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5/08 KzlProleocaPresIClRClDateDuo.indd

SUCCESSFUL AND REPRODUCIBLE BIOACTIVITY WITH C-PEPTIDE VIA
ACTIVATION WITH ZINC

By

Jennifer Meyer

A DISSERTATION
Submitted to
Michigan State University
In partial fulﬁllment of the requirements
for the degree of
DOCTOR OF PHILOSOPHY
Chemistry

2009

ABSTRACT

SUCCESSFUL AND REPRODUCIBLE BIOACTIVITY WITH C-PEPTIDE VIA
ACTIVATION WITH ZINC

By
Jennifer Meyer

Diabetes is considered to be a major health problem throughout the world with
its hallmark feature being hyperglycemia, or high blood glucose. Diabetes is
classiﬁed into 2 general types- type 1 and type 2 diabetes. While both types of
diabetes suffer similar complications, the cause of each type is vastly different. Type
1 diabetes, or juvenile diabetes, generally occurs during childhood or adolescence and
is the result of an autoimmune destruction of the B-cells. Conversely, type 2 diabetes
is the result of various factors including age, obesity, race, and family history.
Patients who suffer from type 1, and in many cases type 2 diabetes, are required to
take insulin injections to maintain proper blood glucose levels.

Insulin is produced in the pancreas as proinsulin and is released into the
bloodstream after an intake of glucose. Insulin, while effective at maintaining proper
blood glucose levels, does not ameliorate many of the complications associated with
diabetes. Proinsulin also consists of C-peptide which, since its discovery, has been
thought to possess little biological activity. Within the past decade, however, there
has been new evidence that C-peptide is indeed a biologically relevant peptide and has
been shown to improve many of the complications associated with diabetes including
neuropathy, nephropathy, retinopathy, and those associated with the microcirculation.
Despite these advances, there has been no successful long-term clinical trial involving

C-peptide replacement therapy, most likely due to difﬁculties with reproducibility.

The lack of a successful long-term clinical trial is one reason C-peptide is not
considered to be a biologically relevant peptide.

As reported here, it has been determined that C-peptide requires proper
preparation, namely the activation by a metal such as zinc, to provide reproducible
results when incubated with the red blood cell (RBC), an unlikely factor in the
pathophysiology of diabetes. Data reported here suggests that incubation of RBCs
with metal-activated C—peptide has the ability to increase the release of ATP, a
recognized stimulus of nitric oxide (a potent vasodilator), via an increase in glucose
transport.

Additional arguments against C-peptide include the lack of a receptor and the
abundance of C-peptide in patients with type 2 diabetes. However, data presented
here suggest that the lack of a known receptor does not mean that C-peptide is
biologically irrelevant. Also, it has been determined during the course of this thesis
that the membrane of the RBC from type 2 patients contains a higher amount of
phosphatidylserine, which may result in repulsion between the membrane and C-
peptide resulting in an apparent “C-peptide resistance”. However, incubation of the
RBCs with the most commonly prescribed drug to treat type 2 diabetes, metforrnin,
prior to the addition of metal-activated C-peptide, appears to correct this resistance.

Overall, data reported here offer solutions to the primary arguments against C-
peptide as a biologically relevant peptide. The results offer potentially clinically
relevant data, which may provide the foundation for a successful clinical trial ofC-

peptide replacement therapy.

 

 

ACKNOWLEDGEMENTS

First and foremost I would like to thank Dana Spence for all of his dedication
and time over the past four years. He was kind enough to let me join his group before
ofﬁcially starting grad school and even after I joined someone else’s group in the fall,
he was kind enough to take me back into his group when the other group didn’t work
out. Out of all of the people in my life, I think that Spence has inspired me the most.
The way he runs his group and the way in which he handles success is very inspiring
and I hope to be the same way some day.

Also, these past four years have been great thanks to my lab mates. I can say
something special about each and every person that has been in the group and is still in
the group. First, however, I should acknowledge Andrea, who has been my best friend
throughout grad school. It is hard to say if I will ever have the connection I have with
her with anyone else. She has been there to support me through so many times
(including near death experiencesEl) and I am genuinely going to miss seeing and
talking to her every day in lab. Thank you for everything you have done for me. I
love you. Chia and Nicole have also been there from the beginning and we have had a
ton of great times together! The rest of the group has been great and I will have great
memories of everyone.

My family also deserves a lot of credit for my success. Throughout the years
they have supported me and always were pushing me to do my best. Finally, I would
like to thank Jill. I know things have gone south with us in the past few months, but if

I had to pick a single person in my life who has given me the most hope and strength it

iv

has been you. You have supported me unconditionally throughout the past nine years
and I am eternally grateful for everything that you have done for me. I h0pe that the
future holds great things for us because we deserve it after everything we have been

through together.

TABLE OF CONTENTS

LIST OF TABLES ..................................................................................................... viii
LIST OF FIGURES ................................................................................................... ix
CHAPTER 1- INTRODUCTION
1.1 Diabetes Mellitus .................................................................................... 1
1.2 C-peptide History and its use as a Potential Therapeutic for Diabetes...3
1 .2.1 Background ............................................................................ 3
1 .2.2 Mechanism ............................................................................. 7
1 .2.3 Neuropathy ............................................................................. 13
1 .2.4 Nephropathy ........................................................................... 15
1.2.5 Retinopathy ............................................................................ 17
1 .2.6 Microcirculation ..................................................................... 18
References ...................................................................................................... 22

CHAPTER 2- DETERMINING EFFECT OF C-PEPTIDE ON ATP RELEASE AND
GLUCOSE TRANSPORT FROM RBCS

2.1 Diabetes and the Red Blood Cell ............................................................ 30

2.2 Mannose as an Inhibitor of Glycolysis ................................................... 32

2.3 Phloretin as an Inhibitor of GLUTl ........................................................ 33

2.4 Experimental ........................................................................................... 33

2.4.1 Collection and Preparation of RBCS ...................................... 33

2.4.2 Preparation of Reagents ......................................................... 35

2.5 Methods ................................................................................................... 36
2.5.1 Chemiluminescence Detection of RBC-derived ATP Release

after Incubation with C-peptide ............................................. 36

2.5.1.1 Flow-based assay ...................................................... 36

2.5.1.2 Determination of C-peptide Metal Content .............. 38

2.5.1.3 Non-ﬂow based assay ............................................... 38

2.5.2 Liquid Scintillation Counting to Determine Glucose Transport

into RBCS in the Presence of C-peptide ................................ 40

2.6 Results.. ................................................................................................... 41

2.7 Discussion ............................................................................................... 52

References ...................................................................................................... 63

CHAPTER 3- THE ROLE OF C-PEPTIDE IN TYPE 2 DIABETES AND THE
CONCEPT OF C-PEPTIDE RESISTANCE

3.1 Arguments Against C-peptide as a Bioactive Peptide ............................ 72
3.2 Experimental ........................................................................................... 75
3.2.1 Collection of RBCS .................................................................. 75

vi

3.2.2 Preparation of Reagents ........................................................... 76

3.3 Methods ................................................................................................... 76
3.3.1 ATP Release from Type 2 and Control Rat RBCS .................. 76
3.3.2 Mass Spectrometry Analysis .................................................... 77
3.3.3 Liquid Scintillation Counting to Determine Glucose
Accumulation in RBCS from Type 2 and Control Rats ........... 77
3.3.4 C-peptide ELISA ..................................................................... 78
3.3.5 Phosphatidyl Serine Determination Using Annexin V ............ 80
3.4 Results ..................................................................................................... 80
3.5 Discussion ............................................................................................... 95
References ...................................................................................................... 101

CHAPTER 4- DETERMINING THE MECHANISM BY WHICH C-PEPTIDE IS

INTERACTING WITH RBCS
4.1 Current Proposed Mechanism of C-peptide In Vivo .............................. 105
4.2 PTX and Mastoparan-7 (MAS-7) and their Ability to Inhibit or Activate
G-proteins ............................................................................................... 107
4.3 Inhibitors of Proteins Involved in the Deformation Induced ATP Release
Mechanism .............................................................................................. 109
4.4 Experimental ........................................................................................... 111
4.4.1 Collection and Preparation of RBCS ........................................ 111
4.4.2 Preparation of Reagents ........................................................... 112
4.4.2.1 Preparation of Antagonists used to Inhibit ATP Release
Pathway ...................................................................... 112
4.5 Methods ................................................................................................... 113
4.5.1 ATP Release from RBCs upon Incubation with Various
Inhibitors .................................................................................. 1 13
4.5.2 Fluorescence Determination of cyclic AMP (CAMP) Levels
Upon Incubation of RBCS with C-peptide ............................... 114
4.5.3 Glucose Transport of RBCS Incubated with C-peptide and
Inhibitors ................................................................................. 1 14
4.6 Results ..................................................................................................... 115
4.7 Discussion ............................................................................................... 123
References ...................................................................................................... 1 30

CHAPTER 5- CONCLUSIONS AND FUTURE DIRECTIONS

5.1 Overall Conclusions ................................................................................ 134
5.2 Future Directions .................................................................................... 139
References ...................................................................................................... 141

vii

1‘ '-

LIST OF TABLES

Table 4.1: Cell signaling elements activated by C-peptide according to cell type ...106

viii

t

LIST OF FIGURES

Figure 1.1 Structure of proinsulin with the C-peptide amino acids labeled. The
arrows are directed at the amino acids that contain a negatively
charged R group at physiological pH ....................................................... 4

Figure 1.2 Mechanism of insulin/C-peptide secretion and the role of zinc ions in
the pancreatic beta cell. Zinc accumulates in the vesicles throughout
the insulin maturation, storage, and secretion processes. ........................ 6

Figure 2.1 The proposed mechanism of deformation-induced ATP release from
RBCS and the resultant mechanism of vasodilation. ............................... 31

Figure 2.2: A) The structure of D-mannose, which was used to inhibit glycolysis
in RBCS. B) The structure of phloretin, which was used to inhibit
GLUTl ..................................................................................................... 34

Figure 2.3 Luciferin/luciferase chemiluminescence reaction between ATP and
luciferin. ................................................................................................... 37

Figure 2.4 The top panel is a schematic of the experimental setup used to
determine ATP release from RBCs using the ﬂow-based assay. The
bottom panel is a schematic of the experimental setup used to
determine the ATP release from RBCS using the nonﬂow-based
assay. ........................................................................................................ 39

Figure 2.5: Determination of ATP release from rabbit RBCS. The data shown
are normalized values from the RBCS of 11 rabbits incubated in the
presence and absence of 1 nM C-peptide. ATP release (determined
by chemiluminescence assay) from cells incubated in C-peptide
(black bars) increased aﬁer 4 hours (p<0.005) and approximately
29-fold over a period of 8 hours compared with RBCS incubated
with no C-peptide (white bars). Error bars are 2’: SEM (n=11) ............... 42

Figure 2.6: RBCS obtained from human patients with diabetes (black bars)
released 64i13 nM ATP at 0 hour. After incubation with C-peptide
for 6 hours, the ATP release increased (p<0.005) to 260i39 nM. The
release of ATP from RBCS obtained from human control patients
(white bars) was 260i60 nM at 0 hour and 480i109 nM at 6 hours
(p<0.005). The C-peptide—metal adduct increased the ATP release
from the RBCS obtained from patients with type 2 diabetes to a value
that is statistically identical to that from RBCS of the healthy control
participants. Error bars areiSEM (n=7) .................................................. 44

ix

Figure 2.7: Nanoelectrospray ionization mass spectrometry analysis of C-
peptide and its metal adducts. Panel A shows a mass spectrum of
freshly prepared C-peptide in its triply charged (around 1007 m/z)
and doubly charged states (around 1510 m/z). Panel B shows a high-
resolution mass spectrum of the [M+3H+]3+ region from panel A
demonstrating that C-peptide contains F e2+ and F e3+ when freshly
prepared .................................................................................................... 45

Figure 2.8: Normalized ATP release from fresh RBCS after incubation with a
C-peptide solution in contact with a metal after 6 and 72 hours. At 6
hours the C-peptide solutions containing F e2+ (white bars) and Cr3+
(hatched bars) had similarly increased (p<0.005) ATP release. After
72 hours, the ATP release from RBCS incubated with the Fe2+
peptide solution decreased to a level similar to that from RBCS
without any peptide (black bars). The Cr3+ peptide solution
maintained its level of activity. Error bars are iSEM (n = 4) ................. 47

Figure 2.9: RBCS incubated with 10 nM Cr3+-activated C-peptide (P+Cr3+)
showed a signiﬁcant (p<0.005) increase in ATP release
(approximately 74%). However, when RBCS were incubated with
the GLUTl inhibitor phloretin (PR) prior to addition of P+Cr3+,
ATP release was only about 63% of the value for the RBCS alone.
Error bars are i SEM (n=8).) ................................................................... 49

Figure 2.10: ATP release of RBCS that had been incubated with mannose (M)
and P+Cr3+ showed a 57% decrease in ATP release against RBCS
incubated with P+Cr3+ alone, bringing ATP release to a level not
signiﬁcantly different from that in RBCS. However, in RBCS
incubated with P+Cr3+ the signal intensity was approximately 64%
higher than in RBCS alone (p<0.005) Error bars are iSEM (n=5). ........ 50

Figure 2.11: RBCS incubated with 1.554 Bq 14C-1abeled glucose in a solution
of PSS containing normal glucose. After 4 hours, the RBCS
incubated with 10 nM Cr3+-activated C-peptide (P+Cr3+) showed
an approximately 31% increase in radioactive counts per min (cpm),
while RBCs incubated with only C-peptide (P), Cr3+-activated C-
peptide plus the GLUTl inhibitor phloretin (PR) or PR alone all
resulted in no increase in CPM. Error bars are iSEM (n=4) .................. 51

Figure 3.1 A schematic representation of the setup used to measure the
radioactivity of RBC samples. ................................................................. 79

Figure 3.2 A schematic representation of the procedure followed to obtain the
C-peptide/RBC membrane interaction data (ELISA). ............................. 81

T ”1-1

Figure 3.3: Electrospray Ionization - Mass Spectrometry (ESI-MS) analysis of
an aqueous solution of 10 uM C-peptide containing 10 uM ZnCl-‘2.
The most abundant ion in the ‘normal’ mass spectrum (panel A)
corresponds to the triply charged precursor ion of Zn-bound C-
peptide ([M+H++Zn2+]3+). High resolution mass spectra of the
triply and doubly charged ions of the Zn-bound c-peptide ions
[M+H++Zn2+]3+ and [M+Zn2+]2+ are shown in panels B and C,
respectively. The isotopic distributions for these charge states are
consistent with those predicted for the Zn-bound C-peptide. .................. 83

Figure 3.4: The normalized ATP release of RBCS from type 2 rats and controls
alone (a), in the presence of 20 nM Zn2+-activated C-peptide (b), C-
peptide without Zn2+ (c), in the presence of 20 nM Zn2+-activated
C—peptide and 30 uM metforrnin (d), and 30 uM metforrnin with
RBCS alone (e). Error bars are :1: SEM (n=8 diabetic, n=4 control)
p< 0.001 ................................................................................................... 84

Figure 3.5: Normalized uptake of 14C-glucose by RBCS from diabetic and
control rats; (a) untreated RBCS, (b) RBCS incubated with 20 nM
Zn2+-activated C-peptide, (c) RBCS incubated with 20 nM Zn2+-
activated C-peptide and 30 uM metfonnin, (d) RBCS incubated with
20 nM Zn2+-activated C-peptide and 30 uM metforrnin and 30 uM
insulin, and (e) RBCS incubated with 30 uM metforrnin alone. Error
bars are :1: SEM (n=4), p< 0.001 .............................................................. 86

Figure 3.6: Normalized ATP release of rabbit RBCs incubated (a) alone, (b)
with 20 nM Zn2+ and C-peptide, (c) with 20 nM Zn2+, C-peptide
and 30uM metforrnin, (d) 30uM metforrnin and 20 nM Zn2+, and
(e) 20 nM Zn2+. Error bars are i SEM (n=3). * represents values
statistically different from RBCS alone, + represents values
statistically different from values indicated by connecting lines. P-

values < 0.001. ......................................................................................... 87

Figure 3.7: Normalized binding of anti-C-peptide antibodies for RBCS obtained
from type 2 and control rats: (a) untreated RBCS, (b) RBCs
incubated in the presence of 20 nM Zn2+-activated C-peptide, (c)
RBCS incubated with 20 nM Zn2+-activated C-peptide and 30 uM
metforrnin, (d) and (e) RBCs incubated with C-peptide (without
Zn2+) with 30 uM metfonnin (d), (e) 30 uM metforrnin with 30 uM
insulin, and (f) RBCS incubated with 20 nM Zn2+-activated C-
peptide, 30 uM metformin, and 30 uM insulin. Error bars are :h
SEM (n=4), p-values < 0.001 ................................................................... 88

Figure 3.8: The top left graphic represents the differences in PS levels of the

diabetic and control RBC membranes and the ability of annexin V to
bind to these membranes. The graphic on the right (top) represents a

xi

 

raw data ﬂuorescence spectrum that is typically obtained when
performing the FITC annexin V-binding experiment. The bottom
panel shows a quantitative determination of the ﬂuorescence
intensities (resulting from annexin V binding to PS) obtained upon
the incubation of diabetic (black bars) and control (gray bars) RBCS
with and without 30 uM metforrnin. Error bars are :1: SE (n=4), p-
values < 0.001. ......................................................................................... 92

Figure 3.9: The normalized ATP release of type 1 rat RBCS incubated in the
presence of Zn2+-activated C-peptide. Error bars are i SEM (n=5).
P values <0.001. ....................................................................................... 93

Figure 3.10: The normalized glucose transport in CPMs of RBCS from type 1
and control rats in the (presence of Zn2+-activated C-peptide. Error
bars are i SEM (n=4). P value <0.01. .................................................... 94

Figure 3.11 Under normoglycemic conditions the Zn2+-activated C-peptide is
able to interact with the cell membrane and stimulate an increase in
glucose transport into the RBC, which increases glutathione levels
and glycolysis in the cell, ultimately increasing the amount of ATP
released by the RBC. However, under hyperglycemic conditions,
the increased glucose concentration may result in an overall decrease
in glutathione levels due to an increased rate of the sorbitol pathway.
An increase in the sorbitol pathway would decrease lutathione,

resulting 1n the externalrzatron of PS, resulting 1n less Zn -act1vated
C-peptide interaction with the membrane. .............................................. 100

Figure 4.1: The proposed mechanism of ATP release from RBCS due to

mechanical deformationg’1 1’1 3 ............................................................... 108

Figure 4.2: Peptide sequence of the G-protein activator mastoparan-7. ................... 110

Figure 4.3: Normalized ATP release from rabbit RBCS in the presence of zinc-
activated C-peptide (10P+10Zn). Resultant ATP release after
incubation with pertussis toxin prior to zinc-activated C-peptide
(PTX+10P+10Zn). ATP release using G-protein activator
mastoparan-7 in combination with pertussis toxin (mas-7+PTX).
Error bars are :t SEM (n=7), p<0.001 ...................................................... 116

Figure 4.4: Normalized ATP release of RBCs incubated with Zn2+-activated
C-peptide (10P+10Zn) and RBCS incubated with Zn2+-activated C-
peptide and the adenylyl cyclase inhibitor MDL12,330a
(10P+IOZn+MDL). Error bars are 3: SEM (n=7), p<0.001 .................... 118

xii

Figure 4.5: Normalized ﬂuorescence intensity obtained using competitive
ELISA for CAMP levels and RBCS containing H-89 (RBCs+H-89),
Zn2+-activated C-peptide (10P+10Zn), and Zn2+-activated C-
peptide and H-89 (10P+IOZn+H-89). Error bars are at SEM (n=6),
p<0.001 .................................................................................................... 120

Figure 4.6: Normalized ATP release of RBCS incubated with Zn2+-activated
C-peptide (10P+10Zn) and Zn2+-activated C-peptide and H-89
(10P+IOZn+H89). Error bars are i SEM (n=4), p<0.001. ..................... 121

Figure 4.7: The normalized counts per minute (CPM) of RBCS incubated with
Zn2+-activated C-peptide (10P+10Zn). The resultant CPM after
inhibition with inhibitors (10P+10P+H89, 10P+IOZn+BIS). Error
bars are d: SEM (n=3) p<0.005. ............................................................... 122

Figure 4.8: Normalized ATP release of RBCS incubated with Zn2+-activated
C-peptide (10P+10Zn), Zn2+-activated C-peptide and BIS
(10P+IOZn+BIS) and RBCs with BIS alone (BIS). Error bars are :E
SEM (n=4), p<0.001. ............................................................................... 124

Figure 4.9: The grams (g) of glucose transported into RBCS using 14C-1abeled
glucose and various amounts of the CFTR inhibitor niﬂumic acid in
the presence of Zn2+-activated C-peptide. Error bars are i SEM
(n=5), p<0.01. .......................................................................................... 125

xiii

.'-

CHAPTER 1- INTRODUCTION

1.1 DIABETES MELLITUS

Currently, diabetes mellitus affects over 23 million Americans, and 180
million people worldwide with these numbers expected to double by the year 2030. In
2005 alone, 1.1 million deaths worldwide resulted from complications associated with
diabetes. Nearly 80% of all deaths associated with diabetes occur in countries with
low to moderate incomes. However, the World Health Organization expects that

deaths in middle to upper income countries will increase nearly 80% over the next 6

1
years.

