leptin. 29,25 Therefore, it is important and necessary to know whether or not leptin the rapy in T1D mice can be applied to T1D patients. Leptin therapy has been limited to diabetic patients with generalized lipodystrophy. 30 - 31 It will be of greater worth to determine its efficacy in the more common T1D. 4.2 Experimental Procedure 4.2.1 ERY Sa mple Preparation Fresh ERYs were collected and then purified either in PSS or albumin - free PSS as previously described in chapter 2. Hematocrit was immediately determined by a microhematocrit centrifugation method with a microcapillary reader. For deformab ility characterization, all fresh ERYs samples were prepared at a final volume of 1 mL with a 5% final hematocrit in corresponding PSS or albumin - free PSS, containing 0 nM Zn 2+ - C - peptide, 10 nM Zn 2+ - C - peptide, 10 nM C - peptide alone, or 10 nM Zn 2+ alone. St ored ERY samples AS - 1N - PSS, AS - 1 - PSSH, and AS - 1 - PSS were obtained in the same manner as described in chapter 3, and re - suspended in corresponding PSS or PSSH to a 5% final hematocrit, containing 0 or 10 nM Zn 2+ - C - peptide. All samples were incubated at 37 º C for two hours, and then cooled at room temperature for 20 minutes before performing the deformability measurement. - derived ATP release, purified, fresh ERY samples were re - suspended in PSS to a final hematocrit of 7% , and incubated with 0, 0.95, 1.9, 3.8, or 7.6 nM leptin for 2 hours at 37 ºC to measure static ATP release. To - derived A TP release in the presence of Zn 2+ - C - 162 peptide, another group of fresh ERY samples were prepared at a 7% hematocrit in PSS containing none, 10 nM Zn 2+ - C - peptide, 3.8 nM leptin alone, 10 nM Zn 2+ - C - peptide + 3.8 nM leptin, 10 nM Zn 2+ + 3.8 nM leptin, or 10 nM C - peptide + 3.8 nM leptin for two hours at 37 ºC. Then, all samples were cooled to room temperature prior to peforming a static ATP measurement. 4.2.2 Characterization of Deformability of ERYs using 3D Printed Cell Filter All samples were allowed to equilibrate to room temperature prior to all deformability measurements, which were performed at room temp erature as well. The deformability of ERYs was characterized using a novel 3D - printed cell filter. Briefly, shown in Fig 4.1, two pieces of 3D - printed slabs with a hole in the center are clamped together with a piece of polycarbonate membrane (pore size of represent the filter. Two o - rings were simultaneously printed on the inside around the hole of the slabs to ensure a tight seal around the membrane and prevent leaking. Tubing for sample introduction was connected to the hole of the top slab, and each ERY sample was driven through the cell filter by a peristaltic pump with a pressure fixed at 5 cm H 2 O (~0.075 psi). A 10 minute filtration period was allowed for collection of each sample into a cuvette. Timing commenced when filtered ERY suspension filled all void volume of the filter. Each filtered ERY suspension was diluted in PSS by 100 fold, followed by a determination of the hematocrit using a hemocytometer (Reichert, Buffalo, NY). No filtration - induced lysis was observ ed. Between tests, the two slabs of the cell filter were detached from the pump and tubing, disassembled and carefully rinsed with DDW first and then PSS. Then, the cell 163 filter was dried with a piece of Kimwipe and then re - assembled with a new membrane. Th e tubing was pumped with DDW first and then PSS to clear the previous ERY sample. The cell filter was re - attached to the tubing to be ready for the next sample. 4.2.3 Measurement of Static ATP Release from ERYs aliquots of each ERY sample or standard with solution was prepared fresh as described in chapter 2. Chemiluminescence intensity from the reaction of ATP and luciferin/luciferase was measured exactly after 18 seconds of mixing using a PMT housed in a light excluding box (built in - house. It is important to ensure adequate mixing of the sample and luciferin/luciferase solution by gently shakin g the cuvette several times without lysing the ERYs. ERY samples were measured in a random order in triplicates. 4.3 Results 4.3.1 Increase of Membrane Deformability of Fresh ERYs by Albumin - Zn 2+ - C - peptide Deformability of fresh ERY samples was first inves tigated in order to confirm the effect from the albumin - Zn 2+ - C - peptide complex before stored ERYs were measured. No cell lysis was detected during the deformability tests using the 3D printed cel l filter. For convenient comparison, cell number counted for the untreated ERY sample (~20 million) was set to 100% deformability, as shown by the first black bar in Fig 4.2, and cell numbers of all other samples were normalized to the untreated ERY sample. Incubation of fresh ERYs with 10 nM Zn 2+ - C - peptide led to about 50% increase of membrane deformability compared to that of untreated, ERY samples, which was not 164 observed for incubation with 10 nM C - peptide or Zn 2+ alone. Conversely, when albumin - free PSS was used, incubation with 10 nM Zn 2+ - C - peptide did not lea d to any significant increase of cell membrane deformability, reconfirming the necessity of albumin for C - Fig 4.1: Schematic diagram of 3D printed cell filter for characterization of ERY cell membrane deformability. a) Design of the cell filter. Two pieces of 3D - printed slabs with a hole in t he center are clamped together with a piece of polycarbonate membrane (pore size = for sample introduction was connected to the hole of the top slab, and each ERY sample was driven through the cell filter by a peristaltic pump with a pressure fixed at 5 cmH2O (~0.075 psi). 10 min ute filtration was allowed for collection of each sample into a cuvette. b) Picture showing each part of the cell filter. c) Picture showing the actual setup of the syst em, including the cell filter, cuvette and pump. An ERY sample is flowing in the tubing and being filtered through the filter and collected into the cuvette. 165 Fig 4.2: The change of deformability of fresh ERYs in the presence of Zn 2+ - C - peptide characte rized by 3D printed cell filter. No cell lysis was detected during the deformability tests. The number of cells counted for the untreated ERY sample in PSS (left - most black bar) was set to deformability of 100%, to which the cell numbers of all other sampl es were normalized. Incubation of fresh ERYs with 10 nM Zn 2+ - C - peptide in PSS led to 50% increase of membrane deformability compared to untreated ERYs. Incubation with 10 nM of either C - peptide or Zn 2+ alone did not lead to any significant change in membra ne deformability. No change in deformability was observed either when ERYs were suspended in albumin - free PSS. Error bars are ±SEM, n = 5. 166 Fig 4.3: Deformability of stored ERYs. Cell counts of day 1 AS - 1N - PSS samples were set to 100%, to which cell co unts of all other samples were normalized. Regardless of the length of storage, AS - 1N - PSS samples (black bars) displayed consistent cell membrane deformability (cell count ~20 million) to a level that was not different than that of fresh ERYs. As compariso n, AS - 1 - PSSH samples (light grey bars) gradually lost their ability to deform while remained in hyperglycemic buffer, which decreased from 80% on day 1 to 60% on day 36. After transferred to physiological PSS, AS - 1 - PSS samples (dark grey bars) displayed 10 0% reversibility in deformability up until day 5, and partial reversibility between day 8 and 12. Beyond day 15, deformability of AS - 1 - PSS samples was statistically the same as that of AS - 1 - PSSH samples and was no longer reversible. Error bars are ±SEM, n = 4. 