IMPLICATIONS OF C-PEPTIDE STIMULATED ATP RELEASE FROM RED BLOOD CELLS IN AUTOIMMUNE DISEASES By Monica Jacobs A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of Comparative Medicine and Integrative Biology – Doctor of Philosophy 2022 ABSTRACT Adenosine triphosphate (ATP) is an energy containing molecule that is essential for numerous physiological processes throughout the body. ATP affects many different systems, such as the central nervous system, the immune system, and the vascular system. Due to ATP’s vital role, alterations in its homeostasis can progress into several pathologies, such as type 1 diabetes (T1D) and multiple sclerosis (MS). Red blood cells (RBCs) play a fundamental role in blood flow regulation through indirectly stimulating vasodilation in the vasculature via secretion of ATP. As glucose levels rise within the circulation, GLUT1 activates in the RBC membrane, which propagates ATP release. This stimulates nitric oxide (NO) to be released from endothelial cells, which results in the relaxation of myosin filaments in the smooth muscle cells on the exterior of the vessel. This relaxation allows for vasodilation and increased blood flow through the vasculature. RBCs have numerous stimuli that result in ATP release and vasodilation, such as hypercapnia, hypoxia, deformation, and C-peptide. C-peptide is a molecule that is secreted by the pancreatic b-cell in response to high levels of glucose. C-peptide has been shown to increase metabolism within RBCs, by binding to RBCs, and stimulating an increase in ATP release. Individuals with T1D have been reported to have hypometabolic RBCs, corresponding to decreased C-peptide binding, GLUT1 activation and ATP release. Whereas individuals with MS have been reported to have hypermetabolic RBCs, corresponding to increased C-peptide binding, GLUT1 activation and ATP release. Understanding C-peptide’s role in decreasing or increasing the metabolic activity of RBCs in both T1D and MS can provide vital information relating to the progression of these diseases and potential therapeutic sites of intervention. The work presented in this dissertation discusses the implications of C-peptide stimulated ATP release in T1D and MS. Methods to study this interaction utilize various binding techniques, as well as ATP measurements, to better understand how C-peptide plays a role in each disease state. These findings will aid in the determination of how these alterations may lead to the progression of T1D and MS, as well as, potentially unveiling novel therapeutic sites of intervention. An additional therapeutic mechanism of action in relation to C-peptide is outlined to determine if current therapies also affect C-peptide binding, transport and downstream physiologic effect on RBCs. The overall intent of the work in this dissertation is to discover and refine therapeutic interventions to improve the lives of individuals with T1D and MS. ACKNOWLEDGEMENTS I want to thank Michigan State University and the National Institutes of Health for funding my research and making the work outlined in this dissertation possible. The Comparative Medicine and Integrative Biology program and Veterinary Medicine school were very supportive at facilitating a smooth graduate school experience, and for that I am very appreciative. I would also like to thank my guidance committee, Dr. Kurt Zinn, Dr. Laura McCabe, and Dr. Daniel Langlois. I appreciate the time and expertise they have given me throughout my time in graduate school. I would also like to thank the Institute for Neuroscience at Memorial Healthcare in Owosso, and specifically, Dr. Rany Aburashed, for supplying blood samples to facilitate the MS project. My advisor, Dr. Dana Spence, has played a vital role molding me into a better scientist, and I could not have asked for a better mentor over the years. Dr. Spence has a way of creating a work environment where you truly feel like family. His open-door policy allowed me to explore ideas that contributed to much of the work discussed in this dissertation. I can honestly say that his guidance and mentorship pushed me to where I am today, and for that, I am very grateful. I will cherish many of the memories made while in the Spence group; namely, group meetings at the Spence household, wallball, sporkle, heardle and much more. I cannot thank the Spence lab members enough for creating a fun, supportive, and encouraging work environment. Dr. Suzanne Summers collected much of the C-peptide data outlined in this dissertation and was always willing to proofread anything I placed in front of her. Dr. Morgan Geiger collaborated with me on many projects throughout graduate school, and I can honestly say there is no better person to work with. Dr. Cody iv Pinger played a pivotal role in the glycated albumin project and paved the way for a rewarding dissertation project. I would also like to thank all other current Spence lab members for their encouragement, expertise and laughs: Nathan Redman, Logan Soule, Lauren Skrajewski, and Stephen Branch. I will miss our lunchtime chats, research “rabbit holes”, and spiderman hidings. Finally, I would like to thank my family and friends for being a presence of love and support throughout graduate school. My parents, Brian and Jill, have always encouraged me to put my best foot forward, in all aspects of life. My six siblings, Luke, Kasey, Alana, Maggie, Sammy and Deano have been a positive reminder that there is life outside of graduate school. My grandfather, “Bumpa,” saw the best in me and always encouraged me to follow my dreams. My fiancé, Austin, always reminded me that I can do anything I set my mind to and has been a calming presence throughout graduate school. My friends, Anna and Elizabeth, have always been my biggest supporters inside and outside of school. I would not have made it through graduate school without all of these people by my side. “I can do all things through Christ who strengthens me.” Philippians 4:13 v TABLE OF CONTENTS LIST OF FIGURES ........................................................................................................ viii Chapter 1: ATP, Diabetes, and Multiple Sclerosis ............................................................1 1.1 ATP .........................................................................................................................1 1.2 Diabetes and Insulin ...............................................................................................6 1.3 Multiple Sclerosis ..................................................................................................17 1.4 Therapeutics, Diabetes, and MS ...........................................................................19 REFERENCES ...............................................................................................................21 Chapter 2: Measuring Zn2+ and C-peptide binding to albumin with increased glycation and fatty acids .......................................................................................................................31 2.1 Introduction ...........................................................................................................31 2.2 Methods ................................................................................................................37 2.3 Results ..................................................................................................................45 2.4 Discussion .............................................................................................................54 REFERENCES ...............................................................................................................60 Chapter 3: Measuring Zn2+ and C-peptide binding and its downstream physiological effects on RBCs with glycation and fatty acids present ..................................................66 3.1 Introduction ...........................................................................................................66 3.2 Methods ................................................................................................................72 3.3 Results ..................................................................................................................76 3.4 Discussion .............................................................................................................83 REFERENCES ...............................................................................................................88 Chapter 4: Measuring Zn2+ and C-peptide binding and its downstream physiological effects on MS RBCs .......................................................................................................92 4.1 Introduction ...........................................................................................................92 4.2 Methods ..............................................................................................................100 4.3 Results ................................................................................................................103 4.4 Discussion ...........................................................................................................110 REFERENCES .............................................................................................................115 Chapter 5: Conclusions and Future Directions .............................................................121 vi 5.1 Diabetes Conclusions .........................................................................................121 5.2 Future binding studies with glycated RBCs ........................................................123 5.3 Future binding studies with increased fatty acids ...............................................124 5.4 Future studies with the addition of exogenous albumin ......................................126 5.5 Future clinical trials with C-peptide therapy ........................................................127 5.6 MS Conclusions ..................................................................................................128 5.7 IFN-β binding to RBCs ........................................................................................130 5.8 Specific dosing frequency of IFN-β after meals ..................................................131 REFERENCES .............................................................................................................133 vii LIST OF FIGURES Figure 1.1 The structure of ATP. ATP is composed of three parts. On the right side of this molecule is an adenine base. This adenine is attached to carbon one on a five carbon ribose sugar. This ribose sugar is attached to three phosphate groups at carbon five. ...1 Figure 1.2 An overview of Cellular Respiration. The first step of cellular respiration is glycolysis, where glucose is broken down into pyruvate. During glycolysis, there is a net gain of 2 ATP molecules and 1 NADH molecule. Pyruvate is broken down into Acetyl- CoA, which enters the second step of cellular respiration, the citric acid cycle, within the mitochondria. During the citric acid cycle, NADH, FADH2, and 2 ATP molecules are created. The electron carriers (NADH and FADH2) produced during glycolysis and the citric acid cycle are cycled into the electron transport chain in the intermembrane space of the mitochondria. This electron gradient activates ATP synthase in the final step of cellular respiration, or oxidative phosphorylation, inside the mitochondria. During this final step, 34 ATP molecules are produced. ............................................................................2 Figure 1.3 Mechanism of ATP release from RBCs. External stimulation of a GPCR on the RBC membrane causes activation of the Gs subunit. Gs activates AC, which produces cAMP and thus activates PKA. Activation of PKA causes the CFTR to open, creating a shuttling of electrons on the exterior of the RBC membrane into the interior, altering the membrane potential. This change in membrane potential, allows ATP created through glycolysis to exit the RBC via Pannexin-1. ATP binds to the P2Y receptor on the endothelial cell. This binding causes Ca2+ channels to open resulting in the subsequent activation of eNOS. eNOS catalyzes the conversion of L-arginine to L-citruline, which produces NO as a biproduct. NO freely diffuses into the smooth muscle cell, activating GC. GC produces cGMP resulting in the relaxation of myosin filaments and vasodilation of the blood vessel. ...........................................................................................................4 Figure 1.4 Proinsulin, Insulin and C-peptide structure. Proinsulin is an 86 amino acid molecule that consists of the A and B chain of insulin, containing 3 disulfide bridges, and C-peptide. In the process of release, Proinsulin is cleaved into a 51 amino acid hormone called insulin and a 31 amino acid peptide called C-peptide. ...........................................7 Figure 1.5 Insulin and C-peptide release from the Pancreatic b-cell. In the rough ER, proinsulin is shuttled into the Golgi apparatus, where it is packed in to b-cell granules. In these b-cell granules, endopeptidases and carboxypeptidases cleave the proinsulin molecule into insulin and C-peptide. Insulin forms hexamers in the b-cell granules with two Zn2+ ions bound, and C-peptide is in a soluble monomer formation. As glucose levels rise outside of the b-cell, glucose enters via the GLUT2 glucose transporter. Glucose is broken down via glycolysis and ATP is created. Increasing concentrations of ATP within the cell results in the closing of ATP dependent K+ channels and subsequent opening of voltage gated Ca2+ channels, which cause the b-cell granule to translocate to the membrane. As the b-cell granule opens to physiological pH, insulin hexamers viii disaggregate into monomers, and insulin, C-peptide and Zn2+ are released into circulation. ........................................................................................................................8 Figure 1.6 The role of C-peptide, Zn2+ and albumin in RBC ATP release. (a) RBC samples were prepared containing either C-peptide, Zn2+ or a combination of both with and without albumin. (b) RBC samples in different conditions were placed in a 3D-printed microfluidic device and were subject to flow conditions. ATP release from RBCs diffused into a channel via a porous membrane and were measured with a photomultiplier tube and luciferin/luciferase assay. (c) Samples containing C-peptide, Zn2+ and albumin resulted in a significant amount of ATP release. All samples without albumin resulted ATP release statistically equal to samples with just RBCs. Samples containing albumin with C-peptide or Zn2+ also resulted in ATP release statistically equal to control conditions with just RBCs. (n=5, p<0.05). Borrowed from Liu et al. ..........................................................................14 Figure 1.7 ATP release from control and MS RBCs. RBCs were subject to flow conditions and ATP was quantified. Control RBCs released 138 ± 2.1 nM ATP. MS RBCs released significantly more ATP than controls, measuring 375 ± 51 nM ATP release. (n = 11 control RBCs, 18 MS RBCs, error = SEM, p<0.001) Borrowed from Letourneau, et. al. ...........17 Figure 2.1 Protein glycation through the Maillard reaction. A glucose molecule binds to a terminal amine group on a protein, forming a Schiff base. This Schiff base can rearrange to form a more stable Amadori product. The final step of glycation involves the irreversible formation of an advanced glycation end product. ...........................................................32 Figure 2.2 Fatty acid binding sites on albumin. A 3D structure of albumin complexed with fatty acids is depicted. There are a total of seven specific binding sites for fatty acids on albumin (3/4 contain one binding site). Each fatty acid binding site has one or more known fatty acids that specifically bind to those areas. .............................................................33 Figure 2.3 High affinity Zn2+ binding site on albumin. Albumin has three different domains depicted in (a). In (b), the Zn2+ ion (depicted in purple) binds to a His 67 and Asn 99 on domain I and a His 247 and Asp 249 on domain II. The fifth ligand to complete this binding site is believed to be water. ............................................................................................34 Figure 2.4 C-peptide binding to normal and glycated HSA. Normal HSA (nHSA) is depicted as a light gray bar and glycated HSA (gHSA) is depicted as dark gray bar. There was statistically equal amount of C-peptide binding in nHSA and gHSA conditions. Results indicate that glycation does not interfere in C-peptide binding to HSA (n=6 for nHSA, n=3 for gHSA, error=SD). Borrowed from Castiaux et al. ...................................36 Figure 2.5 Ultrafiltration device methodologies. Ultrafiltration uses pressure, commonly from a centrifuge, to drive low molecular weight compounds, such as the free ligand, from the bulk of the solution, which contains the receptor ligand complex. If 2-10% of the initial ix volume is in the final ultrafiltrate, equilibrium is not disturbed and therefore, the free ligand concentration in the ultrafiltrate should equal the free ligand concentration in the bulk solution. ..........................................................................................................................37 Figure 2.6 Calculation of the global affinity constant. The overall calculation consists of dividing the bound ligand concentration by the product of the free ligand concentration and the free receptor concentration. In the context of the 65Zn2+ experiments, the bound 65 Zn2+ is divided by the product of the free 65Zn2+ concentration and free HSA concentration. .................................................................................................................44 Figure 2.7 Mass spectrum of control and T1D HSA samples isolated from plasma. Electrospray ionization time of flight mass spectrometry was used to analyze samples from control (a) and T1D (b) donors. Spectral deconvolution was carried out via MaxEnt software before being exported for further analysis. HSA is combined with glucose (G = +162 Da), cysteine (C = +119 Da), and sodium (Na = +23 Da). The control sample is approximately 12.1% glycated, and the T1D sample is approximately 37.6% glycated. 45 Figure 2.8 Differences in glycation percent for control and diabetic HSA isolated from plasma. Control HSA isolated from healthy control plasma had an average glycation percentage of 13 ± 0.5%. gHSA isolated from plasma of people with diabetes had an average glycation percentage of 27 ± 3.0% (n=3 control, n=5 diabetic, *p<0.05, error=SEM). ....................................................................................................................46 Figure 2.9 Fabrication and characterization of 3D-printed ultrafiltration devices. In (a), a pictorial representation of the device cross-section is shown, containing the individual layers of the device. Each membrane is exogenously embedded within the individual Tango layers. In (b), a photograph depiction of a 3D-printed ultrafiltration device is shown within a 1.7 mL centrifugation tube on the left and by itself on the right. In (c), characterization of the ultrafiltration device comparing different centrifugation speeds and times versus the volume of the ultrafiltrate passing through the membrane is shown. Ideal centrifugation speeds and times were deemed 15,000 g for 90 minutes (n=3, error=SD). .......................................................................................................................47 Figure 2.10 Zn2+ binding to nHSA and gHSA. In (a), a full binding curve of the nHSA sample comparing the bound Zn2+ and the free Zn2+ was plotted. Using non-linear regression software, the equilibrium dissociation constant was calculated (Kd = 2.1 ± 0.5 x 10-7 M) and stoichiometry (Bmax = 17 ± 1.2 µM, n = 1.2 ± 0.1). (n=3, error=SD) In (b), a comparison of the binding affinity for nHSA and gHSA is depicted, indicating a 2.3x increase in Kd and a significant decrease in Zn2+ binding for gHSA compared to nHSA (n=5-6, error=SD, *p<0.05). ............................................................................................49 Figure 2.11 Zn2+ binding to various % gHSA samples. A 15% gHSA sample indicated the highest binding affinity to Zn2+ with a nKa value of 10.3 ± 0.06 µM-1. The 24% gHSA x sample had a significant decrease in its binding affinity to Zn2+ (nKa = 5.98 ± 0.06 µM-1) compared to the 15% gHSA. There was a statistical decrease in both 42% gHSA (nKa = 4.0 ± 0.2 µM-1) and 51% gHSA (nKa = 3.6 ± 0.2 µM-1) when comparing the nKa values to both the 15% gHSA and 24% gHSA samples (n=4-8, error=SEM, *p<0.05 to 15% gHSA, **p<0.05 to 24% gHSA). .................................................................................................50 Figure 2.12 Binding curve between Zn2+ and T1D HSA isolated from plasma. Linear regression software was used to plot the free Zn2+ from the bound Zn2+. This software was also used to compute an equilibrium dissociation constant of 3.3 ± 0.5 x 10-7 M and binding stoichiometry (Bmax = 15 ± 0.6 µM) (n=3-4, error=SD). ......................................51 Figure 2.13 C-peptide binding to HSA with and without PA and MA. a) There was no statistical difference in C-peptide binding to HSA with (3.4 ± 0.2 nM free) and without (3.4 ± 0.2 nM free) PA in solution, indicating that C-peptide binding to HSA is not influenced by PA. (n=3, error=SD) b) There is a statistical increase in free C-peptide with MA (3.9 ± 0.3 nM) compared to without MA (2.9 ± 0.2 nM). This indicates that MA interferes in C- peptide binding to HSA. Samples containing PA or MA represent a 1.4x increase in fatty acid concentration compared to control conditions, a ratio that is commonly found in diabetes (n=4, error=SD, *p<0.05). ................................................................................53 Figure 2.14 Zn2+ binding to HSA with and without MA. There was no statistical difference in Zn2+ binding to HSA with (0.55 ± 0.05 nM) and without MA (0.5 ± 0.3 nM). These results indicate that Zn2+ binding to HSA is not influenced by MA. Samples containing MA represent a 1.4x increase in fatty acid concentration compared to control conditions, a ratio that is commonly found in diabetes (n=3, error=SD). .............................................54 Figure 3.1 Structure of carboxylic acid and glutamic acid. In (a), carboxylic acid is depicted, containing a carbonyl group and a hydroxyl group in fatty acids. In (b), glutamic acid is depicted, containing a carboxylic acid residue at both ends of the molecule in C- peptide. At physiological pH, the alpha carboxylic group to the right on glutamic acid is deprotonated, leaving an overall net negative charge. ...................................................67 Figure 3.2 BSA binding to RBCs with and without C-peptide and Zn2+. In (a), the specific binding curves of BSA to RBCs are shown with the black curve representing BSA binding without C-peptide and Zn2+ and the white curve representing BSA binding with C-peptide and Zn2+. (n³4, error=SEM, *p<0.05). In (b), the average increase in BSA binding without C-peptide and Zn2+ (grey) and with C-peptide and Zn2+ (dark gray), depicting a specific binding site for BSA on the RBC and a specific binding site for BSA, C-peptide and Zn2+ on the RBC (n=5, error=SEM). .......................................................................................68 Figure 3.3 Specific binding of nBSA and gBSA to the RBC. Depicted are two specific binding curves of nBSA (11% glycated, triangles) and enriched gBSA (48% glycated; xi circles) binding to the RBCs in the presence of C-peptide and Zn2+ (n³4, error=SEM, *p<0.05). .........................................................................................................................70 Figure 3.4 C-peptide uptake by RBCs with BSA and HSA. C-peptide uptake by RBCs was statistically equal in BSA and HSA, indicating no difference in C-peptide binding (n=3, error=SEM, p=0.08). .......................................................................................................71 Figure 3.5 Mass spectra of nBSA and enriched gBSA isolated using boronate affinity chromatography. Electrospray ionization time of flight mass spectrometry was used to analyzed nBSA (a) and enriched gBSA (b). Depicted, BSA is combined with glucose (G = +162 Da), cysteine (C = +119 Da) and potassium (K = +39 Da). The nBSA sample is approximately 14% glycated and the enriched gBSA sample is approximately 64% glycated. .........................................................................................................................77 Figure 3.6 C-peptide uptake by RBCs with different percent glycated BSA samples. 