Diabetes is a chronic disease that occurs when the body cannot produce or
utilize insulin effectively. A person is considered to be diabetic if their fasting blood
glucose level is above 126 mg/dl or reports a glucose level above 200 mg/dl after a
glucose challenge test. Hyperglycemia or elevated blood glucose levels, is often

associated with uncontrolled diabetes and may require exogenous insulin injections to

maintain proper blood glucose levels.1

Insulin is a peptide hormone that was ﬁrst discovered in 1921 by Canadian
scientists Banting and Best and was isolated from porcine pancreas. Up until their
discovery, diabetes was a virtual death sentence. The ﬁrst patients were administered
insulin a year later, however the quality of insulin was not very good and patients
often suffered complications due to impurities. Several modiﬁcations were made to

insulin, with the addition of the presence of zinc, which allowed for an increase in the

length of the effect of insulin. Eventually human insulin was successﬁrlly synthesized,
with variations of this peptide being used today.1

Diabetes can be categorized into 2 main types. Type 1 diabetes, which
accounts for approximately 10% of all diagnosed cases of diabetes, results from
immune-related destruction of pancreatic B-cells. It is theorized that type 1 diabetes
develops as the result of a virally induced immune response, where the body attacks its
own immune system, destroying the pancreatic B-cells. It has been hypothesized that
the body’s immune response to the Coxsackie virus, or German measles, may result in
an autoimmune reaction that destroys the B-cells. Another hypothesis suggests that
type 1 diabetes may be the result of an autoimmune attack in response to antibodies in
cow’s milk. Despite evidence suggesting a possible environmental cause of diabetes,

there have been over 20 genes discovered that inﬂuence susceptibility to type 1

. 1
diabetes.

People who are diagnosed with type 1 diabetes are often children or
adolescents, although diagnosis can occur at any age, and because of the destruction of
the pancreatic B-cells no insulin is being produced. Therefore these patients are
required to take insulin injections in order to maintain proper blood glucose levels.

Type 2 diabetes, which accounts for over 90% of all diagnosed cases of
diabetes, occurs when the body can no longer effectively use the insulin that is being
produced by the B-cells. Because of this, these patients are often termed “insulin-
resistant”. While most of the people diagnosed with type 2 diabetes have been
overweight adults, it is becoming more common for children and adolescents to be

diagnosed due to an increase in childhood obesity. However, several factors have

been found to attribute to the development of type 2 diabetes including, obesity,
family history, and/or age. 1

Initially these patients are given a low glucose diet in an attempt to maintain
proper blood glucose levels. However, this is often accompanied by exercise and
weight loss, especially in obese patients. The weight loss totals observed from these
patients is modest at best, which results in the need for an oral antidiabetic medication,
some of which aid in the uptake of glucose into various body tissues. As the disease
progresses and the oral antidiabetic drugs fail, hyperglycemia causes a destruction of

the B-cells and these patients are required to take insulin injections to maintain healthy
blood glucose levels.1

Both type 1 and type 2 diabetics suffer similar complications including,
cardiovascular disease, retinopathy, nephropathy, and neuropathy. However, the

severity and occurrence of these complications vary depending on the type of diabetes

and duration of the disease.

1.2 C-PEPTIDE HISTORY AND ITS USE AS A POTENTIAL
THERAPEUTIC FOR DIABETES
1.2.1 Background

C-peptide is a 31 amino acid peptide that is excreted in equimolar amounts to insulin

(ﬁgure 1.1). Since its discovery in 1967,2 it has not gained ground as a biologically

active peptide. Its primary function has been thought to maintain the proper folding of

Go

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096 9/
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G /
o o /
o. - 90.. . 0/
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Figure 1.1: Structure of proinsulin with the C-peptide amino acids labeled. The
arrows are directed at the amino acids that contain a negatively charged R group
at physiological pH.

gm

the A- and B- chains of insulin. However, within the past 15 years there has been
increasing evidence suggesting C-peptide is a biologically active peptide.
More speciﬁcally, when produced in the beta cells of the pancreatic islets,

. . . . . 3-8 2+ .
rnsulrn rs produced as a solrd hexamer around zrnc. There are two Zn rons per
. . 2+ . . .

rnsulrn hexamer, however Zn contrnues to accumulate even after 1t rs bound to the

hexamer. As shown in ﬁgure 1.2, prornsulrn, the peptide contarmng rnsulrn and C-
peptide, is located inside vesicles within the pancreas. Here, the insulin forms a

hexamer around the Zn2+, followed by the addition of more Zn2+ into the vesicle via a

zinc transporter protein. Eventually, the vesicle reaches the surface of the beta cell

where insulin, C-peptide, and Zn2+ are released out of the beta cell.

Interestingly, C-peptide contains several acidic amino acid residues including 4
glutamic acids and 1 aspartic acid residue. The glutamic acids in positions 3, 11, and

27 are well conserved throughout various mammalian species and are thought to play

an important role in cell signaling.9 More speciﬁcally, the glutamic acid present in

position 27 appears to be important for binding to human renal tubular cells,10 while

the glutamic acid in position 3 has been reported to play some role in receptor
interactions. Whether or not C-peptide is able to form a stable secondary structure is
debatable. A report by Ido er al. suggested that the N-terminal and C-terminal
segments of C-peptide were able to form helical segments, while there appeared to be
a non-polar turn-like structure in the middle of the peptide. These ﬁndings were

reported based on predictions from molecular modeling and CD spectroscopy.

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However, a more recent report by Henriksson et al. provide qualitative CD evidence

that C-peptide does not form a state secondary structure when in an aqueous

. ll . .
solution. The reasons for the discrepancy are not clear, although rt has been noted

that C-peptide was able to form a stable secondary structure in an aqueous media

. . . . 11
when subjected to hrgh concentrations of tnﬂuoroethanol. Of course, the presence of

such a high concentration of triﬂuoroethanol will not exist in physiological conditions
and is therefore unlikely to contribute to secondary structure in vivo.

1.2.2 Mechanism

The complete mechanism of how C-peptide exerts its effects in vivo is not fully
understood, with many variations in discoveries between cell types. Due to its

hormone-like actions, much of the recent C-peptide research has focused on ﬁnding a

C-peptide receptor. The ability of 125I--labeled C-peptide to bind to pancreatic B-cells

was ﬁrst demonstrated by Flatt et al. in 1986.12 However, various forms of C-peptide

including retro (reversed order amino acid sequence) and all-D-amino acid
enantiomers resulted in improvements in nerve function or vascular permeability

blood ﬂow, suggesting that C-peptide was not binding in a stereospeciﬁc manner to a

receptor.13 Based on these results it was hypothesized that the biological activity of
C-peptide was established largely on poorly deﬁned membrane interactions that took
place due to structural features related to the C-peptide sequence, but independent of

its direction or chirality.

C-peptide fragments have also been examined for biological activity using

Na+, K+-ATPase activity in rat renal tubular segments.14 The C-peptide carboxy

terminal pentapeptide, EVARQ (rat), elicited 100% of the activity of intact C-peptide,
whereas the remaining portion of the molecule without the last 5 amino acids, was
completely inactive. Using the human C-peptide pentapeptide sequence, EGSLQ, on
a rat model, it was able to elicit 75% activity. The behavior of the C-terminal
pentapeptide was typical of a peptide ligand interacting with a speciﬁc receptor.

In contrast, several C-peptide mid region sequences provided partial activity of
the intact peptide, but exhibited rather different properties. Activity relating to this
region was not seen with the des-(27-3l)-C-peptide, while several unnatural D-amino
acid containing sequences showed some activity. This behavior does not follow the

properties of peptide-receptor interactions, but is similar to the nonspeciﬁc type

. . . . . l3
interactions of C-peptrde wrth plasma membranes as prevrously postulated. How

the C-terminal region and midregion of the peptide manifest in vivo is not well
understood. Other recent work suggests that the presence of conserved glutamic acid

residues at positions 3, 11, and 27 of C-peptide and the presence of helix-promoting
residues in the N-terminal segment may be required for signaling pathways.9 The
overall picture now emerging suggests that the terminal sections are involved in
functional interactions, with the midregion forming a joining segment.9
Rhodamine-labeled human C-peptide binding to a variety of human cell
. . . . 15
membranes has now been earned out usrng ﬂuorescence correlation mrcroscopy.
High-afﬁnity speciﬁc binding of C-peptide was observed with an association rate

constant of approximately 3 nM. The maximal number of binding sites was

approximately 1000-1500 per cell located on human renal tubular cells. Binding could

be displaced by excess intact C-peptide and by the C-terminal pentapeptide, but not
scrambled C-peptide, insulin, or insulin-like grth factor. Binding of C-peptide to
the cell membranes could largely be inhibited by pertussis toxin pretreatment of the
cells, indicating the existence of a speciﬁc GTP-binding protein coupled receptor for

C-peptide, which interacts with the C-terminal pentapeptide region of the C-peptide

molecule.15 Despite these gains in ﬁnding a receptor for C-peptide, gene-cloning

strategies or using proteomic approaches have been unsuccessful thus far.

Much of the mechanistic research on C-peptide has focused on Na+, K+-

ATPase, which is a ubiquitous membrane-associated protein complex that uses energy

from the hydrolysis of ATP to drive the counter-transport of sodium and potassium

across the plasma membrane. It is widely accepted that impaired Na+, K+-ATPase

activity is present in a variety of cell types in diabetes and contributes to the

. . . . . 16-19
pathogenesrs of drabetrc complrcatrons.
. . + + . .
C-peptrde has been shown to activate Na , K -ATPase 1n rat kidney tubules at
. . 20 . . .
low to hrgh nanomolar concentratrons. Thrs effect, however, was abolished 1n the
. . . 2+
presence of pertussrs toxrn, and appeared to be dependent on intracellular Ca

. . . . 2+ . .
concentratrons after Incubation wrth the Ca -calmodulrn-dependent protern

phosphatase 2B inhibitor FK506. Interestingly, when rat kidney proximal tubule

+
segments were exposed to the C-terminal tetra- and pentapeptide fragments, full Na ,

K+-ATPase activity was observed. However, the midregion peptides elicited only

partial activity at best.14 These results, combined with C-peptide binding studies,15

support the emerging concept of C-peptide binding to a speciﬁc receptor resulting in
the activation of enzymes downstream.
In addition to studies performed on animal modes, research has also been

performed on human subjects. It was found that patients with type 1 diabetes with

complete C-peptide deﬁciencies had red blood cell (RBC) Na+, K+-ATPase activity
that was signiﬁcantly lower than healthy controls.21 Interestingly, in patients with

type 2 diabetes, the RBC Na+, K+-ATPase activity was signiﬁcantly lower in patients

treated with insulin compared to those treated with oral hypoglycemic agents,

indicating that the fasting C-peptide level was the only variable that correlated with

Na+, K+-ATPase activity.21 When C-peptide was infused into patients with type 1

diabetes, an increase in plasma cGMP and RBC Na+, K+-ATPase activity was
. 22

observed 1n a dose-dependent manner.

While it has been established that C-peptide increases the activity of Na+, K+-

ATPase, the exact mechanism is still incomplete. Using isolated rat kidney medullary

thick ascending limb tubules, C-peptide was found to activate Na+, K+-ATPase at
physiological concentrations.23 More speciﬁcally, treating these tubules with C-
peptide also resulted in phosphorylation of the Na+, K+-ATPase (Jr-subunit and

translocation of the Ca2+-dependent protein kinase C-a (PKC) to the membrane,

10

indicating activation. Consequently, both of these effects could be inhibited by a

speciﬁc inhibitor of PKC, GF109203X.

In addition to being able to increase the activity of Na+, K+-ATPase, C-peptide

has also been reported to activate endothelial NO synthase (eNOS).24 Early reports

demonstrated the ability of C-peptide to increase glucose utilization in streptozotocin-

induced diabetic rats, however the increase was determined to be sensitive to

inhibition of NOS by coadministration of N-monomethyl-l-arginine.25 Prompted by

these results, Wallerath provided details of the underlying mechanism by which C-

peptide had the ability to increase NO production in endothelial cells.24 Using bovine

aortic endothelial cells, it was reported that at a concentration of 6.6 nM, C-peptide

. . . . 2+
strmulated NO release as a consequence of an increase 1n rntracellular Ca . It was

. . . . . . 2+ . .
speculated that C-peptrde srgnalrng resulted 1n an actrvatron of the Ca -sensrtrve

eNOS, which largely explained the vasodilatory effects of C-peptide observed in vivo.
Additionally, it has also been shown (using the same cell line) that NO release is
enhanced by stimulation with C-peptide and was accompanied by upregulation of
eNOS gene transcription. This effect appeared to be independent of the upstream

phosphorylation and activation of extracellular signal-regulated mitogen activated

protein kinase (MAPK).26

MAPKs phosphorylate multiple targets on serine and threonine, and as a result,
control many critical cell functions such as growth, gene expression, and cell survival

and adaptation. Recently, reports have described the ability of C-peptide to induce

11

phosphorylation and activation of members of the MAPK family. Kitamura et al.
demonstrated the ability of C-peptide to activate ERK (a MAPK family member)

using both immunoblotting of phosphorylated ERK] and ERK2 from Swiss 3T3

27 . . . . . . . .
cells. Mrmmal actrvatron was achreved wrth concentratrons as low as 1 pM, wrth

maximal activation concentration of 1 nM C-peptide. The activation was abolished by
pretreatment of the cells with pertussis toxin. Additionally, neither retro C-peptide nor
all D-amino acid C-peptide stimulated ERK. However, other cell types were tested to

determine an activation of ERK by C-peptide and the results were not universal. No

effect was observed in 3T3-L1 cells, L6E9 muscle cells, HepG2 hepatoma cells,

NG108.15 neuroblastoma cells, or C6 glioma cells.27

Activation of ERK is also involved in eNOS gene transcription. A report by

Kitamura et al. attempted to bridge these 2 phenomena by examining the ability of C-

. . . . 28 . .
peptrde to actrvate transcrrptron factors. C-peptrde was shown to strmulate both p38

and ERK MAPK activities, but not c-Jun N-terminal kinase. In addition, C-peptide
activated the CAMP response element (CRE)-binding protein (CREB)/activating
transcription factor-1 (ATF-l) in a p38 dependent manner. As a result, it was

concluded that enhanced eNOS transcription in bovine aortic endothelial cells

following C-peptide treatment was indeed MAPK-dependent.26

Most recently, C-peptide has been shown to promote translocation of low

molecular weight GTP-binding protein Rho A, from the cytoplasm to the membrane of

human kidney proximal tubular cells.29 This effect was found to be completely

12

dependent upon the upstream activation of phospholipase C. Similar to other studies,
all stimulatory effects of C-peptide were abolished by pretreatment of the cells with
pertussis toxin.

The molecular mechanisms by which C-peptide is activating MAPK can now
be described with some common features. It has been determined that the C-peptide
signal transduction pathway involves: C-peptide binding to a pertussis toxin sensitive

GTP-protein coupled receptor, activation of phopholipase C, a subsequent increase in

intracellular Ca2+ levels stimulating PKC, PKC-dependent activation and

translocation of Rho A to plasma membranes, and phosphorylation and activation of
MAPKs.

1.2.3 Neuropathy

Neuropathy, or disease of the peripheral nervous system, is the most common
complication associated with diabetes and results in the highest morbidity. The
prevalence of diabetic neuropathy varies greatly depending on the variation of the
disease. For example, approximately 10% of patients develop signs of neuropathy
within a year of diagnosis of diabetes; however, this number may increase to as much
as 50% in patients with diabetes for over 25 years. Moreover, the rate of neuropathy

is much higher in type 1 diabetes, as close to 100% of these patients eventually

develop neuropathy.3O

The underlying causes of diabetic neuropathy are many and involve genetic

predispositions and several interrelated metabolic and molecular abnormalities due to

hyperglycemia, as well as insulin and C-peptide deﬁciencies“.36 Despite its

13

frequency and debilitating nature, there is no current sufﬁcient treatment for diabetic
neuropathy.

Recently, it has become apparent that hyperglycemia is not the sole
determinant in the development of diabetic complications, and increasing attention is

being given to insulin and/or C-peptide deﬁciencies. Both peptides have been shown

. . . . 34,37-43
to exert a number of metabolrc, neuroprotectrve, and antrapoptotrc effects.