167 4.3.2 Increase of Membrane Deformability of Stored ERYs by Albumin - Zn2+ - C - peptide Albumin - free PSS was not used in the deformability tests for stored ERYs because initial studies showed that Zn 2+ - C - peptide did not increase deformability of fresh ERYs in the absence of albumin. Fig 4.3 shows the results of deformability studies. Cell counts of AS - 1N - PSS samp les on day 1 (~20 million) were set to 100%, to which cell counts of all other samples were normalized. In Fig 4.3, black bars represent cell membrane deformability of AS - 1N - PSS samples. Regardless of the length of storage, AS - 1N stored ERYs displayed cons istent cell membrane deformability (cell count ~20 million) to a level that was not different than that of fresh ERYs. By comparison, ERYs stored in hyperglycemic AS - 1 solution gradually lost their ability to deform when stored in hyperglycemic PSSH, shown by the light grey bars, which decreased from 80% on day 1 to 60% on day 36. When AS - 1 stored ERYs were transfused to PSS buffer, AS - 1 - PSS samples (dark grey bars) displayed 100% reversibility in deformability up until day 5. On day 8 and day 12, decreasin g partial reversibility in deformability of AS - 1 - PSS samples was observed. However, beyond day 15 of storage, deformability of AS - 1 - PSS samples was statistically the same as that of AS - 1 - PSSH samples and was no longer reversible upon transferring to PSS. S hown in Fig 4.4, in the presence of Zn 2+ - C - peptide, the deformability of AS - 1N - PSS samples consistently increased by 30 - 40% over 5 weeks, without showing a significant decrease with length of storage. The increase was slightly lower than that o f fresh ERYs. Zn 2+ - C - peptide was able to improve the deformability of AS - 1 - PSSH samples by 25% on day 1 and 20% on day 5, followed by a minor increase of 10% in 168 week 2 before the efficacy was abandoned. AS - 1 - PSS samples demonstrated increase of deformabili ty with Zn 2+ - C - peptide that was statistically the same as the increase of AS - 1N - PSS samples on day 1 and day 5, which gradually disappeared afterwards. 4.3.3 Further Increase of ERY - derived ATP Release by Leptin An increase in static ATP release from ERYs was not observed when the cells were incubated with leptin alone, as shown in Fig 4.5. However, incubation with 3.8 nM leptin in the presence of 20 nM Zn 2+ - C - peptide did lead to an additional 30% increase in the chemiluminescence intensity, as shown in Fig 4.6, which resulted from an additional increase of ATP release. In comparison, leptin with either C - peptide or Zn 2+ alone did not lead to a significant increase of ATP release. 4.4 Discussion C - peptide and Zn 2+ at physiological levels can increase ATP re lease from ERYs, however, an exact mechanism for this effect is still lacking. Sprague reported that shear stress induced deformation of ERYs leads to ATP release, indicating a causal link between deformability of ERYs and the subsequent ATP release. There fore, in the first part of this section, the effect that C - peptide and Zn 2+ have on the deformability of fresh ERYs was investigated. Hach et al. reported C - peptide and its C - terminal fragments are equally effective in improving ERY deformability in type 1 diabetes. 32 However, according to data in Fig 4.2, C - peptide significantly increases ERY deformability only in the presence of Zn 2+ . One possible explanation can be that, since the authors mentioned the C - peptide used in that study was 98% pure without fu rther purification, 169 Zn 2+ ions could be in the impurity components and us ed unknowingly with C - peptide. Data in Fig 4.2 demonstrates that the increase of deformability of fresh ERYs by C - peptide and Zn 2+ is an important contributing factor to subsequent i ncrease in flow - induced ATP release. More studies on how Zn 2+ - C - peptide may have changed the cytoskeleton network of the ERY would provide more insights for the understanding of the unidentified mechanism. Additionally, the increase of deformability is als o albumin - dependent. This is because albumin is responsible for the delivery of C - peptide and Zn 2+ to ERYs, the same requirement that must be met for measured increases in ATP release. We then compared the effect of C - peptide and Zn 2+ on deformability of E RYs stored in different glycemic conditions as shown in Fig 4.3 and Fig 4.4. Normoglycemic - stored ERYs show an average of 40% increase in deformability in the presence of C - peptide and Zn 2+ over 36 days of storage. This increase is slightly lower than the increase seen in fresh ERYs. Hyperglycemia stored ERYs do not show an increase of deformability as noticeable as normoglycemia stored ERYs since day 1, consistent with the minor increase in ATP release shown in Fig 3.6 in chapter 3. However, after transfus ion to PSS, hyperglycemia stored ERYs have a 35% increase in deformability i n the presence of C - peptide and Zn 2+ until day 5, which is statistically the same as that of normoglycemia stored ERYs. This increase gradually reduced to 10% during the following week, and was essentially gone after day 15. The results shown in Fig 4.4 shared a lot of similarities in trends with the results in Fig 3.6 in chapter 3. This validates 170 our thought that C - peptide and Zn 2+ may affect ATP release from transfused ERYs via ch anging deformability of the cells. Fig 4.4: Change of deformability of stored ERYs in the presence of Zn 2+ - C - peptide. Cell counts in this figure were all normalized to the cell counts of day 1 AS - 1N - PSS samples in Fig 4.3. In the presence of Zn 2+ - C - peptide, deformability of AS - 1N - PSS samples consistently increased by 30 - 40% over 5 weeks without showing a significant decrease with the length of storage. Zn2+ - C - peptide was able to improve the deformability of AS - 1 - PSSH samples by 25% on day 1 and 20% on day 5, followed by a minor 10 % in week 2 after which the efficacy was totally abolished. AS - 1 - PSS samples demonstrated 30 - 40% increase of deformability with Zn 2+ - C - pepti de that was statistically the same as the increase of AS - 1N - PSS samples on day 1 and day 5, which gradually weakened afterwards. Error bars are ±SEM, n = 4. 171 Fig 4.5: Characterization of static ATP release from fresh and healthy ERYs in the presence of a series of concentrations of leptin. Leptin (0, 0.95, 1.9, 3.8 and 7.6 nM) was added to ERYs. After 2 hour incubation, levels of static ATP release from ERY samples were characterized by chemiluminescence intensity in the luciferin/luciferase enzymatic r eaction. No change of level of static ATP release was observed after ERYs were incubated with any of the chosen concentration of leptin. Error bars are ±SEM, n = 4. 172 Fig 4.6: Further increase of static ATP release from ERYs in the presence of both lepti n and Zn 2+ - C - peptide. Incubation with 3.8 nM leptin in the presence of 20 nM Zn 2+ - C - peptide led to an additional 30% increase in the chemiluminescence intensity, which resulted from an additional increase of ATP release. However, leptin with either C - pepti de or Zn 2+ alone did not lead to a significant increase of ATP release. Error bars are ±SEM, n = 5. 173 3D printing technology proved its excellent performance and convenience in method development for a deformability characterization. In this study, the same 3D printed cell filter was used for deformability measu rements for all samples, and it has successfully shown great potential for practicability, reusability, durability, and reproducibility. Above all, the 3D printed cell filter is a very simple filtration method to use, when compared to common currently used techniques such as optical tweezers 33,34 and magnetic twisting cytometry. 