13% gBSA and 17% gBSA samples had statistically equal amount of C-peptide binding when compared to one another. There was a significant decrease in C-peptide binding when 22% gBSA and 50% gBSA were present compared to both 13% gBSA and 17% gBSA samples (n=4-6, error=SEM, *p<0.05 to 13% gBSA, **p<0.05 to 17% gBSA). ..............78 Figure 3.7 Zn2+ binding to RBCs with different percent glycated BSA samples. Samples signifying control conditions at 14% gBSA had a significant increase in Zn2+ binding compared to other glycation percentages. Samples containing 18% and 23% gBSA has statistically equal Zn2+ binding in comparison to one another. 49% gBSA samples had a 38% decrease in Zn2+ binding to the RBC when compared to 14% gBSA control conditions (n=4, error=SEM, *p<0.05 to 14% gBSA, **p<0.05 to 18% and 23% gBSA). ............................................................................................................................79 Figure 3.8 ATP release from RBCs with different percent glycated BSA samples with and without C-peptide and Zn2+. ATP release was significantly higher in 15% gBSA samples with C-peptide and Zn2+ compared to without. There is a statistical increase in ATP from 18% gBSA samples with C-peptide and Zn2+ compared to 18% gBSA control. There is no statistical increase in ATP for 26% and 56% gBSA samples with or without C-peptide and Zn2+ (n=3-6, error=SEM, *p<0.05 to all gBSA samples, **p<0.05 to all samples except 15% gBSA). ....................................................................................................................81 Figure 3.9 ATP release from RBCs with different concentrations of MA with and without C-peptide and Zn2+. There was a significant increase in ATP release from RBCs when C-peptide and Zn2+ were present for control conditions. All samples containing MA (31- 106 µM) showed a significant decrease in ATP release when compared to the 0 µM C-peptide and Zn2+ sample. All samples containing MA were statistically equal to the control sample without C-peptide and Zn2+ (n=3, error=SEM, *p<0.05 to all bars). .......82 xii Figure 4.1 Comparison of a healthy and MS neuron. On the left, a healthy neuron is depicted. This contains a cell body attached to dendrites and an axon. This axon extends to form synapses to communicate between other neurons. Surrounding the axon is myelin, which encases the axon for more efficient long-distance signaling. On the right, a MS neuron is depicted. This MS neuron is similar to the healthy neuron; however, the axon is encased by damaged myelin, which decreases nerve signaling to other axons. .............................................................................................................................92 Figure 4.2 The blood brain barrier in healthy and MS conditions. On top, a pictorial representation of the BBB is shown. There are tight junctions across a layer of endothelial cells surrounding a blood vessel, inhibiting access of certain molecules within the brain. A standard amount of ATP is released from RBCs, and therefore, a normal amount of NO is released from endothelial cells, inhibiting disruption of the BBB. On the bottom, a pictorial representation of the MS BBB is depicted. Increased ATP from MS RBCs cause an increase in NO release from endothelial cells. Increased levels of NO have been shown to disturb the BBB through breaking tight junctions, which allows for infiltration of T-cells into the brain. T-cells as well as NO have been shown to demyelinate axons, which is a hallmark of MS. ........................................................................................................94 Figure 4.3 Crystallin structure of IFN-b1a. This molecule is a dimer, containing 166 amino acids. IFN-b1a consists of five alpha helices that connect to one another via different loops. There is a glycosylation site at residue Asn-80, specific to IFN-b1a compared to IFNb-1b. Where the two helical clusters interface, there is a specific Zn2+ binding site. In this site, His 121, 93, and 97 form a tetrahedral to stabilize the interaction with Zn2+. ...97 Figure 4.4 The effect of IFN-b on GLUT1 activation. The percent decrease of measurable GLUT1 on control and MS RBCs with C-peptide and Zn2+ is depicted with 0.5 and 10 nM IFN-b. There is a significant decrease in measurable GLUT1 for both 0.5 and 10 nM IFN- b in control and MS RBCs (n=3-5, *p<0.05 to control RBCs C-peptide and Zn2+, **p<0.05 to MS RBCs C-peptide and Zn2+, error=SEM)................................................................98 Figure 4.5 Albumin binding to control and MS RBCs with and without 2 nM IFN-b. All samples contain C-peptide, Zn2+ and either 0 nM IFN-b or 2 nM IFN-b. There is a significant decrease in specific binding of albumin to control and MS RBCs in the presence of C-peptide and Zn2+ with the addition of 2 nM IFN-b (n≥7 control donors, n≥8 MS donors, error=SEM, *p<0.05). ................................................................................100 Figure 4.6 Zn2+ binding to BSA with 2 nM IFN-β. Samples contain either Zn2+ and BSA or Zn2+, BSA and 2 nM IFN-β. There was significantly more Zn2+ binding to BSA without the addition of 2 nM IFN-β, indicating that IFN-β interferes in Zn2+ binding to BSA (n=4, error=SEM, *p<0.05). ...................................................................................................104 xiii Figure 4.7 Zn2+ binding to RBCs with different concentrations of IFN-β. All samples contain C-peptide and Zn2+ with and without IFN-β. There was a statistically equal amount of Zn2+ bound to control RBCs with and without IFN-β. There was significantly more Zn2+ bound to MS RBCs than control RBCs. Significantly less Zn2+ bound to MS RBCs with 1 nM, 2 nM and 4 nM IFN-β compared to MS RBCs without IFN-β (n=3-8 donors, error=SEM, *p<0.05 to 0 nM IFN-β control RBCs, **p<0.05 to 0 nM IFN-β MS RBCs). ..........................................................................................................................105 Figure 4.8 C-peptide binding to BSA with 2 nM IFN-β. Samples contain either C-peptide and BSA or C-peptide, BSA and 2 nM IFN-β. There was significantly more C-peptide binding to BSA without the addition of 2 nM IFN-β, indicating that IFN-β interferes in C- peptide binding to BSA (n=4, error=SEM, *p<0.05)......................................................106 Figure 4.9 C-peptide uptake by control and MS RBCs with difference concentrations of IFN-β. All samples contain C-peptide, Zn2+, no IFN-β or differing concentrations of IFN-β. There is a significant increase in C-peptide binding to MS RBCs compared to control RBCs. Significantly less C-peptide bound to control RBCs with 2 nM IFN-β. Significantly less C-peptide bound to MS RBCs with 1 nM and 2 nM IFN-β (n=4, error=SEM, *p<0.05 to 0 nM IFN-β control RBCs, **p<0.01 to 0 nM IFN-β MS RBCs). ...............................108 Figure 4.10 ATP release from RBCs with difference concentrations of IFN-β. There is a significant increase in ATP for samples containing C-peptide and Zn2+ (stripes) when compared to samples without C-peptide and Zn2+. There is a significant decrease in ATP release from control RBCs with C-peptide and Zn2+ when 1 nM and 2 nM IFN-β are present. There is also a significant decrease in ATP release from MS RBCs with C- peptide and Zn2+ when 1 nM and 2 nM IFN-β are present (n=3-6, error=SEM, *p<0.05 to 0 nM IFN-β control RBC C-peptide and Zn2+, **p<0.05 to 0 nM IFN-β MS RBCs C-peptide and Zn2+, ***p<0.05 to respective RBCs without C-peptide and Zn2+). ........................109 Figure 5.1 Zn2+ and C-peptide binding to RBCs with different concentrations of MA. All samples contain C-peptide, Zn2+ and either PSS with MA or without. a) There was a significant decrease in Zn2+ binding with 31 and 56 µM MA when compared to samples without MA. (n=4-5, error=SEM, *p<0.05 to C-peptide and Zn2+ and 0 µM MA) b) There was a significant decrease in C-peptide binding with 56, 81 and 106 µM MA when compared to samples without MA (n=4-6, error=SEM, *p<0.05 to 0 µM MA). .............125 xiv Chapter 1: ATP, Diabetes, and Multiple Sclerosis 1.1 ATP Adenosine triphosphate (ATP) is an energy containing molecule that is found within all living organisms, and enables proper body function. Commonly referred to as the universal unit of energy, ATP is a nucleoside triphosphate consisting of three separate components; an adenine nitrogenous base, a five carbon ribose sugar, and three phosphate groups, as shown in Figure 1.1.1 Figure 1.1 The structure of ATP. ATP is composed of three parts. On the right side of this molecule is an adenine base. This adenine is attached to carbon one on a five carbon ribose sugar. This ribose sugar is attached to three phosphate groups at carbon five. These phosphate groups are attached to one another via high-energy phosphodiester bonds. Hydrolysis of the third phosphodiester bond converts ATP to adenosine diphosphate (ADP), or an adenine, ribose, and two phosphate groups, which yields energy release. Generally, ATP is created through catabolizing glucose into chemical energy, which is then used in other physiological processes. Within most cells, ATP can be created aerobically or anaerobically.2,3 In cellular respiration, or aerobic respiration, three processes occur to yield the maximum ATP production from glucose. These processes include glycolysis, the citric acid cycle, and oxidative phosphorylation.2,3 1 Figure 1.2 An overview of Cellular Respiration. The first step of cellular respiration is glycolysis, where glucose is broken down into pyruvate. During glycolysis, there is a net gain of 2 ATP molecules and 1 NADH molecule. Pyruvate is broken down into Acetyl-CoA, which enters the second step of cellular respiration, the citric acid cycle, within the mitochondria. During the citric acid cycle, NADH, FADH2, and 2 ATP molecules are created. The electron carriers (NADH and FADH2) produced during glycolysis and the citric acid cycle are cycled into the electron transport chain in the intermembrane space of the mitochondria. This electron gradient activates ATP synthase in the final step of cellular respiration, or oxidative phosphorylation, inside the mitochondria. During this final step, 34 ATP molecules are produced. In the first step of cellular respiration, a glucose molecule is broken down into two pyruvate molecules in the cytoplasm, yielding a net gain of two ATP molecules3, as shown in Figure 1.2. Pyruvate molecules created in glycolysis are oxidized, producing Acetyl-CoA, which enters the mitochondria where the citric acid cycle takes place. During the citric acid cycle, Acetyl-CoA is further oxidized into carbon dioxide and additional 2 reduced electron carriers, NADH and FADH2. In this process, two additional ATP molecules are created.3 Finally, electron transporters embedded within the inner membrane space of the mitochondria, pump high energy electrons from NADH and FADH2 to oxygen (O2).4 As a result, protons are pumped from the matrix of the mitochondria into the intermembrane space. As this process ensues, an electrochemical gradient is produced from the aggregation of protons in the intermembrane space, activating ATP synthase.3,4 ATP synthase uses this proton gradient to shuttle protons back into the mitochondrial matrix. This shuttling of protons down their electrochemical gradient facilitates the addition of a phosphate group to ADP, creating ATP.2–4 This final step of cellular respiration, called oxidative phosphorylation, generates 90% of the total ATP created during cellular respiration, producing of 34 ATP molecules.3–5 ATP plays a role in many essential physiological processes. Some of the most common are neurotransmission, intracellular and purinergic signaling, DNA and RNA synthesis, movement, immune responses, and vessel dilation.6–11 Because ATP supports many functions of the body and plays a vital role in all living organisms, alterations in its homeostasis are associated with severe physiological impairments. Some diseases associated with alterations in ATP production are Alzheimer’s Disease, cancer, diabetes, and Multiple Sclerosis (MS).12–14 Among these diseases, diabetic and MS red blood cells (RBCs) have been reported to have altered ATP release,14,15 which may have implications in disease progression. 1.1.1 Red Blood Cells ATP is readily used by many different cell types such as muscle, bone, and red blood cells (RBCs).16–19 Specifically, RBCs primarily produce ATP anaerobically through 3 glycolysis.20,21 RBCs use ATP to create energy for cellular processes, and importantly, indirectly regulate blood vessel enlargement for blood flow through the release of nitric oxide (NO).10,19 In this mechanism, RBCs produce ATP by uptake of glucose through the glucose transporter, GLUT1. Figure 1.3 describes how glucose undergoes glycolysis, producing ATP, and subsequently releasing it back into the bloodstream via Pannexin-1.10 ATP diffuses to endothelial cells that line the blood vessels where it can bind to the purinergic 2Y (P2Y) receptor, resulting in the shuttling of a voltage gated Figure 1.3 Mechanism of ATP release from RBCs. External stimulation of a GPCR on the RBC membrane causes activation of the Gs subunit. Gs activates AC, which produces cAMP and thus activates PKA. Activation of PKA causes the CFTR to open, creating a shuttling of electrons on the exterior of the RBC membrane into the interior, altering the membrane potential. This change in membrane potential, allows ATP created through glycolysis to exit the RBC via Pannexin-1. ATP binds to the P2Y receptor on the endothelial cell. This binding causes Ca2+ channels to open resulting in the subsequent activation of eNOS. eNOS catalyzes the conversion of L-arginine to L-citruline, which produces NO as a biproduct. NO freely diffuses into the smooth muscle cell, activating GC. GC produces cGMP resulting in the relaxation of myosin filaments and vasodilation of the blood vessel. 4 potassium (K+) channel and the opening of a calcium (Ca2+) channel.10 As Ca2+ enters the endothelial cell, endothelial nitric oxide synthase (eNOS) is activated and converts L-arginine to L-citruline.10 A byproduct of this reaction is gaseous nitric oxide (NO), which is able to freely diffuse into the smooth muscle cells. As NO enters the smooth muscle cells, guanylate cyclase (GC) is activated, which increases cyclic guanosine monophosphate release (cGMP), allowing for vasorelaxation, or enlarging of the blood vessels. There are many different external stimuli that can result in ATP release from the RBC. Hypoxic and hypercapnic conditions, or low O2 and high carbon dioxide (CO2) levels, have been shown to increase ATP release from the RBC.22–24 These conditions cause the RBC to shift production of ATP from glycolysis to the pentose phosphate pathway.23,25 Unlike glycolysis, the pentose phosphate pathway produces more NADPH and the energy containing molecule ribose-5-phosphate (R5P), which can be fed back into the glycolysis pathway to create ATP. In addition, decreased oxygen (O2) leads to active transport of Ca2+ into the RBC, resulting in depolarization of the cell and increased ATP release.24 Rupture of the RBC membrane, or hemolysis, causes release of concentrated ATP into the vasculature due to the expulsion of ATP housed within the cytosol.2,3 Commonly, hemolysis occurs when there is either damage to the membrane, mechanical stress exerted on the membrane, or when there is changes in the tonicity of the RBC. Concentrations of ATP within RBCs range from 1-3 mM,26,27 whereas, extracellular ATP concentrations range from 20-11,000 nM.28–30 The significant difference in intracellular and extracellular ATP due to hemolysis results in significant changes in the vasculature. 5 Certain health conditions have increased rates of RBC hemolysis, such as, thalassemia, sickle cell anemia, infection with specific bacterial strains, mechanical damage, and prolonged exercise.31,32 Increased deformability of RBCs results in an increase in RBC-derived ATP release.19,22,33 Physiologically, RBC deformability enables the cells to flow through vessels with diameters smaller than themselves, thus ensuring O2 delivery to all tissues. This allows for O2 to be transported to all areas of the body, regardless of vascular diameter. Hypoxia and RBC deformability pathologies correlate to one another because RBCs in areas with low O2 become more rigid due to deoxygenated hemoglobin.25 Consequently, RBCs are unable to transport sufficient amounts of O2 throughout the body, leading to a further increase in hypoxic conditions. Diseases associated with hypoxia and decreased deformability of RBCs have been reported to also have decreased ATP release, such as diabetes.34,35 1.2 Diabetes and Insulin People with diabetes have been reported to have less deformable RBCs,36,37 due to decreased ATP release and thus decreased O2 distribution in the body.22,33 The two main types of diabetes are classified as type 1 diabetes (T1D) and type 2 diabetes (T2D). T2D accounts for 90-95% of all cases of diabetes.38 In T2D, individuals are unable to effectively process glucose, and therefore, the body is unable to efficiently use glucose for energy production.39 T1D accounts for 5% of the total diabetes cases.38 T1D is an autoimmune disease in which the immune system attacks the pancreatic b-cells, and therefore, hormones released by these cell types, such as insulin, are no longer 6 produced.40 Because of this, people with T1D commonly rely on exogenous insulin, other glucose regulating drugs, diet, and exercise for survival. Insulin is formed from a molecule called proinsulin in the islets of Langerhans of the pancreas as shown in Figure 1.4.41 These islets are comprised of specialized cell clusters that release specific hormones based on chemical changes in vivo. b-cells are found within the islets of Langerhans and are responsible for the production of insulin. In the endoplasmic reticulum (ER), proinsulin consists of insulin and a connecting peptide, called C-peptide.42 C-peptide connects insulin through the N-terminus of the A chain and C-terminus of the B chain.42 Proinsulin is transported to the Golgi apparatus, where it is packaged into b-cell granules. Within these granules, proinsulin is exposed to a pH of 5.5, which is optimum for activation of pH dependent endopeptidases and Figure 1.4 Proinsulin, Insulin and C-peptide structure. Proinsulin is an 86 amino acid molecule that consists of the A and B chain of insulin, containing 3 disulfide bridges, and C-peptide. In the process of release, Proinsulin is cleaved into a 51 amino acid hormone called insulin and a 31 amino acid peptide called C-peptide. 7 carboxypeptidases.15,43 Proinsulin is cleaved into insulin and C-peptide, where C-peptide is in a soluble protonated formation, and insulin is in a insoluble crystalline hexamer formation with two zinc (Zn2+) ions bound for stability.44 Insulin and C-peptide remain in the b-cell granules until glucose levels rise in circulation. In response to high levels of glucose, commonly after consumption of food, glucose enters the b-cell through the GLUT2 transporter found on the cell membrane (Figure 1.5). Glucose is catabolized through glycolysis within the cell, and ATP is formed. Figure 1.5 Insulin and C-peptide release from the Pancreatic b-cell. In the rough ER, proinsulin is shuttled into the Golgi apparatus, where it is packed in to b-cell granules. In these b-cell granules, endopeptidases and carboxypeptidases cleave the proinsulin molecule into insulin and C-peptide. Insulin forms hexamers in the b-cell granules with two Zn2+ ions bound, and C-peptide is in a soluble monomer formation. As glucose levels rise outside of the b-cell, glucose enters via the GLUT2 glucose transporter. Glucose is broken down via glycolysis and ATP is created. Increasing concentrations of ATP within the cell results in the closing of ATP dependent K+ channels and subsequent opening of voltage gated Ca2+ channels, which cause the b-cell granule to translocate to the membrane. As the b-cell granule opens to physiological pH, insulin hexamers disaggregate into monomers, and insulin, C-peptide and Zn2+ are released into circulation. 8 As ATP concentrations increase within the cytosol, ATP dependent K+ channels close, resulting in depolarization of the b-cell.45 This depolarization allows for voltage gated Ca2+ channels to open, and Ca2+ begins to enter the cytosol.45,46 The increase in Ca2+ causes b-cell granules to move towards and fuse with the cell membrane. As the granules are exposed to physiological pH (7.4), insulin hexamers solubilize, and equal amounts of insulin monomers and C-peptide, accompanied by large amounts of Zn2+, are released into circulation.46 As mentioned previously, individuals with T1D have little to no insulin secretion, and therefore, rely on exogenous insulin for blood glucose control. Despite insulin treatment, microvascular blood flow complications persist, such as retinopathy, neuropathy and nephropathy. Among these complications, retinopathy, or loss of vision, is the most common.47 Increased glucose levels damage blood vessels resulting in decreased blood flow to the eye, resulting in vascular endothelial growth factor (VEGF) to be released, which promotes the production of many small fragile vessels.48 These weak vessels cloud the vitreous of the eye, causing impaired vision. In the central nervous system (CNS), neuropathy, or nerve damage, results from high levels of glucose and low distribution of O2 in the blood vessels, resulting in endoneureal hypoxia.49 These hypoxic conditions within the neurons decrease nerve signaling, resulting in loss of brain function, often seen in numbness to extremities.49,50 Finally, decreased blood flow also causes nephropathy, or kidney failure. Decreased blood flow to the kidney results in partial sclerosis, decreasing filtration rates and increasing protein leakage.51,52 Researchers believe targeting therapies to improve blood circulation could alleviate microvascular complications reported in people with T1D. Despite lacking C-peptide, Zn2+ and insulin 9 secretions following the destruction of the b-cells, insulin is currently the only b-cell derived hormone used as a therapy to treat diabetes. 