More speciﬁcally, C-peptide has been shown to have potential therapeutic value for
29,42,44-57

the prevention and amelioration of type 1 diabetic complications.

It has been reported that C-peptide is able to elicit metabolic effects on various

cell types including renal tubular cells, rat sciatic nerve, pancreatic islets, granulation

22,5 8-60

tissue, and RBCS. Speciﬁcally, it has been reported that C-peptide elicits

concentration-dependent stimulation of Na+K+-ATPase activity, which has been
further supported by C-peptide’s effects on Na+K+—ATPase activity on rat sciatic
nerve Na+K+-ATPase activity in type 1 diabetic BB/W or rats treated with C-peptide

for 8 monthssg’6O This is supported by partial correction of the associated defect in

nerve conductance velocities and paranodal swelling. Additionally, using the BB/W or
rat model, it has been demonstrated that C-peptide replacement can normalize both the

insulin receptor and the insulin growth factor-I (IGF-I) receptor mRNA and protein in

. . . 38
perrpheral nerve and brarn trssue.

C-peptide has also been shown to prevent loss of both motor and sensory nerve

conductance velocities in addition to thermal hyperalgesia (increased sensitivity to

14

5,58

pain), which is a function of C-ﬁbers.3 Furthermore, C-peptide replacement

therapy has also been shown to signiﬁcantly prevent chronic functional abnormalities

. . 58 . .
1n terms of nerve functron. Other recent ﬁndrngs suggest that C-peptrde has a

ubiquitous effect on both myelinated and unmyelinated ﬁber populations; however,

none of these effects were demonstrated in patients who received insulin therapy

44,

alone. 61 Additionally, C-peptide replacement therapy has been reported to improve

axonal degenerative changes and resulted in signiﬁcant protection against myelinated
and unmyelinated ﬁber loss most likely mediated by the corrective effects on the

expression of neurotrophic factors transcending normalization of the expression of key

. . 58
neuroskeletal proterns such as B-tubulrns and neuroﬁlaments.

Moreover, type 1 rat models administered C-peptide exhibited mild changes in
early gene response and expression of neurotrophic factors and their receptors, tubulin,

and neuroﬁlaments in dorsal root ganglion, resulting in normalization of axonal

caliber grth and improvement of the elongation of regenerating ﬁbers.62 These

ﬁndings combined with the report of C-peptide replacement therapy resulting in
prevention of nerve ﬁber loss in type 1 BB/Wor rats58 suggests that impaired nerve
regeneration is more prominent in typel diabetic neuropathy and appears to be mainly
the result of impaired insulin/C-peptide action rather than hyperglycemia.

1.2.4 Nephropathy

Diabetic nephropathy is a progressive kidney disease that is caused by the angiopathy

(disease of the blood vessels) of capillaries in the kidney glomeruli. In many western

15

countries, it is the primary reason for dialysis treatment. In general, it is more
common in patients who have had diabetes for more than 15 years or who are above
age 50. The disease is progressive and may cause death in as little as 2 to 3 years after
diagnosis, with a higher mortality rate among men.

C-peptide has been shown to have a positive effect on renal function in human
subjects. After 3 months of C-peptide and insulin administration, type 1 diabetic

patients exhibited better glycemic control, a reduction of diabetes-induced glomerular
hyperﬁltration and microalbuminuria, 2 classic markers of nephropathy.63 In a similar
study, it was conﬁrmed that proteinuria was signiﬁcantly reduced in the group

receiving C-peptide supplementation compared to healthy controls.61 Similar results
. . 47
were found wrth regard to glomerular ﬁltratron rate.

Using animal models, it has been reported64 that perfusion of C-peptide results

in a signiﬁcant reduction of both proteinuria and diabetes-induced glomerular
hyperﬁltration, which is considered to be the initial state of diabetic nephropathy in

streptozotocin-induced diabetic rats. Also reported was the ability of C-peptide to
. 50,65
restore renal functional reserve and prevent glomerular hypertrophy. It was
determined in a separate study that the C-terminal pentapeptide EVARQ (rat) was
. 49
responsrble for most of the observed effects.

C-peptide has also been shown to improve glomerular and tubular function in
type 1 diabetic rats through in vivo C-peptide supplementation for one month. This

resulted in improved body weight in streptozotocin-induced diabetic rats and

16

decreased urinary sodium wasting.64 Additionally, it has been observed that in vivo

C-peptide supplementation for one month induced no changes in kidney abundance
and transcription status of several tubular sodium transporters including the epithelial
sodium channel (EnaC) and NKCC2/BSC1 cotransporter in diabetic rats, indicating
that C-peptide’s action is most likely not relying on changes in amounts of renal
tubule Na transporters.
1.2.5 Retinopathy
Diabetic retinopathy (DR) affects approximately 45% of people diagnosed with
diabetes, with nearly 100% of type 1 and 60% of type 2 patients developing DR after
20 years of being diagnosed with diabetes. Like other complications associated with
diabetes, hyperglycemia is a determinant in the initiation as well as the progression of
DR.

The various biochemical abnormalities associated with hyperglycemia
including increased oxidative stress, nonenzymatic glycation of proteins, activation of
the polyol pathway, up-regulation of growth factors, vasoactive peptides, and

cytokines, cause pathological changes resulting in the loss of retinal capillary

pericytes.66 The pericytes lead to the formation of capillary ghosts, the thickening of

. . . . 67
the retrnal caprllary basement membrane, and neovascularrzatron. It has also been

reported that basement membrane thickening is accompanied by increased alpha-1

(IV) collagen and ﬁbronectin expression.68

It has been reported that ﬁbronectin is upregulated due to diabetes in addition

to being involved in retinal capillary basement membrane thickening.69 Diabetic

17

BB/W or rats having a diabetic duration of 8 months, displayed increased expression of
total ﬁbronectin as well as oncofetal ﬁbronectin compared to age-matched nondiabetic
controls. Interestingly, C-peptide treatment completely normalized the diabetes-
induced oncofetal ﬁbronectin up-regulation. However, using BBDRZ/W or rats (type

2 diabetes model having normal C-peptide levels) resulted in no alteration in oncofetal

ﬁbronectin expression levels.

1.2.6 Microcirculation

Many of these above complications including peripheral neuropathy, nephropathy, and
retinopathy constitute a component of the microvascular complications also associated
with diabetes. More speciﬁcally, patients with type 1 diabetes have increased
microvascular blood ﬂow, shear stress, and tangential pressure on the microvascular
endothelium. Other factors affecting microvascular blood ﬂow include increased
leukocyte-endothelial adhesion, blood viscosity, and changes to the hemodynarnic
properties of RBCS.

The role of the vascular endothelium for blood ﬂow regulation has been

3

. . 7 . . . .
studied extensrvely over the past decade. In addrtron to servrng as a barrier

between the circulating blood cells and the smooth muscle cells, the endothelium
serves as a facilitator of a complex array of signaling between the vessel wall and the
blood. Several transmitters are released from endothelial cells including NO,
endothelin 1, prostaglandins, thrombin, substance P, bradykinin, and serotonin.

NO release from endothelial cells is particularly important because it has been

identiﬁed as the primary vasodilator released from the these cells.73 Importantly,

18

impaired endothelial function and reduction in endothelial NO release are early
features of type 1 diabetes and thought to be a principal cause of morbidity and
mortality among these patients.

C-peptide, as previously mentioned, is absent among patients with type 1

diabetes. However, C-peptide has been reported to signiﬁcantly enhance the release

of NO from bovine aortic endothelial cells in a dose-dependent manner24’74 in the

physiological range of 1-6 nM. It was hypothesized that C-peptide was causing the

. . . . . . 2+ . .
Increase 1n NO productron by rncreasrng rntracellular Ca concentratrons 1n the

bovine aortic endothelial cells. Another study by Kitamura et al. demonstrated that C-

peptide was able to stimulate NO production in aortic endothelial cells of Wistar rats

by enhancing the MAPK dependent transcription of eNOS.28

The improvements in NO production due to C-peptide can be translated into
improvements in microvascular blood ﬂow. Several studies report C-peptide’s ability

to affect microvascular blood ﬂow and improve nerve or renal function in animal

61 ,75,76

models of type 1 diabetes and in humans with type 1 diabetes. C-peptide,

when given subcutaneously twice daily for 5 weeks in control rats and streptozotocin-
induced diabetic rats, has been reported to reduce the diabetes induced blood ﬂow in
the anterior uvea, retina, and sciatic nerve. These studies were performed using
supraphyiological concentrations of C-peptide near 10 nM. Interestingly, no effect of

C-peptide, neither on microvascular blood ﬂow nor motor nerve conductance velocity,

could be observed in healthy control rats.13

19

Improvements in blood ﬂow have also been demonstrated in the skeletal

muscle of type 1 diabetic patients during exercise.47 Prior to C-peptide therapy, the

blood ﬂow and capillary diffusion capacity of the exercising forearm were
approximately 30% lower compared to healthy controls at baseline. Intravenous
administration of C-peptide increased forearm blood ﬂow by 27% and capillary
diffusion capacity by 52%, similar to levels to observed in healthy controls. No
signiﬁcant changes in blood ﬂow were observed in healthy controls given insulin and
C-peptide or in diabetics receiving placebo infusion.

In addition to improved blood ﬂow in the muscle of the forearm, C-peptide has

also been shown to improve blood ﬂow in the skin.33 While total skin perfusion is

increased in patients with diabetes, nutritional capillary skin blood ﬂow has been

77-79 . . . . .
shown to be reduced. Patients grven short-term rnfusron of C-peptrde saw a

redistribution of microvascular blood ﬂow from the subpapillary thermoregulatory
blood ﬂow into the nutritive capillary bed. C-peptide supplementation in type 1
diabetic patients increased capillary blood ﬂow to a level comparable to that observed
in healthy controls. No such effect of C-peptide supplementation was observed in
non-diabetic subjects.

At a molecular level, it has been determined that the deformability of RBCS

obtained from patients with type 1 diabetes are less deformable compared to healthy

controls.74 The deformability was tested under physiological and supraphysiological

shear stress rates by means of laser diffractoscopy. Incubation of RBCs from type 1

patients and healthy controls with different concentrations of C-peptide restored RBC

20

deformability in type 1 patients, but did not affect the RBCS of non-diabetic controls.
However, pretreatment of RBCS from type 1 diabetic patients with ouabain, pertussis
toxin, or EDTA completely abolished C—peptide effects on RBC deformability.
Despite all of the reports described above suggesting that C-peptide is a
biologically relevant peptide, there are still doubts surrounding its relevance. Namely,
the lack of a successful long-term clinical trial, the lack of efﬁcacy in patients with

type 2 diabetes, and the lack of a deﬁned receptor are all common arguments against

C-peptide being biologically relevant.80 The ultimate goal of this dissertation. is to

better explain these arguments and provide evidence supporting that C-peptide is a

biologically relevant peptide.

21

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46. Hansen A, Johansson B-L, Wahren J, Von Bibra H. C-peptide exerts beneﬁcial
effects on myocardial blood ﬂow and function in patients with type 1 diabetes.
Diabetes. 2002;51:3077-3082.

47. Johansson BL, Sjoeberg S, Wahren J. The inﬂuence of human C-peptide on

renal function and glucose utilization in type 1 (insulin-dependent) diabetic patients.
Diabetologia. 1992;35:121-128.

48. Kunt T, Forst T, Pfutzner A, Beyer J, Wahren J. The physiological impact of
proinsulin C-peptide. Pathophysiology. 1999;5z257-262.

49. Nordquist L, Lai EY, Sjoequist M, Patzak A, Persson AEG. Proinsulin C-
peptide constricts glomerular afferent arterioles in diabetic mice. A potential
renoprotective mechanism. Am J Physiol. 2008;2942R836-R841.

50. Samnegard B, Jacobson SH, Jaremko G, Johansson B-L, Sjoquist M. Effects of
C-peptide on glomerular and renal size and renal function in diabetic rats. Kidney
International. 2001 ;60: 1258-1265.

51. Schmidt Robert E, Dorsey Denise A, Beaudet Lucie N, Parvin Curtis A, Zhang
W, Sima Anders AF. Experimental rat models of types 1 and 2 diabetes differ in

sympathetic neuroaxonal dystrophy. Journal of neuropathology and experimental
neurology. 2004;63:450-460.

52. Shafqat J, Melles E, Sigrnundsson K, et al. Proinsulin C-peptide elicits
disaggregation of insulin resulting in enhanced physiological insulin effects. Cell Mol
Life Sci. 2006;63:1805-1811.

26

53. Sima AAF. Pathological mechanisms involved in diabetic neuropathy: can we
slow the process? Current Opinion in Investigational Drugs (Thomson Scientiﬁc).
2006;7:324-337.

54. Sima AAF, Kamiya H. Insulin, C-peptide, and diabetic neuropathy. Science &
Medicine (N arberth, PA, United States). 2004;9:308-319.

55. Sima Anders AF, Li Z-G, Zhang W. The insulin-like growth factor system and
neurological complications in diabetes. Exp Diabesity Res 2003;4z235-256.

56. Wahren J, Ekberg K, J oemvall H. C-peptide and neuropathy in type 1 diabetes.

Immunology, Endocrine & Metabolic Agents in Medicinal Chemistry. 2007;7:69-77.

57. Zhang W, Kamiya H, Ekberg K, Wahren J, Sima AAF. C-peptide improves
neuropathy in type 1 diabetic BB/Wor-rats. Diabetes/Metabolism Research and
Reviews. 2007;23 :63-70.

58. Sima AAF, Zhang W, Sugimoto K, et a1. C-peptide prevents and improves
chronic Type I diabetic polyneuropathy in the BB/Wor rat. Diabetologia.
2001;44:889-897.

59. Wahren J, Ekberg K, Johansson J, et al. Role of C-peptide in human
physiology. American Journal of Physiology. 2000;278:E759-E768.

60. Zhang W, Yorek M, Pierson CR, Murakawa Y, Breidenbach A, Sima AA.
Human C-peptide dose dependently prevents early neuropathy in the BB/Wor-rat.
International journal of experimental diabetes research. 2001;2:187-193.

61. Johansson BL, Borg K, Femqvist-Forbes E, Kemell A, Odergren T, Wahren J.
Beneﬁcial effects of C-peptide on incipient nephropathy and neuropathy in patients
with type 1 diabetes mellitus. Diabetic Medicine. 2000;17:181-189.

62. Pierson CR, Zhang W, Sima AAF. Proinsulin C-peptide replacement in type 1
diabetic BB/Wor-rats prevents deﬁcits in nerve ﬁber regeneration. Journal of
Neuropathology and Experimental Neurology. 2003;62:765-779.

63. Johansson BL, Kemell A, Sjoberg S, Wahren J. Inﬂuence of combined C-

peptide and insulin administration on renal function and metabolic control in diabetes
type 1. The Journal of clinical endocrinology and metabolism. 1993;77:976-981.

27

“r

.i

64. Rebsomen L, Pitel S, Boubred F, et al. C-peptide replacement improves weight
gain and renal function in diabetic rats. Diabetes & metabolism. 2006;32:223-228.

65. Sjoquist M, Huang W, Johansson B-L. Effects of C-peptide on renal function
at the early stage of experimental diabetes. Kidney International. 1998;54:758-764.

66. Cai J, Boulton M. The pathogenesis of diabetic retinopathy: old concepts and
new questions. Eye. 2002;16:242-260.

67. Tsilibary EC. MicrovasCular basement membranes in diabetes mellitus. J
Pathol. 2003;200:537-546.

68. Evans T, Deng DX, Chen S, Chakrabarti S. Endothelin receptor blockade _
prevents augmented extracellular matrix component mRNA expression and capillary

basement membrane thickening in the retina of diabetic and galactose-fed rats.
Diabetes. 2000;49:662-666.

69. Komblihtt AR, Pesce CG, Alonso CR, et al. The ﬁbronectin gene as a model
for splicing and transcription studies. Faseb J. 1996;10:248-257.

70. Chen S, Khan ZA, Cukiernik M, Chakrabarti S. Differential activation of NF-
kB and AP-l in increased ﬁbronectin synthesis in target organs of diabetic
complications. Am J Physiol. 2003;284zE1089-E1097.

71. Calles-Escandon J, Cipolla M. Diabetes and endothelial dysﬁmction: a clinical
perspective. Endocr Rev. 2001;22:36-52.

72. Kuo L, Davis MJ, Chilian WM. Myogenic activity in isolated subepicardial
and subendocardial 'coronary arterioles. Am J Physiol. 1988;255:H1558-1562.

73. Palmer R, Ferrige MJ, Moncada S. Nitric oxide release accounts for the
biological activity of endothelium-derived relaxation factor. Nature. 1987;327:524-
526.

74. Kunt T, Schneider S, Pfutzner A, et al. The effect of human proinsulin C-
peptide on erythrocyte deformability 1n patients with type I diabetes mellitus.
Diabetologia. 1999; 42: 465-471.

28

75. Cotter MA, Ekberg K, Wahren J, Cameron NE. Effects of proinsulin C-peptide
in experimental diabetic neuropathy: Vascular actions and modulation by nitric oxide
synthase inhibition. Diabetes. 2003 ;52: 1812-1 817.

76. Kamiya H, Zhang W, Ekberg K, Wahren J, Sima Anders AF. C-Peptide
reverses nociceptive neuropathy in type 1 diabetes. Diabetes 2006;55:3581-3587.

77. Jomeskog G, Brismar K, F agrell B. Skin capillary circulation is more impaired
in the toes of diabetic than non-diabetic patients with peripheral vascular disease.
Diabet Med. 1995;12:36-41.

78. Jomeskog G, Brismar K, Fagrell B. Skin capillary circulation severely
impaired in toes of patients with IDDM, with and without late diabetic complications.
Diabetologia. 1995;38:474-480.

79. J omeskog G, Ostergren J, Tyden G, Bolinder J, Fagrell B. Does combined
kidney and pancreas transplantation reverse functional diabetic microangiopathy?
Transpl Int. 1990;3:167-170.

80. Luzi L, Zerbini G, Caumo A. C-peptide: a redundant relative of insulin?
Diabetologia 2007;50:500-502.

29

CHAPTER 2- DETERMINING EFFECT OF C-PEPTIDE ON ATP RELEASE

AND GLUCOSE TRANSPORT FROM RBCS

2.1 DIABETES AND THE RED BLOOD CELL (RBC)

RBCs have generally considered to be bags of hemoglobin, having the primary
function to carry oxygen to locations throughout the body. RBCs can be characterized
as being biconcave disc-shaped cells ranging in size between 6-8 pm in diameter and
having a thickness of approximately 2 um, which is signiﬁcantly smaller than other
mammalian cells. These cells contain no nucleus or other organelles, a fact that has
rendered these cells to have no other function than to carry oxygen throughout the
body.