35 Data in Fig 4.5 and Fig 4.6 suggests that secretion from adipocytes can also affect ATP release from ERYs, under the condition of the co - existence of Zn 2+ - C - peptide. The mechanism of how leptin has an impact on ER Y metabolism is not understood. Because ERYs are known to release ATP in response to stimuli including deformability, change of pH, hypoxia, and chemicals, the increase of static ATP release in the presence of Zn 2+ - C - pept ide, and further increase of ATP release with leptin may result from a different mechanism than deformability, change of pH or hypoxia. Even though leptin may have activated a different pathway to further enhance static ATP release from ERYs, it does not n ecessarily mean that leptin does not further increase the membrane deformability of ERYs. To prove this, results of flow - induced ATP release and change of deformability will be helpful. Leptin concentration in the bloodstream increases as a person becomes more obese. Very similar to insulin resistance in T2D, obese people can develop leptin resistance as well, whose brains cannot respond to leptin properly to control food intake. 36 It will be interesting to obtain ERYs from obese people and investigate whet her or not there is leptin resistance in their ERYs affecting 174 ATP release. Even though leptin does not increase static ATP release from ERYs by itself, whether or not leptin can increase the intracellular ATP production has not been investigated. This can be done by measuring the lysis product from the ERYs. In addition, glucose uptake by ERYs may be another piece of useful information to this question since an increase of glycose uptake may indicate increase of intracellular ATP production. Moreover, to ha - derived ATP release, it will also be helpful to investigate flow - induced ATP release from ERYs and change in deformability after incubation with leptin alone. Response of stored ERYs to leptin can be anoth er research area of importance in terms of evaluating the health of stored cells from an aspect of proper response to healthy organs, as has been done for - cell secretion in chapter 3. 175 REFERENCES 176 REFERENCES 1. S prague, R. S.; Ellsworth, M. L.; Stephenson, A. H.; Kleinhenz, M. E.; Lonigro, A. J., Deformation - induced ATP release from red blood cells requires CFTR activity. The American journal of physiology 1998, 275 (5 Pt 2), H1726 - 32. 2. Sprague, R.; Ellsworth, M .; Stephenson, A.; Lonigro, A., Increases in flow rate stimulate adenosine triphosphate release from red blood cells in isolated rabbit lungs. Exp Clin Cardiol 1998, 3 , 73 - 77. 3. Ellsworth, M. L.; Forrester, T.; Ellis, C. G.; Dietrich, H. H., The erythrocy te as a regulator of vascular tone. The American journal of physiology 1995, 269 (6 Pt 2), H2155 - 61. 4. Bergfeld, G. R.; Forrester, T., Release of ATP from human erythrocytes in response to a brief period of hypoxia and hypercapnia. Cardiovascular research 1992, 26 (1), 40 - 7. 5. Light, D. B.; Capes, T. L.; Gronau, R. T.; Adler, M. R., Extracellular ATP stimulates volume decrease in Necturus red blood cells. The American journal of physiology 1999, 277 (3 Pt 1), C480 - 91. 6. Zhang, Y.; Proenca, R.; Maffei, M. ; Barone, M.; Leopold, L.; Friedman, J. M., Positional cloning of the mouse obese gene and its human homologue. Nature 1994, 372 (6505), 425 - 32. 7. Ahima, R. S.; Prabakaran, D.; Mantzoros, C.; Qu, D.; Lowell, B.; Maratos - Flier, E.; Flier, J. S., Role of le ptin in the neuroendocrine response to fasting. Nature 1996, 382 (6588), 250 - 2. 8. Kimura, K.; Tsuda, K.; Baba, A.; Kawabe, T.; Boh - oka, S.; Ibata, M.; Moriwaki, C.; Hano, T.; Nishio, I., Involvement of nitric oxide in endothelium - dependent arterial relaxa tion by leptin. Biochemical and biophysical research communications 2000, 273 (2), 745 - 9. 9. Furchgott, R. F.; Zawadzki, J. V., The obligatory role of endothelial cells in the relaxation of arterial smooth muscle by acetylcholine. Nature 1980, 288 (5789), 373 - 6. 10. Buga, G. M.; Gold, M. E.; Fukuto, J. M.; Ignarro, L. J., Shear stress - induced release of nitric oxide from endothelial cells grown on beads. Hypertension 1991, 17 (2), 187 - 93. 11. Rubanyi, G. M.; Romero, J. C.; Vanhoutte, P. M., Flow - induced rel ease of endothelium - derived relaxing factor. The American journal of physiology 1986, 250 (6 Pt 2), H1145 - 9. 12. Sprague, R. S.; Stephenson, A. H.; Dimmitt, R. A.; Weintraub, N. L.; Branch, C. 177 A.; McMurdo, L.; Lonigro, A. J., Effect of L - NAME on pressure - f low relationships in isolated rabbit lungs: role of red blood cells. The American journal of physiology 1995, 269 (6 Pt 2), H1941 - 8. 13. Sprague, R. S.; Ellsworth, M. L.; Stephenson, A. H.; Lonigro, A. J., ATP: the red blood cell link to NO and local contr ol of the pulmonary circulation. The American journal of physiology 1996, 271 (6 Pt 2), H2717 - 22. 14. Fischer, D. J.; Torrence, N. J.; Sprung, R. J.; Spence, D. M., Determination of erythrocyte deformability and its correlation to cellular ATP release usin g microbore tubing with diameters that approximate resistance vessels in vivo. The Analyst 2003, 128 (9), 1163 - 8. 15. Sprung, R.; Sprague, R.; Spence, D., Determination of ATP release from erythrocytes using microbore tubing as a model of resistance vessel s in vivo. Analytical chemistry 2002, 74 (10), 2274 - 8. 16. Moehlenbrock, M. J.; Price, A. K.; Martin, R. S., Use of microchip - based hydrodynamic focusing to measure the deformation - induced release of ATP from erythrocytes. The Analyst 2006, 131 (8), 930 - 7. 17. Price, A. K.; Fischer, D. J.; Martin, R. S.; Spence, D. M., Deformation - induced release of ATP from erythrocytes in a poly(dimethylsiloxane) - based microchip with channels that mimic resistance vessels. Analytical chemistry 2004, 76 (16), 4849 - 55. 18. Price, A. K.; Martin, R. S.; Spence, D. M., Monitoring erythrocytes in a microchip channel that narrows uniformly: towards an improved microfluidic - based mimic of the microcirculation. Journal of chromatography. A 2006, 1111 (2), 220 - 7. 19. Monti, V.; Carl son, J. J.; Hunt, S. C.; Adams, T. D., Relationship of ghrelin and leptin hormones with body mass index and waist circumference in a random sample of adults. Journal of the American Dietetic Association 2006, 106 (6), 822 - 8; quiz 829 - 30. 20. Morton, G. J.; Cummings, D. E.; Baskin, D. G.; Barsh, G. S.; Schwartz, M. W., Central nervous system control of food intake and body weight. Nature 2006, 443 (7109), 289 - 95. 21. Flier, J. S., Clinical review 94: What's in a name? In search of leptin's physiologic role. The Journal of clinical endocrinology and metabolism 1998, 83 (5), 1407 - 13. 22. Heymsfield, S. B.; Greenberg, A. S.; Fujioka, K.; Dixon, R. M.; Kushner, R.; Hunt, T.; Lubina, J. A.; Patane, J.; Self, B.; Hunt, P.; McCamish, M., Recombinant leptin for weigh t loss in obese and lean adults: a randomized, controlled, dose - escalation trial. Jama 1999, 282 (16), 1568 - 75. 23. Boozer, C. N.; Leibel, R. L.; Love, R. J.; Cha, M. C.; Aronne, L. J., Synergy of sibutramine and low - dose leptin in treatment of diet - induce d obesity in rats. Metabolism: clinical and experimental 2001, 50 (8), 889 - 93. 178 24. Ahima, R. S., Revisiting leptin's role in obesity and weight loss. The Journal of clinical investigation 2008, 118 (7), 2380 - 3. 