1.2.1 C-peptide In the 1960s C-peptide was discovered as a molecule that was needed for the proper folding of insulin in the pancreatic b-cell.42 Following this discovery, researchers began evaluating C-peptide for a possible role in diabetes; yet, no biological activity was determined in vivo.53 However, researchers did find that C-peptide had a half-life of 30 minutes, compared to insulin’s half-life of 4-6 minutes.54 Therefore, C-peptide became a biomarker for researchers to determine overall b-cell function, due to its ability to be detected in human serum longer than insulin. In the 1980s a study entitled the “Diabetes Control and Complications trial” was performed, which studied diabetic complications overtime. In these trials, 1,441 people with T1D were subject to different tests corresponding to glucose maintenance over a 6.5 year period.55–57 Results indicated that strict adherence to insulin, diet and exercise improved diabetic complications compared to conventional therapy usage alone. In addition, these results suggested that people with T1D who had residual b-cell activity had less diabetic complications than those with no b-cell activity, regardless of adherence to therapies.57,58 This indicates the importance of the additional b-cell secretions in alleviating diabetic complications that insulin cannot improve alone. After this finding, many groups investigated the potential of C-peptide as a possible therapeutic to treat diabetic complications. Specifically, Johansson et. al. reported improved blood flow and capillary diffusion in the skeletal muscle of people with T1D infused with C-peptide.59 Researchers infused C-peptide or a sodium chloride placebo 10 into healthy controls or people with T1D for 60 minutes. Blood flow and capillary diffusion were measured using a chromium-EDTA and indocyanine green indicator. Improvements in vascular blood flow and capillary diffusion were measured in people with T1D taking C-peptide therapy and found to be statistically equal to healthy controls. These results indicate the potential role of C-peptide in improved microvascular blood flow in diabetes. Researchers have also measured increased deformability in T1D RBCs when treated with C-peptide.60,61 Kunt et. al. used laser diffractoscopy to measure differences in RBC deformability on healthy and T1D RBCs.60 RBCs were treated with differing concentrations of C-peptide for 4 hours and measurements were performed over a period of 8 hours. Results indicate that all concentrations of C-peptide improved deformability of T1D RBCs to levels statistically equal to healthy controls.60 In addition, these increases were reported for all measurements throughout the period of 8 hours, suggesting a role for C-peptide in improved deformability of T1D RBCs. The Spence lab reported an increase in C-peptide stimulated ATP release and an increase in the activation of GLUT1 on RBCs obtained from people with T2D and controls treated with C-peptide.62 RBCs were treated with varying concentrations of C-peptide and allowed to incubate for 6 hours. Samples prepared for GLUT1 studies also contained a GLUT1 phloretin inhibitor to block GLUT1 activity and subsequent ATP release. ATP release was measured using luciferin/luciferase stimulated chemiluminescence detected with a photomultiplier tube. Results indicated an increase in ATP release from T2D and control RBCs with all administered concentrations of C-peptide.62 In addition, ATP release was decreased when GLUT1 was inhibited, suggesting that C-peptide’s effect on the cells involved GLUT1 in the RBC membrane.62 The increase in ATP and GLUT1 after 11 C-peptide stimulation also suggest a possible mechanistic role of C-peptide in subsequent NO release and vasodilation (Figure 1.3). Following numerous discoveries demonstrating C-peptide’s possible role in ameliorating diabetic complications in vitro,44,59–62 in 2011, Cebix pharmaceuticals was approved for a human C-peptide clinical trial. In this trial, 250 participants used a once weekly injection of mono-pegylated C-peptide therapy called Ersatta or a saline-solution placebo.63,64 This study consisted of a 12 month clinical trial to determine the effects of C-peptide on neuropathy in people with T1D. Unfortunately, this trial failed in phase IIb due to the inability of C-peptide to improve sural sensory nerve conduction in people with T1D compared to controls.53,63,64 The failure of Ersatta in patients with T1D was never discussed by Cebix in any publication. However, this failed human C-peptide trial may be due to a number of factors that may have been overlooked during the study, such as inconsistencies in in vivo and in vitro models.65,66 Commonly, researchers use streptozotocin (STZ) to mimic T1D in rodent models. STZ is a chemical that is cytotoxic to pancreatic b-cells, therefore, rodent models with STZ administration no longer secrete insulin after a few says of STZ dosing.66,67 STZ-induced rodent models typically rapidly gain weight and have overall poor health. The most common side effect in STZ mice is the development of diabetic neuropathy, with decreased sensory response and conduction.65 Thus, in some respects, STZ creates a decent phenotypic model of diabetes. STZ is generally given to mice 5-7 days prior to treatment with insulin and/or C-peptide, therefore rodent models transition from having endogenous secretion of insulin and C-peptide to having it injected exogenously within a short period of time. This regimen 12 may have implications when studying C-peptide’s effects in such models, as rodents do not go without C-peptide therapy for an extended period of time. In contrast, people with T1D post diagnosis live the duration of their lives with external insulin injections and completely without C-peptide. As described previously, without C-peptide, blood flow and hyperglycemic complications arise. Specifically, hyperglycemic conditions can cause glycation of cells and proteins.68–71 The process of glycation can take months to years to complete, therefore, STZ-induced rodent models do not accurately mimic long-term effects of glycation in T1D. Glycation alters the transport, conformation, and overall function of cells72 and proteins69,73 which may complicate C-peptide’s transport and delivery to the RBC. Additional research into C-peptide’s overall mechanism in vivo is essential for utilization of C-peptide as a therapeutic. 1.2.2 Zn2+ and Albumin Another possible reason for the failure of the Ersatta clinical trials was an improper understanding of basic chemical stoichiometry and the presence of contaminates in the C-peptide sample. Early studies performed by the Spence group involving C-peptide were performed by directly mixing commercially available C-peptide (98% purity) with RBCs. However, these results were not always reproducible; specifically, an effect on the RBCs could always be measured within 24 hours of dissolving the C-peptide in water. However, beyond that approximate time frame, no effects could be measured. A mass spectrometry experiment revealed an impurity in the C-peptide, iron (Fe2+). After dissolving C-peptide in water, the Fe2+ was oxidizing to Fe3+ and all C-peptide activity was lost. Therefore, using high performance liquid chromatography (HPLC), C-peptide was purified to >99% 13 pure to improve results. In studies following this purification step, C-peptide no longer increased ATP release from RBCs, suggesting that the metal was required for activity on the RBC.74 As described previously, within the b-cell granule, high concentrations of Zn2+ are found, therefore, it was hypothesized that Zn2+ may be the transition metal needed for C-peptide to elicit ATP release from RBCs.44 Following this discovery, the effect of C-peptide stimulated ATP release on the RBC with and without Zn2+ was measured and reported.75 In these experiments, RBCs were treated in the presence and absence of various formulations of C-peptide and Zn2+ as shown in Figure 1.6a. The individual RBC samples were then subject to flow conditions in a 3D-printed fluidic device in Figure 1.6b. ATP released from RBCs diffused into wells Figure 1.6 The role of C-peptide, Zn2+ and albumin in RBC ATP release. (a) RBC samples were prepared containing either C-peptide, Zn2+ or a combination of both with and without albumin. (b) RBC samples in different conditions were placed in a 3D-printed microfluidic device and were subject to flow conditions. ATP release from RBCs diffused into a channel via a porous membrane and were measured with a photomultiplier tube and luciferin/luciferase assay. (c) Samples containing C-peptide, Zn2+ and albumin resulted in a significant amount of ATP release. All samples without albumin resulted ATP release statistically equal to samples with just RBCs. Samples containing albumin with C-peptide or Zn2+ also resulted in ATP release statistically equal to control conditions with just RBCs. (n=5, p<0.05). Borrowed from Liu et al. 14 separated from the channel by a membrane, and ATP was measured using a luciferin luciferase assay. In Figure 1.6c, results indicate that samples containing Zn2+ and C-peptide yielded a significant amount of ATP release from RBCs, whereas samples containing just C-peptide without Zn2+ did not. These results demonstrate the importance of Zn2+ in C-peptide stimulated ATP release.62,76 Interestingly, numerous other studies have also demonstrated the importance of metals in C-peptide’s biological activity.15,44,62,74,77–79 A few years after Zn2+ was found to be a key determinant in C-peptide activity, an additional discovery was made by the Spence group. Specifically in preparation for ATP experiments with RBC samples, physiological salt solution (PSS) is utilized to mimic the contents of the blood stream. Aside from various salts and glucose, PSS also contains high concentrations of bovine serum albumin (BSA). Albumin is the most concentrated protein found in the bloodstream, totaling 60% of the total protein content.80 Albumin is characterized as a binding protein used to transport many different molecules, hormones, drugs, ions, metals and more throughout the body.80–83 In Figure 1.6c, Lui, et al. measured C-peptide stimulated ATP release with buffer containing albumin and buffer without albumin. In buffer without albumin, there was no significant increase in ATP release from RBCs regardless of C-peptide and Zn2+ addition.75 However, in samples containing albumin, Zn2+ and C-peptide there was a significant amount of ATP release from RBCs.75 These studies indicate the importance of Zn2+, C-peptide and albumin to elicit ATP release from RBCs. Since these findings, our group and others have demonstrated that both Zn2+ and C-peptide specifically bind albumin.75,84–86 In the body, 75-85% of Zn2+ is bound to 15 albumin.87 Albumin has been shown to have a specific binding site for Zn2+ where domain I and II interface.84,85,87 Albumin also has two lower affinity binding pockets for Zn2+, but these are commonly unoccupied in vivo.84 Using isothermal titration calorimetry (ITC), C-peptide binding to albumin was measured, and C-peptide was shown to have a binding affinity of 1.75 ± 0.64 x 105 M-1 and a binding stoichiometry of 0.53 ± 0.03, denoting that two C-peptide molecules bind one albumin.76 The binding of Zn2+ and C-peptide together to albumin was also measured with a binding affinity of 5.08 ± 0.98 x 107 M-1 for Zn2+ and 2.66 ± 0.25 x 105 M-1 for C-peptide, indicating that C-peptide and Zn2+ can both bind albumin simultaneously.76 The association between Zn2+, C-peptide, albumin and the RBC have also been measured.76,88 Specifically, Geiger, et al. measured binding of the albumin, C-peptide and Zn2+ complex to RBCs.89 In these studies, BSA was radiolabeled using technetium and subsequent gamma emission detection was utilized to determine the amount of 99m 99m Tc-BSA bound to the RBC. Results indicate that in Tc-BSA samples without C-peptide and Zn2+, there were approximately 13,900 receptor molecules on the RBC.89 99m In Tc-BSA with C-peptide and Zn2+, there were approximately 17,900 receptor molecules on the RBC.89 Interestingly, the difference in 99m Tc-BSA binding in samples with and without C-peptide and Zn2+ correlate to how many C-peptide molecules bound to the RBC in earlier studies.76,89 The increase in 99mTc-BSA binding when C-peptide and Zn2+ are present indicate that there is a separate receptor on RBCs for a C-peptide, Zn2+, albumin complex. Since these findings, additional cells from various disease states have been reported to bind C-peptide and Zn2+ with differing affinities, such as MS. 16 1.3 Multiple Sclerosis MS is an autoimmune disease in which the myelin sheath surrounding axons in the CNS become damaged.90,91 Demyelination of axons leads to lesion formation in the brain, which slows nerve signals. The mechanism of this destruction is unknown; however, researchers speculate that this may be due to increased NO levels reported in the MS lesions found within the brain.92–94 NO has toxic effects in axonal conduction, demyelination and oligodendrocyte injury.92,95,96 Neuronal damage can cause complications in both movement and cognition,97 therefore, people with MS have an average lifespan of 25 to 35 years post diagnosis.98 Researchers have found that RBCs from people with MS bind more C-peptide than healthy controls.99 As described previously, C-peptide has been shown to increase Figure 1.7 ATP release from control and MS RBCs. RBCs were subject to flow conditions and ATP was quantified. Control RBCs released 138 ± 2.1 nM ATP. MS RBCs released significantly more ATP than controls, measuring 375 ± 51 nM ATP release. (n = 11 control RBCs, 18 MS RBCs, error = SEM, p<0.001) Borrowed from Letourneau, et. al. 17 GLUT1 levels and ATP release from RBCs, which causes NO release from endothelial cells.19 In addition to increased C-peptide binding, MS RBCs have also been reported to release nearly three times the amount of ATP compared to controls.14 As shown in Figure 1.7, using a syringe pump and capillary to mimic blood flow through a capillary, control RBCs released 138 ± 2.1 nM ATP whereas MS RBCs released 375 ± 51 nM ATP. In addition, increased deformability of RBCs has been shown to increase ATP release from RBCs and people with MS have been suggested to have increased deformability compared to healthy controls.100 This increased deformation and thus ATP release could contribute to downstream toxic amounts of NO release in the MS brain. Researchers have also reported increased Zn2+ concentrations in RBC membranes of people with MS,101 which may further explain the increase in RBC derived ATP. In addition, epidemiologists have indicated an increase in the incidence of MS in cluster populations that were exposed to high Zn2+ contamination in water and soil, due to Zn2+ manufacturing plants near each of these locations.102–104 Since these findings, numerous researchers have investigated a potential role of Zn2+ in the progression of MS.101,105 However, many conclude that increased Zn2+ levels alone do not lead to the diagnosis of MS.101,102 Therefore, other medications are utilized to slow progression of this disease. Common medications prescribed for MS act to suppress the immune system. Some of these therapies include glatiramer acetate, fingolimod, natalizumab, mitoxantrone, teriflunomide, and interferon-b.106–108 Unfortunately, most of these therapies have mechanisms of action that are not well understood and have varying efficacies in vivo.107,109 The most common therapy for MS is interferon-beta (IFN-b); 18 however, this only reduces relapses in 33% of patients over a 2 year duration.110 Researchers speculate that the varying effects of MS therapies may be due to the unknown pathophysiology causing axon damage in MS, accompanied by the unknown mechanisms of certain therapies. However, increased C-peptide binding and ATP release in MS RBCs may have implications in its pathophysiology, as well as provide sites of therapeutic intervention. 1.4 Therapeutics, Diabetes, and MS In both T1D and MS, ATP release from RBCs has been reported to be altered. ATP has been shown to play a vital role in many physiological processes, therefore, alterations in its homeostasis can be damaging. In addition, ATP has a known role in regulating blood flow in the vasculature through its release from RBCs which indirectly propagates vasodilation. The work in this dissertation aims to examine how alterations in C-peptide stimulated ATP release for T1D and MS RBCs occur. As well as, investigate how these changes may contribute to the overall pathophysiology of these diseases, to serve as a platform for therapeutic intervention. Various binding techniques and ATP measurements will be utilized in order to determine the relationship between C-peptide, Zn2+, albumin and the RBC. Specifically, in Chapter 2, fabrication of novel 3D-printed ultrafiltration devices to specifically measure the interaction between the Zn2+, C-peptide and albumin complex was utilized in order to understand how these molecules associate with one another in T1D. 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How effective is glatiramer acetate (Copaxone) in the treatment of relapting-remitting multiple sclerosis (MS) (RRMS). 30 Chapter 2: Measuring Zn2+ and C-peptide binding to albumin with increased glycation and fatty acids 2.1 Introduction A common characteristic of diabetes is elevated blood glucose levels.1,2 In individuals with type 1 diabetes (T1D), pre-prandial blood glucose levels are commonly higher than 7 mM, whereas in non-diabetic individuals, levels typically do not exceed 5-6 mM.3 Blood glucose levels that range in between these two values are considered pre-diabetic, therefore, careful monitoring of these levels is important for prognosis. Other ways to monitor and diagnose diabetes consist of measuring hemoglobin A1C (HbA1c) levels. Hemoglobin (Hb) is a protein found in all red blood cells (RBCs) and is known for its ability to bind and transport oxygen (O2) throughout the body. When exposed to a high glucose environment, glucose reacts with one or both N-terminal valine groups on Hb and becomes a ketoamine, or HbA1c.4 Due to the approximate 120 day lifespan of RBCs, HbA1c levels are measured in T1D to evaluate average glycemic control over the period of 2-3 months. Therefore, HbA1c is an important biomarker for how well an individual with T1D is maintaining their blood glucose levels.5 However, some researchers speculate that alterations in the RBC lifespan and other blood diseases may interfere in this measurement,6,7 leading investigators to explore other glycated molecules within the bloodstream that are correlated to average glucose levels in T1D.2,5,8 Albumin is a prominent carrier protein found in the plasma accounting for 60% of the total protein content.9 This molecule is known for its extensive ligand binding capabilities, transporting fatty acids, hormones, drugs, ions, metals and more throughout the body.10,11 When exposed to high glucose environments, albumin undergoes glycation, 31 leading to structural and functional alterations.12–14 People with diabetes have been reported to have 2-5 times more glycated albumin than healthy controls.15 In addition, glycated albumin has been a useful biomarker for measurement of intermediate (2-3 week) glucose control due to the 14-20 day half-life of albumin.5 The glycation process involves a series of non-enzymatic steps, known as the Maillard reaction in Figure 2.1, in which glucose binds to a terminal amine group on a protein, commonly a lysine or arginine residue.16 This molecule forms a Schiff base, which is a reversible intermediate. If glycation ensues, a more stable rearrangement can develop, forming an Amadori product. A further irreversible rearrangement can occur, forming an advanced glycation end product. This final irreversible process commonly results in functional changes to the cells, tissues and proteins. These changes have been reported to lead to apoptosis, promotion of oxidative stress, formation of blood clots and inflammation.17–19 Figure 2.1 Protein glycation through the Maillard reaction. A glucose molecule binds to a terminal amine group on a protein, forming a Schiff base. This Schiff base can rearrange to form a more stable Amadori product. The final step of glycation involves the irreversible formation of an advanced glycation end product. In addition to increased glucose levels in diabetes, increased levels of fatty acids have also been reported.20 Albumin contains seven specific fatty acid binding sites21,22 to 32 transport and deliver them to various organs throughout the body.22,23 Alterations in this homeostasis have been attributed to pathologies such as insulin resistance, hypertension, and altered fibrinolysis.24–26 The structure of fatty acids consists of a carboxylic acid head bound to a fatty acyl carbon chain. The negatively charged carboxylic acid head has been reported to bind to the seven fatty acid binding sites on albumin with differing affinities due to differing hydrophobicities of each pocket,22 as shown in Figure 2.2. The total basal plasma concentration of fatty acids bound and unbound in the plasma is 250-500 µM.27 Interestingly, individuals with diabetes have been reported to have three times this concentration.28 1 9 2 5 7 3/4 6 8 Figure 2.2 Fatty acid binding sites on albumin. A 3D structure of albumin complexed with fatty acids is depicted. There are a total of seven specific binding sites for fatty acids on albumin (3/4 contain one binding site). Each fatty acid binding site has one or more known fatty acids that specifically bind to those areas. 33 As discussed in Chapter 1, the importance of C-peptide’s delivery was first discovered by Liu et al., where these authors described ATP release from healthy RBCs when treated with C-peptide, Zn2+ and a buffer with and without albumin.29 Results indicated that a significant amount of ATP was released only when C-peptide, Zn2+ and albumin were present in the sample. Since this discovery, both C-peptide (Kd = 2.4 ± 0.3 x 10-6 M)30 and Zn2+ (Kd = 2.1 ± 0.5 x 10-7 M)29 have been shown to specifically bind to albumin. Albumin is responsible for transport of approximately 75-85% of Zn2+ throughout the body.31 Zn2+ contributes to many physiological processes, such as immune cell function, platelet coagulation, cell division and differentiation32,33 Therefore, Zn2+ transport throughout the body is of vital importance. Albumin contains one high-affinity binding site for Zn2+ where domain I and II interface and two lower-affinity binding sites, as shown in Figure 2.3.27,34,35 In the high affinity binding site, a His 67 and Asn 99 on domain I and a Domain I Domain III Domain II Figure 2.3 High affinity Zn2+ binding site on albumin. Albumin has three different domains depicted in (a). In (b), the Zn2+ ion (depicted in purple) binds to a His 67 and Asn 99 on domain I and a His 247 and Asp 249 on domain II. The fifth ligand to complete this binding site is believed to be water. 34 His 247 and Asp 249 on domain II are bound to Zn2+, with a third exogenous ligand believed to be water.27,34 Alterations in Zn2+ homeostasis have detrimental effects on such organ systems, such as the central nervous system (CNS), gastrointestinal system, epidermis, skeletal system, and reproductive system.32,33 A specific binding site for C-peptide on albumin has yet to be discovered. However, the C-terminus of C-peptide has been shown to contribute to overall bioactivity, therefore, researchers speculate that this region may hold importance for possible receptor ligand interactions.36,37 This terminus consists of 5 amino acid residues (EGSLQ) and importantly, a glutamic acid at position 27.36–39 When this glutamic acid is substituted with an alanine, C-peptide loses its bioactivity.38 It is speculated that this negatively charged glutamic acid may be important in the binding and delivery of C-peptide to its target. It is important to note that the structure of glutamic acid consists of a carboxylic acid group. Interestingly, the head portion of fatty acids shown to bind to albumin also contain a carboxylic acid group. Therefore, increased levels of fatty acids could lead to competition with C-peptide for the same binding site on albumin, which may result in altered C-peptide binding. Preliminary experiments utilizing ultrafiltration enabled measurement of C-peptide binding to normal human serum albumin (nHSA) and glycated human serum albumin (gHSA).30 Castiaux et al. indicated that C-peptide binding to gHSA (21 ± 7.4% free) was statistically equal to nHSA (22 ± 6.2% free), as shown in Figure 2.4. These results indicate that glycation does not interfere in C-peptide binding to albumin, therefore, the binding between glycated albumin and C-peptide are not outlined in this chapter. However, similar binding techniques are utilized to measure the association of Zn2+ and albumin in glycated 35 conditions, as well as C-peptide and Zn2+ binding to albumin in increased fatty acid conditions. Figure 2.4 C-peptide binding to normal and glycated HSA. Normal HSA (nHSA) is depicted as a light gray bar and glycated HSA (gHSA) is depicted as dark gray bar. There was statistically equal amount of C-peptide binding in nHSA and gHSA conditions. Results indicate that glycation does not interfere in C-peptide binding to HSA (n=6 for nHSA, n=3 for gHSA, error=SD). Borrowed from Castiaux et al. Specifically, a custom ultrafiltration device was fabricated using 3D-printed technologies. Ultrafiltration utilizes centrifugal pressure to push low molecular weight compounds into the filtrate through a size exclusion membrane, while retaining high molecular weight compounds above the membrane in the retentate.40 As depicted in Figure 2.5, the membrane enables separation of free ligand from the receptor ligand complex. For all ultrafiltration techniques, contingent upon 2-10% of the initial volume in the ultrafiltrate, equilibrium should not be disturbed.30,40 Therefore, the amount of free 36 ligand in the ultrafiltrate should equate to the free ligand in the bulk sample above the size exclusion membrane in the rententate.30,40 Quantification of free ligand in the ultrafiltrate can be measured using various techniques (immunoassays, calorimetric assays, radiochemistry, etc.) and equilibrium binding constants can be calculated. Figure 2.5 Ultrafiltration device methodologies. Ultrafiltration uses pressure, commonly from a centrifuge, to drive low molecular weight compounds, such as the free ligand, from the bulk of the solution, which contains the receptor ligand complex. If 2-10% of the initial volume is in the final ultrafiltrate, equilibrium is not disturbed and therefore, the free ligand concentration in the ultrafiltrate should equal the free ligand concentration in the bulk solution. 