However within the past 2 decades, RBCs have been shown to release

adenosine triphosphate (ATP), a recognized stimulus of nitric oxide (NO), which is a
potent vasodilator.1 When RBCS are subjected to hypoxia, a decrease in pH,
molecular stimuli, or mechanical deformation, high nanomolar to low micromolar

amounts of ATP are released?4 The mechanism of ATP release from RBCs is well

established and includes activation of a heterotrimeric G protein, which activates
adenylyl cyclase. Adenylyl cyclase converts ATP into cyclic AMP (CAMP), which

activates protein kinase A (pka), phosphorylating the cystic ﬁbrosis transmembrane

conductance regulator (CFTR) protein as shown in ﬁgure 2.1.5 The exact manner in

which ATP is released from the RBC is still unknown; however the previously

mentioned factors are required.

30

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31

Once released from the RBCS, ATP has the ability to activate the sz receptors on the

. . . 4,6,7 . . .. . . .
endothelral cells lrmng the vascular wall. Thrs rnrtrates nrtrrc oxrde synthase

(NOS) activation using L-arginine along with NADPH and 02 as substrates to produce
the relaxing factor, NO, which then diffuses to the underlying smooth muscle cells and
subsequently triggers vessel dilation. Studies showed that when using NG-nitro-L-
arginine methyl ester (L-NAME), a NOS inhibitor, along with RBCs in the pulmonary

circulation, the arterial pressure increased. This suggests that NO is an important

. . . . 8
factor in deterrnrnrng vascular resrstance.

Despite the lack of a known exact mechanism, there is an abundance of
evidence pointing to C-peptide being a biologically active peptide, particularly the
evidence of C-peptide improving microcirculation.9-12 As previously discussed in
chapter 1, a report by Kunt et al.10 reported that incubation of RBCs from patients
with type 1 diabetes, who typically have RBCS that are less deformable than healthy

human controls,13 with C-peptide resulted in an increase in the deformability of the

RBC over a period of 8 hours. Previous work in our group has shown that when an
RBC is under considerable oxidative stress, as such is the case with the RBCS from

patients with type 1 diabetes, the ATP release from the RBCs are lower than those of

healthy human controls.14 Taken collectively, it was hypothesized that incubation of

RBCS with C-peptide may lead to an increase in ATP release from these RBCS.

2.2 MANNOSE AS AN INHIBITOR OF GLYCOLYSIS

32

D-mannose (ﬁgure 2.2a) was chosen to inhibit glycolysis because mannose does not
interfere with other cellular processes, and while it is able to be converted into D-

mannose-6-phosphate by hexokinase, it is not able to be converted further by the more

speciﬁc enzyme glucose-6-phosphate dehydrogenase.15

2.3 PHLORETIN AS AN INHIBITOR OF GLUTl

Phloretin (ﬁgure 2.2b), a compound that has been reported to inhibit L-type Ca2+

channels, has also been shown to inhibit GLUTl, the most common glucose

transporter present in RBCS.16 The mechanism of how phloretin inhibits GLUTl is

poorly understood, however it has been determined that phloretin docks to a binding

site near the extracellular opening of the transport channel. I 7

2.4 EXPERIMENTAL

2.4.1 Collection and Preparation of RBCs

Rabbits (male New Zealand whites, 2.0-2.5 kg) were anaesthetised with ketamine (8
ml/kg, i.m.) and xylazine (1 mg/kg, i.m.) followed by pentobarbital sodium (15 mg/kg
iv.) A cannula was placed in the trachea and the animals were ventilated with room
air, having a ﬂow rate of 20 breaths per min (10 cc air). A catheter was placed into a
carotid artery for administration of heparin and for phlebotomy. After heparin (500
units, iv), the animals were exsanguinated. Human blood was obtained by
venipuncture without the use of a tourniquet (antecubital fossa) and collected into a
heparinized syringe. Blood was centrifuged for 10 min at 500 g and 4°C. The plasma

and buffy coat were discarded. The RBCS were resuspended and washed three times

33

 

OH

HO OH

OH 0

Figure 2.2: A) The structure of D-mannose, which was used to inhibit glycolysis
in RBCS. B) The structure of phloretin, which was used to inhibit GLUTI.

.34

in a physiological salt solution (PSS) (in mM: 4.7 KCl, 2.0 CaClz, 140.5 NaCl, 12
MgSO4, 21.0 tris[hydroxymethyl]aminomethane, 11.1 dextrose with 5% w/v bovine
serum albumin [ﬁnal pH 7.4]). Cells were prepared on the day of use and
experiments were ﬁnished within 8 hours of removal from the animal or human
participants. All studies were approved by the Animal Investigation Committee at
Wayne State University.

2.4.2 Preparation of Reagents

Human C-peptide (American Peptide, Sunnyvale, CA, USA), was prepared by
dissolving 0.25 mg (molecular mass 3020), in 10 ml puriﬁed water (DDW, 18 M0) to

yield a concentration of 83 nM.
. 3+ 2+ . . .
The solutrons of Cr and Fe (derrved from chromrc chlorrde hexahydrate and

ferrous ammonium sulphate hexahydrate, respectively) were prepared in DDW
(80uM). The metal solutions were then added in equimolar amounts to the C-peptide
solution (also in puriﬁed water) through a series of dilutions.

Mannose was prepared by dissolving 9.9 mg of D-mannose in PSS, yielding a 5.5
mM solution of D-mannose.

Phloretin was prepared by dissolving 0.100 g (Sigma-Aldrich) in an appropriate
amount (approximately 1.5 m1) of dirnethyl sulfoxide, followed by dilution to 50.0 ml
in phosphate-buffered saline (PBS) yielding a ﬁnal concentration of 0.5 mM.

For the glucose transport studies, experiments were performed using RBCs that
had been washed in a low glucose concentration (0.55 mM) PSS. The solution

preparation for C-peptide and all inhibitors remained the same as for previous studies.

35

r “m ﬂ

Additionally, 1.554 Bq 14C-labeled glucose were added to the PSS. This created a

ratio of 14C-labeled glucose : non-radiolabeled glucose of 1:10.

Luciferin/luciferase was prepared by dissolving 2 mg of D-luciferin in 5 ml of
DDW. This solution was then added to a vial of ﬁreﬂy tail lantern extracts (50 mg) to

increase sensitivity, which already contained luciferase (ﬁgure 2.3).

2.5 METHODS
2.5.1 Chemiluminescence Detection of RBC-derived ATP Release after
Incubation with C-peptide
2.5.1.1 Flow-based Assay
To determine ATP release from RBCS, a 500 pl syringe was ﬁlled with the
luciferin/luciferase mixture. RBCs were loaded into a second 500 pl syringe. The
RBC suspension was prepared by placing an appropriate aliquot of packed RBCS into
a centrifuge tube followed by dilution with an appropriate volume of PSS, resulting in
an RBC suspension with a hematocrit of 7%. Attached to each syringe were 30 cm
segments of fused silica microbore tubing (Polymicro, Phoenix, AZ) having an
internal diameter of 50 um. Each syringe was positioned onto a syringe pump
(Harvard Apparatus, Hollinston, MA) set at 6.70 til/min. Each mixture was pumped
through the tubing where the solutions were combined in a light-excluding box at a
mixing T-junction having an internal volume of 560 nl (Upchurch Scientiﬁc, Oak
Harbor, WA). Once combined, the solution was continued over a segment of
microbore tubing having an internal diameter of 75 pm. This larger diameter section

had its external polyimide coating removed to allow for the resulting light from the

36

w > < i ..I~l ‘1’
NO x“ S S i O O 0
m-B-o-lf—o-lé-o-cuz
o

 

  

Luciferin
a. . OH
ATP
. Fireﬂy
Reaction 1 Luciferase H:
+ M92+ / N
k, .>
/ N 5' (if: g I
. H 0- +043“:
'. \_ s 8 ”.H (I) PPi
. . H H

Adenyl-Iuciferin

. 0
Reaction 2 2 _
Luminescence

o
N Nj/o
.O‘sHsl + H20 + 002

Figure 2.3: Luciferin/luciferase chemiluminescence reaction between ATP and
luciferin.

37

chemiluminescence reaction to be detected by a photomultiplier tube (PMT)
(Hamamatsu Corporation, Hamamatsu, Japan) as shown in ﬁgure 2.4.

For RBC samples containing C-peptide, 60 pl of the 83 nM working C-peptide
solution were added to a centrifuge tube, followed by the addition of 4440 ul PSS to
buffer the aqueous C-peptide solution, followed by approximately 500 pl of RBCS to
result in a suspension containing a 1 nM C-peptide solution in an RBC suspension
having a hematocrit of 7%. The RBC suspension was incubated for a total of 8 hours
with the ATP release measured at 2 hour intervals. The syringe was rotated
periodically to ensure a homogenous mixing of the RBCs.
2.5.1.2 Determination of C-peptide Metal Content
Experiments were performed using a Thermo model LTQ linear ion trap mass
spectrometer (Thermo, San Jose, CA, USA). Samples were prepared by dissolving
synthetic C-peptide in DDW at a concentration of 8.3 nM. The spray voltage was
maintained at 2.0 kV. The heated capillary temperature was 250°C.
2.5.1.3 Non-ﬂow based Assay
To determine the ATP released from RBCS under non-ﬂow conditions, a plastic
cuvette containing 200 pl of RBCS followed by the immediate addition of 100 pl of
luciferin/luciferase was gently shaken for 15 seconds and placed over the PMT inside
of a light-excluding box, where the resulting light from the chemiluminescence
reaction was detected (ﬁgure 2.4).

The RBC suspensions were prepared by adding 600 pl of the 83 nM C-peptide

working solution and 490 pl of the 102 nM Cr3+ solution to a 15 ml centrifuge tube.

Next, 3410 ul of PSS was added to the aqueous C-peptide/Cr3+ solutions, followed by

38

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39

the immediate addition of RBCS, resulting in an RBC suspension with a 10 nM C-
peptide/metal concentration and a hematocrit of 7%. The RBC solution was allowed
to incubate for a period of 6 hours prior to the measurement of ATP release. To
determine the ability of mannose to inhibit ATP release from RBC, 500 pl of a 5.5

mM solution of D-mannose that had been prepared in the PSS was added to the 7%

hematocrit RBCs suspensions with and without the 10 nM C-peptide and Cr3+. The

resultant ATP release was measured after 6 hours using the chemiluminescence assay
described above.

For the GLUTl inhibition studies, 1.5 ml of the 0.5 mM phloretin solution
were added to 1090 ul of DDW containing 10 nM C-peptide and metal; after the
addition of 500 pl of RBCS to the phloretin/peptide mixture, the solution was diluted

using 1910 a] of PSS to a ﬁnal volume of 5 ml, resulting in a 7% suspension of RBCs

in the presence of 10 nM C-peptide and Cr3+. This solution was allowed to incubate

for a period of 6 hours, followed by measurement of the ATP release from the RBCS

using the same luciferase assay for ATP as described above.

2.5.2 Liquid Scintillation Counting to Determine Glucose Transport into RBCS
in the Presence of C-peptide

Experiments were performed using RBCS that had been washed in a low glucose

concentration (0.55 mM) PSS. The solution preparation for C-peptide and all

inhibitors remained the sarrre as for previous studies. Additionally, 1.554 Bq 14C-

labeled glucose were added to the PSS. This created a ratio of 14C-labeled glucose :

non-radiolabeled glucose of 1:10 to allow for fair competition between the labeled and

40

’_—"’l

the non-radiolabeled glucose. RBCS were added to create a 7% hematocrit solution
that was allowed to incubate for a period of 4 hours. After centrifugation (at 500 g),
the RBCs were combined with a scintillation cocktail (1 ml) and the radioactivity was

measured using a scintillation counter over a period of 3 min for each sample.

2.6 RESULTS

The ATP released by RBCS was measured using an established chemiluminescence

,

assay described above.1 Aliquots of RBC sample were incubated in the presence

or absence of 1 nM C-peptide and the resultant ATP release was measured every 2
hours for a period up to 8 hours. Figure 2.5, which shows the normalized values of
ATP released from the RBCs of n=11 rabbits in the presence and absence of C-
peptide, indicates that the addition of C-peptide resulted in an increased release of
ATP from the RBCS. The increase in RBC-derived ATP observed over the 8 hour
time period was almost three times greater than that of the RBCS incubated with a
control (buffer without C-peptide). The increase in measured extracellular ATP release

was not due to cell lysis, based on the inhibition of ATP release when the RBCS were
incubated in glibenclamide, a substance known to inhibit ATP release from RBCS.l9

Under these conditions, if cell lysis was occurring, the glibenclamide would have no

affect on the lysed RBCS ATP release.
It has been reported that the ATP released from RBCS obtained from the whole

blood of patients with type 2 diabetes is approximately only 50% of that released from

the RBCs of healthy control patients. 14 It was concluded that the RBCS of the patients

41

 

 

 

Normalized ATP Release
N

 

 

 

 

 

 

 

 

 

 

 

O I I I l 1
0 2 4 6 8
Time (Hours)

 

 

Figure 2.5: Determination of ATP release from rabbit RBCS. The data shown are
normalized values from the RBCS of 11 rabbits incubated in the presence and
absence of 1 nM C-peptide. ATP release (determined by chemiluminescence
assay) from cells incubated in C-peptide (black bars) increased after 4 hours
(p<0.005) and approximately 2.9-fold over a period of 8 hours compared with
RBCs incubated with no C-peptide (white bars). Error bars are :1: SEM (n=11).

42

with diabetes may have released less ATP due to oxidative stress within the RBCS

leading to a less deformable cell. A decrease in RBC deformability is a recognized

. . . . . 20,21 .
trart of the RBCS obtarned from patients wrth diabetes. Moreover, rt has recently

been reported that a decreased release of ATP from diabetic RBCs may be due to an

. . . . . 22 . . . .
rnactrvatron of the G, protern subunit. Due to the abrlrty of the C-peptrde to increase

ATP release from the RBCS of healthy rabbits, it was anticipated that the C-peptide
may be able to increase the ATP release from the RBCS of patients with type 2
diabetes. The data in ﬁgure 2.6 show that, not only does the C-peptide have the ability
to increase the ATP released (63.6 i: 12.6 nM ATP at 0 hours) from the RBCs of
patients with type 2 diabetes, but it also has the ability to restore ATP levels (256.1 i

38.7 nM, n = 7) to a value that is statistically equivalent to that of healthy control

. l 4
patients.

Interestingly, the C-peptide exhibited signiﬁcantly reduced activity, with
respect to its ability to increase the concentrations of RBC-derived ATP, after a period
of 24-36 hours following its preparation in DDW . Analysis of the C-peptide by
electrospray ionization mass spectrometry (ESI-MS) indicated that the peptide had not
undergone any signiﬁcant modiﬁcation or degradation during this time, even after

remaining in solution for periods > 30 days (ﬁgure 2.7 top). In addition, there have

. . . . . 23,24
been no reports 1ndrcat1ng the formatron of secondary structures of C-peptrde.

Figure 2.7 (bottom panel) shows the high resolution mass spectrum obtained following

analysis of an 8.3 uM solution of C-peptide freshly prepared in

43

{ML-r.

 

0.7 _J___‘
0.6
III
E 0.5 l
‘5'
g 0.4
2
g 0.3
.‘h
< 0.2
0.1
l ,

 

 

 

O
3'
_.

6hr

Figure 2.6: RBCS obtained from human patients with diabetes (black bars)
released 64213 nM ATP at 0 hour. After incubation with C-peptide for 6
hours, the ATP release increased (p<0.005) to 260289 nM. The release of
ATP from RBCS obtained from human control patients (white bars) was
260:1:60 nM at 0 hour and 480i109 nM at 6 hours (p<0.005). The C-
peptide—metal adduct increased the ATP release from the RBCS obtained
from patients with type 2 diabetes to a value that is statistically identical to
that from RBCS of the healthy control participants. Error bars areiSEM
(n=7).

44

 

 

100 — A [M+3H+]3’r
8 _
g [M+2H+Na + ]3+ [M+2H+]2+
a _
g [M+2H+1<+ 13+ [M+H+Na+ 12+
> —r
E
2
“ L
1] .LI .Lrli A] .11. Jud .. .l I. “J L r
l l l I | l l l l I

l
400 600 800 1000

 

I l l
1200 1400 1600 1800

 

 

+ 3+
100 [M+3H ]
[M +2H+K+] 3+
3 * [M+2H+Na +13+ W+W2Na+ 13+
g a [M+H+ Fe 2+] 3‘? [M+Fe 3+] 3+
'2 [M+H+ Na + K + 13+
0
g — [M+3Na+ ] 3+
3 ,
°\° T l!
‘
Ill l l‘ ll
1000 1010 1020 1030 1040 1050

m/z

Figure 2.7: Nanoelectrospray ionization mass spectrometry analysis of C-peptide
and its metal adducts. Panel A shows a mass spectrum of freshly prepared C-
peptide in its triply charged (around 1007 rrr/z) and doubly charged states (around

1510 m/z). Panel B shows a high-resolution mass spectrum of the [M+3H+] +

region from panel A demonstrating that C-peptide contains Fe + and Fe3+ when
freshly prepared.

45

DDW, which revealed that the peptide had formed adducts with various ions including
Na+, K+, and Fe2+.
Based on the spectra shown in ﬁgure 2.7 where the freshly prepared C-peptide

. 2+ . . . . . . 2+
rs shown bound to Fe , RBCS were 1ncubated 1n C-peptrde solutrons contarnrng Fe

3+ . 3+ . . . . 25-28
or Cr (due to the extensrve role that Cr plays 1n glycemrc control 1n drabetes)

and their subsequent ability to release ATP was determined (ﬁgure 2.8). Although the
Fe2+-bound C-peptide had the ability to increase the ATP derived from the RBCS, its
activity was observed to decrease after about 24 hours. For example, the activity of
the C-peptide solution 72 hours after the addition of F e2+ generally showed no

statistical difference from that of RBCs alone (ﬁgure 2.8). In contrast to that observed

for the C-peptide in the presence of Fe2+, the increase in RBC-derived ATP release for

the C-peptide solution 72 hours after the addition of Cr3+ was observed to be
essentially the same as that for the 6 hour time period, indicating that the activity of
the C-peptide could be extended to at least 3 days upon binding to Cr3+.