25. Wang, M. Y.; Chen, L.; Clark, G. O.; Lee, Y.; Stevens, R. D.; Ilkayeva, O. R.; Wenner, B. R.; Bain, J. R.; Charron, M. J.; Newgard, C. B.; Unger, R. H., Leptin therapy in insulin - deficient type I diabetes. Proceedings of the National Academy of Sciences of the United States of America 2010, 107 ( 11), 4813 - 9. 26. Ness, G. C.; Zhao, Z.; Wiggins, L., Insulin and glucagon modulate hepatic 3 - hydroxy - 3 - methylglutaryl - coenzyme A reductase activity by affecting immunoreactive protein levels. The Journal of biological chemistry 1994, 269 (46), 29168 - 72. 27 . Larsen, J.; Brekke, M.; Sandvik, L.; Arnesen, H.; Hanssen, K. F.; Dahl - Jorgensen, K., Silent coronary atheromatosis in type 1 diabetic patients and its relation to long - term glycemic control. Diabetes 2002, 51 (8), 2637 - 41. 28. Orchard, T. J.; Olson, J. C.; Erbey, J. R.; Williams, K.; Forrest, K. Y.; Smithline Kinder, L.; Ellis, D.; Becker, D. J., Insulin resistance - related factors, but not glycemia, predict coronary artery disease in type 1 diabetes: 10 - year follow - up data from the Pittsburgh Epidemiolog y of Diabetes Complications Study. Diabetes care 2003, 26 (5), 1374 - 9. 29. Liu, H. Y.; Cao, S. Y.; Hong, T.; Han, J.; Liu, Z.; Cao, W., Insulin is a stronger inducer of insulin resistance than hyperglycemia in mice with type 1 diabetes mellitus (T1DM). The Journal of biological chemistry 2009, 284 (40), 27090 - 100. 30. Oral, E. A.; Simha, V.; Ruiz, E.; Andewelt, A.; Premkumar, A.; Snell, P.; Wagner, A. J.; DePaoli, A. M.; Reitman, M. L.; Taylor, S. I.; Gorden, P.; Garg, A., Leptin - replacement therapy for lip odystrophy. The New England journal of medicine 2002, 346 (8), 570 - 8. 31. Park, J. Y.; Chong, A. Y.; Cochran, E. K.; Kleiner, D. E.; Haller, M. J.; Schatz, D. A.; Gorden, P., Type 1 diabetes associated with acquired generalized lipodystrophy and insulin re sistance: the effect of long - term leptin therapy. The Journal of clinical endocrinology and metabolism 2008, 93 (1), 26 - 31. 32. Hach, T.; Forst, T.; Kunt, T.; Ekberg, K.; Pfutzner, A.; Wahren, J., C - peptide and its C - terminal fragments improve erythrocyte deformability in type 1 diabetes patients. Experimental diabetes research 2008, 2008 , 730594. 33. Ashkin, A., Acceleration and trapping of particles by radiation pressure. Physical Review Letters 1970, 24 , 156 - 9. 34. Svoboda, K.; Block, S. M., Biological applications of optical forces. Annual review of biophysics and biomolecular structure 1994, 23 , 247 - 85. 35. Wang, N.; Butler, J. P.; Ingber, D. E., Mechanotransduction across the cell surface and through the cytoskeleton. Science 1993, 260 (5111), 1124 - 7. 179 36. Myers, M. G., Jr.; Leibel, R. L.; Seeley, R. J.; Schwartz, M. W., Obesity and leptin resistance: distinguishing cause from effect. Trends in endocrinology and metabolism: TEM 2010, 21 (11), 643 - 51. 180 Chapter 5 Conclusions and Future Directions 5.1 Co nclusions 5.1.1 C - peptide Replacement Therapy Potentially Complements Insulin Therapy for T1D C - peptide is a 31 amino acid peptide which is co - secreted with insulin from - cells. Since its discovery in the 1960s, C - peptide was thought a byproduct of insulin synthesis that facilitated the structure formation of proinsulin. Its biological activity was not observed until 1990s, where C - peptide replacement therapy displayed ability in reducing diabetic neuropathy, retinopathy, and nephropathy. 1 - 3 T1D patients are characterized by progressive immune - - cells r esulting in absolute insulin deficiency in the bloodstream. 4 Currently, T1D patients who are treated solely with insulin replacement therapy also tend to develop severe diabetic complications, indicating glucose control is not enough to solve the health is sue of diabetes. 5 - 6 However, C - peptide replacement therapy is not an approved therapy for diabetes and one main obstacle is an unknown mechanism of action. One hypothesis is that C - peptide replacement improves vasodilation and blood flow, leading to a decr ease in diabetic microvascular complications mentioned above. 7 - 9 NO is a well - known vasodilator in the bloodstream, and an important of source of NO comes from endothelium production in response to ERY - derived ATP stimulation. 10 - 11 ERYs produce mM level of ATP via anaerobic glycolysis, 12 and release ATP upon multiple stimuli including lower pH, 13 hypoxia, 14 change in osmotic pressure, 15 and mechanical deformation. 16 - 17 181 Meyer et. al. were the first to report significant increase of ATP release by ERYs in th e presence of C - peptide activated by specific metals including Zn 2+ , Fe 2+ or Cr 3+ . 18 - 19 Zn 2+ has the highest potential to interact with and activate C - peptide due to the fact that Zn 2+ is the most abundant metal ion in the pancreas, where C - peptide is synt hesized. 20 And Zn 2+ has been used in al l later studies. In this dissertation, the presence of albumin is found to be another requirement for C - 21 The role of albumin was first realized in the binding study of albumin and C - peptide using is othermal titration calorimetry. It was observed that two human C - peptide molecules can specifically bind to one human serum albumin molecule at physiological pH condition, with a binding affinity around 1.75 x 10 5 M - 1 . Next, the interaction between albumin and C - peptide and Zn 2+ was studied by ITC, and resulted suggested albumin is able to bind to C - peptide and Zn 2+ at the same time. According to ITC data, albumin has a higher affinity for Zn 2+ ions (Ka~10 7 M - 1 ) than C - peptide, and the measured Ka value agr ees with previously reported values in the literature. 22,23 However, these ITC data does not indicate whether or not Zn 2+ interacts with C - peptide when they are bound to albumin. ITC data does show that in the absence of albumin, C - peptide and Zn 2+ inte ract through weak electrostatic attraction without evidence of specific binding. Albumin in the circulating serum is a well - known transporter for fatty acids, hormones, metal ions, and drug molecules. 24 A three - 182 molecule system of albumin/C - peptide/ Zn 2+ ha s been hypothesized where albumin functions as the plasma transporter for both C - peptide and Zn 2+ after they are secreted - cells and delivers them to the sites of action, including the ERY for biological effects. The three - molecule system of albumin/C - peptide/ Zn 2+ explains how C - peptide may circulate in the bloodstream, and also shed s light on the mechanism of how C - peptide and Zn 2+ interact with ERYs and lead to increase of ATP release. Interaction with ERYs results in equimolar cellular uptake of C - peptide and Zn 2+ (~1800 molecules or ions per ERY), which does not occur in the absen ce of albumin. In addition, C - peptide uptake by the ERY is zinc - independent; whereas Zn 2+ uptake by the ERY requires C - peptide. This may suggest that albumin facilitates the ERY to take up C - peptide, which then in turn facilitates zinc transport into the c ells. The uptake of C - peptide and Zn 2+ increases ERY deformability by 40 - 50%, which was characterized by a 3D printed cell filter which functions by measuring the number of cells that pass a porous membrane under a fixed pressure during a fixed amount of t ime. This 3D printed cell filter has proved very durable, reproducible and cost - effective in this study. Incubation with C - peptide and Zn 2+ leads to significant increase in static and flow - induced ATP release from ERYs. As mentioned above, mechanical defor mation is one of the stimuli