2.2 Methods 2.2.1 Plasma collection Whole human blood was drawn via venipuncture into heparinized tubes and centrifuged at 500 g for 5 minutes. The plasma was subsequently collected into a 15 mL 37 tube and aliquoted into 1 mL samples. The aliquoted plasma samples were stored at -20°C until use. 2.2.2 Isolation and characterization of HSA from plasma Human serum albumin (HSA) was isolated from plasma using antibody coated magnetic beads (PureProteomeTM, Millipore, Burlington, MA) using a previously reported method.41 A 1:10 dilution of plasma with 18 MW distilled deionized water (DDI H2O) was placed into a tube containing antibody beads and placed on an orbital shaker for 20 minutes at room temperature, allowing the HSA to bind to the beads. The HSA bound to the antibody beads was then separated from the plasma using a magnet (EasySepTM, Stemcell Technologies, Cambridge, MA), and the HSA was detached from the antibody bead using a glycine buffer (0.1 M glycine, pH 3.0). The HSA solution was purified using Amicon Ultra-15 ultrafiltration centrifugal filter units (10 kDa MWCO, Millipore) through the addition DDI H2O and seven washing steps to ensure the glycine was removed. The purified HSA solution was then lyophilized and stored at -20°C. 2.2.3 Isolation of nHSA and gHSA using boronate affinity chromatography Wash buffer (50 mM HEPES, 0.5 M NaCl, pH 8.0-8.5) and elution buffer (100 mM sorbitol, 50 mM HEPES, 0.5 M NaCl, pH 8.0-8.5) were prepared the day of experimentation. A gravity-flow column (Takarta, Mountain View, CA) was placed in the upright position, followed by the addition of glycoprotein enrichment resin (Takarta). An HSA/wash buffer solution was added to the column and incubated at room temperature for 20 minutes on an orbital shaker (300 rpm) enabling the boronate groups within the glycoprotein enrichment resin to bind to the hydroxyl groups within the glucose. The gravity-flow column was opened and the solution containing normally glycated HSA 38 (nHSA), was collected. Subsequently, the column was washed four times with wash buffer to remove the remaining nHSA. The elution buffer was added, and the enriched-glycated HSA (gHSA) was eluted off the column and collected. The HSA solutions was purified from buffer contaminates using methodologies as described in section 2.2.2. Both nHSA and enriched gHSA were lyophilized and stored at -20°C. 2.2.4 Mass spectrometry analysis of HSA samples All HSA samples were analyzed and characterized using a Waters Xevo G2-XS time of flight mass spectrometer (TOF-MS). Using MassLynx software, a spectrum was processed, and mass to charge (m/z) ratios were accounted for to create a mass spectrum in the range of 66,000-69,000 Da. The peaks were centered, and the spectra was exported for further analysis. Glycation percent was assessed by computing the total ion counts of the whole spectrum and dividing that by the individual centroided peaks to calculate a percent for each peak. The peaks that were shifted +162 Da from the parent peak were characterized as glycation adducts, based off of the molecular weight of glucose (180 Da) after the removal of an oxygen and two hydrogens occurring in the process of glycation. After summing the total amount of glycation adduct ion counts, this was then divided by the total ion counts to compute a glycation percentage. Other HSA adducts were accounted for such as cystenylated-albumin (+119 Da) and sodiated-albumin (+23 Da). 2.2.5 3D-printed centrifuge-enabled ultrafiltration devices 2.2.5.a Design of centrifuge-enabled ultrafiltration devices This device was fabricated to fit into a 1.7 mL microcentrifugation tube, therefore the diameter of the devices could not exceed 6.0 cm. The device was engineered through 39 computer aided design (CAD) software (Autodesk Inventor Professional, San Rafael, CA) in three separate stereolithography (STL) files. A stacked-printing method created by former Spence lab members was incorporated, enabling different materials to be integrated along the z-axis without the addition of support material.30,42 Generally, 3D-PolyJet printers add sacrificial support material within any voids of the device or when there are material changes along the z-axis. Support material is waxy and may clog the membrane pores or contaminate the sample of interest, potentially altering experimental results. Therefore, the parameters were altered to print in a support-free mode. This was accomplished by changing carpet height, carpet protector z, and setting the support thickness of pedestal to 0 mm prior to printing. The bulk of the device was made in VeroClear, which is a clear, hard plastic, allowing for impact resistance. The first file contained a 1.0 mm layer of VeroClear with four small holes in the middle to allow fluid to pass. The following file, and the next three prints, consisted of a 0.1 µm thick Tango material, in a rubber-like O-ring formation. This enabled two membranes to be embedded within the device, while creating a water-tight seal to prevent leaking. Lastly, the final layer was made from VeroClear, comprised of a 15 mm hollow, cup-like structure with four holes on the bottom to retain the bulk liquid sample while allowing minimal fluid past (Figure 2.9a). 2.2.5.b Preparation of membranes Size exclusion membranes (12 kDa MWCO, 150 mm sheets, SpectraPor, Waltham, MA) and polycarbonate membranes (0.1 µm, 75 mm sheets, Sterlitech Corporations, Auburn, WA) were cut into 1 cm diameter circles using a commercially available single hand, steel hole punch. 40 2.2.5.c Fabrication of the centrifuge-enabled ultrafiltration device Prior to printing, the Stratasys PolyJet 3D-printer was put in support-free mode using the parameters mentioned previously. The first file containing a 1.0 mm VeroClear layer was opened and printed. Subsequently, a second file containing 0.1 µm Tango was printed atop the previous VeroClear layer. The 0.1 µm polycarbonate membrane was manually placed on the Tango layer using forceps and arranged such that the membrane covered the device, without overlapping the sides. Another layer of 0.1 µm Tango was printed to completely seal the polycarbonate membrane in place. This process was repeated with a size-exclusion, 12 kDa MWCO membrane, followed by an additional 0.1 µm Tango layer. Finally, a 15 mm VeroClear layer was printed atop the previous layers, concluding the printing of the device. Prior to each print, the build tray height was corrected for by lowering the tray based upon the height of the previous print. 2.2.6 Determination of HSA concentration Ultrafiltration buffer (10 mM tris (hydroxymethyl) aminomethane (Invitrogen, Carlsbad, CA) and 150 mM NaCl (Sigma Aldrich, St. Louis, MO), pH 7.4) was prepared the day of experimentation and used to prepare a 75 µM HSA stock solution. The concentration of isolated or commercially available Sigma HSA stock solution was determined through Pierce bicinchoninic (BCA) assay (ThermoFisher Scientific, Waltham, MA) preceding the preparation of all samples. Each HSA stock concentration was diluted to fit within the standard curve. Working reagent was created by adding reagent A and regent B in a 1:50 ratio. Following the addition of 25 µL of the standards and samples were added to a 96-well clear plate, an additional 200 µL of working reagent were added to each well and incubated at 37°C for 30 minutes. After incubation, the 41 96-well plate was allowed to cool to room temperature and absorbance was measured on a FlexStation-3 spectrophotometer (Molecular Devices, San Jose, CA) at 562 nm. The stock concentration of HSA was calculated from the external standard calibration curve and utilized to prepare samples. 2.2.7 Sample preparation for C-peptide binding to HSA Isolated or commercially available HSA stocks were prepared by weighing the HSA and diluting with albumin-free physiological salt solution (AF-PSS, 4.7 mM KCl (Fisher Scientific, Waltham, MA), 2.0 mM CaCl2 (Fisher Scientific), 140.5 mM NaCl, 12.0 mM MgSO4 (Fisher Scientific), 21.0 mM tris (hydroxymethyl) aminomethane, and 5.5 mM dextrose (Sigma Aldrich) pH at 7.40) to make a physiological salt solution (PSS) containing 75 µM HSA (Sigma Aldrich). A 2.5 mM stock solution of commercially available fatty acid (myristic acid, MA, or palmitic acid, PA, Sigma-Aldrich) was dissolved in 100% ethanol. C-peptide was diluted from an 8 µM stock to an 800 nM working solution, with the eventual concentration of C-peptide in the HSA-containing samples equating to 20 nM.29 Samples representing glycated HSA were prepared to contain 20 nM C-peptide and ultrafiltration buffer with 75 µM isolated HSA from either a sample from a person with diabetes or a control. Samples representing increased fatty acids contained 20 nM C-peptide, increasing concentrations of fatty acids, and ultrafiltration buffer with 75 µM commercially available HSA. 3D-printed ultrafiltration devices were placed into 1.7 mL centrifugation tubes, and 200 µL of each HSA sample was transferred into their respective devices. These were centrifuged at 15,000 g for 90 minutes. Following centrifugation, the ultrafiltrate was removed, and the concentration of C-peptide was determined through a human C-peptide enzyme-linked immunosorbent assay (ELISA). 42 2.2.8 Sample preparation for Zn2+ binding to HSA A stock solution of 91 µM 65Zn2+ (PerkinElmer, Waltham, MA) was used to prepare all samples. A stock solution of fatty acids (MA or PA) was prepared as described in section 2.2.7. Ultrafiltration buffer was utilized to prepare a 15 µM stock solution of HSA. The concentration of isolated or commercially available HSA stock solution was determined through Pierce BCA assay preceding the preparation of all samples as described in section 2.2.6. Samples with increased glycation were prepared containing ultrafiltration buffer with 15 µM isolated gHSA and increasing concentrations of 65 Zn2+. Whereas, samples with increased free fatty acids contained ultrafiltration buffer with 15 µM commercially available HSA, 65Zn2+ and increasing concentrations of fatty acids. The samples were placed into the 3D-printed ultrafiltration devices within 1.7 mL tubes and centrifuged as previously described in section 2.2.7. The ultrafiltrate was removed and a 10 µL aliquot was pipetted into a 1.7 mL tube. 2.2.9 C-peptide binding analysis External C-peptide standards were prepared in the concentration of 0-15 nM and further diluted 1:50. The free C-peptide ultrafiltrate was removed from each sample tube and diluted 1:20 in DDI H2O. A C-peptide ELISA was used to measure the concentration of free C-peptide in the ultrafiltrate. A standard curve was created and utilized to determine the concentration of free C-peptide in each sample. The concentration of free C-peptide calculated was then used to compute the amount of C-peptide bound, by subtracting the free C-peptide measured from the total C-peptide added. 43 2.2.10 Zn2+ binding analysis External standards with volumes of 10 µL and concentrations ranging from 0.13-8.0 µM 65 Zn2+ were prepared and placed into a 1.7 mL tubes. The ultrafiltrate, consisting of the free 65Zn2+, was removed from the bottom of the 1.7 mL tube and 10 µL of the ultrafiltrate was placed into another 1.7 mL tube. All samples and standards were placed into the 2480 WIZARD2 automatic gamma counter (PerkinElmer, Waltham, MA) 65 and quantified using gamma emission detection specific for Zn2+ (1116 keV) for 5 minutes each. The concentration of free 65Zn2+ was determined by comparing the counts per minute (cpm) from each sample to the external standard calibration curve. The free 65 Zn2+ concentration was used to compute the bound 65 Zn2+ by subtracting the 65 concentration of free Zn2+ from the concentration of 65 Zn2+ originally added to each sample. Full binding curves were computed from these values by plotting the free 65Zn2+ 65 concentration versus the bound Zn2+ concentration. Non-linear regression software (SigmaPlot 13.0) was used to compute Kd and Bmax values. A single-point (global) binding 65 affinity (nKa) was also computed by dividing the concentration of bound Zn2+ Figure 2.6 Calculation of the global affinity constant. The overall calculation consists of dividing the bound ligand concentration by the product of the free ligand concentration and the free receptor concentration. In the context of the 65 Zn2+ experiments, the bound 65Zn2+ is divided by the product of the free 65Zn2+ concentration and free HSA concentration. 44 65 concentration by the product of the free Zn2+ concentration and the free HSA concentration (Figure 2.6). 2.3 Results 2.3.1 Mass spectrometry analysis of control and T1D HSA The percent glycation of HSA was analyzed using electrospray ionization TOF-MS. Figure 2.7a shows a mass spectrum of control HSA isolated from healthy control plasma, with an overall glycation percentage of 12.1%. A mass spectrum of gHSA isolated from T1D plasma, with a glycation percentage of 37.6% is shown in Figure 2.7b. The gHSA sample contained numerous additional glycation peaks when compared to the control HSA sample, indicating that the sample is more glycated. Figure 2.7 Mass spectrum of control and T1D HSA samples isolated from plasma. Electrospray ionization time of flight mass spectrometry was used to analyze samples from control (a) and T1D (b) donors. Spectral deconvolution was carried out via MaxEnt software before being exported for further analysis. HSA is combined with glucose (G = +162 Da), cysteine (C = +119 Da), and sodium (Na = +23 Da). The control sample is approximately 12.1% glycated, and the T1D sample is approximately 37.6% glycated. 2.3.2 Glycation % of HSA separated from control and diabetic plasma Albumin was isolated from plasma using antibody coated magnetic beads and analyzed using electrospray ionization TOF-MS. Results in Figure 2.8, indicate that the 45 glycation of control HSA samples ranged from 12-14% and diabetic HSA samples ranged from 21-38%. The average of the control HSA samples (13 ± 0.5%) were significantly lower than the diabetic HSA samples (27 ± 3.0%). Figure 2.8 Differences in glycation percent for control and diabetic HSA isolated from plasma. Control HSA isolated from healthy control plasma had an average glycation percentage of 13 ± 0.5%. gHSA isolated from plasma of people with diabetes had an average glycation percentage of 27 ± 3.0% (n=3 control, n=5 diabetic, *p<0.05, error=SEM). 2.3.3 Fabricating ultrafiltration devices Previously published additive manufacturing methods were used to enable material changes and placement of membranes without the addition of support material.30,42 A stacked-printing method was also utilized to allow for integration of membranes within the device, as shown in Figure 2.9a. After placement of each of the 46 Figure 2.9 Fabrication and characterization of 3D-printed ultrafiltration devices. In (a), a pictorial representation of the device cross-section is shown, containing the individual layers of the device. Each membrane is exogenously embedded within the individual Tango layers. In (b), a photograph depiction of a 3D-printed ultrafiltration device is shown within a 1.7 mL centrifugation tube on the left and by itself on the right. In (c), characterization of the ultrafiltration device comparing different centrifugation speeds and times versus the volume of the ultrafiltrate passing through the membrane is shown. Ideal centrifugation speeds and times were deemed 15,000 g for 90 minutes (n=3, error=SD). 47 membranes or printing of the individual layer, the print tray was lowered based on the previous print height and an additional layer was printed atop of the previous layer. This device was characterized by measuring the volume of ultrafiltrate pushed through the membrane pores upon exposure to different centrifugation times and g-forces. The 3D-printed ultrafiltration devices were placed into 1.7 mL centrifugation tubes as shown in Figure 2.9b, and 200 µL of DDI H2O was placed into each device. These devices were subsequently centrifuged at different speeds and times, and the volume of the ultrafiltrate was determined by massing the volume pushed through the membranes. The ideal centrifuge time was contingent upon 10-13 µL in the ultrafiltrate, due to the low volume inhibiting equilibrium disruption, while retaining enough detectable analyte to quantify. The preferred setting was determined to be 15,000 g for 90 minutes as shown in Figure 2.9c. 2.3.4 Zn2+ binding to normal and enriched glycated HSA To characterize the ability of the device to measure binding affinities, the interaction between Zn2+ and commercially available nHSA was measured, as depicted in Figure 2.10a. The binding curve saturated at a Bmax value of 17 ± 1.2 µM and had a binding affinity of Kd = 2.1 ± 0.5 x 10-7 M, which are values similar to what is seen in the literature.43 The extent of glycation was measured using TOF-MS, where nHSA had a glycation percentage of 12%, a value commonly reported in the literature for non-diabetic, healthy controls.15 Following these findings, it was determined that the devices were able to accurately measure the binding of Zn2+ to HSA, and therefore, the devices were used in subsequent binding experiments. To assess interaction between HSA and Zn2+ with increased glycation, boronate 48 affinity chromatography was utilized to isolate enriched gHSA from the nHSA in Figure 2.10a. In this separation technique, HSA is isolated into an enriched gHSA fraction containing a majority of the albumin that is glycated and a lesser glycated fraction, nHSA. The average glycation percentage of gHSA was approximately 66% compared to the nHSA at 12% glycated. The binding stoichiometry of gHSA was statistically equal to nHSA, with a Bmax value of 18 ± 1.1 uM (Figure 2.10b, n = 1.2 ± 0.1). Importantly, the Kd values were statistically different than one another, with the gHSA exhibiting a 2.3-fold decrease in binding capabilities (Kd of 4.8 ± 0.8 x 10-7 M) compared to nHSA. Because glycation was the only experimental factor altered, these results confirm that the association between HSA and Zn2+ is decreased by glycation alone. Figure 2.10 Zn2+ binding to nHSA and gHSA. In (a), a full binding curve of the nHSA sample comparing the bound Zn2+ and the free Zn2+ was plotted. Using non-linear regression software, the equilibrium dissociation constant was calculated (Kd = 2.1 ± 0.5 x 10-7 M) and stoichiometry (Bmax = 17 ± 1.2 µM, n = 1.2 ± 0.1). (n=3, error=SD) In (b), a comparison of the binding affinity for nHSA and gHSA is depicted, indicating a 2.3x increase in Kd and a significant decrease in Zn2+ binding for gHSA compared to nHSA (n=5-6, error=SD, *p<0.05). 2.3.5 Zn2+ binding to HSA with different glycation levels To further determine the role of HSA glycation on Zn2+ binding, different glycation 49 percentages of HSA were made from the nHSA and gHSA stock samples isolated using boronate affinity chromatography. Using different ratios of nHSA (15% glycated) to gHSA (51% glycated), four HSA stocks were created, with glycation percentages of 15%, 24%, 42% and 51%. In Figure 2.11, the 15% gHSA sample had the highest binding affinity (10.3 ± 0.06 µM-1) when compared to all other glycation percentages. There is a significant decrease in Zn2+ binding to 24% gHSA (nKa = 5.98 ± 0.06 µM-1) when Figure 2.11 Zn2+ binding to various % gHSA samples. A 15% gHSA sample indicated the highest binding affinity to Zn2+ with a nKa value of 10.3 ± 0.06 µM-1. The 24% gHSA sample had a significant decrease in its binding affinity to Zn2+ (nKa = 5.98 ± 0.06 µM-1) compared to the 15% gHSA. There was a statistical decrease in both 42% gHSA (nKa = 4.0 ± 0.2 µM-1) and 51% gHSA (nKa = 3.6 ± 0.2 µM-1) when comparing the nKa values to both the 15% gHSA and 24% gHSA samples (n=4-8, error=SEM, *p<0.05 to 15% gHSA, **p<0.05 to 24% gHSA). 50 compared to the 15% gHSA. In addition, the 42% (nKa = 4.0 ± 0.2 µM-1) and 51% gHSA (nKa = 3.6 ± 0.2 µM-1) samples had a significant decrease in Zn2+ binding compared to both the 15% and 24% gHSA samples. Results indicate that as glycation of HSA increases Zn2+ binding decreases. 2.3.6 Zn2+ binding to isolated T1D HSA The interaction between Zn2+ and HSA isolated from T1D plasma was measured using the same binding characteristics and conditions as previously described. The glycation percentage of the T1D HSA was determined to be 24%, a glycation percentage that is between the nHSA and gHSA samples isolated from plasma in Figure 2.8 and is Figure 2.12 Binding curve between Zn2+ and T1D HSA isolated from plasma. Linear regression software was used to plot the free Zn2+ from the bound Zn2+. This software was also used to compute an equilibrium dissociation constant of 3.3 ± 0.5 x 10-7 M and binding stoichiometry (Bmax = 15 ± 0.6 µM) (n=3-4, error=SD). 51 one of the glycation percentages depicted in Figure 2.11. In Figure 2.12, a full saturation binding curve is depicted. Similar to both the nHSA and gHSA samples in Figure 2.10, the binding curve saturated at 14.5 ± 0.6 µM (n = 1.03 ± 0.04), which indicates binding of one Zn2+ ion to one HSA molecule. However, with T1D HSA, there was approximately a 1.5-fold decrease (Kd = 3.3 ± 0.5 x 10-7 M) in Zn2+ binding affinity to HSA when compared to the nHSA sample and a 1.5-fold increase in Zn2+ binding affinity to HSA when compared to the gHSA sample in Figure 2.10b. Interestingly, the T1D HSA binding affinity falls in between nHSA and gHSA samples, in addition to showing similar binding characteristics as the 24% gHSA sample created using boronate affinity chromatography in Figure 2.11. These results confirm that physiologically relevant glycation percentages seen in T1D HSA also show decreased binding capabilities to Zn2+. 2.3.7 C-peptide binding to HSA with fatty acids The interaction between C-peptide and HSA with increased fatty acids was determined utilizing similar methodologies as described previously with gHSA samples, corresponding to utilization of ultrafiltration devices and quantification through C-peptide ELISAs. Samples containing either PA or MA represent a 1.4-fold increase in fatty acid concentrations, which are ratios that are commonly seen in vivo. In Figure 2.13a, the binding of C-peptide to HSA with and without PA is depicted. These samples had statistically equal amounts of C-peptide binding with (3.4 ± 0.7 nM free) and without PA (3.4 ± 0.2 nM free), indicating that PA does not interfere in C-peptide binding to HSA. In Figure 2.13b, the association between C-peptide and HSA with or without MA was measured. In samples that contained MA, there was a significant increase in the concentration of free C-peptide with MA (3.9 ± 0.3 nM) when compared to without (2.9 ± 52 0.2 nM). These results indicate that C-peptide binding to HSA is inhibited when MA is present. Figure 2.13 C-peptide binding to HSA with and without PA and MA. a) There was no statistical difference in C-peptide binding to HSA with (3.4 ± 0.2 nM free) and without (3.4 ± 0.2 nM free) PA in solution, indicating that C-peptide binding to HSA is not influenced by PA. (n=3, error=SD) b) There is a statistical increase in free C-peptide with MA (3.9 ± 0.3 nM) compared to without MA (2.9 ± 0.2 nM). This indicates that MA interferes in C-peptide binding to HSA. Samples containing PA or MA represent a 1.4x increase in fatty acid concentration compared to control conditions, a ratio that is commonly found in diabetes (n=4, error=SD, *p<0.05). 2.3.8 Zn2+ binding to HSA with fatty acids To determine if MA also decreases Zn2+ binding to HSA, the association between Zn2+ and HSA with and without MA was measured, equating to concentrations used in the C-peptide and fatty acid binding experiments (i.e. 106 µM). In Figure 2.14, there was no statistical difference in Zn2+ binding to HSA with samples containing MA (0.55 ± 0.05 nM) and samples without MA (0.5 ± 0.3 nM), indicating that Zn2+ binding to HSA was not altered with MA in solution. PA was not utilized in these experiments due to previous experiments indicating that C-peptide binding to HSA is not affected with PA. 53 Figure 2.14 Zn2+ binding to HSA with and without MA. There was no statistical difference in Zn2+ binding to HSA with (0.55 ± 0.05 nM) and without MA (0.5 ± 0.3 nM). These results indicate that Zn2+ binding to HSA is not influenced by MA. Samples containing MA represent a 1.4x increase in fatty acid concentration compared to control conditions, a ratio that is commonly found in diabetes (n=3, error=SD). 2.4 Discussion The decreased ligand binding abilities of glycated albumin in T1D and its corresponding health consequences in disease states have been explored for decades.10,15,44 Further assessing glycated albumin as it pertains to increased free Zn2+ and C-peptide in circulation is integral for understanding microvascular complication progression associated with T1D and additional diseases that are associated with altered Zn2+ homeostasis. 