The proposed mechanism for this ATP release is based on work by Sprague,

where in reports, cellular pathways explaining the mechanism for the release of ATP

from RBCs have been proposed.29 While there have been subtle differences, each

reported mechanism involves activation of adenylyl cyclase and subsequent
production of cyclic adenosine monophosphate (CAMP) from ATP. Glycolysis is the

main, if not only route, by which RBCs produce ATP. Therefore, experiments were

46

so:
25$ - -

205

Normalized ATP Release
01

 

 

 

 

 

 

 

00‘ . .
6h 72h

Figure 2.8: Normalized ATP release from fresh RBCS after incubation
with a C-peptide solution in contact with a metal after 6 and 72 hours. At

6 hours the C-peptide solutions containing Fe2+ (white bars) and Cr3+
(hatched bars) had similarly increased (p<0.005) ATP release. After 72

hours, the ATP release from RBCS incubated with the Fe2+ peptide
solution decreased to a level similar to that from RBCS without any

peptide (black bars). The Cr3+ peptide solution maintained its level of
activity. Error bars are iSEM (n = 4).

47

performed to determine if the increased ATP release from the RBC in the presence of
the C-peptide-metal complex was due to enhanced glycolytic ATP production. To test

this hypothesis, RBCs were incubated in the presence and absence of phloretin, a

GLUTI inhibitor, and C-peptide activated with Cr3+. As shown in ﬁgure 2.9, the

Cr3+-activated C-peptide resulted in an increase of ATP release of 70 %. This

increase was not obtained when the RBCS were incubated with phloretin prior to

. . 3+ . .
rntroductron of the Cr -actrvated C-peptrde.

These results suggest that the ATP release from the RBCS is dependent upon
the ability of the C-peptide to increase glycolysis within the RBC via increased
cellular glucose uptake through the GLUTI transporter. However, phloretin has also
been shown to be a Chloride ion channel inhibitor and, therefore, may also inhibit ATP
release from the RBCS. To provide further proof that C-peptide was causing an
increase in ATP due to an increase in glycolysis, D-mannose was employed to inhibit
RBC glycolysis. While the exact mechanism of this inhibition is not known, it most
likely occurs once the D-mannose-6-phosphate is produced. Upon addition of a
mannose solution to RBCS containing 10 nM C-peptide, the ATP release was reduced
to levels of RBCS alone, while the RBCS with metal-activated C-peptide saw increased

levels of ATP as shown in ﬁgure 2.10. To provide further evidence that the

metal-activated C-peptide facilitates glucose transport into the RBC, 14C-labeled

glucose was included in the PSS at a 1:10 ratio with unlabeled glucose creating a
competition between the different glucose isotopes. As shown in ﬁgure 2.11, the

amount of glucose entering the RBC increased by 31% when the RBC solution

48

 

2.5 --

 

2.0 ~-

1.5 -~

 

0.5 +

Normalized ATP Release

 

 

0.0 -

RBCs 1=>+czr3+ P+Cr3’+PR PR

Figure 2.9: RBCs incubated with 10 nM Cr3+-activated C-peptide (P+Cr3+)
showed a signiﬁcant (p<0.005) increase in ATP release (approximately 74%).
However, when RBCs were incubated with the GLUTl inhibitor phloretin (PR)

prior to addition of P+Cr3+, ATP release was only about 63% of the value for
the RBCs alone. Error bars are :1: SEM (n=8).

49

20%
18%
16%
14%
i2%
10%
08%
06%
04%
02%
00%

Normalized ATP Release

 

 

RBCs M M+P+Cr3+ P+Cr“’+

Figure 2.10: ATP release of RBCs that had been incubated with mannose (M) and

P+Cr + showed a 57% decrease in ATP release against RBCs incubated with P+Cr +
alone, bringing ATP release to a level not signiﬁcantly different from that in RBCS.

However, in RBCs incubated with P+Cr3+ the signal intensity was approximately 64%
higher than in RBCs alone (p<0.005) Error bars are :ESEM (n=5).

50

1.6 —_
1.4%
1.2%
1.0%
0.8%

0.6 -%

Nomralized CPM

0.4 E

0.2 %

 

 

0.0 -‘

 

RBCS P+Cr3+ P P+Cr3++PR PR

Figure 2.11: RBCs incubated with 1.554 Bq 14C-labeled glucose in a solution
of PSS containing normal glucose. After 4 hours, the RBCs incubated with 10
nM Cr +-activated C-peptide (P+Cr3+) showed an approximately 31% increase
in radioactive counts per min (cpm), while RBCs incubated with only C-peptide
(P), Cr3+-activated C-peptide plus the GLUTl inhibitor phloretin (PR) or PR
alone all resulted no increase in CPM. Error bars are :l:SEM (n=4).

51

. 3+ . . . . . .
contarned Cr -act1vated C-peptrde. Whrle thrs 1ncrease 1n glucose uptake was not as

high as the ATP released from the RBCs, it should be noted that the ratio of radio-

labeled glucose to unlabeled glucose was not stoichiometrically equivalent.

2.7 DISCUSSION

C-peptide originates in the proinsulin form from the B cells of the islets of Langerhans
and, when released, is in equimolar amounts to insulin.30 Since its discovery, it has

been thought to play no signiﬁcant physiological role in viva. Generally, the function

of C-peptide was seen as solely facilitating the formation of the disulﬁde bonds of the

A- and B-Chain of the proinsulin molecule.31 However, recent reports have provided

evidence suggesting a biological role for C-peptide. For example, it has been

established that C-peptide increases renal function in patients with type 1 diabetes.32

Also, C-peptide has been shown to increase the microvascular blood ﬂow in the skin

of type 1 diabetic patients.9 Taken collectively, the reports on blood ﬂow

improvement, amelioration of certain diabetic complications, and increased glucose
transport in endothelial cells suggest that C-peptide may have the ability to increase
the levels of ATP release from RBCs due to increased glycolysis. The ability to
release increased levels of ATP from the RBCs is veriﬁed by the data in ﬁgure 2.5,
which shows an increase in ATP release from RBCs that were subjected physiological
concentrations of C-peptide.

The results involving RBCs obtained from patients having type 2 diabetes are

also of interest. Recently,l4 Carroll et al reported that the ATP released from the

52

RBCs of patients with type 2 diabetes was approximately 50% of that ATP released
from healthy controls. Sprague demonstrated that decreased ATP levels were released

from the RBCs of type 2 diabetic patients due to improper accumulation of CAMP

levels in the RBCs of these patients.29 The ATP release from RBCs obtained from a

sample set of patients with type 2 diabetes is shown to revert to normal levels in the
presence of active C-peptide (ﬁgure 2.6). These results suggest that C-peptide may be
a determinant in the control of blood ﬂow in vivo due to its ability to stimulate the
release of a recognized stimulus of NO production (ATP).

Another interesting aspect of the results discussed here are those involving

activation of the C-peptide through metal ion binding. As a product of insulin

production, C-peptide has a direct relationship to diabetes. Reports by Wahren”.34

provide a direct veriﬁcation of this role for C-peptide. The concept of metal-
activation required for activity is not without precedent. One of the main reasons why
C-peptide has never had a successful long-term clinical trial is due to lack of
reproducibility. Prior to the knowledge of needing a metal to elicit an ATP release
from RBCs, our group was unable to provide reproducible data as well. As veriﬁed by
mass spectrometry in ﬁgure 2.7, freshly prepared C-peptide shows an adduct with a
transition metal, which is dissociated after 24 hours. In addition, there have been
countless reports over the years discussing the role that metals play in diabetes. Many

metals including, selenium, vanadium, chromium, and zinc have all been shown to

. . . . . . 25,27,35-41
help amelrorate complrcatrons assocrated wrth type 1 and type 2 drabetes.

53

More speciﬁcally, we Chose to add Chromium to C-peptide because chromium
has gained popularity as a carbohydrate burning supplement as well as a nutritional
supplement for diabetic patients. While its role as a micronutrient has been known for
over four decades, progress eluding its exact role has been slow. There are several

different oxidation states of Chromimn, although the biologically important form of

. . . 3+
Chromrum IS the trivalent form Cr .

During the 1950’s, Mertz and Schwarz discovered that rats fed a Torula yeast

. . . . . 42
dret developed rmparred glucose tolerance when grven an rntravenous glucose load.

This resulted in the identiﬁcation of a new dietary requirement called the glucose

43 . . . . .
tolerance factor. Thrs phenomenon was reversed when grven a dret that was rrch 1n

Chromium or when given inorganic chromium salts. These researchers determined the

. . . 3+ 44
actrve mgredrent of the glucose tolerance factor to be Cr . Natural sources of

glucose tolerance factor were identiﬁed in brewer’s yeast and acid-hydrolyzed porcine
kidney powder. Concentrates of the natural sources had the ability to restore proper
glucose metabolism in deﬁcient rats. While the separation and puriﬁcation protocol
was not speciﬁc, glucose tolerance factor was determined to be water-soluble,
extractable with phenol and isobutanol, and absorbable on charcoal and ion exchange

resins. Several additional inorganic compounds containing nearly all other transition

44
metals were tested and none could restore glucose tolerance.
The mechanism of action has been hypothesized to involve the interaction of

+ . . . . .
Cr3 wrth the actrvrty of 1nsulrn. Several attempts were made over the course of

54

decades to try to synthesize and characterize glucose tolerance factor. It was
postulated that the glucose tolerance factor is a glutathione complex; however several
inorganic chromium containing complexes seem to improve glucose tolerance.

During the 1980’s a breakthrough occurred in establishing the mechanism of

Chromium at a molecular level. Wada, Yamamoto and coworkers discovered a low

. . . . . 45
molecular werght Chromium bmdrng substance termed Chromodulrn. It was

determined to be a peptide containing only glycine, cysteine, glutamate, and aspartate

with carboxylates composing more than half of the amino acid residues.46’47 This

susbstance was determined to have a molecular weight of only 1500 kDa. Despite

this, the peptide is able to bind four equivalents of chromic ions. To date this

oligopeptide has been isolated and puriﬁed from rabbit liver,48 bovine liver,46 porcine

kidney and kidney powder,49 dog liver,50 and mouse and rat liver.51 Based on the

isolation and puriﬁcation data, it appears that chromodulin is widely distributed
among mammals. The most novel attribute of Chromodulin is its ability to potentiate

the effects of insulin during the conversion of glucose into carbon dioxide or lipid as
. . . . 52-54 . . . . . .
deterrnrned usrng rsolated rat adrpocytes. Thrs strmulatron of 1nsulrn rs
accomplished without Changing that concentration of insulin required, suggesting that
. . . . . . 55
Chromodulrn plays an rntrrnsrc role 1n the adrpocytes.

Chromodulin has also been implicated in signal transduction. Recently

chromodulin has been shown to result in the activation of a membrane

. 56 . . . . . . . .
phosphotyrosme phosphatase and an rnsulrn-sensrtrve strmulatron of 1nsulrn receptor

55

. . . . 52,57 . . . .
tyrosrne krnase actrvrty. The actrvatron of these srgnal transductron pathways was

determined to be dependent on the chromium content of the oligopeptide. Substituting
chromium for another transition metal was shown to be ineffective in restoring the
ability of apochromodulin to stimulate kinase activity, indicating that the activation is
chromium speciﬁc.

9

Chromodulin has also been suggested to play a role in insulin signaling.5

It has been reported that Chromodulin is stored in the cytosol of insulin-sensitive cells

and is able to move from the blood to insulin-dependent cells in the presence of

i

. . . . 5 0 . .
1ncreasrng plasma 1nsulrn. It has been suggested that thrs movement rs due to the

metal transport protein transferrin.

With all of the data suggesting a role for Chromium in glucose metabolism,
Chromium has been marketed as a nutritional supplement, used frequently by during
weight training and exercise. It is sold as chromium picolinate, which has become
extremely proﬁtable and is available over the counter in the form of pills, chewing
gum, sports drinks, and nutrition bars. Chromium picolinate is absorbed relatively

well in the body with 2-5% efﬁciency compared to dietary Chromium, which is

absorbed with only 0.5% efﬁciencym’62 While chromium picolinate has been

proposed to be the biologically active form of chromium,63 it has not been shown to

exist in vivo.
During a double-blind crossover study that involved volunteers taking 200 11g
of Chromium as chromium picolinate per day, it was reported that the volunteers had

decreased total Cholesterol, LDL cholesterol, and apolipoprotein B and increased

56

. . 64 . .
apolrpoprotern A. Studres performed usrng cultured rat skeletal muscle myoblasts

found increased insulin internalization and increased glucose and leucine uptake when
exposed to media containing chromium picolinate. These results were not obtained

when using the same concentration of Chromic Chloride, Chromium nicotinate, or zinc

. . 63
prcolmate.

Recently, studies have made a link between type 2 diabetes and chromirun.

Speciﬁcally, individuals with type 2 diabetes have lower serum and higher urine levels

65,66

of Chromium. However, many early reports suggest that Chromium has the

ability to lower blood glucose levels or to have an effect on Cholesterol, triglycerides
. . 67,68 . .
or 1nsulrn after several weeks. More recently, a study performed 1n Chrna
demonstrated the ability of Chromium picolinate to lower fasting serum glucose levels
and hemoglobin A re levels with increasing chromium.69 It has been suggested that a
similar study needs to be performed in the United States as the body mass indexes of

the Chinese patients differs signiﬁcantly from American type 2 diabetics.7O A

comprehensive study on the effectiveness of Chromium supplementation as it pertains
to diabetes by Balk concluded that chromium supplementation did not have an effect

on patients without diabetes; however Chromium supplementation signiﬁcantly

. . . . . 71
1mproved glycemra among patients wrth drabetes.

Taken collectively, the previous research concerning chromium and diabetes

indicated to us that chromium may be the metal ion that activates C-peptide in vivo.

The data presented here indicates that when C-peptide binds to Fe2+ there is activity

57

. . 3+ . .
that lasts for about 24 hours. However, when bmdrng to Cr , the actrvrty lasts for at

least 3 days (Figure 2.8). These results are interesting when one considers that the

exchange rates for Fe2+ binding to most ligands is generally faster than the exchange
3+ . . . . . . . 2+ .

rates for Cr specres bmdrng to srmrlar lrgands. In other words, 1f the Fe brnds to

the C-peptide, its activity lasts for a short period but upon replacement of the Fe2+ (for

example, with Na+ or K+), the C-peptide has no activity.

Perhaps the most important aspect of the results shown here is the ability of the
metal-activated C-peptide to enhance glucose transport into the RBC. There are not
insulin receptors on the RBC, and glucose transport into the RBC mainly occurs
through the GLUTl transporter, which is not activated by insulin. Therefore, the
ﬁnding that metal-activated C-peptide has the ability to clear extracellular glucose
from the bloodstream may have profound effects. First, for those diabetic patients not
producing appreciable amounts of insulin, and therefore not producing appropriate
amounts of C-peptide (e.g., a type 1 diabetic), the addition of C-peptide may help

reduce blood glucose levels and relieve some of the pressure from insulin alone having

+
to maintain glucose levels. Secondly, due to the long-lasting activity of the Cr3 -

activated C-peptide, the need for multiple injections or pump-administered insulin
may be reduced.

The end result of the glucose transport into the RBC (ATP) release may also be a
major determinant in the maintenance of proper vascular caliber. ATP is a recognized

stimulus of NO production in the endothelium and in platelets. The beneﬁts of

58

endothelium-derived NO are well-understood. In addition, platelet-derived NO is also

. . . 72 .
known to reduce platelet recrtutment upon actrvatron. Such reductions may help

reduce the incidence of thrombus formation and subsequent vaso-occlusion.

Despite all of the evidence that points to Chromium as being biologically
relevant to the treatment of diabetes, it is unlikely that Chromium is the metal that
would likely activate C-peptide in vivo. A more likely choice would be zinc. As
described in chapter 1, insulin is secreted by the B cell both tonically and as a spike in
response to an increase in blood glucose concentrations. It is well established that
there is a relationship between insulin and zinc. Before any evidence of a relationship
between zinc and insulin in the B cell, it was known that the addition of zinc to insulin
would extend the effect of a given dose of insulin. When insulin was ﬁrst being
prescribed as a treatment for type 1 diabetes, zinc was being added in vitro to prolong
the duration of the action of insulin by delaying its absorption from the subcutaneous
injection site.

When produced in the B cells of the pancreatic islets, insulin is produced in a

. . 73-78 . . . .
solrd hexamer around zrnc. The levels of 21m 1n the [3 cells at any grven trme are

near millimolar levels, although most of this zinc is not available in the free form.

2+ . . . 2+ .
There are two Zn rons per 1nsu11n hexamer, however Zn contrnues to accumulate
. . 78 . .
even after rt rs bound to the hexamer. The zrnc transporter, ZnT-8, rs a
. .. 2+ . .
transmembrane transporter protern that facrlrtates Zn rnto the vesrcles. Here, the

+ .. 2+ .
insulin forms a hexamer around the Zn2 , followed by the addrtron of more Zn rnto

59

the vesicle. Eventually, the vesicle reaches the surface of the B cell where insulin, C-
peptide, and Zn2+ are released into the [3 cell. Insulin is secreted in equimolar

amounts to C-peptide, although the concentration present in the bloodstream may not
be the same as C-peptide, it may be hypothesized that the biologically active form of
C-peptide is likely to be bound to zinc, however this has not been determined to be
true. The presence of high levels of zinc in the B cells, zinc was used to activate C-
peptide for all future measurements.

Beyond the physical conformational data demonstrating the importance of zinc
in relationship to insulin, there has been data suggesting that the conformational

changes that occur in the presence of zinc also affect receptor binding and antigenic

. . . 79 . . . . . . .
propertres of 1nsulrn. In addrtron, data suggests that 1nsulrn brnds to rsolated lrver

membranes to a greater extent and that there is less degradation when zinc is co-
administered with insulin.

Patients with diabetes often suffer from hypozincemia, which may be the result

. . . . 80 . . .
of hyperzrncurra or decreased absorption of zrnc, or both. Whrle 1t rs Clear that
patients with diabetes secret more zinc in the urine than individuals without diabetes,
. . . . . 81

there rs less data suggesting that the there rs malabsorptron of zrnc. It appears that
hyperzincuria is a result of hyperglycemia more than any speciﬁc effect of insulin.
80,82-84

There is conﬂicting data whether insulin treatment reduces hyperzincuria.

Elevated levels of zinc have been found in the liver, muscle and kidney in

. . . . 85 . . . . .
streptozotocrn-rnduced drabetrc rats. Thrs rs s1 gnrﬁcant because these are the trssues

60

that are responsive to insulin-mediated glucose transport. However, when using rats

that spontaneously become diabetic, hyperglycemia was associated with reduced

levels of zinc in the liver, kidney, and muscle.