that induce ATP release from ERYs, therefore a causal relationship between increase in ERY cell membrane deformability by C - peptide and Zn 2+ and subsequent increase of 183 ATP release is built. The flow - induced ATP release from ERY s was measurement using a 3D printed fluidic device designed by the Spence group. Using the same device, my collaborator Chengpeng Chen also investigated downstream NO production from the endothelial cells. Data suggests that significantly higher levels of NO are produced by the endothelium when ATP release from the ERY is increased by C - peptide and Zn 2+ (data not shown here). This further provides strong evidence for the potential of albumin/C - peptide/ Zn 2+ in improving vasodilation and blood flow, and red ucing diabetes complications when provided to T1D patients. Collectively, delivery of C - peptide and Zn 2+ to ERYs by albumin through specific binding appears to be the first requirement for subsequent biological effects of the peptide, including increasing deformability and ATP releasing ability of ERYs. The idea of the three - molecule system of albumin/C - peptide/ Zn 2+ would be further confirmed if structural information for binding could be gathered and binding sites identified. In this study, Glu27 of C - pep tide has been recognized as an indispensable site for C - peptide/albumin binding, since replacement of Glu27 with Ala completely abolished albumin binding and cellular uptake of C - peptide and subsequent increase in deformability and ATP release. This indica tes that Glu27, probably its carboxylate group may play a key role in interaction with albumin and delivery of zinc. More structural studies will reveal more information about other binding sites and structure change involved in forming of the three - compon ent ensemble. Finally, albumin and zinc are found crucial for the bioactivity of C - peptide in this work, which has not been mentioned much in other previously reported C - peptide 184 studies in the literature. A review of these publications reveals that albumin - containing buffers and C - peptide that was not re - purified after purchased were used in most of the studies. 25 And zinc could exist as impurities in C - peptide. Therefore, revisiting the studies with strictly controlled presence of albumin and purity of C - p eptide would be helpful to confirm their necessity. 5.1.2 Normolgycemic Storage Condition Reduces ERY Storage Lesions Blood transfusion plays an important role in modern critical healthcare and saving transfused into patients who need blood because of trauma, for surgery or as therapeutic treatment of diseases, such as sickle cell disease, anemia and as a result of chemotherapy. 26 However, the existing post - transfusion complications have long been a ma jor concern of the safety of blood transfusion industry, and can put patients at increased risks of morbidity and mortality. Common post - transfusion complications include transfusion - transmitted infections (e.g., malaria, HIV, hepatitis), 27 acute transfus ion reactions (with transfusion - related acute lung injury (TRALI) being the most common cause of transfusion - related fatality), 28,29 and recently reported insufficient nitric oxide bioavailability (INOBA). 30 INOBA can be briefly stated as a reduction of local NO bioavailability after ERY transfusions that is insufficient to meet metabolic demands leading to morbidity and mortality in the recipient. INOBA can further lead to insufficient vasodilation and inadequate blood flow and O 2 delivery to organs and tissues, which can cause multiorgan failure and death in the worst case. 30 Some studies have pointed 185 out that patients who receive transfusion of ERYs that have been stored for longer than 14 days have had higher risks for in - hospital mortality, complicat ions occurrence, and ERYs units. 31,32 Unfortunately, the causes of post - transfusion complications and the two - week time mark for the health of stored blood components re main unclear. As we know, NO is a well - known vasodilator that maintains blood flow. ERY - derived ATP is capable of inducing nitric oxide production from vascular endothelium and platelets. In addition, a s mentioned in the previous section, C - peptide togethe r with Zn 2+ can further increase ATP release from ERYs and help avoid vascular diseases. Therefore the normal function of stored ERYs to release ATP can have an impact on the health of blood circulation environment of patients who receive transfusion of ER Ys. Even though it is not proved if INOBA is caused by a decrease of ERY - stimulated NO, researchers have noticed some physiological damage of ERYs (e.g., oxidative stress 33 and advanced glycation endproducts 34 ) during storage, known as ERY storage lesions. 35 Importantly, such lesions are also found in ERYs obtained from diabetic patients. 36 The Spence group recently reported that, according to calculation, ERYs after collection in CPD and storage in AS - 1, are actually exposed to a glucose environment of ~40 mM. 37 Given that a heathy individual has a bloodstream glucose concentration of 4 - 6 mM, and a diabetic patient of 7 - 9 mM, 38 the glucose concentration in CPD and AS - 1 may adversely affect ERY functions during storage. To prove the hypothesis that hyperglyc emic processing and storage condition may cause adverse 186 effects on functions of ERYs, normoglycemic versions of CPD and AS - 1 (referred to as CPDN and AS - 1N, respectively) were created and tested for collection of whole blood and storage of ERYs by Y. Wang et al. in Spence group. Results have shown a significant decrease of ATP release levels in hyperglycemia stored ERYs, which can be avoided in normoglycemia stored ERYs. In addition, the ERY - derived ATP is having a significant impact on endothelium - derived NO, which strongly suggests that maintenance of normoglycemic levels in collection and storage of ERYs may help increase NO bioavailability during and after transfusion. 37 In Y. Wang et al. measurements were performed. In this dissertation, stored ERYs were transfused to physiological buffers (PSS) prior to ATP measurements in order to better mimic a real transfusion process and investigate any reversibility in the loss of ATP release due to hypergly cemi c storage upon transfusion. Results of 5 week storage indicate that after transfused to PSS buffers, normoglycemia stored ERYs can maintain a level of ATP release that is not different than that of fresh ERYs. Whereas regarding hyperglycemia stored ERYs, t he reduction of ATP release can be fully reversed upon transfusion to PSS if the cells are stored for less than 5 days, and partially reversed if stored for no longer than 15 days. No reversibility can be observed at any time beyond this 15 day time mark, suggesting some permanent change has occurred to hyperglycemia stored which damages ATP releasing ability. In addition, investigation of ERY membrane deformability reveals similar trend to the reduction and reversibility of stored ERYs before and after transfusion to PSS buffers, which indicates a correlation between hyperglycemia storage induced damage to ERY deformability and subsequent 187 ATP releasing ability. Due to the profound biological significance of C - peptide and Zn 2+ in maintaining vascul ar health, the response of stored ERYs to C - peptide and Zn 2+ after transfusion to PSS buffers has also been studied in this dissertation. Data suggests that normoglycemia stored ERYs behave no differently than fresh ERYs regarding response to C - peptide and Zn 2+ . Results from C - peptide uptake study show consistent approximately 2.1 pmol uptake of C - peptide by normoglycemia stored ERYs throughout the storage period, which then leads to subsequent increase of deformability and ATP release. In contrast, C - pepti de uptake and subsequent increase in deformability and ATP release by hyperglycemia stored ERYs all tend to have a decreasing trend with length of storage, even after transfusion to PSS. The reduction also displays full to partial reversibility in if the s torage is less than 15 days, and no reversibility beyond 15 day storage. In conclusion, studies in the Spence group have shown multiple functional defects of ERYs after stored in the current FDA - approved AS - 1 storage solution, including reduction of deform ab ility, ATP release to induce NO production, and response to C - - cell secretion. Such reduction of ERY function may be fully or partially reversed upon transfusion if the cells have been stored for less than 15 days, but these functi onal defects can become irreversible after 15 days of storage, which is consistent with some reported clinical observations that reports adverse effects on weeks. By usin g the modified normoglycemic storage solution for ERY storage, the 188 - cell secretion that are seen in hyperglycemia stored ERYs can all be avoided. These data indicates the hyperglycemic processing an d storage condition for ERYs may be an important source of ERY storage lesion, a weak link in current transfusion medicine. And by applying a normoglecemic condition other types of storage lesion may be avoided as well, which may decrease the chances for p ost - transfusion complications in patents. 5.1.3 Leptin May Affect Energy Balance through Interaction with ERYs Leptin is a hormone secreted from adipocytes, 39 which functions by inhibiting food intake and increasing energy expenditures. 39,40 Plasma levels of leptin linear to body mass index (BMI, a measure of body fat based on weight and height), and higher levels of leptin are found in rodents and humans that have obesity. 41 In recent years, another interesting area of study about leptin is leptin therapy in insulin - deficient T1D. Some researchers have reported that leptin, either alone or combined with insulin therapy, provides equivalent or superior glycemic stability without the increase in body fat and up - regulation of cholesterologenic and lipogenic tr anscription factors and enzymes observed with insulin monotherapy in T1D. 42 In another study, leptin shows ability to prevent ectopic fat deposition in liver and muscle and insulin resistance in T1D mice caused by insulin therapy. 43,42 Importantly, it has also been pointed out that since leptin therapy has been by far limited to diabetic patients with generalized lipodystrophy, 44,45 it will be of greater worth to determine its efficacy in the more common T1D. 189 Driven by these findings, preliminary research o n the possible effect of leptin on ERYs that may bring beneficial effects to T1D associated complications has been investigated. Data suggests that with the co - existence of Zn 2+ and C - peptide, leptin is able to further increase static ATP release from ERYs obtained from healthy donors. This further increase in ATP release may suggest further elevated glucose uptake by the ERY, and a further enhancement of ERY membrane deformability. Leptin does not increase static ATP release form ERYs by itself, however, w hether or not leptin can increase the intracellular ATP production and flow - induced ATP release has not been investigated and can provide useful information in understanding the mechanism of action of leptin on ERY metabolism, and shed light on using lepti n as a therapy for the balance of energy. 5.2 Future Directions In chapter 2 the discovery of simultaneous binding of C - peptide and Zn 2+ to serum albumin by isothermal titration calorimetry (ITC) has been discussed, which helps explain how C - peptide is tra nsported in the bloodstream. Glu27 is an indispensable binding site on C - peptide. By testing specific binding of other C - peptide mutants and albumin by ITC will provide information on other possible binding sites on C - peptide, however, this would require l arge amount of peptide and albumin, and cannot provide information on the binding sites on albumin, which may possibly help explain how C - peptide interacts with the ERY. X - ray crystallography can be a useful tool for identifying binding sites of C - peptide by knowing the crystal structure of the albumin/C - peptide/ Zn 2+ conjugate. Preliminary experiment on obtaining crystals for the three - molecule 190 conjugate indicated a more successful forming of crystals at higher protein and peptide concentrations. The solu bility of albumin in aqueous solution is outstanding, and the limited solubility of C - peptide in aqueous solution by itself can be overcome through binding with albumin. In spite of the well accepted bioactivity of C - peptide and its potential in reducing d iabetes - associated complications, C - peptide replacement is not an approved therapy for T1D most due to its unidentified mechanism. It has been hypothesized that there is a G - protein coupled receptor (GPCR) receptor for C - peptide on ERY membrane. 46 Besides , a GPCR mediated mechanism has been reported to be involved in the interaction of C - peptide and cell types including renal tubular cells, 47 and kidney proximal tubular cells. 48 However, there has been a lacking of direct evidence to support this hypothesi s. Previous data in Spence group has indicated that metal - activated C - peptide can increase the glucose uptake in the ERY, 18 suggesting the involvement of the only glucose transporter, GLUT1 on ERY membrane in C - biological effects. This involvemen t may be a result of increased activity of GLUT1, or increased amount of membrane GLUT1 trigger by C - peptide/ Zn 2+ uptake, or both. The change in the amount of GLUT1 protein on ERY membrane can be studied by SDS - PAGE with lysed ERY membrane. Even though the re is difficulty in finding the exact mechanism of C - peptide, it will also be important to prove the effect of albumin/C - peptide/ Zn 2+ on ERYs from T1D patients, to prove the idea of C - peptide/ Zn 2+ replacement therapy from the aspect of 191 improving ATP releas e and subsequent NO availability and blood flow. 3D printed fluidic device can be a n excellent platform for this study. ERYs from T1D patients can be introduced onto the device for measurement of flow - induced ATP release. Meanwhile, inserts cultured with endothelial cells can be placed in wells of row G, so that ATP - induced NO production from endothelial cells in G wells can be monitored at the same time when flow - induced ATP release is measured fr om E wells. In addition, 3D printed Y - shaped injection port can be connected to the injection end of the device where albumin/C - peptide/ Zn 2+ can be quantitatively injected into the channels of flowing E RYs in a controlled manner, to better mimic the proces s of C - peptide/ Zn 2+ replacement therapy and may also obtain PKPD profile of C - peptide/ Zn 2+ as a drug. On the other hand, to better understand the process of increase of ERY - derived ATP release, an investigation of time profile of ATP release would be neces sary other that sampling at 2 hour time point alone. This time profile may also be helpful for understanding the mechanism of C - peptide. Zn 2+ /C - peptide after being exposed to a hyperglycemic environment. As a result, C - peptide uptake by ERYs is significantly reduced when glucose concentration in PSS was raised to 20 mM. 49 It has been reported that in diabetes, elevated blood glucose concentrations can cause glycation of serum alb umin, which involves non - enzymatic addition of glucose of degradation products of glucose to amine groups on albumin. 