54 Various methodologies can explore the binding of proteins and ligands, such as equilibrium dialysis, surface plasmon resonance, and isothermal titration calorimetry. Ultrafiltration was chosen for these experiments due to its rapid, reproducible, inexpensive and high-throughput applications for determining the interaction between proteins and ligands.40,45–47 Although commercially available, ultrafiltration devices have a limited selection of pore sizes and may contain contaminants (i.e. glycerol). Therefore, novel 3D-printed technologies were utilized to custom fabricate ultrafiltration devices to specifically fit the parameters of these experiments to measure the binding of HSA to Zn2+ and C-peptide. Customizability of these devices allows for integration of membranes with virtually any pore size. This allows for measurement of numerous interactions with proteins and ligands that have differing molecular weights. Aside from protein-ligand interactions, contingent upon a significant difference in the molecular weights between the molecules of interest, essentially any interaction between biomolecules could be measured using these technologies. Another key feature of this ultrafiltration device is the ability to fabricate 20 devices in less than 90 minutes, permitting these devices to be printed the day of experimentation. In addition, due to the small-scale size of the ultrafiltration device, 24 of these devices can be centrifuged at a time, allowing construction of numerous binding curves in one run time. There are also additional benefits to this device in respect to cost (<20% of commercial devices), low volume requirements (200 µL) and the ability to measure binding constants and affinities that are statistically equal to literature values.43 After fabrication, these devices were used to measure the interaction between 55 Zn2+, C-peptide and HSA in glycated and increased fatty acid conditions. To determine physiologically relevant glycated albumin percentages for subsequent binding studies, HSA was isolated from plasma of healthy, control individuals and individuals with diabetes. The glycation percentages of HSA isolated from plasma corresponded to reported values in the literature,8,48 and therefore, these percentages were utilized in subsequent studies. Previously, the interaction between C-peptide and gHSA was measured.30 Results indicate that there was no statistical difference in the binding abilities of nHSA and gHSA to C-peptide,30 and therefore, it was not further explored here. The binding of nHSA and gHSA to Zn2+ was determined using nHSA and enriched gHSA that were isolated using boronate affinity chromatography. Shown in Figure 2.10b, there was a significant decrease in binding of enriched gHSA at 66% glycation, and therefore, lower glycation percentages were explored to determine if lower glycation HSA percentages also resulted in decreased Zn2+ binding. It was determined that there is a steady decrease in Zn2+ binding after 15% glycation, with a significant decrease at 24% glycation, as shown in Figure 2.11. To better understand the physiological relevance of these findings, Zn2+ binding to T1D HSA (24% glycated) that was isolated from plasma, and therefore glycated in vivo, was measured in Figure 2.12. It is important to note that this glycation percentage was statistically equal to the glycation percentages measured in gHSA isolated from diabetic plasma (Figure 2.8) and the glycation percentage where Zn2+ binding was significantly decreased (Figure 2.11). In comparison with nHSA at 12%, in Figure 2.10a, the equilibrium binding constant of the T1D HSA was statistically higher, equating to a weaker 56 binding affinity. Collectively, these results indicate that as glycation of HSA increases, Zn2+ binding decreased. These findings may have implications in C-peptide’s therapeutic effects, as well as the pathophysiological consequences of increased free Zn2+ in circulation. To further determine the effect of diabetic HSA conditions on C-peptide and Zn2+ delivery, increased fatty acids were utilized. Both PA and MA are saturated fatty acids and were chosen due to their upregulation in T1D.49 Generally, concentrations of fatty acids in diabetes are 2-5x more concentrated than what is seen in healthy controls,24,50 therefore, concentrations to mimic these conditions were utilized. There was no decrease in C-peptide binding to HSA when PA was in solution, however, there was a decrease in C-peptide binding to HSA when MA was in solution (Figure 2.13). Therefore, MA was used in subsequent experiments to measure Zn2+ binding to HSA. Interestingly, Zn2+ binding to HSA was statistically equal whether or not MA was in solution (Figure 2.14), indicating that MA only inhibited C-peptide binding to HSA and not Zn2+ binding to HSA. Numerous diseases are associated with altered Zn2+ homeostasis. For example, increased levels of Zn2+ have been measured in the cerebrospinal fluid in patients with Alzheimer’s disease (AD), leading to pathological consequences.51,52 Increased levels of unbound Zn2+ have also been reported to contribute to the formation of plaques in the brain and has been shown to cause neurotoxicity.53,54 Commonly, AD is referred to as type 3 diabetes because of its similarities in respect to increased levels of glycated proteins. Glycated albumin’s inability to bind to Zn2+ may explain the overall pathophysiology of AD and aid in therapeutic development to inhibit its progression. In addition to AD, increased free Zn2+ levels have also been attributed to 57 atherothrombosis,27,55 which is one of the main causes of death in people with T1D. In this mechanism, free Zn2+ is able to bind to histidine-rich glycoproteins (HRG), which indirectly causes the formation of blood clots.27 Finally, Zn2+ binding to HSA is required for C-peptide stimulated ATP release from RBCs,29 and alterations in this association could alter downstream therapeutic effects of C-peptide in respect to improvements in microvascular complications. Without C-peptide stimulated ATP release, this may result in a decrease in NO release from endothelial cells, and therefore inhibition of NO’s vasodilating effects. Vasodilation is essential for normal blood flow through the vasculature, and therefore, inhibition of his cascade through decreased Zn2+ binding may have implications in microvascular problems seen in T1D. Overall, these findings may explain the pathophysiological role of glycated albumin and in the progression of T1D, AD, and other diseases associated with altered Zn2+ homeostasis. Increased concentrations of free C-peptide with increased MA indicate a potential problem in C-peptide therapy for people with T1D. As mentioned previously, there are increased levels of fatty acids in T1D and most of these fatty acids in circulation are bound to HSA.23 Zn2+, C-peptide and HSA are necessary for the therapeutic release of ATP from RBCs for regulation of blood flow,29 however, if C-peptide is unable to bind to HSA in individuals with T1D, no effect of C-peptide will be measured. There have been human C-peptide clinical trials, however, all have failed, having no effect on diabetic complications in T1D when compared to healthy controls.56–58 The findings corresponding to decreased C-peptide binding to HSA with increased MA explain a potential rationale as to why the results in human clinical trials had unexpected outcomes. In addition, these 58 results provide a platform for new C-peptide therapy research, in discovering how C-peptide can bind to HSA to alleviate microvascular complications in T1D. 59 REFERENCES (1) Forbes, J. M.; Cooper, M. E. Mechanisms of Diabetic Complications. Physiol. Rev. 2013, 93 (1), 137–188. https://doi.org/10.1152/physrev.00045.2011. (2) Kim, K. J.; Lee, B. W. The Roles of Glycated Albumin as Intermediate Glycation Index and Pathogenic Protein. Diabetes Metab. J. 2012, 36 (2), 98–107. https://doi.org/10.4093/dmj.2012.36.2.98. (3) CDC. Type 1 Diabetes. (4) Freeman, V. S. Glucose and Hemoglobin A1c. Lab Med. 2014, 45 (1), e21–e24. https://doi.org/10.1309/LMNSU432YJWCWZKX. (5) Ciobanu, D. M.; Bogdan, F.; Pătruț, C. I.; Roman, G. Glycated Albumin Is Correlated with Glycated Hemoglobin in Type 2 Diabetes. Med. Pharm. Reports 2019, 92 (2), 134–138. https://doi.org/10.15386/mpr-1247. (6) Eames, J.; Rupert, J. How Accurate Is HbA1c in Diagnosing Diabetes in Children. 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Diabetes 2003, 52 (2), 536–541. https://doi.org/10.2337/diabetes.52.2.536. 65 Chapter 3: Measuring Zn2+ and C-peptide binding and its downstream physiological effects on RBCs with glycation and fatty acids present 3.1 Introduction As discussed in chapter 2, the binding of the albumin, zinc (Zn2+) and C-peptide complex was discussed in association with increased glycation of albumin and fatty acid environments. The binding of normally glycated human serum albumin (nHSA) and glycated human serum albumin (gHSA) to Zn2+ was explored, indicating that Zn2+ binding was decreased in the presence of gHSA.1 Previous Spence lab members measured the association between C-peptide and gHSA and found that the binding of C-peptide to gHSA was not decreased when compared to nHSA.2 Further investigation regarding the binding of the albumin, Zn2+ and C-peptide complex to the red blood cell (RBC) in conditions that mimic diabetic conditions (i.e. increased glycation and fatty acids) may facilitate the development of a C-peptide based therapy for type 1 diabetes (T1D). In T1D, glycation occurs due to increased glucose levels, commonly ranging between 7-11 mM.3 These hyperglycemic levels can result in glycation to proteins through a non-enzymatic reaction called the Maillard reaction (Figure 2.1).4,5 Albumin is a protein that maintains osmotic pressure in vessels, pH in the bloodstream, and importantly for studies in this chapter, acts as a binding and transporting molecule throughout the body.6– 9 Glycation of albumin has been reported to alter the structure of the protein, therefore, modifying the overall function.10–12 Modifications in albumin’s structure and function has been reported to lead to many complications, such as nephropathy, neuropathy, retinopathy, and vascular disease.13 In the literature, glycated albumin has been shown to have decreased ligand binding capabilities10–12 which may have implications in 66 C-peptide’s transport and delivery to cells. In addition to nHSA and gHSA binding to Zn2+, the previous chapter also showed the association between Zn2+, C-peptide and HSA in increased fatty acid environments. Individuals with T1D have been reported to have a 3.5-fold increase in fatty acid concentration when compared to healthy, controls.14,15 This is commonly due to irregular insulin concentrations in vivo, therefore, the body begins to break down adipose tissue for energy metabolism in the form of fatty acids.15–17 Albumin contains seven fatty acid binding sites, and when occupied, may have implications in C-peptide’s transport.18,19 This measurement is especially important for C-peptide binding due to the similarities in structure of fatty acid carboxylic acid head groups and a glutamic acid residue on C-peptide (Figure 3.1). Although C-peptide contains four glutamic acid residues, the glutamic acid found near the C-terminus at position 27 has been shown to play a vital role 20–22 in its bioactivity. Specifically, when this glutamic acid was changed to an alanine, C-peptide’s binding and transport was shown to decrease.23 C-peptide binding to HSA Figure 3.1 Structure of carboxylic acid and glutamic acid. In (a), carboxylic acid is depicted, containing a carbonyl group and a hydroxyl group in fatty acids. In (b), glutamic acid is depicted, containing a carboxylic acid residue at both ends of the molecule in C-peptide. At physiological pH, the alpha carboxylic group to the right on glutamic acid is deprotonated, leaving an overall net negative charge. 67 with palmitic acid (PA) and myristic acid (MA) was shown in Chapter 2, however, only MA decreased C-peptide binding to HSA. Therefore, MA was utilized for subsequent HSA and Zn2+ binding experiments, where Zn2+ binding to HSA was statistically equal with and without the addition of MA. A specific binding site for C-peptide on the red blood cell (RBC) has yet to be reported, however, previous efforts to isolate this receptor have involved a C-peptide receptor alone. Recent reports suggest that this receptor may be an albumin, C-peptide and Zn2+ complex receptor,23 rather than just a C-peptide receptor alone. Geiger, et al. 99m radiolabeled bovine serum albumin (BSA) with Tc and measured the association of BSA to the RBC without C-peptide and Zn2+ and with C-peptide and Zn2+ (Figure 3.2).24 In control conditions, results indicated that BSA specifically bound the RBC, saturating at 14,021 ± 1489 BSA molecules per RBC, with an equilibrium dissociation constant of 1.14 Figure 3.2 BSA binding to RBCs with and without C-peptide and Zn2+. In (a), the specific binding curves of BSA to RBCs are shown with the black curve representing BSA binding without C-peptide and Zn2+ and the white curve representing BSA binding with C-peptide and Zn2+. (n³4, error=SEM, *p<0.05). In (b), the average increase in BSA binding without C-peptide and Zn2+ (grey) and with C-peptide and Zn2+ (dark gray), depicting a specific binding site for BSA on the RBC and a specific binding site for BSA, C-peptide and Zn2+ on the RBC (n=5, error=SEM). 68 ± 0.07 x 10-7 M and a Bmax of 1.94 ± 0.02 x 10-8. These values equate to approximately 13,900 receptor molecules for BSA on the RBC without the addition of C-peptide and Zn2+.24 In samples containing C-peptide and Zn2+, specific binding saturated at 16,695 ± 1,479 BSA molecules per RBC with an equilibrium dissociation constant of 2.00 ± 0.05 x 10-7 M and a Bmax value of 2.50 ± 0.01 x 10-8.24 These values represent 17,900 receptor molecules for 99mTc-BSA on the RBC with the addition of C-peptide and Zn2+.24 In Figure 3.2b, the average increase of 1606 ± 492 BSA molecules per RBC with the addition of C-peptide and Zn2+ indicate both a specific binding site for BSA on the RBC alone, as well as a specific binding site for a BSA, C-peptide, Zn2+ complex. Our group has shown that healthy albumin, with low glycation and fatty acid levels, is essential for binding and transport of Zn2+ and C-peptide to the red blood cell (RBC), for increased ATP release,23 and thus, downstream NO release from endothelial cells (Figure 1.3). Without proper binding and transport of C-peptide, its downstream therapeutic effect of increasing ATP release from RBCs will not ensue, which may result in complications associated with altered blood flow in T1D.25,26 Measuring the association between albumin, C-peptide and Zn2+ to the RBC in glycated and fatty acid conditions is integral to understand C-peptide as a therapeutic, in addition to opening an unpresented area of study, investigating possible homeostatic disruptions corresponding to albumin modifications in T1D. In Figure 3.3, nBSA (11% glycated) and enriched gBSA (48% glycated) were radiolabeled with technetium (99mTc) to determine specific biding to the RBC in the presence of C-peptide and Zn2+. Results indicate that nBSA binding to the RBC saturated at 15,222 ± 627 nBSA molecules per RBC, showing 18,500 receptor molecules per RBC. 69 Figure 3.3 Specific binding of nBSA and gBSA to the RBC. Depicted are two specific binding curves of nBSA (11% glycated, triangles) and enriched gBSA (48% glycated; circles) binding to the RBCs in the presence of C-peptide and Zn2+ (n³4, error=SEM, *p<0.05). The gBSA saturated at 17,011 ± 732 BSA molecules per RBC, showing 19,500 receptor molecules per RBC. These results indicate that average gBSA specific binding saturated higher than nBSA, or that more gBSA molecules bound to the RBC than nBSA. These findings indicate the need to explore C-peptide and Zn2+ binding to the RBC in glycated conditions to better understand if gBSA delivers C-peptide and Zn2+ to the RBC in a similar manner as nBSA. If gBSA is unable to deliver C-peptide and Zn2+ to the RBC as seen with nBSA, the observed increased in gBSA binding to RBCs may not hold importance in C-peptide’s overall effect on RBCs in T1D. Findings in this chapter will increase our understanding of how C-peptide, Zn2+ and BSA bind to RBCs in individuals with T1D for expertise in how to utilize this therapy in a clinical setting. In the previous chapter, HSA was used for all studies to measure the association 70 between Zn2+, C-peptide and albumin, however in this chapter, BSA is utilized in its place. The structure of HSA and BSA consists of one tryptophan (Trp) difference in amino acid sequence.27,28 In HSA, there is one Trp 214 located in the first hydrophobic binding pocket, whereas BSA contains Trp 134 on the surface and Trp 212 in the hydrophobic binding pocket of domain 1.27 However, both Trp 214 in HSA and Trp 212 in BSA are in similar locations to one another.27 There is also a difference in valine (Val) amino acid residues in HSA compared to BSA, where HSA contains 45 Val residues and BSA contains 35.27 However, neither Trp or Val differences in HSA and BSA result in a difference in fluorescent signaling.28 Figure 3.4 C-peptide uptake by RBCs with BSA and HSA. C-peptide uptake by RBCs was statistically equal in BSA and HSA, indicating no difference in C-peptide binding (n=3, error=SEM, p=0.08). 71 Previous unpublished research by the Spence lab has indicated that both HSA and BSA are able to carry C-peptide to the RBC in a statistically the same manor. In Figure 3.4, when C-peptide was carried by HSA this resulted in 1.36 ± 0.10 nM C-peptide uptake by RBCs, whereas when C-peptide was carried by BSA, this resulted in 1.75 ± 0.13 nM C-peptide uptake by RBCs. These results indicate statistically equal binding characteristics to one another, with a p value of 0.08. Therefore, BSA is used in subsequent studies due to the cost difference of BSA compared to HSA. 3.2 Methods 3.2.1 RBC collection and purification Whole blood was collected via venipuncture into heparinized tubes from consenting donors. Whole blood was centrifuged at 500 g for 10 minutes. The plasma and buffy coat were subsequently removed via aspiration, and the packed RBCs were placed into a 15 mL polypropylene tube. The RBCs were then washed by adding physiological salt solution (PSS, 4.7 mM KCl, 2.0 mM CaCl2,140.5 mM sodium chloride, 12.0 mM MgSO4, 21.0 mM tris (hydroxymethyl) aminomethane, 5.5 mM dextrose and 0.5% BSA, pH at 7.40) and then placing the tube into a centrifuge at 500 g for 10 minutes. The buffer was aspirated from the packed RBCs, and this washing process was repeated twice for a total of three washing steps. The hematocrit of the RBC solution was determined via a StatSpin MP microhematocrit centrifuge (Beckman Coulter, Brea, CA) and a hematocrit reader (StatSpin CritSpin, Beckman Coulter). 3.2.2 Isolation of nBSA and gBSA using boronate affinity chromatography This method is similar to what is shown in section 2.2.3, however, BSA was used in place of HSA. Stock solutions of nBSA and enriched gBSA were isolated, lyophilized 72 and stored at -20°C until further experimentation. 3.2.3 Mass spectrometry analysis of BSA samples This method is similar to what is shown in section 2.2.4, however, BSA was analyzed in place of HSA. Glycation percentage was determined for each BSA fraction and other BSA adducts were accounted for such as potassium (K = 39 Da) and cysteine (C = 119 Da). 3.2.4 Preparation of myristic acid solution A 500 mL solution of 150 mM NaCl was prepared to dissolve MA (Sigma Aldrich, Burlington, MA). 100 mL of the 150 mM NaCl buffer were added to a pre-warmed beaker containing a thermometer and placed on a stir plate ensuring the temperature was at 37°C. 2.267 g of BSA was added to the stirring NaCl buffer and until completely dissolved. The BSA/NaCl solution was sterile filtered using a vacuum filter system, and 50 mL of this solution were diluted with 150 mM NaCl to make a 0.17 mM BSA/NaCl stock control. The BSA/NaCl stock control pH was adjusted to 7.4 and placed into glass vials in 4 mL aliquots. The other 50 mL fraction of the BSA/NaCl solution was transferred to a 250 mL beaker prewarmed in a water bath, stirring. While the BSA/NaCl was stirring, 30.7 mg of MA was added to 44 mL of 150 mM NaCl in a 50 mL Erlenmeyer flask. This flask was covered with parafilm, placed in a water bath, stirred and heated to 70°C; MA typically dissolving between 50°C and 60°C. After the MA dissolved and thermometer reached 70°C, 40 mL of the MA solution was added to the stirring 50 mL BSA/NaCl solution, in 5 mL aliquots. The final volume was adjusted to 100 mL for a final concentration of 1,239 µM MA stock. The MA stock was adjusted to pH 7.4 and aliquoted into 4 mL stock 73 samples in glass vials. The MA stock samples and the BSA/NaCl control samples were stored at -20°C. 3.2.5 Preparation of BSA stocks BSA stocks of different glycation percentages were prepared from the nBSA and enriched gBSA fractions isolated through boronate affinity chromatography using phosphate buffered saline (PBS; 10.1 mM Na2HPO4 (Sigma Aldrich), 2.7 mM KCl, 136.9 mM NaCl, 1.8 mM KH2PO4 (Sigma Aldrich) at pH 7.40). Different ratios of nBSA and gBSA were used to prepare solutions containing 13-15% gBSA, 17-18% gBSA, 22-16% gBSA and 49-56% gBSA stock at 1 mg/µL. Determination of the BSA stock concentration was described in section 2.2.6, however, BSA was utilized instead of HSA. 3.2.6 Sample preparation for C-peptide binding and analysis A C-peptide and Zn2+ stock was diluted to 800 nM working solutions with 18 MW distilled deionized water (DDI H2O). All stock solutions of 13-15% gBSA, 17-18% gBSA, 22-16% gBSA and 49-56% gBSA were used to create 20 µM working solutions of each glycation percentage in albumin free (AF)-PSS (PSS without the addition of 0.5% BSA). Samples were prepared to contain 20 nM Zn2+, 20 nM C-peptide, or DDI H2O, BSA with various glycation percentages and 7% RBCs. Standards were prepared with C-peptide in concentrations ranging from 4.8-25.6 nM. These samples and standards were incubated at 37°C for 2 hours, inverting each sample at 1 hour. After incubation, the samples were centrifuged at 500 g for 5 minutes, and the supernatant was removed. The standards and supernatant were diluted 1:50 in DDI H2O. The concentrations of diluted standards and supernatant were determined by a human C-peptide enzyme linked immunosorbent assay (ELISA; ALPCO, Salem, NJ). Standards were used to determine the concentration 74 of free C-peptide in the supernatant, which was used to determine the amount of C-peptide bound to the RBCs by subtracting the free C-peptide from the amount of C-peptide added to each sample. 3.2.7 Sample preparation for Zn2+ binding and analysis An 800 nM C-peptide stock was created as described in section 3.2.6. 65 Radioisotopic ZnCl2 (Perkin Elmer, Waltham, MA) was diluted in DDI H2O to create an 800 nM working solution. Stock solutions of various BSA glycation percentages were created as described in section 3.2.5. BSA glycation samples were prepared to contain 20 nM 65Zn2+, 20 nM C-peptide, or DDI H2O, BSA with various glycation percentages and 65 7% RBCs. Standards were prepared with Zn2+ in concentrations ranging from 1.25-40 nM. Samples and standards were incubated at 37°C for 2 hours on an orbital shaker (260 rpm, Talboys Professional, Thorofare, NJ). After incubation, the samples were centrifuged at 500 g for 5 minutes, and the supernatant was removed. The standards and packed RBCs were analyzed using a 2480 WIZARD2 automatic gamma counter (PerkinElmer, 65 Waltham, MA), utilizing a 5-minute protocol specific to Zn2+. The gamma counter recorded counts per minute (cpm), which were compared to the known concentrations of the standards to create a standard curve. The individual samples cpm values were analyzed and used to compute concentration of 65Zn2+ on RBCs. 3.2.8 Sample preparation for C-peptide stimulated ATP release Stock solutions and samples with increased glycation were created and incubated as described in section 3.2.6. Stock solutions and samples with increased fatty acids were created by thawing stock solutions of MA and BSA:NaCl control 37°C. The 1,239 µM MA stock was used to create 31 µM, 56 µM, 81 µM and 106 µM MA stocks in PSS. The 75 BSA:NaCl stock control was utilized to create a control sample stock solution without MA, utilizing equal ratios of BSA:NaCl stock to PSS as used in 106 µM MA stock. Fatty acid samples were prepared to contain 20 nM Zn2+, 20 nM C-peptide, or DDI H2O controls, PSS with various fatty acid concentrations and 7% RBCs. Following a 2 hour incubation, each sample was placed into a 1 mL syringe. This syringe was secured to a 250 µm inner diameter capillary and placed on a syringe pump with a flow rate of 4,000 µL/hr to simulate blood flow through a capillary. Each sample was collected into a 1.7 mL tube and centrifuged at 500 g for 5 minutes to remove and collect the supernatant. ATP standards were created ranging in the concentration of 20-640 nM. Luciferin luciferase was created by dissolving 5 mg of potassium luciferin (Gold Biotechnology Inc., Olivet, MO) in 5 mL of DDI H2O. 5 mL of the potassium luciferin were utilized to further dissolve 100 mg firefly lantern extract (Sigma Aldrich, Burlington, MA), to create a luciferin luciferase solution. The luciferin luciferase was placed into a 15 mL tube, stored at 4°C, and used for up to 3 weeks. Standards and supernatant in volumes of 100 µL were placed into a 96 well black bottom plate with 50 µL of luciferin luciferase and a timer was started. When the timer reached 20 seconds, chemiluminescence was measured on a FlexStation-3 spectrophotometer (Molecular Devices, San Jose, CA). The standards were used to create a calibration curve and determine the concentration of each sample. 