Zinc has also been implicated in the cellular oxidant status of people with

. 86 . . .
drabetes. Because type 1 drabetes 1s thought to be the result of an autormmune

attack on the [3 cell, the destruction of these cells results in less bioavailable zinc for

use in important antioxidant enzymes. It has been proposed that the lack of zinc

available for use in the enzymes may contribute to the tissue damage observed in

diabetes.”
Zinc also plays a role in the progression of type 2 diabetes. While there is no 0

solid evidence for oxidative stress as a major factor in the development of either

insulin deﬁciency or islet cell damage, there is evidence of increased levels of insulin

. . . . 88 . .
secretron and therefore zrnc) 1n the early stages of the drsease. It rs possrble for the
pancreas to synthesize more insulin; however, the pancreas cannot synthesize more
zinc, which will result in the depletion of zinc from the pancreas. Without zinc, the
pancreas may not function correctly and could result in complete destruction of the
islet. This hypothesis provides a potential mechanism by which zinc deﬁciency may

affect the progress of type 2 diabetes.

The data presented here shows that C-peptide, when activated by a metal such

as Fe2+ or Cr3+, has the ability to reproducibly elicit an ATP release from RBCs after

incubation. To date, no other work has been performed using a metal to activate C-

peptide. As discussed by Luzi,89 one of the main reasons for C-peptide not being

61

accepted as a biologically relevant peptide is due to lack of a long-term clinical study.
However, as shown here, adding a metal allows for the successful reproducibility of a
biologically relevant molecule (ATP) from RBCs. Future studies, as discussed in
chapter 3, will address another argument given against C-peptide being a biologically

relevant peptide- the presence of C-peptide in patients with type 2 diabetes.

62

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46. Davis CM, Vincent JB. Isolation and characterization of a biologically active
chromium oligopeptide from bovine liver. Archives of Biochemistry and Biophysics.
1997;339:335-343.

47. Yamamoto A, Wada 0, Suzuki H. Puriﬁcation and properties of biologically
active chromium complex from bovine colostrum. Journal of Nutrition. 1988;118:39-
45.

48. Yamamoto A, Wada O, Ono T. Isolation of a biologically active low-
molecular—mass Chromium compound from rabbit liver. European Journal of
Biochemistry. 1987;165:627-631.

49. Sumrall KH, Vincent JB. Is glucose tolerance factor an artifact produced by
acid hydrolysis of low-molecular-weight Chromium-binding substance? Polyhedron.
1997;16:4171-4177.

50. Wada 0, Wu GY, Yamamoto A, Manabe S, Ono T. Puriﬁcation and
chromiurn-excretory function of low-molecular-weight, Chromium-binding substances
from dog liver. Environmental Research. 1983;32:228-239.

67

51. Yamamoto A, Wada O, Ono T. Distribution and Chromium-binding capacity of
a low-molecular-weight, chromium-binding substance in mice. Journal of Inorganic
Biocherrristry. 1984;22:91-102.

52. Davis CM, Vincent JB. Chromium Oligopeptide Activates Insulin Receptor
Tyrosine Kinase Activity. Biochemistry. 1997;36:4382-4385.

53. Mertz W, Roginski EE, Schwarz K. Effect of trivalent Chromium complexes

on glucose uptake by epididymal fat tissue of rats. Journal of Biological Chemistry.
1961;236:318-322.

54. Vincent JB. Relationship between glucose tolerance factor and low-molecular-
weight chromium-binding substance. The Journal of nutrition. 1994;124:117-119.

55. Yamamoto A, Wada O, Manabe S. Evidence that Chromium is an essential
factor for biological activity of low-molecular-weight, Chromium-binding substance.
Biochemical and Biophysical Research Communications. 1989;163:189-193.

56. Davis CM, Sumrall KH, Vincent JB. A Biologically Active Form of
Chromium May Activate a Membrane Phosphotyrosine Phosphatase (PTP).
Biochemistry. 1996;35:12963-12969.

57. Davis CM, Royer AC, Vincent JB. Synthetic Multinuclear Chromium
Assembly Activates Insulin Receptor Kinase Activity: Functional Model for Low-
Molecular-Weight Chromium-Binding Substance. Inorganic Chemistry.
1997;36:5316-5320.

58. Vincent JB. Mechanisms of chromium action: low-molecular-weight
Chromium-binding substance. Journal of the American College of Nutrition.
1999;18:6-12.

59. Morris BW, Gray TA, Macneil S. Glucose-dependent uptake of Chromium in
human and rat insulin-sensitive tissues. Clinical Science. 1993;84:477-482.

60. Morris BW, MacNeil S, Stanley K, Gray TA, Fraser R. The inter-relationship
between insulin and Chromium in hyperinsulinemic euglycemic Clamps in healthy
volunteers. Journal of Endocrinology. 1993;139:339-345.

68

61. Anderson RA, Bryden NA, Polansky MM, Gautschi K. Dietary Chromium
effects on tissue Chromium concentrations and Chromium absorption in rats. Journal of
Trace Elements in Experimental Medicine. 1996;9z11-25.

62. Olin KL, Stearns DM, Armstrong WH, Keen CL. comparative
retention/absorption of 51Chromium (51Cr) from 51Cr Chloride, 51Cr nicotinate and
51Cr picolinate in a rat model. Trace Elements and Electrolytes. 1994;11:182-184.

63. Evans GW, Bowman TD. Chromium picolinate increases membrane ﬂuidity

and rate of insulin internalization. Journal of Inorganic Biochemistry. 1992;46:243-
250.

64. Press RI, Geller J, Evans GW. The effect of chromium picolinate on serum
cholesterol and apolipoprotein fractions in human subjects. West J Med FIELD Full
Journal TitlezThe Western journal of medicine. 1990;152:41-45.

65. Morris BW, Grifﬁths H, Kemp GJ. Effect of glucose loading on concentrations

of Chromium in plasma and urine of healthy adults. Clinical Chemistry (Washington,
DC, United States). 1988;34:1114-1116.

66. Morris BW, Kemp GJ, Hardisty CA. Plasma Chromium and chromium
excretion in diabetes. Clinical Chemistry. 1985;31:334-335.

67. Sherman L, Glennon JA, Brech WJ, Klomberg GH, Gordon ES. Failure of
trivalent chromium to improve hyperglycemia in diabetes mellitus. Metabolism:
clinical and experimental. 1968;17:439-442.

68. Uusitupa MI, Kumpulainen JT, Voutilainen E, et al. Effect of inorganic
chronriurn supplementation on glucose tolerance, insulin response, and serum lipids in
noninsulin-dependent diabetics. The American journal of Clinical nutrition.
1983;38:404-410.

69. Anderson RA, Cheng N, Bryden NA, et al. Elevated intakes of supplemental
Chromium improve glucose and insulin variables in individuals with type 2 diabetes.
Diabetes. 1997;46:1786-1791.

70. Hellerstein MK. Is chromium supplementation effective in managing type II
diabetes? Nutrition reviews. 1998;56:302-306.

69

71. Balk EM, Tatsioni A, Lichtenstein AH, Lau J, Pittas AG. Effect of Chromium
supplementation on glucose metabolism and lipids: A systematic review of
randomized controlled trials. Diabetes Care. 2007;30:2154-2163.

72. Freedman JE, Loscalzo J, Barnard MR, Alpert C, Keaney JF, Jr., Michelson
AD. Nitric oxide released from activated platelets inhibits platelet recruitment. Journal
of Clinical Investigation. 1997;100:350-356.

73. Zalewski PD, Millard SH, Forbes IJ, et al. Video image analysis of labile zinc
in viable pancreatic islet cells using a speciﬁc ﬂuorescent probe for zinc. Journal of
Histocherrristry and Cytocherrristry. 1994;42:877-884.

74. Baker EN, Blundell TL, Cutﬁeld JF, et al. The structure of 2-zinc pig insulin
crystals at 1.5 .ANG. resolution. Philosophical Transactions of the Royal Society of
London, Series B: Biological Sciences. 1988;319:369-456, 365 plates.

75. Brader ML, Dunn MF. Insulin hexamers: new conformations and applications.
Trends in Biochemical Sciences. 1991;16:341-345.

76. Brange J, Langkjoer L. Insulin structure and stability. Pharmaceutical
biotechnology. 1993;5z315-350.

77. Derewenda U, Derewenda Z, Dodson GG, Hubbard RE, Korber F. Molecular
structure of insulin: the insulin monomer and its assembly. British Medical Bulletin.
1989;45:4-18.

78. Chimienti F, Favier A, Seve M. ZnT-8, A Pancreatic B-Cell-Speciﬁc Zinc
Transporter. BioMetals. 2005;18:313-317.

79. Arquilla ER, Thiene P, Brugrnan T, Ruess W, Sugiyama R. Effects of zinc ion
on the conformation of antigenic determinants on insulin. Biochemical Journal.
1978;175:289-297.

80. Garg VK, Gupta R, Goya] RK. Hypozincemia in diabetes mellitus. The Journal
of the Association of Physicians of India. 1994;42:720-721.

81. Kinlaw WB, Levine AS, Morley JE, Silvis SE, McClain CJ. Abnormal zinc
metabolism in type II diabetes mellitus. The American journal of medicine.
1983;75:273-277.

70

82. el-Yazigi A, Harman N, Raines DA. Effect of diabetic state and related
disorders on the urinary excretion of magnesium and zinc in patients. Diabetes
research (Edinburgh, Scotland). 1993;22:67-75.

83. Honnorat J, Accominotti M, Broussolle C, F leuret AC, Vallon JJ, Orgiazzi J.
Effects of diabetes type and treatment on zinc status in diabetes mellitus. Biological
trace element research. 1992;32:311-316.

84. Isbir T, Tamer L, Taylor A, Isbir M. Zinc, copper and magnesium status in
insulin-dependent diabetes. Diabetes research (Edinburgh, Scotland). 1994;26:41-45.

85. Cordova A. Zinc content in selected tissues in streptozotocin-diabetic rats after
maximal exercise. Biological Trace Element Research. 1994;42:209-216.

86. DiSilvestro RA. Zinc in relation to diabetes and oxidative disease. Journal of
Nutrition. 2000;130:15098-15118.

87. Rabinovitch A, Suarez WL, Thomas PD, Strynadka K, Simpson I. Cytotoxic
effects of cytokines on rat islets: evidence for involvement of free radicals and lipid
peroxidation. Diabetologia. 1992;3 5:409-413.

88. Sprietsma JE, Schuitemaker GE. Diabetes can be prevented by reducing
insulin production. Medical hypotheses. 1994;42:15-23.

89. Luzi L, Zerbini G, Caumo A. C-peptide: a redundant relative of insulin?
Diabetologia 2007;50:500-502.

71

CHAPTER 3- THE ROLE OF C-PEPTIDE IN TYPE 2 DIABETES AND THE

CONCEPT OF C-PEPTIDE RESISTANCE

3.1 ARGUMENTS AGAINST C-PEPTIDE AS A BIOACTIVE PEPTIDE
C-peptide has historically been considered to be a biologically inert peptide. Despite
mounting evidence demonstrating the biological effects of C-peptide, critics of C-
peptide often have 3 main arguments against the notion of C-peptide being
biologically active including: the lack of a speciﬁc receptor, the lack of a successful
long-term Clinical trial, and the lack of an effect in type 2 diabetes where there is an
excess of C-peptide and insulin.

To be Classiﬁed as an independent hormone, a speciﬁc receptor is typically
required. However, there has been no such receptor identiﬁed to date for C-peptide.
Mechanistically, reports have shown that C-peptide was exerting its biological effects

on certain cell types via activation of G proteins therefore implying the receptor to be

a G-protein coupled receptor."2 Despite these ﬁndings, no speciﬁc G-protein coupled

receptor has been identiﬁed. Another report has shown that C-peptide’s

insulinomimetic effect is most likely mediated through the modulation of the tyrosine

kinase coupled receptor of insulin.3 Thus C-peptide is not universally considered to

be biologically active, due to the lack of conclusive evidence on the exact location of
the receptor.

Another common criticism of C-peptide being a biologically relevant peptide
is the fact that there has been no successful long-term Clinical trial. To date, the

longest Clinical studies have only lasted a few months, where the longitudinal effect of

72

C-peptide on the development and progression of diabetic complications in humans

. 4 .
were studred. Logrcally, there should be no reason for a lack of successful long-term

studies, however as described in Chapter 2, it was recently discovered that C-peptide

required metal-activation in order to be biologically active consistently in erythrocytes

(RBCs).5 It was hypothesized that the lack of a successful long-term Clinical trial may

be due to the lack of proper handling of the C-peptide prior to infusion (lack of metal
activator).

The last argument against C-peptide being a biologically relevant peptide is the
lack of an effect on patients with type 2 diabetes, who often have excessive levels of
C-peptide and insulin, especially during the early stages of the disease. Patients with
type 2 diabetes typically develop complications associated with diabetes at the same
rate as patients with type 1 diabetes. Because it has been asserted that C-peptide
therapy can help patients with type 1 diabetes avoid diabetic complications, it would
be expected that patients affected by type 2 diabetes would remain largely without
these complications.

A possible reason for the lack of an effect on patients with type 2 diabetes is
the concept of C-peptide resistance among these patients. As will be shown here,
RBCs from type 2 rat models display an apparent resistance to the effects of C-peptide
compared to healthy controls. C-peptide resistance may stem from high levels of
oxidative stress within the RBCs of patients with type 2 diabetes.

Oxidative stress is caused by an imbalance between the production of reactive
oxygen species, such as free radicals, and a biological system’s ability to readily

detoxify the species or easily repair the resulting damage. Free radicals and oxidative

73

stress are among the factors involved in the pathogenesis of diabetes and appear to be

involved in B-Cell destruction and development of Chronic complications of

. 6,7 . .
drabetes. Hrgh levels of exogenous or endogenous free radrcals could lead to

destruction of major components of cellular structure, including nucleic acids,
proteins, amino acids, lipids, and carbohydrates and would affect various cell

functions such as membrane function, metabolism and even gene expression, and they

. . . 8,
cause a number of other pathological condrtrons.

In diabetic patients, glucose auto-oxidation and non-enzymatic glycation of
proteins are known as important reactive oxygen species producing factors.
Moreover, hyperglycemia leads to aldose reductase activation and NADPH depletion,

which in turn causes disorders in the glutathione (GSH) reduction cycle and

. . . 10,11 . . .
exacerbates oxrdatrve stress 1n cells. Measurement of total antroxrdant capacrty

and particular antioxidants such as glutathione and glutathione peroxidase can be used
for evaluating oxidative stress in patients.

RBCs contain a high concentration of GSH and is synthesized in human RBCs

with a half life of 4 days.12 The synthesis is catalyzed by 2 enzymes, 7-

glutamylcysteine synthase and GSH synthetase. In human RBCs there is a GSH redox
cycle catalyzed by GSH peroxidase and GSH reductase. GSH is transported out of
RBCs in the oxidized form, GSSG.

An early report by Murakarni et al. showed that there was a decreased ratio of
GSH/GSSG in patients with diabetes. Moreover, there was also a decrease in the

activity of y-glutamylcysteine synthase, which accounts for the decrease in the levels

74

of GSH.13 In addition, it was reported that the transport rate of GSSG is decreased in

RBCs from patients with diabetes. The decreased activity of GSH reductase in
diabetics together with the decreased transport rate of GSSG indicates that
regeneration and transport systems, which decrease intracellular GSSG, are impaired

in diabetics when RBCs are exposed to oxidative stress in vitro.

In a report by Dumaswala et a1,14 RBCs that were exposed to high levels of

oxidative stress underwent a phospholipid rearrangement of the outer membrane of the
cell. More speciﬁcally, phosphatidylserine (PS), which is usually only found in the
inner leaﬂet of the RBC membrane, was translocated to the outer leaﬂet of the
membrane when exposed to high levels of oxidative stress. The increased exposure of
PS exposure is signiﬁcant because PS is considered to be a more negatively charged
phospholipid compared to phosphatidylcholine, which is the phospholipid exposed
under conditions of low oxidative stress or normal conditions. Below, evidence is
provided suggesting that RBCs from type 2 rat models may be resistant to C-peptide
due to increased PS exposure on the cell membrane resulting in an increase in Charge-

charge repulsions between the RBC membrane and the highly negatively Charged C-

peptide.

3.2 EXPERIMENTAL
3.2.1 Collection of RBCs

Whole blood was obtained by cardiac stick on nine 5-month type 2 diabetic male
BB/ZDR/Wor-rats (Biomere InC., Springﬁeld, MA) (blood glucose 24.0 i 1.9 mM),

and ﬁve age- and sex-matched control rats (blood glucose 5.0 i 0.4 mM). Whole

75

blood was centrifuged for 10 min at 500 g and RBCs were resuspended and washed

three times in physiological salt solution (PSS) (in mM: 4.7 KCl, 2.0 CaClz, 140.5

NaCl 12 MgSO4, 21.0 tris[hydroxymethyl]aminomethane, 11.1 dextrose with 5% w/v

bovine serum albumin [ﬁnal pH 7.4])

3.2.2 Preparation of Reagents

Synthetic human C-peptide was commercially purchased (Genscript, Piscataway, NJ)
and puriﬁed in house using liquid chromatographic methods. Its content was veriﬁed
using electrospray mass spectrometry. A stock solution of 8.3 11M was prepared by
dissolving 0.25 mg of the puriﬁed peptide in 10 ml of distilled deionized water
(DDW).

A solution of zinc was prepared by dissolving 5.5 mg of zinc (II) chloride in
500 ml of DDW yielding a stock concentration of 80 11M. From this stock, 31.8 111
was diluted in 25 ml of DDW to yield a working solution with a concentration of 102
nM.

A solution of 1,1-dimethylbiguanid hydrochloride (metforrnin, Aldrich,
Steinheim, Germany) was prepared by dissolving 0.0207 g of metformin in 10 ml of
DDW, resulting in a concentration of 12.5 mM.

Insulin obtained from porcine pancreas (Sigma Aldrich, St. Louis, MO, USA)

was prepared by creating a 100 ml volume of 12.5 mM stock.

3.3 METHODS

3.3.1 ATP Release from Type 2 and Control Rat RBCs

76

To determine the ATP released from RBCs under non-ﬂow conditions, a plastic
cuvette containing 200 111 of RBCs followed by the immediate addition of 100 111 of
luciferin/luciferase was gently shaken for 15 seconds and placed over the PMT inside
of a light-excluding box, where the resulting light from the Chemiluminescence
reaction was detected.

The RBC suspensions were prepared by adding 600 111 of the 83 nM C-peptide

working solution and 490 111 of the 102 nM Zn2+ solution to a 15 ml centrifuge tube.

Next, 3410 111 of PSS was added to the aqueous C-peptide/Zn2+ solutions, followed by

the immediate addition of RBCs, resulting in an RBC suspension with a 10 nM C-
peptide/metal concentration and a hematocrit of 7%. The RBC solution was allowed
to incubate for a period of 6 hours prior to the measurement of ATP release.

Metformin (3011M) was incubated with RBCs at room temperature for 2 hours,

followed by the addition of C-peptide/Zn2+/PSS and incubated for an additional 4

hours.