50 Since amine groups on albumin can potentially be binding sites for carboxyl groups on 192 C - peptide through hydrogen bonding, glycation of albumin may result in failure of C - - peptide in the previous experiments. ITC can be a proper technique to prove the failure of C - peptide binding to glycated albumin. In this case, albumin/C - pe ptide/ Zn 2+ must be provided as a conjugate drug if given to T1D patients as a replacement therapy. The effect of leptin on the ERY is a new area of interest. In this dissertation, leptin has been shown to further increase static ATP release from ERYs in th e presence of Zn 2+ - C - peptide, whereas leptin by itself, or leptin with either C - peptide or Zn 2+ alone does not seem to have any effect. The process of ERY releasing ATP is thought to be a result of cell membrane deformation, a physical change that occurs to flowing ERYs in the bloodstream. Therefore, it will be necessary to investigate flow - induced ATP release and change of deformability of ERYs after incubation with leptin, and find causal link to explain how leptin further enhances ATP release from ERYs. The 3D printed fluidic device used in this dissertation has realized high throughput analysis of ATP; however, the 3D printed cell filter for ERY deformability measurement can be further improved towards high throughput purposes. For example, because the cell filter has shown great reproducibility and durability, multiple same cell filters can be printed and used simultaneously on one same pump, so that the volume of sample handling is increased. The stress exerted by the pump to drive the ERYs to pass the porous membrane in the cell filter needs to be improved too, so is the pore size. The idea of the cell filter is to measure the number of cells that pass through a porous membrane during a fixed amount of time. The stress and membrane pore size can both a ffect the result and difference between samples. A lower pressure and membrane with smaller 193 por e size can be tested for performance since they both have potential to amplify the performance, especially for deformability characterization for stored ERYs, since the d iameter of the cells tend to shrink with the length of storage. In order to store and maintain ERYs at a normoglycemic condition, periodic glucose feeding by opening storage bags and manually adding glucose feeding solution continued to be used in this dis sertation. To make the feeding procedure simpler, a design of an automatic feeding device can be promising and bring potential for the realization of ERY normoglycemic storage. Besides, usually ERYs are stored for blood banking after leukocytes are removed by filtration. Due to the small volumes of ERYs stored in our lab that the large dead volume of commercially available leukocyte filters are difficult to match, 3D printed leukocyte filters with reduced dead volume can be a solution for this issue. As als o mentioned in chapter 4, proper response of stored ERYs to healthy organs and tissues may be another important part of evaluation of the health of stored cells and transfusion safety, it will continue to provide valuable information to investigate the res - cells, such as the response to leptin. 194 REFERENCES 195 REFERENCES 1. Ekberg, K.; Brismar, T.; Johansson, B. L.; Lindstrom, P.; Juntti - Berggren, L.; Norrby, A.; Be rne, C.; Arnqvist, H. J.; Bolinder, J.; Wahren, J., C - Peptide replacement therapy and sensory nerve function in type 1 diabetic neuropathy. Diabetes care 2007, 30 (1), 71 - 6. 2. Sjoquist, M.; Huang, W.; Johansson, B. L., Effects of C - peptide on renal functi on at the early stage of experimental diabetes. Kidney international 1998, 54 (3), 758 - 64. 3. Klein, R.; Klein, B. E.; Moss, S. E., The Wisconsin Epidemiologic Study of Diabetic Retinopathy. XVI. The relationship of C - peptide to the incidence and progressi on of diabetic retinopathy. Diabetes 1995, 44 (7), 796 - 801. 4. Association, A. D., Diagnosis and classification of diabetes mellitus. Diabetes care 2009, 32 Suppl 1 , S62 - 7. 5. Sima, A. A.; Kamiya, H., Insulin, C - peptide, and diabetic neuropathy. Science Me d 2003, 9 , 308 - 19. 6. Grunberger, G.; Qiang, X.; Li, Z.; Mathews, S. T.; Sbrissa, D.; Shisheva, A.; Sima, A. A., Molecular basis for the insulinomimetic effects of C - peptide. Diabetologia 2001, 44 (10), 1247 - 57. 7. Fernqvist - Forbes, E.; Johansson, B. L.; E riksson, M. J., Effects of C - peptide on forearm blood flow and brachial artery dilatation in patients with type 1 diabetes mellitus. Acta physiologica Scandinavica 2001, 172 (3), 159 - 65. 8. Lindstrom, K.; Johansson, C.; Johnsson, E.; Haraldsson, B., Acute effects of C - peptide on the microvasculature of isolated perfused skeletal muscles and kidneys in rat. Acta physiologica Scandinavica 1996, 156 (1), 19 - 25. 9. Ido, Y.; Vindigni, A.; Chang, K.; Stramm, L.; Chance, R.; Heath, W. F.; DiMarchi, R. D.; Di Cera, E.; Williamson, J. R., Prevention of vascular and neural dysfunction in diabetic rats by C - peptide. Science 1997, 277 (5325), 563 - 6. 10. Bogle, R. G.; Coade, S. B.; Moncada, S.; Pearson, J. D.; Mann, G. E., Bradykinin and ATP stimulate L - arginine upta ke and nitric oxide release in vascular endothelial cells. Biochemical and biophysical research communications 1991, 180 (2), 926 - 32. 11. Furchgott, R. F.; Zawadzki, J. V., The obligatory role of endothelial cells in the relaxation of arterial smoo th muscle by acetylcholine. Nature 1980, 288 (5789), 373 - 6. 12. Miseta, A.; Bogner, P.; Berenyi, E.; Kellermayer, M.; Galambos, C.; Wheatley, D. N.; Cameron, I. L., Relationship between cellular ATP, potassium, sodium and magnesium concentrations in mammal ian and avian erythrocytes. Biochimica et 196 biophysica acta 1993, 1175 (2), 133 - 9. 13. Ellsworth, M. L.; Forrester, T.; Ellis, C. G.; Dietrich, H. H., The erythrocyte as a regulator of vascular tone. The American journal of physiology 1995, 269 (6 Pt 2), H21 55 - 61. 14. Bergfeld, G. R.; Forrester, T., Release of ATP from human erythrocytes in response to a brief period of hypoxia and hypercapnia. Cardiovascular research 1992, 26 (1), 40 - 7. 15. Light, D. B.; Capes, T. L.; Gronau, R. T.; Adler, M. R., Extracellul ar ATP stimulates volume decrease in Necturus red blood cells. The American journal of physiology 1999, 277 (3 Pt 1), C480 - 91. 16. Sprague, R. S.; Ellsworth, M. L.; Stephenson, A. H.; Kleinhenz, M. E.; Lonigro, A. J., Deformation - induced ATP release from r ed blood cells requires CFTR activity. The American journal of physiology 1998, 275 (5 Pt 2), H1726 - 32. 17. Sprague, R. S.; Ellsworth, M. L.; Stephenson, A. H.; Lonigro, A. J., ATP: the red blood cell link to NO and local control of the pulmonary circulati on. The American journal of physiology 1996, 271 (6 Pt 2), H2717 - 22. 18. Meyer, J. A.; Froelich, J. M.; Reid, G. E.; Karunarathne, W. K.; Spence, D. M., Metal - activated C - peptide facilitates glucose clearance and the release of a nitric oxide stimulus via the GLUT1 transporter. Diabetologia 2008, 51 (1), 175 - 82. 19. Meyer, J. A.; Subasinghe, W.; Sima, A. A.; Keltner, Z.; Reid, G. E.; Daleke, D.; Spence, D. M., Zinc - activated C - peptide resistance to the type 2 diabetic erythrocyte is associated with hypergl ycemia - induced phosphatidylserine externalization and reversed by metformin. Molecular bioSystems 2009, 5 (10), 1157 - 62. 