3.3 Results 3.3.1 Mass spectrometry analysis of normal BSA and glycated BSA Time of flight mass spectrometry was utilized to determine the percent glycation of each BSA sample. Figure 3.5 shows a nBSA and enriched gBSA sample isolated using 76 boronate affinity chromatography. The main mass peak is labeled as BSA and all additional peaks are adducts onto the main BSA peak. Three additional adducts, potassium (K = +39 Da), cysteine (C = +119 Da) and glucose (G = +162 Da) are shown. The total peak ion counts from additional glucose adducts were summed and divided by the total ion counts to determine the percent glycation for each BSA sample. The average glycation was approximately 14% for the nBSA sample (a) and 64% for the enriched gBSA sample (b). Figure 3.5 Mass spectra of nBSA and enriched gBSA isolated using boronate affinity chromatography. Electrospray ionization time of flight mass spectrometry was used to analyzed nBSA (a) and enriched gBSA (b). Depicted, BSA is combined with glucose (G = +162 Da), cysteine (C = +119 Da) and potassium (K = +39 Da). The nBSA sample is approximately 14% glycated and the enriched gBSA sample is approximately 64% glycated. 3.3.2 C-peptide binding to RBCs with normal and glycated BSA To determine the effect of C-peptide binding to the RBC with albumin glycation present, samples were prepared to contain C-peptide, Zn2+ and differing glycation percentages of BSA. The 13% gBSA, depicting glycation levels in non-diabetic controls, delivered 1.2 ± 0.2 nM C-peptide molecules to the RBC (Figure 3.6). The 17% gBSA sample delivered a statistically equal amount of C-peptide to the RBC as the 13% gBSA, 77 Figure 3.6 C-peptide uptake by RBCs with different percent glycated BSA samples. 13% gBSA and 17% gBSA samples had statistically equal amount of C-peptide binding when compared to one another. There was a significant decrease in C-peptide binding when 22% gBSA and 50% gBSA were present compared to both 13% gBSA and 17% gBSA samples (n=4-6, error=SEM, *p<0.05 to 13% gBSA, **p<0.05 to 17% gBSA). with values equating to 1.2 ± 0.1 nM C-peptide molecules bound to the RBC. The 22% gBSA (0.8 ± 0.1 nM bound) and 50% gBSA (0.5 ± 0.1 nM bound) samples resulted in a significant decrease in C-peptide binding to the RBC compared to both the 13% gBSA and 17% gBSA samples. These results indicate that as glycation of albumin approaches 22% and above, C-peptide binding to the RBC is decreased. 78 3.3.3 Zn2+ binding to RBCs with normal and glycated BSA The association between Zn2+ and the RBC was measured with increasing glycation percentages of BSA to determine if the glycation of BSA affects Zn2+ binding to the RBC. Zn2+ samples were prepared to contain C-peptide, radioactive 65 Zn2+, differing glycation percentages of BSA and 7% RBCs. Results indicate that Zn2+ binding decreases as glycation of BSA increases. In Figure 3.7, samples representing control BSA glycation conditions (14% glycated), resulted in 3.09 ± 0.06 nM Zn2+ ions bound to Figure 3.7 Zn2+ binding to RBCs with different percent glycated BSA samples. Samples signifying control conditions at 14% gBSA had a significant increase in Zn2+ binding compared to other glycation percentages. Samples containing 18% and 23% gBSA has statistically equal Zn2+ binding in comparison to one another. 49% gBSA samples had a 38% decrease in Zn2+ binding to the RBC when compared to 14% gBSA control conditions (n=4, error=SEM, *p<0.05 to 14% gBSA, **p<0.05 to 18% and 23% gBSA). 79 the RBC. There was a statistical decrease in Zn2+ binding to the RBC when compared to control conditions with 18% gBSA (2.6 ± 0.1 nM bound), 23% gBSA (2.38 ± 0.04 nM bound) and 49% (1.9 ± 0.1 nM bound) gBSA. In addition to decreases in Zn2+ binding compared to control conditions, there was also a statistical decrease in Zn2+ binding to the RBC for the 49% gBSA sample when compared to the 18% gBSA sample. Collectively, the data shows that as glycation of BSA increases to 18% and above, Zn2+ binding to the RBC decreases. 3.3.4 C-peptide stimulated ATP release from RBCs with normal and glycated BSA To determine the downstream physiological effect of decreased C-peptide and Zn2+ binding as glycation of albumin increases, ATP release from RBCs was measured with samples containing C-peptide, Zn2+, various glycation percentages of BSA and 7% RBCs. RBCs were subject to flow conditions within a capillary to better mimic blood flow through the vasculature in vivo, and chemiluminescence was detected by a spectrophotometer. In Figure 3.8, samples representing control conditions (15% gBSA) showed a significant increase in ATP release when C-peptide and Zn2+ (101 ± 13 nM) were added to the samples compared to control conditions (47 ± 3 nM). In 18% gBSA samples, there was a significant 37% increase in ATP release in samples with C-peptide and Zn2+ (59 ± 2 nM) compared to control conditions (43 ± 2 nM), however, the increase in ATP was lower than what was shown in 15% gBSA samples. In the 26% gBSA sample, there was a statistically similar amount of ATP release with C-peptide and Zn2+ (35 ± 9 nM) and without (31 ± 7 nM), indicating no effect of C-peptide and Zn2+ at this glycation percentage. Similarly, in the 56% gBSA samples with C-peptide and Zn2+ (35 ± 6 nM) and without (38 ± 1 nM), there was a statistically equal amount of ATP release from RBCs. 80 These results indicate that as glycation percentages reach 26% and higher, there is no significant increase in ATP release from RBCs with C-peptide and Zn2+, therefore no downstream physiological effect of C-peptide. Figure 3.8 ATP release from RBCs with different percent glycated BSA samples with and without C-peptide and Zn2+. ATP release was significantly higher in 15% gBSA samples with C-peptide and Zn2+ compared to without. There is a statistical increase in ATP from 18% gBSA samples with C-peptide and Zn2+ compared to 18% gBSA control. There is no statistical increase in ATP for 26% and 56% gBSA samples with or without C-peptide and Zn2+ (n=3-6, error=SEM, *p<0.05 to all gBSA samples, **p<0.05 to all samples except 15% gBSA). 3.3.5 C-peptide stimulated ATP release from RBCs with MA To determine if MA interferes in C-peptide stimulated ATP release from RBCs, samples were prepared with C-peptide, Zn2+ and BSA with increasing concentrations of MA. All MA concentrations utilized in these experiments are BSA to fatty acid ratios 81 Figure 3.9 ATP release from RBCs with different concentrations of MA with and without C-peptide and Zn2+. There was a significant increase in ATP release from RBCs when C-peptide and Zn2+ were present for control conditions. All samples containing MA (31-106 µM) showed a significant decrease in ATP release when compared to the 0 µM C-peptide and Zn2+ sample. All samples containing MA were statistically equal to the control sample without C-peptide and Zn2+ (n=3, error=SEM, *p<0.05 to all bars). commonly reported in vivo.29 RBCs were subject to flow conditions and measured as described in section 3.2.8. In Figure 3.9, results indicate that in control conditions, samples with C-peptide and Zn2+ resulted in a statistically significant increase in ATP release (91 ± 19 nM) from RBCs compared to samples without C-peptide and Zn2+ (29 ± 10 nM). There was a statistically significant decrease in ATP release from RBCs with C-peptide and Zn2+ at 31 µM MA (46 ± 14 nM), 56 µM MA (35 ± 7 nM), 81 µM MA (36 ± 82 10 nM), and 106 µM MA (28 ± 11 nM) when compared to control samples with C-peptide and Zn2+. All samples containing MA (31-106 µM) were statistically similar to control samples without C-peptide and Zn2+. Results indicate that samples containing MA significantly decreased ATP release from RBCs, demonstrating that C-peptide stimulated ATP release from RBCs is decreased when MA is present. 3.4 Discussion Previous reports have indicated that albumin is a key molecule for C-peptide and Zn2+ transport to the RBC to increase ATP release and improve blood flow.30 In Chapter 2, the effect of increased glycation and fatty acids on Zn2+ and C-peptide binding to HSA was measured, indicating decreased Zn2+ binding to gHSA and decreased C-peptide binding to HSA with increased MA. In addition, previous results indicate that the gBSA specific binding curve saturated higher than nBSA, indicating that there are more receptors for gBSA compared to nBSA (Figure 3.3).31 However, the increased receptors may be due to the increased glycoproteins on albumin facilitating binding. Therefore, in this chapter, the binding of the C-peptide, Zn2+ and BSA complex to the RBC in T1D conditions (i.e. increased glycation and fatty acids) was measured to further understand how these conditions may implicate C-peptide’s delivery. According to the literature, glycation of albumin is generally 2-5x that of what is measured in healthy, control individuals.32 Albumin isolated from plasma in Chapter 2 (Figure 2.8) indicated that the average glycation of healthy, controls ranged from 12.1-13.5% and average glycation of individuals with diabetes ranged from 20.8-37.6% glycated. Utilizing boronate affinity chromatography, two fractions of purchased BSA were isolated into healthy, control gBSA, at an average glycation level of 14.0%, and enriched 83 gBSA, at an average glycation level of 51.7%. These two fractions were combined and utilized to create varying glycation percentages of BSA to mimic the conditions shown in previous experiments (Figure 2.8) and in vivo. Specifically, BSA with glycation percentages averaging 14.0% indicated control conditions, BSA with a glycation percentage averaging 17.7% indicated a person with T1D that has well controlled glucose levels, and BSA with a glycation percentage averaging 23.7% indicated a person with T1D that has average glucose control. These fractions were utilized to measure C-peptide binding, Zn2+ binding and ATP release from RBCs. C-peptide binding to the RBC was statistically similar in samples representing healthy controls (13% gBSA) and individuals with T1D that have well controlled glucose levels (17% gBSA), in Figure 3.6. As glycation of BSA approached 22% and higher, C-peptide binding to the RBC significantly decreased. Results indicate that individuals with T1D that have moderately controlled glucose levels (i.e. 22%) are not able to carry C-peptide to the RBC the same as lower glycation percentages of BSA, which may correspond to failed C-peptide clinical trials discussed in Chapter 1 and 2.33 Findings in this Chapter indicate that for C-peptide to provide a therapeutic effect, exogenous non-glycated and fatty acid free albumin may need to be administered concurrently. After C-peptide binding to the RBC was explored, Zn2+ binding to the RBC using similar glycation percentages of BSA was investigated, in Figure 3.7. Results indicated that at 18% gBSA and above, there was a significant decrease in Zn2+ binding to the RBC. These results show that even with well controlled glucose levels, Zn2+ cannot bind to the RBC in a manner similar to what is measured in healthy, control individuals. However, for both C-peptide and Zn2+ binding to RBCs, as glycation levels approached ~23%, binding 84 of both ligands decreased. There are numerous sites on albumin that bind glucose2,11,34 and as glucose molecules bind to albumin, there are less available binding sites for other molecules. Therefore, as glycation approaches moderately controlled glucose levels, albumin may not be able to bind C-peptide and Zn2+ with as high of an affinity, disrupting its transport to the RBC. To determine the physiological effect of decreased C-peptide and Zn2+ binding to the RBC as glycation of BSA increases, ATP release from RBCs was also measured with similar glycation percentages (Figure 3.8) as shown in C-peptide and Zn2+ binding experiments. At control glycation percentages (15% gBSA), there was a 115% increase in ATP release from RBCs with the addition of C-peptide and Zn2+ compared to without. As glycation of BSA increased to 18% gBSA, there was a significant 37% increase in ATP release from RBCs with the addition of C-peptide and Zn2+, however, this increase was much less than what was seen at healthy, control glycation percentages. As glycation percentages approached 26%, there was no statistical increase in ATP with the addition of C-peptide and Zn2+ when compared to samples without C-peptide and Zn2+, indicating that at average glycation percentages seen in T1D, the effect of C-peptide on ATP release from RBCs is altered, which may have implications in the potential therapeutic effect of C-peptide in vivo. In addition to increased glycation, increased levels of fatty acids are also reported in T1D.16,17,29,35 Previous results in Chapter 2 show a decrease in C-peptide binding to HSA when MA is present (Figure 2.13b). Therefore, MA was utilized in this chapter to determine its effect on C-peptide, Zn2+, BSA and the RBC. Concentrations of BSA in vivo are 600 µM6 and concentrations of fatty acids in vivo have been shown to range from 85 250-900 µM,15 and therefore, these ratios of BSA to MA were scaled to albumin concentrations normally utilized in experiments. Results indicate that there is a significant increase in ATP for control conditions, without MA (Figure 3.9). However, as MA concentration increased to 31 µM, there is a significant decrease in ATP release from RBCs. These results indicate that even at lower concentrations of MA, ATP release from RBCs is inhibited. As discussed in Chapter 1, C-peptide is a molecule that has been shown to increase deformability and ATP release from RBCs.21,36,37 Individuals with T1D have little to no circulating levels of C-peptide, which may contribute to the increase in microvascular complications reported in this disease. For decades, researchers have attempted to re-administer C-peptide therapies to improve diabetic complications attributed to poor blood flow. Numerous attempts at C-peptide re-administration into people with T1D involved C-peptide alone.33 However, the Spence lab has shown that a C-peptide, Zn2+ and albumin complex is necessary for the transport and downstream physiologic effect of C-peptide on the RBC.23 Therefore, measuring the transport of C-peptide and Zn2+ to the RBC, in addition to ATP release from RBCs in conditions seen in T1D is integral for the future success of C-peptide therapy. Our group has shown that as albumin glycation increases, more albumin specifically binds to the RBC.31 Results in Chapter 2 indicate that although there is more albumin binding to the RBC, there is less C-peptide and Zn2+ bound to albumin. Furthermore, results in this Chapter confirm these results, indicating that less C-peptide and Zn2+ are bound to RBCs as glycation of albumin increases. Although there is more specific binding of glycated albumin to RBCs,31 there is less C-peptide and Zn2+ bound to 86 albumin, and therefore, less C-peptide and Zn2+ delivered to RBCs. As mentioned previously and in the literature,24,30 C-peptide, Zn2+ and albumin need to be bound to one another and transported to the RBC for C-peptide to elicit its therapeutic effect in increasing ATP release. Therefore, additional steps are necessary for this therapy to have an effect in individuals with T1D. Research in this chapter suggests that re-administration of C-peptide therapy to individuals with T1D may not succeed if the albumin is either glycated or in an environment with increased levels of fatty acids. Rather than re-administering C-peptide to individuals with T1D alone, C-peptide may have to be administered alongside exogenous healthy albumin. Additional in vitro studies may hold importance to determine optimal preparation of this therapeutic. 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Multiple Fatty Acid Binding to Albumin in Human Blood Plasma. Eur. J. Biochem. 1990, 189 (2), 343–349. https://doi.org/10.1111/j.1432-1033.1990.tb15495.x. (19) Ascenzi, P.; di Masi, A.; Fanali, G.; Fasano, M. Heme-Based Catalytic Properties of Human Serum Albumin. Cell Death Discov. 2015, 1 (1), 1–7. https://doi.org/10.1038/cddiscovery.2015.25. (20) Hills, C. E.; Brunskill, N. J. Cellular and Physiological Effects of C-Peptide. Clin. Sci. 2009, 116 (7), 565–574. https://doi.org/10.1042/CS20080441. (21) Hach, T.; Forst, T.; Kunt, T.; Ekberg, K.; Pfützner, A.; Wahren, J. C-Peptide and Its C-Terminal Fragments Improve Erythrocyte Deformability in Type 1 Diabetes Patients. Exp. Diabetes Res. 2008, 2008, 730594. https://doi.org/10.1155/2008/730594. (22) Wahren, J.; Jörnvall, H. C-Peptide Makes a Comeback. Diabetes. Metab. Res. Rev. 2003, 19 (5), 345–347. https://doi.org/10.1002/dmrr.403. (23) 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 89 Diabetes Explored with a 3D-Printed Fluidic Device. Integr. Biol. 2015, 7, 534–543. https://doi.org/10.1039/C4IB00243A. (24) Geiger, M.; Janes, T.; Keshavarz, H.; Summers, S.; Pinger, C.; Fletcher, D.; Zinn, K.; Tennakoon, M.; Karunarathne, A.; Spence, D. A C-Peptide Complex with Albumin and Zn2+ Increases Measurable GLUT1 Levels in Membranes of Human Red Blood Cells. Sci. Rep. 2020, 10 (1), 1–9. https://doi.org/10.1038/s41598-020- 74527-6. (25) Johansson, B. L.; Linde, B.; Wahren, J. Effects of C-Peptide on Blood Flow, Capillary Diffusion Capacity and Glucose Utilization in the Exercising Forearm of Type 1 (Insulin-Dependent) Diabetic Patients. Diabetologia 1992, 35 (12), 1151– 1158. https://doi.org/10.1007/BF00401369. (26) Leighton, E.; Sainsbury, C. A.; Jones, G. C. A Practical Review of C-Peptide Testing in Diabetes. Diabetes Ther. 2017, 8 (3), 475–487. https://doi.org/10.1007/s13300- 017-0265-4. (27) Belatik, A.; Hotchandani, S.; Carpentier, R.; Tajmir-Riahi, H. A. Locating the Binding Sites of Pb(II) Ion with Human and Bovine Serum Albumins. PLoS One 2012, 7 (5). https://doi.org/10.1371/journal.pone.0036723. (28) Steinhardt, J.; Krijn, J.; Leidy, J. G. Differences between Bovine and Human Serum Albumins: Binding Isotherms, Optical Rotatory Dispersion, Viscosity, Hydrogen Ion Titration, and Fluorescence Effects. Biochemistry 1971, 10 (2), 4005–4015. (29) Fagot-Campagna, A.; Balkau, B.; Simon, D.; Warnet, J. M.; Claude, J. R.; Ducimetière, P.; Eschwège, E. High Free Fatty Acid Concentration: An Independent Risk Factor for Hypertension in the Paris Prospective Study. Int. J. Epidemiol. 1998, 27 (5), 808–813. https://doi.org/10.1093/ije/27.5.808. (30) 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. Integr. Biol. (United Kingdom) 2015, 7 (5), 534–543. https://doi.org/10.1039/c4ib00243a. (31) Jacobs, M. J.; Geiger, M. K.; Summers, S. E.; DeLuca, C. P.; Zinn, K. R.; Spence, D. M. Albumin Glycation Affects the Delivery of C-Peptide to the Red Blood Cells. ACS Meas. Sci. Au 2022, 2 (3), 278–286. https://doi.org/10.1021/acsmeasuresciau.2c00001. (32) Vernon Roohk, H.; Zaidi, A. R. A Review of Glycated Albumin as an Intermediate Glycation Index for Controlling Diabetes. J. Diabetes Sci. Technol. 2008, 2 (6), 1114–1121. https://doi.org/10.1177/193229680800200620. 90 (33) Cision. Cebix’s Once-Weekly Ersatta for Diabetic Peripheral Neuropathy Well Tolerated in Clinical Study; 2012. (34) 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. Clin. Chim. Acta 2013, 425, 64–76. https://doi.org/10.1016/j.cca.2013.07.013. (35) Castro-Correia, C.; Sousa, S.; Norberto, S.; Matos, C.; Domingues, V. F.; Fontoura, M.; Calhau, C. The Fatty Acid Profile in Patients with Newly Diagnosed Diabetes: Why It Could Be Unsuspected. Int. J. Pediatr. 2017, 2017, 1–5. https://doi.org/10.1155/2017/6424186. (36) Richards, J. P.; Bowles, E. A.; Gordon, W. R.; Ellsworth, M. L.; Stephenson, A. H.; Sprague, R. S. Mechanisms of C-Peptide-Mediated Rescue of Low O2-Induced ATP Release from Erythrocytes of Humans with Type 2 Diabetes. Am. J. Physiol. - Regul. Integr. Comp. Physiol. 2015, 308 (5), R411–R418. https://doi.org/10.1152/ajpregu.00420.2014. (37) Medawala, 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. Rev. Diabet. Stud. 2009, 6 (3), 148– 158. https://doi.org/10.1900/RDS.2009.6.148. 91 Chapter 4: Measuring Zn2+ and C-peptide binding and its downstream physiological effects on MS RBCs 4.1 Introduction Multiple sclerosis (MS) is an autoimmune disease of the central nervous system (CNS), where the myelin sheath surrounding axons become damaged.1–4 This process results in decreased nerve signaling, which often manifests itself in tremors, difficulty walking, blurred vision, and slurred speech.3,5 Although it is considered a rare disease, in the United States alone, 1 million people live with MS. However, MS cases equate to 2.3 million people globally.6 Figure 4.1 Comparison of a healthy and MS neuron. On the left, a healthy neuron is depicted. This contains a cell body attached to dendrites and an axon. This axon extends to form synapses to communicate between other neurons. Surrounding the axon is myelin, which encases the axon for more efficient long-distance signaling. On the right, a MS neuron is depicted. This MS neuron is similar to the healthy neuron; however, the axon is encased by damaged myelin, which decreases nerve signaling to other axons. 