3.3.2 Mass Spectrometry Analysis

Mass spectra were acquired with a Thermo Scientiﬁc model LTQ linear ion trap mass
spectrometer (San Jose, CA), using ‘normal’ and ‘ultrazoom’ (high resolution)
resonance ejection scan modes. Samples were introduced to the instrument by infusion
at a ﬂow rate of 1 til/min. The spray voltage was maintained at 2.5 kV. The heated
capillary temperature was 275°C.

3.3.3 Liquid Scintillation Counting to Determine Glucose Accumulation in

RBCs from Type 2 and Control Rats

77

Experiments were performed using RBCs that had been washed in a low glucose

concentration (0.55 mM) PSS. Additionally, 1.554 Bq 14C-labeled glucose was added

to the PSS. This created a ratio of 14C-labelled glucoseznon-radiolabeled glucose of

1:10. Similar to the ATP studies, RBCs were incubated in the presence of metforrnin

for 2 hours prior to the addition of C-peptide/Zn2+. For studies involving insulin,

RBCs were incubated with insulin in the presence of metformin (30 11M), or insulin

alone (3011M). After the addition of C-peptide/Zn2+, the RBC suspension was

centrifuged and washed 2 times with the low-glucose PSS to remove any excess 14C-

labeled glucose. Packed RBCs were then lysed using 1 ml of bleach and were allowed
to equilibrate for 30 minutes. After equilibration, 200 111 of each lysed RBC sample
was combined with 100 111 of a scintillation cocktail on a 96 well plate and the
radioactivity was measured in triplicate using a liquid scintillation counter. All data
involving the scintillation counting were averages obtained from 1 minute of counting
(ﬁgure 3.1).

3.3.4 C-peptide ELISA

C-peptide binding to the RBCs was determined using ELISA. The RBCs were

incubated with 20 nM Zn2+-activated C-peptide at room temperature for 2 hours, or

for 2 additional hours with metforrnin (30 11M). After incubation, the suspensions

were centrifuged and washed twice at 500 g for 10 minutes to remove any excess

physiological salt solution and Zn2+-activated C-peptide. Packed RBCs were added to

an appropriate volume of DDW to lyse them, followed by the addition of 150 pl of the

78

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79

Figure 3.1:

A schematic representation of the setup used to measure the

radioactivity of RBC samples.

lysate to an ELISA plate (Millipore, Billerica, MA), where the instructions included
with the ELISA were subsequently followed. The resultant signals were measured
with a UV-Vis absorbance plate reader (Molecular Devices, Sunnyvale, CA) as shown
in ﬁgure 3.2.

3.3.5 Phosphatidyl Serine Determination Using Annexin V

In order to evaluate the effect of metforrnin, each RBC sample (from both control and
diabetic samples) was divided in to two equal portions and an appropriate voltune of
metformin was added resulting in an RBC suspension having a 30 pM metforrnin

concentration, while adding PSS to the set of controls. After 2 hrs of incubation, 2 pl

of the 7 % RBCs were diluted in 498 pl of IX binding buffer (100 mM HEPES/
NaOH, pH 7.5 containing 1.4 M NaCl and 25 mM CaClz) provided with an annexin

V-FITC apoptosis detection kit (Sigma Aldrich, St. Louis, MO) yielding a cell

suspension of approximately 1)( 106 cells/ml . To this, 5 pl of annexin labeled with the

ﬂuorescence dye FITC was added. After incubation for 10 min, the samples were
centrifuged (>< 500 g) for 5 min and the cells were rewashed and resuspended prior to
measuring the ﬂuorescence emission using a Horiba-Jobinyvon Fluromax-4

spectroﬂuorometer (excitation 487 nm; emission 515 nm).

3.4 RESULTS

The concentration of zinc in pancreatic B-cells is estimated to be in the millimolar
range and therefore it is reasonable that metal activation of C-peptide would occur due
to an interaction with zinc. Mass spectrometric data demonstrates that zinc forms an

adduct with C-peptide. The mass spectrum of puriﬁed C-peptide includes a triply-

80

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81

charged species appearing at roughly 1,007 mass units (ﬁgure 3.3a). The addition of
zinc to the C-peptide results in a peak at ~l,029 mass units, consistent with zinc
binding to the C-peptide (ﬁgure 3.3b, doubly Charged species 3.3C).

Studies were subsequently performed to determine if the zinc-activated C-

peptide would stimulate an increase of ATP release from RBCs. Previously, it was

reported that metal activation of C-peptide with Fe2+ or Cr3+ stimulates ATP release

5 . 2+ . .
from RBCs. Usrng Zn as the metal activator, RBCs obtarned from control rats and

type 2 BB/ZDB rats were incubated with C-peptide in the presence and absence of

Zn2+ for 4 h prior to measuring the ATP released from the cells. The ATP release

from the RBCs obtained from the diabetic rats (31.2 i 4.0%) was observed and was

signiﬁcantly less (p < 0.001) than that of the control RBCs (78.4 :t 4.9%; ﬁgure 3.4).
Incubation with C-peptide alone (i.e., in the absence of Zn2+) did not increase ATP
release from the RBCs of either animal group.

The ability of Zn2+-activated C-peptide to stimulate the release of ATP from
RBCs is important because ATP stimulates endothelial nitric oxide synthase
(eNOS),15 which produces nitric oxide (N O), a potent vasodilator. Moreover, recent
studies have shown that RBCs obtained from human type 2 diabetes release less ATP

5,16,17
In the

in response to pharmacological stimuli and shear-induced deformation.

latter study, the reduction in deformation was thought to occur due to a decrease in the

. . 16
overall antr-oxrdant status of the RBC.

82

 

 

     

 

- A [M+H++Zn2+]3+

- [M+2H++K+]2.+

_‘ [M+3H+]3+i‘ [M+ H+]2+
D ‘ [M+2H++Zn2+]4+ ; M+an+]2+
‘é - [M+4H+]4+-.
IO 1 4.1 41.21 . .J
g irr|rrilrrrjr111ﬁiﬁrllIIIIIIIIII]
g 400 600 800 1000 1200 1400 1600 1800 2000
g ‘ B [M+H‘“+Zn2+]3+ C [M+Zn2‘L]2+
E _
g2 _

1027 1029 1031 1540 1542 1544 1546

m/z

Figure 3.3: Electrospray Ionization — Mass Spectrometry (ESI-MS) analysis
of an aqueous solution of 10 pM C-peptide containing 10 pM ZnClz. The
most abundant ron in the ‘normal’ mass spectrum (panel A) corresgronds to the
triply charged precursor ion of Zn- bound C-peptide ([M+H +Zn ]3 ). High
resolution mass spectra of the triply and doubly 2charged Ions of the Zn-bound
+

C-peptide rons [M+H+ +Zn 2+]3 and [M+Zn2 +2] are shown 1n panels B and
C, respectively. The isotopic distributions for these Charge states are
consistent with those predicted for the Zn-bound C-peptide.

83

p__

 

 

 

3.0 %

 

- Type 2

1 Control
2.5 j *

 

2.0

H*j_+
]——l*

 

A A A A

1.5 1

1.0j FF. ‘ r—

Normalized ATP Release

0.5 {

 

 

 

 

 

 

 

 

 

 

 

 

 

0.0: 1
(a) (b) (c) (d) (e)

Figure 3.4: The normalized ATP release of RBCs from type 2 rats and controls
alone (a), in the presence of 20 nM Zn2+-activated C-peptide (b), C-peptide

without Zn2+ (c), in the presence of 20 nM Zn2+-activated C-peptide and 30 pM
metformin (d), and 30 pM metforrnin with RBCs alone (e). Error bars are :E SEM
(n=8 diabetic, n=4 control), p< 0.001.

84

In addition to the studies investigating the release of ATP from RBCs incubated in the

2+ . . . . .
presence and absence of Zn -act1vated C-peptrde, srmrlar studres were performed to

. 2+ . . .
deterrnrne the effect of Zn -act1vated C-peptrde on glucose transport rnto RBCs.

Similar to the results obtained for ATP release, RBCs from diabetic rats did not

respond as well to C-peptide induced increase in glucose uptake as those RBCs

obtained from controls (ﬁgure 3.5). l4C-Labeled glucose uptake in the diabetic RBCs

increased by 35.8 i 1.3% when incubated in the Zn2+-activated C-peptide, a

signiﬁcantly lower value when compared to the 64.3 i 5.1% increase obtained using
RBCs obtained from controls (p < 0.001). Incubation with metforrnin increased the

glucose uptake to values that were greater than those of the control RBCs. However,

. . . . . . . .. . 2+

srmrlar studres performed wrth RBCs from rabbrts drsplay no actrvrtres wrth Zn
2+ I O I 0

alone or Zn wrth metforrnrn (ﬁgure 3.6). These results are also consrstent wrth a

. . . . . 24 .
prevrous report 1nvolvrng metal-actrvated C-peptrde. An 1mmunoassay for

exogenously added C-peptide bound to RBCs was performed to address the possibility

that differences in C-peptide binding might explain the decreased RBC-derived ATP
release from, and 14C-labeled glucose transport into, the RBCs obtained from the type
2 rat model. After the addition of Zn2+-activated C-peptide to RBCs obtained from

the sample and control groups, the RBCs were centrifuged and assayed for bound C-
peptide. As shown in ﬁgure 3.7, a signiﬁcant decrease (p<0.001) in C-peptide was

found in RBCs obtained from the diabetic group (32.5 d: 8.2%) in comparison to the

85

 

f‘

f‘

.265 002.2502

 

 

 

 

 

3.0 _ — Type 2
12:: Control *

 

 

Normalized CPM
GI
'11
j—lx-

1.0: 1-1

0.5 {

 

 

 

 

 

 

 

 

 

 

 

 

 

 

0.0: r . *
(a) (b) (c) (d) (e)

Figure 3.5: Normalized uptake of 14C-glucose by RBCs from diabetic and
control rats; (a) untreated RBCs, (b) RBCs incubated with 20 nM Zn2+-
activated C-peptide, (c) RBCs incubated with 20 nM Zn2+-activated C-

peptide and 30 pM metforrnin, (d) RBCs incubated with 20 nM Zn2+-
activated C-peptide and 30 pM metformin and 30 pM insulin, and (e)
RBCs incubated with 30 pM metforrnin alone. Error bars are 2 SEM
(n=4), p< 0.001.

86

Normalized ATP Release

2.5 -
2.05
1.5
1.0

0.5

 

   

0.0 3
(a) (b) (C) (d) (8)

Figure 3.6: 2Normalized ATP release of rabbit RBCs incubated (a) alone, (b) with
+

20 nM Zn and C-peptide, (C) with 20 nM Zn2+, C-peptide and 30pM

metforrnin, (d) 30pM metforrnin and 20 nM Zn2+, and (e) 20 nM Zn +. Error

bars are :E SEM (n=3). * represents values statistically different from RBCs

alone, + represents values statistically different from values indicated by

connecting lines. P-values < 0.001.

87

2'0 % - Type2
- Control *

0.0 i l l 1*"
(a) (b

) (C) (d) (e) (f)

.5
01
l n

n A. 1

Normalized Absorbance
8 3

 

 

 

 

 

 

 

Figure 3.7: Normalized binding of anti-C-peptide antibodies for RBCs
obtained from type 2 and control rats: (a) untreated RBCs, (b) RBCs

incubated in the presence of 20 nM Zn2+-activated C-peptide, (c) RBCs
incubated with 20 nM Zn2+-activated C-peptide and 30 pM metforrnin, (d) and

(e) RBCs incubated with C-peptide (without Zn2+) with 30 pM metforrnin (d),
(e) 30 pM metforrnin with 30 pM insulin, and (f) RBCs incubated with 20 nM

Zn2+-activated C-peptide, 30 pM metfonnin, and 30 pM insulin. Error bars
are i SEM (nﬁ), p-values < 0.001.

88

control group (64.4 i 10.3%). Importantly, in the absence of added Zn2+ to the C-

peptide, the difference in C-peptide bound to the RBCs was not statistically different

from the basal levels of C-peptide for either group of rats (p > 0.001) suggesting that

. . . . 2+
rnteractrons between the C-peptrde and the RBC requrre the presence of the Zn .

Collectively, the data presented in ﬁgures 3.4, 3.5, and 3.7 suggest that the

. . . 2+ . .
drabetrc RBC may be somewhat resrstant to Zn -act1vated C-peptrde. In an attempt

to reduce this apparent resistance, RBCs were incubated with metformin, a biguanide.
Metformin is one of only two drugs currently approved by the World Health
Organization for the treatment of type 2 diabetes (along with glibenclamide) and is
currently the most widely prescribed oral blood glucose-lowering agent for the
treatment of type 2 diabetes.

The primary function of metformin is to improve metabolic control by offering

similar or greater glucose-lowering efﬁcacy compared with other oral antidiabetic

9

9 . . . . .
agents. Metforrnrn does not strmulate 1nsulrn secretron although the presence of

insulin is required for the effectiveness in diabetic states, however efﬁcacy does not
appear to directly correspond to insulin concentration. Metformin is turique in that it
does not cause weight gain, so it is often prescribed for patients that are overweight or
obese, although it is still effective in patients who are not overweight.

At a macromolecular level, metforrnin has been shown to improve or reduce
several cardiometabolic risk factors ascribed to type 2 diabetes. Most notably,

metforrnin has been shown to counter insulin resistance or behave as an “insulin

. . . . . 20 . . .
sensrtrzer” 1n lrver, muscle and other vascular trssues. Srmrlarly, metforrnrn has also

89

been shown to increase glucose transport into RBCs of patients with type 2 diabetes

. . 21,22 .
compared to glucose transport wrthout metformrn. Metformrn has also been

shown to increase RBC membrane ﬂuidity,23 allowing the RBC to release more ATP

than a less deformable RBC, which would require more glucose intake into the cell as
the only mechanism by which RBCs can create ATP is through glycolysis.

Metformin also appears to inhibit cellular apoptosis by inhibiting the cellular

respiratory Chain complex 1.24 Here, the data presented in Figures 3.4, 3.5, and 3.7

also suggest that metforrnin has the ability to serve as a C-peptide sensitizer.

Speciﬁcally, the addition of metformin to the RBCs 3 hours prior to the addition of the

2+ . . . . .
Zn -actrvated C-peptrde, resulted 1n an 1ncrease 1n ATP release, glucose uptake, and

binding to the RBCs that was statistically equivalent to that of the control RBCs.

In an attempt to elucidatethe mechanism by which metforrnin was exerting its
effects on the diabetic RBC, a closer examination of the diabetic RBC membrane was
performed. Additionally, it has been shown that in vitro exposure of RBCs to

hyperglycemic buffers results in a subsequent translocation of the phosphatidylserine

9

(PS) lipid from the inner leaﬂet of the plasma membrane to the outer monolayer.

The overall Charge on the PS lipid is negative, in contrast to the zvviterionic
phospholipids (phosphatidylcholine and sphingomyelin) that typically comprise the
majority of lipids in the outer leaﬂet. At physiological pH, the carboxylic acid-
containing groups on C-peptide (4 glutamic acid sites and 1 aspartic acid site) would

be negatively charged carboxylate groups. This suggests that RBCs having more PS

90

exposed to the extracellular ﬂuid may be more resistant to C-peptide binding due to
charge-based repulsions.

To measure the amount of PS exposed on the outer leaﬂet of the membrane,
control and diabetic RBCs were incubated with ﬂuorescent (FITC) labeled annexin V,
a protein that is known to selectively bind to PS and is commonly used as an indicator
for cell apoptosis and cell aging induced exposure of PS. Indeed, more annexin V
binding is observed in RBCs obtained from the diabetic rats in comparison to controls
(ﬁgure 3.8).

Metformin has been reported to inhibit cellular apoptosis in various cell

types.24 To determine if the positive effects of metforrnin shown in ﬁgures 3.4, 3.6,

and 3.7 were associated with blocking PS, RBCs from both animal sets were incubated
with metfonnin, centrifuged, washed and then exposed to FITC annexin V. The

decrease in annexin binding to the RBCs of the diabetic rats suggests that metforrnin

may be facilitating the interactions of the Zn2+-activated C-peptide by blocking the

effects of the PS.

These results suggest that RBCs exposed to hyperglycemia (as is the case with
type 2 diabetes), do not respond as well to treatment with C-peptide due to
extemalization of PS to the outer leaﬂet of the membrane. However, these data
suggest that results obtained from RBCs in a normoglycemic type 1 rat would behave
similarly to control rats. As shown in ﬁgures 3.9 and 3.10, the normalized ATP
release and glucose transport for RBCs obtained from type 1 rats upon incubation
with metal-activated C-peptide are near the same levels as those obtained from

controls.

91

Control Diabetic
Membrane Membrane 15000 1

+ + 14000 1
12000 1
10000 1
8000 i

6000 1

Fluorescence Intensity

4000 1

+ +

+ 20001 -22.
490 500 510 520 530 540 550

‘1’ Annexin Wavelength (nm)

 

 

O Phosphatidylcholine
O Phosphatidylserine

1400 i
- Control
1200 - Type 2

It)!

1000 i
800 1
600 f
.00

Fluorescence lntens

200 i

 

 

0 SA..-

-Met +Met

Figure 3.8: The top left graphic represents the differences in PS levels of the
diabetic and control RBC membranes and the ability of annexin V to bind to these
membranes. The graphic on the right (top) represents a raw data ﬂuorescence
spectrum that is typically obtained when performing the FITC annexin V-binding
experiment. The bottom panel shows a quantitative determination of the
ﬂuorescence intensities (resulting from annexin V binding to PS) obtained upon the
incubation of diabetic (black bars) and control (gray bars) RBCs with and without
30 pM metforrnin. Error bars are i SE (n=4), p-values < 0.001.

92

Normalized ATP Release

5 -
l — Control
: 1:211 Type1

 

 

 

 

    

 

RBCS 10P+1OZn

Figure 3.9: The normalized ATP release of type 1 rat RBCs incubated in

the presence of Zn2
values <0.001.

+-activated C-peptide. Error bars are r SEM (n=5). P

93

 

Normalized CPM

1'8 — Control
1.6 Type1
1.4
12
1.0
03
0.6
0.4
0.2

0.0 ,

 

 

 

 

 

 

RBCs

10P+1OZn

Figure 3.10: The normalized glucose transport in CPMs of RBCs from

type 1 and control rats in the presence of Zn2+-activated C-peptide.
Error bars are :I: SEM (n=4). P value <0.01.

94

DISCUSSION

In the pancreatic B-cell granules, C-peptide coexists with insulin in its hexameric

form.26 This hexamer is comprised of 6 insulin molecules and 2 Zn2+ atoms. Indeed,
2+ . . . . .