20. M edawala, W.; McCahill, P.; Giebink, A.; Meyer, J.; Ku, C. J.; Spence, D. M., A Molecular Level Understanding of Zinc Activation of C - peptide and its Effects on Cellular Communication in the Bloodstream. The review of diabetic studies : RDS 2009, 6 (3), 148 - 58. 21. Liu, Y.; Chen, C.; Summers, S.; Medawala, W.; Spence, D. M., C - peptide and zinc delivery to erythrocytes requires the p resence of albumin: implications in diabetes explored with a 3D - printed fluidic device. Integrative biology : quantitative biosciences from nano to macro 2015, 7 (5), 534 - 43. 22. Andre, C.; Guillaume, Y. C., Zinc - human serum albumin association: testimony of two binding sites. Talanta 2004, 63 (2), 503 - 8. 23. Bal, W.; Christodoulou, J.; Sadler, P. J.; Tucker, A., Multi - metal binding site of serum albumin. Journal of inorganic biochemistry 1998, 70 (1), 33 - 9. 24. Fanali, G.; di Masi, A.; Trezza, V.; Marino, M.; Fasano, M.; Ascenzi, P., Human 197 serum albumin: from bench to bedside. Molecular aspects of medicine 2012, 33 (3), 209 - 90. 25. Hach, T.; Forst, T.; Kunt, T.; Ekberg, K.; Pfutzner, A.; Wahren, J., C - peptide and its C - terminal fragments improve erythrocyt e deformability in type 1 diabetes patients. Experimental diabetes research 2008, 2008 , 730594. 26. Liumbruno, G.; Bennardello, F.; Lattanzio, A.; Piccoli, P.; Rossetti, G., Recommendations for the transfusion of red blood cells. Blood transfusion = Trasfu sione del sangue 2009, 7 (1), 49 - 64. 27. Bihl, F.; Castelli, D.; Marincola, F.; Dodd, R. Y.; Brander, C., Transfusion - transmitted infections. Journal of translational medicine 2007, 5 , 25. 28. Kleinman, S.; Caulfield, T.; Chan, P.; Davenport, R.; McFarland , J.; McPhedran, S.; Meade, M.; Morrison, D.; Pinsent, T.; Robillard, P.; Slinger, P., Toward an understanding of transfusion - related acute lung injury: statement of a consensus panel. Transfusion 2004, 44 (12), 1774 - 89. 29. Fatalities reported to FDA foll owing blood collection and transfusion: annual summary for fiscal year 2009. March 22, 2010. 2010 . 30. Roback, J. D.; Neuman, R. B.; Quyyumi, A.; Sutliff, R., Insufficient nitric oxide bioavailability: a hypothesis to explain adverse effects of red blood c ell transfusion. Transfusion 2011, 51 (4), 859 - 66. 31. Koch, C. G.; Li, L.; Sessler, D. I.; Figueroa, P.; Hoeltge, G. A.; Mihaljevic, T.; Blackstone, E. H., Duration of red - cell storage and complications after cardiac surgery. The New England journal of me dicine 2008, 358 (12), 1229 - 39. 32. Hebert, P. C.; Wells, G.; Blajchman, M. A.; Marshall, J.; Martin, C.; Pagliarello, G.; Tweeddale, M.; Schweitzer, I.; Yetisir, E., A multicenter, randomized, controlled clinical trial of transfusion requirements in cri tical care. Transfusion Requirements in Critical Care Investigators, Canadian Critical Care Trials Group. The New England journal of medicine 1999, 340 (6), 409 - 17. 33. Sharifi, S.; Dzik, W. H.; Sadrzadeh, S. M., Human plasma and tirilazad mesylate pr otect stored human erythrocytes against the oxidative damage of gamma - irradiation. Transfusion medicine 2000, 10 (2), 125 - 30. 34. Mangalmurti, N. S.; Chatterjee, S.; Cheng, G.; Andersen, E.; Mohammed, A.; Siegel, D. L.; Schmidt, A. M.; Albelda, S. M.; Lee, J. S., Advanced glycation end products on stored red blood cells increase endothelial reactive oxygen species generation through interaction with receptor for advanced glycation end products. Transfusion 2010, 50 (11), 2353 - 61. 35. Kor, D. J.; Van Buskirk , C. M.; Gajic, O., Red blood cell storage lesion. Bosnian journal of basic medical sciences / Udruzenje basicnih mediciniskih znanosti = 198 Association of Basic Medical Sciences 2009, 9 Suppl 1 , 21 - 7. 36. Wautier, J. L.; Zoukourian, C.; Chappey, O.; Wautier, M. P.; Guillausseau, P. J.; Cao, R.; Hori, O.; Stern, D.; Schmidt, A. M., Receptor - mediated endothelial cell dysfunction in diabetic vasculopathy. Soluble receptor for advanced glycation end products blocks hyperpermeability in diabetic rats. The Journal of clinical investigation 1996, 97 (1), 238 - 43. 37. Wang, Y.; Giebink, A.; Spence, D. M., Microfluidic evaluation of red cells collected and stored in modified processing solutions used in blood banking. Integrative biology : quantitative biosciences from n ano to macro 2014, 6 (1), 65 - 75. 38. Engelgau, M. M.; Narayan, K. M.; Herman, W. H. , Screening for type 2 diabetes. Diabetes care 2000, 23 (10), 1563 - 80. 39. Zhang, Y.; Proenca, R.; Maffei, M.; Barone, M.; Leopold, L.; Friedman, J. M., Positional cloning o f the mouse obese gene and its human homologue. Nature 1994, 372 (6505), 425 - 32. 40. Ahima, R. S.; Prabakaran, D.; Mantzoros, C.; Qu, D.; Lowell, B.; Maratos - Flier, E.; Flier, J. S., Role of leptin in the neuroendocrine response to fasting. Nature 1996, 38 2 (6588), 250 - 2. 41. Monti, V.; Carlson, J. J.; Hunt, S. C.; Adams, T. D., Relationship of ghrelin and leptin hormones with body mass index and waist circumference in a random sample of adults. Journal of the American Dietetic Association 2006, 106 (6), 82 2 - 8; quiz 829 - 30. 42. Wang, M. Y.; Chen, L.; Clark, G. O.; Lee, Y.; Stevens, R. D.; Ilkayeva, O. R.; Wenner, B. R.; Bain, J. R.; Charron, M. J.; Newgard, C. B.; Unger, R. H., Leptin therapy in insulin - deficient type I diabetes. Proceedings of the National Academy of Sciences of the United States of America 2010, 107 (11), 4813 - 9. 43. Liu, H. Y.; Cao, S. Y.; Hong, T.; Han, J.; Liu, Z.; Cao, W., Insulin is a stronger inducer of insulin resistance than hyperglycemia in mice with type 1 diabetes mellitus (T1DM) . The Journal of biological chemistry 2009, 284 (40), 27090 - 100. 44. Oral, E. A.; Simha, V.; Ruiz, E.; Andewelt, A.; Premkumar, A.; Snell, P.; Wagner, A. J.; DePaoli, A. M.; Reitman, M. L.; Taylor, S. I.; Gorden, P.; Garg, A., Leptin - replacement therapy fo r lipodystrophy. The New England journal of medicine 2002, 346 (8), 570 - 8. 45. Park, J. Y.; Chong, A. Y.; Cochran, E. K.; Kleiner, D. E.; Haller, M. J.; Schatz, D. A.; Gorden, P., Type 1 diabetes associated with acquired generalized lipodystrophy and insul in resistance: the effect of long - term leptin therapy. The Journal of clinical endocrinology and metabolism 2008, 93 (1), 26 - 31. 46. Yosten, G. L.; Kolar, G. R.; Redlinger, L. J.; Samson, W. K., Evidence for an interaction between proinsulin C - peptide and GPR146. The Journal of endocrinology 199 2013, 218 (2), B1 - 8. 47. Rigler, R.; Pramanik, A.; Jonasson, P.; Kratz, G.; Jansson, O. T.; Nygren, P.; Stahl, S.; Ekberg, K.; Johansson, B.; Uhlen, S.; Uhlen, M.; Jornvall, H.; Wahren, J., Specific binding of proinsuli n C - peptide to human cell membranes. Proceedings of the National Academy of Sciences of the United States of America 1999, 96 (23), 13318 - 23. 48. Al - Rasheed, N. M.; Willars, G. B.; Brunskill, N. J., C - peptide signals via Galpha i to protect against TNF - a lpha - mediated apoptosis of opossum kidney proximal tubular cells. Journal of the American Society of Nephrology : JASN 2006, 17 (4), 986 - 95. 49. Medawala, W., Mechanics and Functional Studies of Zinc (II) Activation of C - Peptide and Its Effect on Re d Blood Cell Metabolism. Ph.D. Dissertation. Michigan State University. 2011 . 50. Anguizola, J.; Matsuda, R.; Barnaby, O. S.; Hoy, K. S.; Wa, C.; DeBolt, E.; Koke, M.; Hage, D. S., Review: Glycation of human serum albumin. Clinica chimica acta; internation al journal of clinical chemistry 2013, 425 , 64 - 76.