92 The average human brain contains 86 billion neurons.7 Shown in Figure 4.1, neurons consist of a cell body, dendrites, an axon, and axon synapses.8 Axons extend from the cell body and act as a communicator between neurons.8,9 Electrical impulses move from the neuron cell body to either the dendrites or the axon, where the axon branches to smaller axon collaterals that make synapses.9,10 Synapses and dendrites are the two points at which neurons communicate with one another by releasing electrical signals that are converted to chemical signals in the form of neurotransmitters.9,10 Some axons contain myelin, a component of the white matter of the brain, which allows axons to transmit signals over longer distances.8 Because of this extended transmission, axons encased by myelin are generally connected to neurons that are found in different regions of the brain, rather than local areas of the brain.8 Damage to the myelin sheath results in decreased nerve signaling and communication, which is a distinctive feature of MS.1,11,12 The sole cause of MS is currently not completely understood. It is speculated that progression of this disease may be due to alterations in the blood brain barrier (BBB).13– 15 The BBB is a compilation of different cell types that separate the brain from the bloodstream, shown in Figure 4.2. This barrier prevents diffusion of pathogens, large molecules, hydrophilic molecules, and certain solutes into the cerebrospinal fluid of the brain.16 Increased permeability of the BBB could allow for diffusion of certain destructive neurotoxins into the CNS, which may result in myelin sheath degradation, and therefore, the pathophysiological progression of MS.13–15 Increased levels of nitric oxide (NO) in lesions, cerebrospinal fluid, blood and urine of patients with MS has been reported.13,17–20 These findings have led to examination of the possible role of NO in the progression of MS.17,21 NO is a molecule that is readily 93 Figure 4.2 The blood brain barrier in healthy and MS conditions. On top, a pictorial representation of the BBB is shown. There are tight junctions across a layer of endothelial cells surrounding a blood vessel, inhibiting access of certain molecules within the brain. A standard amount of ATP is released from RBCs, and therefore, a normal amount of NO is released from endothelial cells, inhibiting disruption of the BBB. On the bottom, a pictorial representation of the MS BBB is depicted. Increased ATP from MS RBCs cause an increase in NO release from endothelial cells. Increased levels of NO have been shown to disturb the BBB through breaking tight junctions, which allows for infiltration of T-cells into the brain. T-cells as well as NO have been shown to demyelinate axons, which is a hallmark of MS. 94 produced in the CNS for proper function.22,23. At normal levels, NO contributes to blood flow regulation and transmission between synapses.22,23 However, when NO is in excess, it can act as a neurotoxin and reactive nitrogen species, resulting in increased permeability of the BBB.13,17,19 Shown in Figure 4.2, when excess NO is able to cross the BBB and levels are increased in the brain, it has been shown to act as a free radical. Formation of free radicals can result in mitochondrial impairment, protein nitro-tyrosination, oligodendrocyte injury, and axonal degeneration.13,24 Production of NO in the CNS requires nitric oxide synthase (NOS).25 There are three types of NOS, neuronal (nNOS), endothelial (eNOS) and inducible (iNOS).19,21,25 Both nNOS and eNOS are stimulated through an increase in intracellular calcium (Ca2+) and produce low levels of NO.25,26 iNOS is stimulated independently of Ca2+ increase, therefore, NO is produced in a continuous manner, resulting in higher production of NO.19,25,26 Under normal conditions, nNOS and eNOS are found in the CNS, contributing to regular function of the brain.25 However, iNOS is only found in the CNS when iNOS mRNA and protein are expressed.21 iNOS mRNA and protein have been found in inflammatory lesions that are expressed in individuals with MS, resulting in elevated levels of NO in the brain, which may contribute to the pathophysiology of MS.19,21 Increased permeability and destruction of the BBB is hypothesized to be directly affected by the production and release of NO.18,19 Excess NO in the brain has been shown to result in many destructive mechanisms associated with progression of MS. Specifically, infiltration and demyelination of axons by T cells (Figure 4.2) is primarily caused by an increase in the permeability of the BBB allowing immune cells to enter the CNS.12,27 Also, NO has been shown to facilitate demyelination through the destruction of 95 oligodendrocytes, which are specialized cells that maintain and assemble the myelin sheath.13,28 Without oligodendrocytes to repair the damaged myelin sheath on axons in MS, further progression of this disease ensues. As discussed in previous chapters, NO is essential in vasodilation of vessels (Figure 1.3).29–31 Vasodilation is necessary in circulation and in the CNS, however, excess vasodilation can also be harmful.32,33 Zinc (Zn2+), C-peptide and albumin play an integral role in vasodilation of the vasculature.34,35 The complex of C-peptide, Zn2+ and albumin is necessary to cause the GLUT1 glucose transporter to activate in the red blood cell (RBC) membrane, resulting in an increase adenosine triphosphate (ATP) release, which is then able to stimulate NO release from endothelial cells, with resultant vessel enlargement.34,35 Dissimilar to the hypometabolism of RBCs shown in previous chapters regarding type 1 diabetes (T1D), MS RBCs are in a state of hypermetabolism, where RBCs of people with MS have been reported to release more ATP release than healthy, control RBCs (Figure 4.2).36 We have previously reported more C-peptide binding to MS RBCs37,38 and more GLUT1 activation on the MS RBC membrane,39 all of which contribute to an increase in ATP release, as discussed in previous chapters. There are currently no medications to specifically target the hypermetabolic activity of MS RBCs. Rather, drug therapies prescribed for MS target the immune system; however, there have been recent efforts to target the CNS.40–42 Some of the most common therapies for MS consist of glatiramer acetate, fingolimod, teriflunomide, dimethyl fumarate, cladribine, and interferon-beta (IFN-b).41–43 Unfortunately, many of the therapies prescribed for MS have incomplete mechanisms of action and conflicting dosing regimens, motivating a need for additional research.40,41,44 IFN-b was the first MS disease 96 modifying therapy to be released and is known for its ability to suppress the immune system.43,45–48 IFN-b is an endogenously created cytokine that is released by macrophages, dendritic cells, fibroblasts and endothelial cells when exposed to foreign invaders in vivo.49,50 Under these conditions, IFN-b is released to signal promotion or inhibition of the immune system depending on the circumstance.49 In some instances, IFN-b has been shown to induce the production of pro-inflammatory and anti-inflammatory cytokines, presenting its immunomodulating effects.43,51,52 There are two types of exogenous IFN-b used for MS therapy, IFN-b1a and IFN-b1b.43 Shown in Figure 4.3, IFN-b1a is glycosylated, containing an enzymatically Figure 4.3 Crystallin structure of IFN-b1a. This molecule is a dimer, containing 166 amino acids. IFN-b1a consists of five alpha helices that connect to one another via different loops. There is a glycosylation site at residue Asn-80, specific to IFN-b1a compared to IFNb-1b. Where the two helical clusters interface, there is a specific Zn2+ binding site. In this site, His 121, 93, and 97 form a tetrahedral to stabilize the interaction with Zn2+. 97 bound glucose, and created by a mammalian cell line expressed in Chinese hamster ovary cells.53 IFN-b1b is not glycosylated and expressed in genetically modified Escherichia coli.54 Both IFN-b1a and IFN-b1b have a 33% efficacy in lowering relapse rate, but have complex, unknown mechanisms of action, resulting in debated dosing regimens.43,45,53–55 Reports suggest that both IFN-b1a and IFN-b1b decrease T and B lymphocyte proliferation, inhibit antigen presentation on immune cells and decrease new MS lesion formation.43,48 In addition, IFN-b1a has been shown to specifically bind to Zn2+ (Figure 4.3),47 which may have implications in inhibiting the C-peptide, Zn2+ and albumin Figure 4.4 The effect of IFN-b on GLUT1 activation. The percent decrease of measurable GLUT1 on control and MS RBCs with C-peptide and Zn2+ is depicted with 0.5 and 10 nM IFN-b. There is a significant decrease in measurable GLUT1 for both 0.5 and 10 nM IFN-b in control and MS RBCs (n=3-5, *p<0.05 to control RBCs C-peptide and Zn2+, **p<0.05 to MS RBCs C-peptide and Zn2+, error=SEM). 98 complex from being delivered to RBCs. This would further decrease GLUT1 activation and ATP release from RBCs which may decrease NO release from endothelial cells, alleviating complications associated with increased NO levels in MS. We recently measured GLUT1 activation on RBC membranes of people with MS and healthy controls with the addition of IFN-b39 and found a decrease in measurable GLUT1 in control RBCs with 0.5 nM IFN-b (20.5 ± 7.4%) and 10 nM IFN-b (25.7 ± 5.5%) when compared to control RBCs without IFN-b (Figure 4.4). In addition, there was a decrease in measurable GLUT1 in MS RBCs with 0.5 IFN-b (20.2 ± 3.1%) and 10 nM IFN-b (24.2 ± 2.4%) when compared to MS RBCs without IFN-b. These results indicate that IFN-b decreases measurable GLUT1 in control and MS RBCs, which may result in decreased ATP release from RBCs. In addition to GLUT1 measurements, the Spence lab also measured bovine serum albumin (BSA) binding to control and MS RBCs in the presence and absence of IFN-b to determine if the delivery of the Zn2+, C-peptide and albumin complex is inhibited with IFN-b.39 In Figure 4.5, results indicate that with samples containing C-peptide, Zn2+, BSA and 2 nM IFN-b, there is a significant decrease in BSA molecules bound to MS RBCs (13,538 ± 314 BSA molecules/RBC), when compared to samples without IFN-b (17,866 ± 452 BSA molecules/RBC). There was a similar trend measured in control RBCs, where in samples containing C-peptide, Zn2+, BSA and 2 nM IFN-b there was a significant decrease in in BSA molecules bound to MS RBCs (14,462 ± 223 BSA molecules/RBC), when compared to samples without IFN-b (15,615 ± 378 BSA molecules/RBC). The decreases in BSA binding to control and MS RBCs in the presence of C-peptide and Zn2+ indicates that in addition to IFN-b decreasing GLUT1 activation in the membrane, it also 99 decreases BSA binding to MS RBCs when C-peptide and Zn2+ are present, which may hold importance in a new mechanism of action for IFN-b. Further research regarding C-peptide, Zn2+ and albumin binding and ATP release in control and MS RBCs with the addition of IFN-b are outlined in this chapter to better understand the role of IFN-b in ameliorating complications associated with MS. Figure 4.5 Albumin binding to control and MS RBCs with and without 2 nM IFN-b. All samples contain C-peptide, Zn2+ and either 0 nM IFN-b or 2 nM IFN-b. There is a significant decrease in specific binding of albumin to control and MS RBCs in the presence of C-peptide and Zn2+ with the addition of 2 nM IFN-b (n≥7 control donors, n≥8 MS donors, error=SEM, *p<0.05). 4.2 Methods 4.2.1 RBC collection and purification This method is the same as described in section 3.2.1. 100 4.2.2 Preparation of IFN-β solution IFN-b1a (0.43 µM, PBL Assay Science, Piscataway, NJ) was ordered and thawed to room temperature in a beaker containing cold 18 MW distilled deionized water (DDI H2O). The IFN-b solution was split into 15 mL tubes with aliquot volumes of 5 µL, 9 µL or 14 µL, and stored at -20°C until the day of experimentation. Tubes containing IFN-b were thawed to room temperature in a beaker containing cold DDI H2O. Physiological salt solution (PSS, 4.7 mM KCl, 2.0 mM CaCl2, 140.5 mM NaCl, 12.0 mM MgSO4, 21.0 mM tris (hydroxymethyl) amino methane, 5.5 mM glucose and 0.5% BSA, pH at 7.40) was added to the thawed IFN-b to make 1 nM, 2 nM or 4 nM IFN-b stocks to be used in subsequent experiments. 4.2.3 Sample preparation and analysis for C-peptide binding to RBCs with IFN-β An 800 nM C-peptide working solution was created from an 8 µM C-peptide stock. The 1 nM and 2 nM IFN-b stocks created in section 4.2.2 were utilized in preparation of samples. Samples were prepared with DDI H2O or 20 nM C-peptide and Zn2+, PSS with either 1 nM or 2 nM IFN-b or PSS alone and control or MS RBCs. These samples were incubated at 37°C for 2 hours, inverting at the hour. The samples were centrifuged at 500 g for 5 minutes. The supernatant was removed and collected into a 1.7 mL tube and stored at -20°C until quantification. External C-peptide standards were prepared from the 800 nM working solution (0, 4.8, 9.6, 15.2, 20.0, 20.6 nM). Both samples and standards were diluted 1:50 the day of quantification. A C-peptide enzyme linked immunosorbent assay (ELISA) was performed on the diluted samples and standards to quantify the amount of free C-peptide in solution. This value was then compared to the total amount of C-peptide that was originally added to determine the bound C-peptide on RBCs. 101 4.2.4 Sample preparation and analysis for Zn2+ binding to RBCs with IFN-β An 800 nM 65 Zn2+ working solution was prepared from a 91 µM 65 Zn2+ stock solution. The 1 nM, 2 nM and 4 nM stocks of IFN-b created in section 4.2.2 were utilized in preparation of samples. Samples were prepared with DDI H2O or 2 nM C-peptide and 65 Zn2+, PSS with either 1 nM, 2 nM, or 4 nM IFN-b or PSS alone and control or MS RBCs. The samples were incubated at 37°C for 2 hours on an orbital shaker (260 rpm). The samples were centrifuged at 500 g for 5 minutes, and the supernatant was removed. External 65Zn2+ standards were prepared from the 800 nM working solution. The packed RBCs and standards were placed on a 2480 WIZARD2 automatic gamma counter (Perkin 65 Elmer, Waltham, MA) and quantified using a 5-minute Zn2+ protocol. The amount of 65 Zn2+ bound to the RBCs were directly quantified by comparing the counts per minute (cpm) and the external standards to quantify the bound 65Zn2+ concentration. 4.2.5 Preparation of 3D-printed ultrafiltration devices This method is the same as described in section 2.2.5a-c. 4.2.6 Sample preparation and analysis for C-peptide and Zn2+ binding to BSA with IFN-β Ultrafiltration buffer (10 mM Tris, 150 mM NaCl, pH 7.4) was prepared the day of experimentation and utilized in subsequent studies for sample preparation. This method is similar to sections 2.2.7 and 2.2.8, however, IFN-b and BSA (instead of HSA) were utilized. A 2 nM stock of IFN-b was prepared in ultrafiltration buffer containing 15 µM BSA for Zn2+ binding or 2.7 µM BSA for C-peptide binding experiments. Concentrations of 65 C-peptide, Zn2+ and BSA were utilized to mimic previous experiments with 3D-printed ultrafiltration devices.34,56 C-peptide binding samples were prepared with 20 nM 102 C-peptide, 2.7 µM BSA in ultrafiltration buffer with 2 nM IFN-b or 2.7 µM BSA in ultrafiltration buffer alone. Zn2+ binding samples were prepared with 352 nM 65 Zn2+, 15 µM BSA in ultrafiltration buffer with 2 nM IFN-b or 15 µM BSA in ultrafiltration buffer alone. 200 µL of each sample were placed into 3D-printed ultrafiltration devices within 1.7 mL centrifugation tubes. These tubes were centrifuged at 15,000 g for 90 minutes. Following centrifugation, 10 µL of the ultrafiltrate was removed and placed into another 1.7 mL tube. 65 External C-peptide and Zn2+ standards were prepared to quantify the amount of C-peptide or 65Zn2+ in the ultrafiltrate. C-peptide binding was quantified using a C-peptide ELISA and parameters that are outlined in section 4.2.3. 65Zn2+ binding was quantified on a gamma counter using parameters described in section 4.2.4. 4.2.7 Sample preparation for C-peptide stimulated ATP release from RBCs with IFN-β Samples and solutions were prepared as described in section 4.2.3. Samples were incubated and subjected to flow conditions, standards were prepared, reagents were created, and samples were measured as described in section 3.2.8. External standards were prepared and utilized to quantify ATP released from RBCs. 4.3 Results 4.3.1 Zn2+ binding to BSA with IFN-β To determine the effect of Zn2+ binding to BSA with and without IFN-b, samples 65 were prepared with Zn2+ and BSA buffer in the presence and absence of 2 nM IFN-b. In Figure 4.6, Zn2+ binding to BSA without IFN-b is similar to what has been reported in the literature,56 with 333 ± 2 nM Zn2+ bound to the BSA. The global affinity constant (nKa) of Zn2+ bound to BSA was also similar to values reported in the literature56 with an affinity 103 of 1.24 ± 0.05 µM-1. There was a significant decrease in Zn2+ binding to BSA when 2 nM IFN-b was present, with 322 ± 2 nM Zn2+ bound to BSA. There was also a significant 23% decrease in the affinity of Zn2+ to BSA when 2 nM IFN-b was present, with a global affinity constant of 0.96 ± 0.12 µM-1. Results indicate that Zn2+ binding to MS RBCs is decreased when IFN-b is present. Figure 4.6 Zn2+ binding to BSA with 2 nM IFN-β. Samples contain either Zn2+ and BSA or Zn2+, BSA and 2 nM IFN-β. There was significantly more Zn2+ binding to BSA without the addition of 2 nM IFN-β, indicating that IFN-β interferes in Zn2+ binding to BSA (n=4, error=SEM, *p<0.05). 4.3.2 Zn2+ binding to RBCs with IFN-β To determine if the association between Zn2+ and the RBC is inhibited with IFN-b, 65 samples were prepared to contain C-peptide, Zn2+ and RBCs in the presence or absence of IFN-b. In Figure 4.7, Zn2+ binding to control RBCs correlated to values seen 104 Figure 4.7 Zn2+ binding to RBCs with different concentrations of IFN-β. All samples contain C-peptide and Zn2+ with and without IFN-β. There was a statistically equal amount of Zn2+ bound to control RBCs with and without IFN-β. There was significantly more Zn2+ bound to MS RBCs than control RBCs. Significantly less Zn2+ bound to MS RBCs with 1 nM, 2 nM and 4 nM IFN-β compared to MS RBCs without IFN-β (n=3-8 donors, error=SEM, *p<0.05 to 0 nM IFN-β control RBCs, **p<0.05 to 0 nM IFN-β MS RBCs). in the literature39,56 with 2.6 ± 0.1 nM Zn2+ bound to control RBCs or 2,035 ± 910 Zn2+ ions bound to control RBCs. There is a significant increase in Zn2+ binding to MS RBCs (3.3 ± 0.2 nM, 2553 ± 158 ions/RBC) when compared to control RBCs, resulting in an additional 520 Zn2+ ions bound to MS RBCs. There is not a significant decrease in Zn2+ binding to control RBCs with the addition of 1 nM IFN-b (2.4 ± 0.1 nM, 1,889 ± 845 ions/RBC) and 2 nM IFN-b (2.65 ± 0.07 nM, 2,053 ± 1,030 ions/RBC). However, there is a significant decrease in Zn2+ binding to MS RBCs with 1 nM IFN-b (2.24 ± 0.3 nM, 1,738 105 ± 210 ions/RBC), 2 nM IFN-b (2.6 ± 0.3 nM, 2,039 ± 499 ions/RBC) and 4 nM IFN-b (2.2 ± 0.2 nM 1,687 ± 144 ions/RBC). All MS RBC samples with IFN-b were statistically equal to one another and to all control RBC samples. Results indicate that IFN-b decreases Zn2+ binding in MS RBCs to levels statistically equal to control RBC conditions. 4.3.3 C-peptide binding to BSA with IFN-β To determine the effect of C-peptide binding to BSA with IFN-b, samples were prepared to contain C-peptide and BSA buffer in the presence and absence of 2 nM IFN-b. In Figure 4.8, C-peptide binding to BSA without IFN-b correlates to literature Figure 4.8 C-peptide binding to BSA with 2 nM IFN-β. Samples contain either C-peptide and BSA or C-peptide, BSA and 2 nM IFN-β. There was significantly more C-peptide binding to BSA without the addition of 2 nM IFN-β, indicating that IFN-β interferes in C-peptide binding to BSA (n=4, error=SEM, *p<0.05). 106 values, with 18.3 ± 0.3 nM C-peptide bound to BSA.34 The global affinity constant of C-peptide binding to BSA was also similar to what is seen in the literature, with an affinity of 4.8 ± 1.1 µM-1.34 There was a significant decrease in C-peptide binding to BSA when IFN-b was present, with 16.0 ± 0.9 nM C-peptide bound to BSA. There was also a statistically significant 63% decrease in binding affinity when IFN-b was present, with a global affinity of 1.8 ± 0.4 µM-1. Results indicate that IFN-b decreases C-peptide binding to BSA, which may have implications in C-peptide’s transport to the RBC for ATP release. 4.3.4 C-peptide binding to RBCs with IFN-β To determine the effect of IFN-b on the binding of C-peptide to the RBCs, samples were prepared to contain C-peptide, Zn2+, and differing concentrations of IFN-b or without IFN-b. In Figure 4.9, C-peptide binding to control RBCs correlate to values seen in the literature36 with 1.64 ± 0.2 nM C-peptide bound or 1,274 ± 157 molecules of C-peptide bound to control RBCs. There was a statistically significant 106% increase in C-peptide binding to MS RBCs (3.38 ± 0.1 nM, 2,617 ± 102 molecules/RBC) when compared to control RBCs. In control RBCs, there was a 62% statistically significant decrease in C-peptide binding when 2 nM IFN-b was present (0.62 ± 0.3 nM, 479 ± 208 molecules/RBC), resulting in a difference of 795 ± 66 C-peptide molecules bound to RBCs when compared to control RBCs. However, there was no significant difference in samples containing 1 nM IFN-b (1.2 ± 0.2 nM, 927 ± 164 molecules/RBC) compared to control RBCs without IFN-b. In MS RBCs, there was a statistically significant 46% decrease in C-peptide binding in samples containing 1 nM IFN-b (1.8 ± 0.3 nM, 1,425 ± 213 molecules/RBC) and a statistically significant 54% decrease in C-peptide binding in 107 Figure 4.9 C-peptide uptake by control and MS RBCs with difference concentrations of IFN-β. All samples contain C-peptide, Zn2+, no IFN-β or differing concentrations of IFN-β. There is a significant increase in C-peptide binding to MS RBCs compared to control RBCs. Significantly less C-peptide bound to control RBCs with 2 nM IFN-β. Significantly less C-peptide bound to MS RBCs with 1 nM and 2 nM IFN-β (n=4, error=SEM, *p<0.05 to 0 nM IFN-β control RBCs, **p<0.01 to 0 nM IFN-β MS RBCs). samples containing 2 nM IFN-b (1.6 ± 0.3 nM, 1,206 ± 211 molecules/RBC). Results indicate that in both control RBCs and MS RBCs, IFN-b decreases binding of C-peptide to the RBC. 4.3.4 C-peptide stimulated ATP release from RBCs with IFN-β To assess the functionality of the observed decreases in C-peptide, Zn2+ and BSA binding to one another and to RBCs, ATP release from control and MS RBCs was 108 Figure 4.10 ATP release from RBCs with difference concentrations of IFN-β. There is a significant increase in ATP for samples containing C-peptide and Zn2+ (stripes) when compared to samples without C-peptide and Zn2+. There is a significant decrease in ATP release from control RBCs with C-peptide and Zn2+ when 1 nM and 2 nM IFN-β are present. There is also a significant decrease in ATP release from MS RBCs with C-peptide and Zn2+ when 1 nM and 2 nM IFN-β are present (n=3-6, error=SEM, *p<0.05 to 0 nM IFN-β control RBC C-peptide and Zn2+, **p<0.05 to 0 nM IFN-β MS RBCs C-peptide and Zn2+, ***p<0.05 to respective RBCs without C-peptide and Zn2+). measured. Samples were prepared to contain C-peptide, Zn2+, PSS with or without differing concentrations of IFN-b and control or MS RBCs. In Figure 4.10, there is a statistically significant increase in ATP release from control RBCs (35 ± 4 nM) with the addition of C-peptide and Zn2+ (68 ± 14 nM). With control RBC samples containing C-peptide and Zn2+ and 1 nM (41 ± 7 nM) or 2 nM (30 ± 4 nM) IFN-b, there is a statistically significant decrease in ATP release when compared to control RBC samples without IFN-b. There is also a statistically significant increase in ATP release from MS RBCs (70 109 ± 11 nM) with the addition of C-peptide and Zn2+ (108 ± 12 nM) when compared to MS RBCs without C-peptide and Zn2+. Similar to control RBCs, there is a statistically significant decrease in ATP release from MS RBCs with C-peptide and Zn2+ and 1 nM (49 ± 6.2 nM) or 2 nM (48 ± 6 nM) IFN-b. In all RBC samples (i.e. control and MS RBCs) containing C-peptide, Zn2+ and IFN-b, IFN-b decreased ATP release to levels statistically equal to control RBCs without the addition of C-peptide and Zn2+. There is a 56% decrease in ATP release in control RBCs with C-peptide, Zn2+ and 2 nM IFN-b when compared to control RBCs with C-peptide and Zn2+. Similarly, there is a 56% decrease in ATP release in MS RBCs with C-peptide, Zn2+ and 2 nM IFN-b when compared to MS RBCs with C-peptide and Zn2+. Results indicate that IFN-b decreases ATP release when C-peptide and Zn2+ are present in both control and MS RBCs. 4.4 Discussion Our group has reported increased ATP release from MS RBCs,36 which may result in increased NO release, and therefore, may contribute to the destruction of the myelin sheath on axons in MS.17,19 Currently, there are no MS therapies aimed at specifically decreasing ATP release from RBCs, however, there are multiple therapies with incompletely understood mechanisms of action, including IFN-b therapy. Because IFN-b has a Zn2+ specific binding site47 and Zn2+ is an integral component to stimulate increased ATP release from RBCs,35 this drug was chosen for subsequent studies to further investigate its potential role in increased NO levels reported in MS patients. Zn2+ binding to BSA and to the RBC was explored to investigate if IFN-b is interfering in Zn2+ binding to the BSA complex, to the RBC or both. In Figure 4.6, results showed a significant decrease in Zn2+ binding to BSA with IFN-b, indicating that Zn2+ 110 cannot bind to BSA in the same manner with IFN-b compared to without IFN-b. Interestingly, in Figure 4.7, there was no decrease in Zn2+ binding to control RBCs with IFN-b, however, Zn2+ binding to MS RBCs was decreased to levels seen in control RBCs with and without IFN-b. RBCs have been reported to have one high affinity and one low affinity binding site for Zn2+.57 MS RBCs bind more Zn2+ than control RBCs, therefore, it is likely that with excess Zn2+ binding to MS RBCs, both the high affinity and low affinity Zn2+ binding sites are occupied, whereas, in control RBC conditions, it is likely that only the high affinity Zn2+ binding pocket is occupied. Because of this, IFN-b may only have the ability to remove Zn2+ bound to the lower affinity binding pocket on the RBC. Interestingly, when comparing Zn2+ binding to MS RBCs and control RBCs, there was a 21% decrease in Zn2+ ions bound (520 ± 70 ions per RBC). Similarly, when comparing Zn2+ binding to MS RBCs with and without 2 nM IFN-b, there was also a 21% decrease in Zn2+ ions bound (510 ± 70 ions per RBC). These results indicate that there was a statistically equal decrease (i.e. 21%) in Zn2+ ions bound to MS RBCs when compared to control RBCs and MS RBCs with 2 nM IFN-b. Therefore, these results indicate that IFN-b may only have the ability to remove excess Zn2+ ions, presumably in the lower affinity binding site on the RBC. Conversely, when normal amounts of Zn2+ are bound to control RBCs, presumably in the higher affinity binding site, IFN-b is unable to remove these Zn2+ ions. After investigating the effect of IFN-b on Zn2+ binding to BSA and RBCs, C-peptide binding to BSA and RBCs with IFN-b was measured to determine if IFN-b also inhibited C-peptide transport and delivery to the RBC. In Figure 4.8, results indicated that C-peptide binding to BSA was decreased when IFN-b was present. Similarly, in Figure 111 4.9, results indicated that IFN-b was able to significantly decrease C-peptide binding to control and MS RBCs. Similar to Zn2+ binding experiments, IFN-b was only able to decrease C-peptide binding in MS RBCs to levels statistically equal to control RBCs with C-peptide and Zn2+. Collectively, these results indicate that C-peptide binding to both BSA and to MS RBCs is inhibited in the presence of IFN-b. To determine the downstream physiological effect of IFN-b therapy on RBCs, ATP release from control and MS RBCs was measured. In Figure 4.10, results indicate that there was a significant decrease in ATP release from both control and MS RBCs with C-peptide, Zn2+ and IFN-b when compared to their respective controls with C-peptide and Zn2+. As shown in both C-peptide, Zn2+ and RBC binding experiments, IFN-b was only able to decrease ATP release from MS RBCs to levels statistically equal to control RBCs with C-peptide and Zn2+. This data is also consistent with previous C-peptide, Zn2+ and BSA binding findings, indicating that if any component of the C-peptide, Zn2+ and albumin complex is not present, ATP release from RBCs is inhibited.58 We recently reported that both GLUT1 and albumin binding is decreased when IFN-b is in present.39 This data compiled with the data outlined in this chapter indicates that IFN-b alleviates complications associated with MS due to its ability to inhibit the Zn2+, C-peptide and BSA complex from being delivered to the RBC, which decreases ATP release, levels that are otherwise increased in MS.36 IFN-b has the ability to decrease C-peptide and Zn2+ binding to albumin, in addition to, inhibiting this C-peptide, Zn2+ and albumin complex from being delivered to the cells. Without the binding of the C-peptide complex to the RBC membrane, GLUT1 stays inactive in the RBC membrane, corresponding to a decrease in ATP release. In all studies outlined in this chapter, IFN-b 112 was only able to decrease C-peptide binding, Zn2+ binding and ATP release to levels statistically equal to control RBC conditions. Currently, IFN-b is an early intervention immunotherapy used to treat MS. This therapy has been shown to inhibit the formation of new MS lesions through suppression of the immune system, however, it has an incomplete mechanism of action.45,46 Findings in this chapter explain a possible mechanism of action for this therapy, as well as provide information regarding the beneficial effects of IFN-b therapy. In addition to its unknown mechanism of action, the effect of IFN-b on treating people with MS is uncertain due to the inability to determine proper dosing and dose frequency that alleviates symptoms and progression of MS.45,46 Due to issues in efficacy, IFN-b is administered at differing doses and frequencies via intramuscular or subcutaneous injection.43,46,59 Results discussed in this chapter indicate that IFN-b decreases C-peptide binding to MS RBCs, and therefore decreases ATP release, which is a direct stimulus of NO. After glucose levels rise post-meal, C-peptide levels increase and remain in circulation for about 30 minutes in vivo.60 Because IFN-b therapy directly affects C-peptide binding, these results loosely suggest that increased efficacy of IFN-b may be possible if administered before a meal, to be present when C-peptide levels are at their peak. Results in this chapter indicate that IFN-b directly affects C-peptide binding and delivery to control and MS RBCs, inhibiting ATP release. ATP has been shown to increase NO release from endothelial cells, and NO has been shown to be upregulated within plasma, urine and brain lesions of people with MS.13,17–20 IFN-b’s ability to decrease ATP release from RBCs via the C-peptide pathway indicates a new mechanism of action for 113 this therapy, as well as suggests possible dose frequency changes of IFN-b administration. 114 REFERENCES (1) Steinman, L. Multiple Sclerosis: A Coordinated Immunological Attack against Myelin in the Central Nervous System. Cell 1996, 85 (3), 299–302. https://doi.org/10.1016/S0092-8674(00)81107-1. (2) Janes, T. M.; Spence, D. M. Steroid Inhibition of Erythrocyte-Derived ATP Reduces Endothelial Cell Production of Nitric Oxide in a 3D-Printed Fluidic Model. Anal. Methods 2018, 10 (27), 3416–3422. https://doi.org/10.1039/c8ay00870a. 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Diabesity Res. 2004, 5 (1), 7–14. https://doi.org/10.1080/15438600490424389. 120 Chapter 5: Conclusions and Future Directions 5.1 Diabetes Conclusions Type 1 diabetes (T1D) is an autoimmune disease in which the pancreatic β-cells are destroyed.1 In the pancreatic β-cells, equimolar concentrations of insulin and C-peptide are found in addition to high levels of Zn2+.2 Despite depletion of all three of these molecules in T1D, insulin is currently the only endogenous molecule that is re-administered as an exogenous therapy. Even with insulin therapy, diabetic complications arise (i.e. retinopathy, neuropathy, and nephropathy),3–7 which have been attributed to poor microvascular blood flow.7–9 Insulin is readily used as a therapy for T1D to improve glucose regulation within the circulation by stimulating uptake of glucose via the GLUT4 glucose transporter on muscle, fat and liver cells. Insulin is transported to these cells within the bloodstream, however, only has a half-life in the bloodstream for 4-6 minutes. Interestingly, blood cells primarily contain the GLUT1 glucose transporter, suggesting a need for an additional therapy to directly affect these cells and improve the microvascular complications in T1D that insulin cannot.10,11 C-peptide has been shown to affect GLUT1 containing cells in the bloodstream.10,11 Activation of GLUT1 results in increased adenosine triphosphate (ATP) release from red blood cells (RBCs),12 which results in nitric oxide (NO) release, a potent vasodilator. Because of C-peptide’s role in this cascade, multiple groups have examined this molecule with hopes to improve microvascular complications associated with T1D. There have been many in vitro studies that denote C-peptide’s importance in RBC deformability, improved blood flow and increased kinase activity.10,13 Additionally, there have been many in vivo studies using animal models that confirm the above results 121 involving the importance of C-peptide in improving microcirculation.14,15 Due to these preliminary findings, a large scale clinical trial was performed in humans using a mono- pegylated C-peptide analog, called Ersatta, in people with T1D.16,17 Unfortunately, these small scale clinical trials failed due to the results in individuals with T1D with C-peptide therapy and the placebo being statistically equal. Since these studies, the Spence lab has shown that C-peptide is only able to bind and affect the RBC when in combination with albumin, a plasma binding protein, and Zn2+, a biologically active ion and additional content of the β-cell.18 The use of pegylated C-peptide in the Ersatta clinical trial involved adding polyethylene glycol (PEG) to the end of the peptide to prolong its half-life in vivo. The addition of PEG to C-peptide could potentially block the binding site on C-peptide to albumin or RBCs which would inhibit its therapeutic effect. This hypothesis could explain its decreased efficacy and failure of C-peptide therapy in human models. Additionally, individuals with T1D have been reported to have decreased albumin concentrations in vivo,19,20 and researchers in the Ersatta C-peptide clinical trials did not re-introduce albumin into the therapy formulation. It is also widely recognized that individuals with T1D have increased glucose concentrations that have been correlated with high levels of glycated albumin.21–23 Furthermore, dysregulation of insulin administration and production leads to increased concentrations of fatty acids in the plasma of people with T1D, which readily associate with albumin.24–26 In Chapters 2 and 3, it was shown that both glycation of albumin and increased fatty acids disrupt the binding, transport, and downstream effect of C-peptide. Because albumin is essential in the transport of C-peptide to RBCs, there may be a benefit to re-introducing healthy 122 albumin with C-peptide therapy to ensure that individuals with T1D can properly deliver C-peptide to the RBC. Additional experiments are necessary to determine how the formulation will function in individuals with T1D in order to utilize this therapy in a clinical setting. 5.2 Future binding studies with glycated RBCs In Chapters 2 and 3, glycated albumin was utilized to measure the binding of C-peptide and Zn2+ to albumin and C-peptide and Zn2+ to the RBC. To measure the association between C-peptide, Zn2+ and the RBC, healthy, control RBCs were utilized. Results indicate that there is decreased binding of C-peptide and Zn2+ to albumin and decreased C-peptide and Zn2+ delivery to healthy, control RBCs. However, in addition to glycated albumin, RBCs can also be glycated.27–31 Researchers have found increased concentrations of glycated RBCs in people with T1D due to the elevated glucose concentrations found within their circulation.32–34 Elevated levels of glycated RBCs may further decrease C-peptide binding to the RBC, inhibiting C-peptide’s therapeutic effect. To ensure that the C-peptide can be transported to the RBC, bind and elicit its downstream therapeutic effect in individuals with T1D, future experiments exploring C-peptide binding to glycated RBCs need to be explored. To mimic conditions in vivo, experiments must be performed utilizing healthy and glycated albumin with healthy and glycated RBCs. Healthy and glycated albumin will be isolated as described in section 2.2.3. Whole blood will be drawn from healthy, controls and individuals with T1D. RBCs would then be washed and isolated as described in section 3.2.1. Glycated albumin percent would then be measured using time of flight mass spectrometry as described in section 2.2.4 and RBC glycation will be determined using 123 an over-the-counter glucometer. C-peptide and Zn2+ binding experiments would utilize different combinations of glycated and control albumin and glycated and control RBCs to determine if C-peptide and Zn2+ can bind to glycated RBCs using methods described in sections 3.2.6 and 3.2.7. In addition, ATP release will be measured using the above conditions from glycated and control RBCs using methods described in section 3.2.8. These results will provide insight into how C-peptide will be transported and delivered in individuals with T1D for better understanding of how C-peptide should be administered for increased clinical efficacy. 5.3 Future binding studies with increased fatty acids In Chapters 2 and 3, C-peptide and Zn2+ binding to albumin was measured in common disease states in T1D, such as increased glycation of albumin and increased fatty acid concentrations. Specifically, the association between C-peptide and albumin in the presence of the fatty acid myristic acid (MA) was measured, and it was determined that C-peptide binding to albumin was decreased in the presence of MA (Figure 2.13b). This resulted in a further decrease of ATP release from RBCs (Figure 3.9) with all concentrations of MA, thus could contribute to microvascular complications in T1D. These preliminary experiments measuring C-peptide binding and ATP release from RBCs may hold importance in understanding how increased fatty acids interfere within C-peptide’s mechanism of action in T1D. Preliminary experiments were performed to investigate the decrease in ATP release from RBCs at various MA concentrations (Figure 3.9). These experiments measured Zn2+ and C-peptide binding to RBCs at concentrations of MA utilized in previous ATP experiments, representing different physiologic ratios of MA to bovine 124 Figure 5.1 Zn2+ and C-peptide binding to RBCs with different concentrations of MA. All samples contain C-peptide, Zn2+ and either PSS with MA or without. a) There was a significant decrease in Zn2+ binding with 31 and 56 µM MA when compared to samples without MA. (n=4-5, error=SEM, *p<0.05 to C-peptide and Zn2+ and 0 µM MA) b) There was a significant decrease in C-peptide binding with 56, 81 and 106 µM MA when compared to samples without MA (n=4-6, error=SEM, *p<0.05 to 0 µM MA). serum albumin (BSA). Zn2+ samples were prepared and measured similar to what was described in section 3.2.7. In Figure 5.1a, Zn2+ binding to RBCs with differing concentrations of MA is measured. Results indicate that Zn2+ binding to RBCs is decreased with MA concentrations of 31 and 56 µM when compared to control conditions without MA. These results need to be repeated and confirmed using fresh radioactive 65 Zn2+, as the studies used a 65 Zn2+ stock that had already gone through two half-lives, which may affect measurements. The association between C-peptide and the RBC was also measured as shown in Figure 5.1b. Samples were prepared and measured as described in section 3.2.8. Results indicate that C-peptide binding to RBCs is decreased with 56, 81 and 106 µM MA when compared to control conditions without MA. There is a decrease of 2624 ± 167 C-peptide molecules bound to RBCs in 106 µM MA samples compared to samples without MA. 125 Additional studies need to be performed to confirm results in Figure 5.1b due to quantitation issues with recent enzyme linked immunosorbent assay (ELISA, PBL Assay Science, Piscataway, NJ) kits. Results in Figure 5.1b are depicted as the change in C-peptide concentration compared to control conditions without MA due to more C-peptide found in the supernatant sample than C-peptide added to the original sample (20 nM). To correct for this, all samples were compared to the control, and the concentration increase of free C-peptide was plotted. All ELISA sample runs held the same trend as depicted in Figure 5.1b. 5.4 Future studies with the addition of exogenous albumin Re-administration of healthy albumin is regularly used in clinical settings. This is commonly prescribed for pathologies such as hypovolemia, acute pulmonary edema, acute renal failure, hypoalbuminemia and excessive bleeding.35,36 Currently, there is an FDA approved albumin formulation called AlbuRx, which consists of a 5% or 25% albumin solution administered via intravenous infusion.37 Additional studies utilizing AlbuRx in regards to measurements in C-peptide binding, albumin binding, RBC binding and ATP release in the presence of glycated albumin will allow for determination of how external albumin supplementation may facilitate improvements in C-peptide’s effect in individuals with T1D. Preliminary experiments will involve samples prepared as described in sections 2.2.7 and 2.2.8 and analyzed as described in sections 2.2.9 and 2.2.10 for albumin binding, however BSA will be utilized instead of human serum albumin (HSA). Samples indicating C-peptide and Zn2+ delivery to RBCs were prepared and analyzed as described in sections 3.2.6 and 3.2.7. For preliminary experiments with albumin and RBC binding 126 studies, 5% and 25% commercially available Sigma-Aldrich albumin will represent healthy, non-glycated albumin. Various percentages of glycated albumin will represent different T1D glucose conditions in vivo using boronate affinity chromatography (section 2.2.3). Primary studies will use the FDA approved 5% or 25% AlbuRx to replicate the potential formulation of C-peptide therapy that would contain healthy albumin for clinical use. Various percentages of glycated albumin will be utilized to represent different albumin glycation percentages seen in T1D. These samples will be subject to C-peptide and Zn2+ binding studies, to both albumin and to the RBC. The downstream physiological effect will be measured, by quantifying ATP release from RBCs. These results will aid in determining if healthy albumin supplementation in addition to C-peptide therapy can improve C-peptide binding, transport, and downstream therapeutic effects in T1D. 5.5 Future clinical trials with C-peptide therapy The ultimate goal of all of the experiments outlined in this dissertation is to produce a C-peptide therapy to alleviate microvascular complications in T1D and improve patients’ lives. In order to do this, different formulations of Zn2+, C-peptide, and albumin need to be tested in people with T1D within a clinical trial. There will be two different cohorts, consisting of a group of individuals that have HbA1c levels above 7% and below 7% to ensure that this therapy works in individuals with variable glucose levels.38 The therapeutic C-peptide dose utilized in these clinical trials will equate to 0.7-1.2 mg of C-peptide, a value that corresponds to the amount of insulin administered in T1D. Within the two cohorts described above, there will be three different dosing regimens, where C-peptide administration will equate to 10%, 30% and 100% of the C-peptide formulation containing Zn2+ and albumin. To test the efficacy of the drug, clinical monitoring including 127 a physical exam, blood glucose measurements, HbA1c, and monofilament vibration testing will be performed. These tests will coincide with laboratory testing, such as GLUT1 RBC activation, performed through Western Blotting using methodologies described in previous Spence lab publications.10,11,39 RBC rigidity will also be measured, by a pressure transducer, using methodologies also previously reported in Spence lab publications.40 It is expected that the drug will show no signs of toxicity and will improve all measurements described above. These results will advance this drug to further clinical trials with the end goal of manufacturing it on a global scale. 5.6 MS Conclusions Multiple sclerosis (MS) is a progressive autoimmune disease in which the myelin sheath found on axons in the central nervous system become damaged.41,42 A hallmark of MS is the formation of brain lesions, comprised of demyelinated axons, which result in slowed communication between nerve cells.42–44 This disease commonly leads to both physical and cognitive decline, accompanied by neurological problems which are commonly demonstrated through tremors, fatigue, visual impairments and more.45 Although the mechanism of MS onset is unclear, researchers speculate that nitric oxide (NO) may play a role in demyelination due to increased levels of NO in lesions within the brains of people with MS.46–48 As described in throughout, ATP is a molecule that has been shown to stimulate NO release from endothelial cells. The Spence lab has shown that a combination of Zn2+, C-peptide and albumin can activate GLUT1 in the RBC membrane, resulting in an increase in ATP release.18 Interestingly, RBCs of people with MS have been shown to bind more C-peptide49 and release more ATP than healthy, control RBCs.50 Increased 128 ATP release from MS RBCs may result in an increase in NO release, which may contribute to formation of MS lesions and progression of MS.47,51,52 Therapies used to treat MS, such as interferon-beta (IFN-β), glatiramer acetate, natalizumab, mitoxantrone, fingolimod and teriflunomide, are immunosuppressive drugs that inhibit myelin degradation and lesion formation.53–56 Many of these therapies are experimental or have incompletely understood mechanisms of action, which lead to dosing and efficacy problems.53,54,56 Specifically, IFN-β is an early intervention therapy that has been shown to reduce new lesion formation.57,58 Although this mechanism is not fully understood, researchers have found that IFN-β decreases T-cell proliferation, immune cell function, and antigen presentation.58–61 In addition to mechanistic issues, the efficacy of IFN-β is also largely undetermined due to the inability to determine proper dosing.58,62 Because of this, IFN-β is given both subcutaneously or intramuscularly at differing doses and frequencies in attempts to increase efficacy.58,63,64 Interestingly, IFN-β has been shown to have a specific binding pocket for Zn2+.65 Decreasing Zn2+ binding to RBCs in people with MS was hypothesized to have implications in ATP release from RBCs, thus decreasing the cytotoxic effects of increased NO release from endothelial cells. Previous research by our group has indicated that albumin binding and GLUT1 activation in MS RBCs is decreased in the presence of IFN-β.39 To understand the relationship between Zn2+ binding and IFN-β, studies measuring the association between Zn2+ and C-peptide binding to albumin and to the RBC with IFN-β therapy were performed. In Chapter 4, it was shown that Zn2+ binding to albumin and to MS RBCs is decreased in the presence of IFN-β. Similarly, it was shown 129 that C-peptide binding to albumin and to MS RBCs is decreased in the presence of IFN-β. Finally, ATP release was also decreased in MS RBCs in the presence of IFN-β. These results indicate a possible new mechanism of action for IFN-β and allows research into potential MS therapies that target the hypermetabolic activities of RBCs. These results also provide insight into the proper dosing regimen of IFN-β, possibly administered just before a meal to coincide with a C-peptide spike, to increase efficacy. Additional research into the mechanism of action for this therapy is integral to increasing the efficacy of this drug, allowing for more comprehensive use in people with MS. 5.7 IFN-β binding to RBCs Previous experiments outlined in Chapter 4 indicate that IFN-β may be blocking the albumin/C-peptide/Zn2+ complex from binding to the RBC; therefore decreasing ATP release from RBCs and the possible toxic effects of increased NO in people with MS.39 Additional studies to determine if IFN-β itself binds to RBCs will provide further evidence for this hypothesis. Current experiments are underway to determine this interaction. In order to investigate the relationship between IFN-β and RBCs, a commercially available ELISA to measure IFN-β will be used. Samples will be prepared to contain C-peptide, Zn2+, albumin free physiologic salt solution (AF-PSS, 4.7 mM KCl, 2.0 mM CaCl2, 140.5 mM NaCl, 12.0 mM MgSO4, 2.01 mM tris (hydroxymethyl) amino methane, 5.5 mM glucose, pH at 7.40) and 7% RBCs with or without 2 nM IFN-β. An additional sample will be created to contain AF-PSS, 2 nM IFN-β and RBCs without C-peptide and Zn2+ to represent control conditions. Samples will be incubated at 37°C for 2 hours on an orbital shaker. RBC samples will be centrifuged, and the supernatant will be removed to measure the concentration of free IFN-β in solution via ELISA. IFN-β concentration will 130 be measured by comparing the sample value to a standard curve. If the concentrations of IFN-β in the supernatant is less than what was added (i.e., 2 nM), this will indicate binding of IFN-β to RBCs. This will provide further evidence that IFN-β is blocking the albumin/C-peptide/Zn2+ receptor on RBCs. If IFN-β concentration is unchanged, this would indicate that IFN-β does not specifically bind to the RBC; rather, IFN-β is interfering in the albumin/C-peptide/Zn2+ complex itself. Additional studies with different experimental parameters may increase our understanding of this interaction, such as using both control and MS RBCs and the addition of albumin for possible competitive inhibition. 5.8 Specific dosing frequency of IFN-β after meals Due to an incompletely understood mechanism of action and unknown efficacy of IFN-β, it is given in a multitude of different dose concentrations and frequencies.58,63,64 Avonex, one of the commercially available IFN-β1a therapies, is given intramuscularly once a week in four different dose concentrations of 7.5, 15.0, 22.5, and 30.0 µg.66 Rebif is another IFN-β1a therapy given subcutaneously three times a week at two different dose concentrations of 44.0 and 22.0 µg.63,66 While regimes vary by individual, both IFN-β therapies are recommended to be administered in the late afternoon or evening due to decreased side effects.66 Experimental results in Chapter 4 suggest that administering lower concentrations of IFN-β prior to a meal may counteract the postprandial spike of C-peptide in circulation. This is due to the reduced transport and binding of C-peptide to RBCs in the presence of IFN-β. As described previously, MS RBCs have been shown to have increased C-peptide binding50 and increased ATP release49 compared to healthy controls. This postprandial 131 increase in C-peptide concentration may be especially harmful to people with MS because increased ATP has been correlated to an increase in NO release. Administering IFN-β prior to a C-peptide increase may inhibit excess C-peptide from binding to the RBC, therefore, further inhibiting the harmful effects of NO in people with MS. To determine the effect of IFN-β post-meal, individuals with MS will be recruited to participate in dose escalation clinical trials similar to what is outlined in section 5.5. There will be two cohorts, consisting of individuals below the age of 35 and above the age of 35, due to onset of disease averaging 35 years of age.67 The dose of IFN-β will be 2.0 µg in saline buffer injected before every meal. There will be three groups within each cohort, with administration of 10%, 30% and 100% of the IFN-β formulation. Individuals with MS enrolled in these studies will visit a neurologist every three months to monitor changes in physical capabilities and every six months to monitor lesion formation using magnetic resonance imaging (MRI). Laboratory studies may also confirm these findings by measuring ATP release from subject RBCs. Decreased ATP release would indicate that the therapy may be improving functionality of the RBCs. The expected outcome of this therapy would be improvements in physical impairments, decreases in lesion formation and possibly decreases in existing lesion size. 132 REFERENCES (1) Center for Disease Control and Prevention. National Diabetes Statistics Report: Estimates of Diabetes and Its Burden in the United States. U.S. Department of Health and Human Services (2020). (2) Steiner, D. F., Cunningham, D., Spigelman, L. & Aten, B. 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