Zn transporter proteins eXist in the beta cells keeping these concentrations at
. . . . . . 27,28 .

millimolar levels. The pH of these granules lS highly ac1d1c rendering the

granular C-peptide protonated and, as such, it would not readily bind to Zn2+.
However, exposed to physiological pH in the bloodstream, it was anticipatedthat C-
peptide would become de-protonated and competitively bind and remove Zn2+ from

hexamerized insulin. Native insulin exists as a hexamer that can become
depolymerized by C-peptide into its monomeric form. As shown here, albeit in the

absence of any insulin hexamer, mass spectrometry reveals that C-peptide effectively

. 2+ . . .
binds Zn . These results are encouraging Since C-peptide has been reported to

disaggregate hexameric insulin. This interaction between hexameric insulin and C-

peptide has been reported to be greatly affected by pH, Zn ions and Zn chelators such

as EDTA.29

In contrast to previous reports in which the presence of insulin was necessary
for the beneﬁcial effects of C-peptide in vivo, the data reported here were obtained

without effects from insulin. Instead C-peptide was activated exogeneously with

Zn2+. The addition of Zn2+ to C-peptide was required for increased ATP release and

glucose uptake by RBCs, without which C-peptide exhibited no biological effect.

95

Moreover, it is worth mentioning that previous reports requiring the co-administration

of insulin with C-peptide for beneﬁcial effects may have actually been providing a

2+- . . . . . . . . .
source of Zn Without realizmg it as most forms of insulin are Zinc-containing.

Increased release of ATP from RBCs by Zn2+-activated C-peptide should not

be considered simply as an increase in the release of a biomarker. ATP release from

RBCS is a recognized stimulus for NO production in platelets30 and endothelium“

resulting in reduced platelet activity. While C-peptide itself has been shown to have

no direct effects on platelet function, the ability of RBC to affect platelet activity and

adhesion to endothelium via ATP release has been reported”-34 It is well established

that type 2 diabetic individuals have hyperactive platelets,35’36 with potential

consequences as to cardiovascular and cerebro-vascular disorders. Such patients

demonstrate deﬁcient ATP-release from RBCs in response to mechanical and

. . . 5,l6,l7
pharmacological stimuli.

Although Zn2+-activated C-peptide increased ATP release from type 2 diabetic

RBCs, these increases were still less than those obtained from non-diabetic RBCs. To
further explore the underlying reasons for these discrepancies, the effects of
metforrnin were explored. The primary function of metformin is to improve metabolic

control by offering similar or greater glucose-lowering efﬁcacy compared with other

9

. . . l 9 . . . . .
oral antidiabetic agents. Metformin does not stimulate insulin secretion although

the presence of insulin is required for the effectiveness in diabetic states, however

efﬁcacy does not appear to directly correspond to insulin concentration.

96

At a macromolecular level, metforrnin has been shown to improve or reduce
several cardiometabolic risk factors ascribed to type 2 diabetes. Most notably,

metforrnin has been shown to counter insulin resistance or behave as an “insulin

. . ,, . . . 20 . . .
senSitizer in liver, muscle and other vascular tissues. Similarly, metforrnin has also

been shown to increase glucose transport into RBCs of patients with type 2 diabetes

9

. . 2 .
compared to glucose transport Without metforrnin. Metformin has also been

shown to increase RBC membrane ﬂuidity,23 allowing the RBC to release more ATP

than a less deformable RBC, which would require more glucose intake into the cell as
the only mechanism by which RBCs can create ATP is through glycolysis. Moreover,
this biguanide molecule would be expected to display positive character when exposed
to physiological conditions.

While it is intriguing that metforrnin may display some activity in combination

with C-peptide, an explanation for such effects currently does not exist in the

. . . . . . . 37
literature. Metformm has been reported to display interactions With cations and also

. . . . . 24 . . .
display antiapoptotic actiVity. The latter work 18 of importance here as it has been

demonstrated that hyperglycemia results in translocation of PS from the inner leaﬂet

9

of the plasma membrane to the outer leaﬂet.1 25 Such extemalization of the PS lipid

is often associated with cell apoptosis. The charge of the PS lipid is negative in
contrast to the zwitterionic phosphatidylcholine and splingomyelin, which normally

constitute the majority of lipids in the outer leaﬂets. Hence, it may be anticipated that

97

the negatively charged PS would further repulse the highly negatively charged C-
peptide.

The demonstration of decreased annexin binding to diabetic RBCs in the
presence of metforrnin suggests that metforrnin is affecting the exposure of the
negatively charged PS lipid, thereby facilitating C-peptide’s interaction with the
diabetic RBC membrane. While its exact mechanism is currently not completely
understood, it does have a biological effect on the normalization of the ATP release in
diabetic and control RBCs, and enhanced C-peptide association with diabetic RBC
following the addition of metforrnin. Interestingly, insulin was not a signiﬁcant
determinant on the latter effect, again providing evidence that C-peptide is able to
exert biological effects in the absence of insulin.

The results reported here suggest that the type 2 diabetic RBC may be resistant
to the effects of C-peptide due to lipid extemalization under hyperglycemic conditions

(see ﬁgure 3.11). The present study has also demonstrated that the apparent resistance

to Zn2+-activated C-peptide displayed by the type 2 diabetic RBC can be overcome
with the simultaneous incubation with metforrnin, thereby normalizing glucose uptake
and ATP release from RBCs. RBC-derived ATP release has been shown to affect NO
production in the endothelium and play a role in platelet adhesion. In this construct,

the present ﬁndings may have therapeutic implications for type 2 diabetic patients

with complications associated with poor blood ﬂow. Importantly, the present data

2+ . . . . . .
demonstrate a novel effect of Zn -activated C-peptide that 18 independent of insulin.

RBCs do not possess insulin receptors and hence the demonstrated effects on glucose

uptake, ATP production and release must be independent of insulin signalling. The

98

 

results demonstrate that metforrnin, often regarded as an insulin sensitizer, also has the
ability to sensitize the effects of C-peptide. Collectively, data presented in this chapter
provides data that suggests a possible reason why people who have diabetes may not

beneﬁt ﬁom the presence of C-peptide.

99

 

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REFERENCES

l. Al-Rasheed NM, Willars GB, Brunskill NJ. C-peptide signals via G.alpha.i to
protect against TNF-a-mediated apoptosis of opossum kidney proximal tubular cells.
Journal of the American Society of Nephrology. 2006;17:986-995.

2. Rigler R, Pramanik A, Jonasson P, et al. Speciﬁc binding of proinsulin C-
peptide to human cell membranes. Proceedings of the National Academy of Sciences
of the United States of America. 1999;96:13318-13323.

3. Grunberger G, Qiang X, Li Z, et al. Molecular basis for the insulinomimetic
effects of C-peptide. Diabetologia. 2001 ;44: 1247-1257.

4. Johansson BL, Borg K, Femqvist-Forbes E, Kernell A, Odergren T, Wahren J.
Beneﬁcial effects of C-peptide on incipient nephropathy and neuropathy in patients
with type 1 diabetes mellitus. Diabetic Medicine. 2000;17:181-189.

5. Meyer JA, Froelich JM, Reid GE, Karunarathne WKA, Spence DM. Metal-
activated C-peptide facilitates glucose clearance and the release of a nitric oxide
stimulus via the GLUTl transporter. Diabetologia. 2008;51:175-182.

6. Baynes JW. Role of oxidative stress in development of complications in
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7. Strain JJ. Disturbances of micronutrient and antioxidant status in diabetes. Proc
Nutr Soc. 1991;50:591-604.

8. Young IS, Woodside JV. Antioxidants in health and disease. J Clin Pathol.
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9. Gutteridge JMC. Lipid peroxidation and antioxidants as biomarkers of tissue
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10. Gaskin RS, Estwick D, Peddi R. G6PD deﬁciency: its role in the high
prevalence of hypertension and diabetes mellitus. Ethnicity & disease. 2001;11:749-
754.

11. Manuel y Keenoy B, Vertommen J, De Leeuw I. Divergent effects of different
oxidants on glutathione homeostasis and protein damage in erythrocytes from diabetic

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patients: effects of high glucose. Molecular and Cellular Biochemistry. 2001;225:59-
73.

12. Daimant E, Landberg, E., London, I.M. The Metabolic Behavior of Reduced

Glutathione in Human and Avian Erythrocytes. Journal of Biological Chemistry.
1955;213:769-776.

l3. Murakarni G. The glucose metabolism of erythrocytes. Hirosaki Igaku.
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14. Dumaswala UJ, Wilson MJ, Wu YL, et al. Glutathione loading prevents free

radical injury in red blood cells after storage. Free Radical Research. 2000;33:517-
529.

15. Sprague RS, Ellsworth ML, Stephenson AH, Lonigro AJ. ATP: the red blood
cell link to NO and local control of the pulmonary circulation. Am J Physiol.
1996;271 :H2717-H2722.

16. Carroll J, Raththagala M, Subasinghe W, et al. An altered oxidant defense
system in red blood cells affects their ability to release nitric oxide-stimulating ATP.
Molecular BioSystems. 2006;2z305-31 1.

l7. Sprague RS, Stephenson AH, Bowles EA, Stumpf MS, Lonigro AJ. Reduced
expression of Gi in erythrocytes of humans with type 2 diabetes is associated with
impairment of both cAMP generation and ATP release. Diabetes. 2006;55:3588-3593.

18. Nathan DM, Buse JB, Davidson MB, et al. Management of hyperglycaemia in
type 2 diabetes: a consensus algorithm for the initiation and adjustment of therapy. A
consensus statement from the American Diabetes Association and the European
Association for the Study of Diabetes. Diabetologia. 2006;49:1711-1721.

l9. Krentz AJ, Bailey CJ. Oral antidiabetic agents: Current role in type 2 diabetes
mellitus. Drugs. 2005;65:385-411.

20. Faure P, Rossini E, Wiemsperger N, Richard MJ, Favier A, Halimi S. An
insulin sensitizer improves the free radical defense system potential and insulin
sensitivity in high fructose-fed rats. Diabetes. 1999;48:353-357.

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21. Rapin JR, Lespinasse C, Yoa R, Wiemsperger N. Erythrocyte glucose
consumption in insulin-dependent diabetes: effect of metforrnin in vitro. Diabete &
Metabolisme. 1991 ;17: 164-167.

22. Yoa RG, Rapin JR, Wiemsperger NF, Martinand A, Belleville I.
Demonstration of defective glucose uptake and storage in erythrocytes from non-
insulin dependent diabetic patients and effects of metformin. Clinical and
experimental pharmacology & physiology. 1993;20:563-567.

23. Muller S, Denet S, Candiloros H, et al. Action of metformin on erythrocyte

membrane ﬂuidity in vitro and in vivo. European Journal of Pharmacology.
1997;337:103-110.

24. Guigas B, Detaille D, Chauvin C, et al. Metformin inhibits mitochondrial
permeability transition and cell death: a pharmacological in vitro study. Biochem J.
2004;382:877-884.

25. Daleke DL. Regulation of phospholipid asymmetry in the erythrocyte
membrane. Current Opinion in Hematology. 2008;] 5: 191-195.

26. Chimienti F, Favier A, Seve M. ZnT—8, A Pancreatic Beta-Cell-Speciﬁc Zinc
Transporter. BioMetals. 2005;18:313-317.

27. Hutton JC. The internal pH and membrane potential of the insulin-secretory
granule. Biochem J. 1982;204:171-178.

28. Orci L, Halban P, Perrelet A, Amherdt M, Ravazzola M, Anderson RGW. pH-
independent and -dependent cleavage of proinsulin in the same secretory vesicle. J
Cell Biol. 1994;126:1149-1156.

29. Shafqat J, Melles E, Sigmundsson K, et al. Proinsulin C-peptide elicits
disaggregation of insulin resulting in enhanced physiological insulin effects. Cell Mol
Life Sci. 2006;63:1805-181 1.

30. Freedman JE, Loscalzo J, Barnard MR, Alpert C, Keaney JF, Jr., Michelson
AD. Nitric oxide released from activated platelets inhibits platelet recruitment. Journal
of Clinical Investigation. 1997;100:350-356.

103

 

31. Kotsis DH, Spence DM. Detection of ATP-induced nitric oxide in a

biomimetic circulatory vessel containing an immobilized endothelium. Anal Chem.
2003;75:145-151.

32. Carroll JS, Ku C-J, Karunarathne W, Spence DM. Red Blood Cell Stimulation
of Platelet Nitric Oxide Production Indicated by Quantitative Monitoring of the
Communication between Cells in the Bloodstream. Analytical Chemistry
(Washington, DC, United States). 2007;79:5133-5138.

33. Ku C-J, D'Amico Oblak T, Spence DM. Interactions between Multiple Cell
Types in Parallel Microﬂuidic Channels: Monitoring Platelet Adhesion to an
Endothelium in the Presence of an Anti-Adhesion Drug. Anal Chem (Washington,
DC, U S). 2008;80:7543-7548.

34. Ku C-J, Karunarathne W, Kenyon S, Root P, Spence D. Fluorescence
Determination of Nitric Oxide Production in Stimulated and Activated Platelets.

Analytical Chemistry. 2007;79:2421-2426.

35. O'Sullivan BP, Linden MD, Frelinger AL, 111, et a1. Platelet activation in cystic
ﬁbrosis. Blood. 2005;105:463 5-4641.

36. Vinik AI, Erbas T, Park TS, Nolan R, Pittenger GL. Platelet dysfimction in
type 2 diabetes. Diabetes Care. 2001;24:1476-1485.

37. Abu-El-Wafa SM, El-Ries MA, Ahmed FH. Formation of metformin
complexes with some transition metal ions: their biological activity. Inorg Chim Acta.
1987;136:127-131.

104

 

CHAPTER 4- DETERMINING THE MECHANISM BY WHICH C-PEPTIDE

IS INTERACTING WITH RBCS

4.1 CURRENT PROPOSED MECHANISM OF C-PEPTIDE IN VIVO

C-peptide is not universally accepted to be a bioactive peptide, one reason being there

. . l . .
has never been an identiﬁed receptor. Despite this, recent efforts have shown that C-

peptide binds stereospeciﬁcally to a variety of cells including renal tubular,

. . . '2
endothelial, skin ﬁbroblasts, mesangial, and neuroblastoma. However, Ido et al has
. . + + . . .
suggested that some of the increases in Na , K - ATPase act1V1ty 18 not a result of C-

. . . . 3 . . + +
peptide binding stereospeCiﬁcally to a receptor, based on increases in Na , K -

ATPase activity upon activation using a retro C-peptide sequence as well as an all D-
amino acid C-peptide enantiomer. The results of this study indicate that the biological
activity of C-peptide was based on membrane interactions related to the structure of
the C-peptide, but independent of its direction or chirality.

As described in detail in chapter 1 and shown in table 4.1, the signaling
pathway of C-peptide is not understood, despite evidence of many possible
mechanisms in vivo. Shown in table 4.1 is a summary of the signaling elements

activated by C-peptide. As shown in the table, the only signaling element that has

been determined for erythrocytes (RBCs) is the increase of Na+, K+- ATPase activity.

An increase in Na+, K+- ATPase activity after incubation with C-peptde has also been

shown in renal tubular cells, which is abolished in the presence of pertussis toxin

105

 

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106

(PTX), therefore suggesting the involvement of a Gi/Go-linked protein.4-7 This is
signiﬁcant because it has been established that the mechanism for the deformation-
induced release of ATP from RBCs involves the heterotrimeric Gi protein.8 This,
paired with a report from Kunt stating that C-peptide treatment results in an increase

in RBC deformability, indicates that ATP released in response to incubation With C-

peptide may be G-protein linked.

Additionally, Na+/K+-ATPase activity in RBCs from people with type 1 diabetes

is reduced, which can lead to secondary effects including decreased deformability of

the RBCs and altered rheological properties. However, both of these defects have

. 9,10 . .
been reported to be corrected after exposure to C-peptide. Based on this eVidence,

it was hypothesized that the ATP release from RBCs after incubation with C-peptide

may be the result of activation of a G-protein coupled receptor, resulting in activation

of the previously reported signaling cascade for the release of ATP (ﬁgure 4.1).l H3

4.2 PTX AND MASTOPARAN-7 (MAS-7) AND THEIR ABILITY TO

INHIBIT OR ACTIVATE G-PROTEINS

PTX is a protein based AB5-type exotoxin produced by the bacterium Bordetella

pertussis, which causes whooping cough. When PTX binds to a cell membrane
receptor, it is taken up by an endosome where it undergoes retrograde transport to the

trans-Golgi network and endoplasmic reticulum. From here, PTX catalyzes the ADP-

ribosylation of the a—subunit of the heterotrimeric G-proteins, Gi, G0, and G. This

107

Mechanical
Deformation

l

a,"
2 cm H

cAMP

\‘CGDj ATP

 

 

 

 

Red Blood Cell

 

Figure 4.1: The proposed mechanism of ATP release from RBCs due to

. . 8,1 1,13
mechanical deformation.

108

prevents the G-proteins from interacting with the G-protein coupled receptors located

. . . . . 14
on the cell membrane, interfering With cellular communication.

Mastoparan (ﬁgure 4.2) is a toxic peptide found in wasp venom and has been

shown to cause a secretion of histamine from various cell types, resulting in an

. . . 2+ . . . . .
increase in cytosolic Ca , which in turn causes an increase in the intracellular second

messenger inositol-l,4,5-triphosphate (1P3). Increases in Ca2+ mediated by 1P3 have

been shown to regulate phospholipase C via G proteins. Based on this mechanism, it

is reported that mastoparan has the ability to activate G-proteins by mimicking

15 . . . .
receptors. MAS-7 is an analog of mastoparan, and is conSIdered to be 10 times

more potent than mastoparan alone.

4.3 INHIBITORS OF PROTEINS INVOLVED IN DEFORMATION
INDUCED ATP RELEASE MECHANISM

H-89 or N-[2-bromocinnamylaminoethyl]-5-isoquinoline sulfonamide is considered to

be a selective and potent inhibitor of PKA. It is thought to inhibit PKA by

competitively competing with ATP. While it is considered to be selective as an

inhibitor for PKA, at higher concentrations (above 10 iiM) it has been shown to inhibit
other kinases.16

MDL-l2,22OA or [N-(cis-2-phenylcyclopentyl)azacyclotridecan-Z-imine HCl]

is a compound that has been reported to inhibit activated adenylyl cyclase

109

 

H - lle-Asn - Leu- Lys-Ala- Leu -A|a-Ala- Leu-
Ala - Lys - Ala - Leu - Leu - NH2

 

Figure 4.2: Peptide sequence of the G-protein activator mastoparan-7.

110

activity with IC50 values ranging from 10-100 nM.”-ZO The mechanism of action is

. . . . . . . 2+ . . 21
believed to be mediated Via inhibition of Ca inﬂux Via slow channels.

BIS is one member of a family of bisindoylmaleimdes that have synthesized
and characterized as selective and potent inhibitors of PKC. These molecules were
synthesized to provide for a more selective inhibition of PKC compared to the most
potent PKC inhibitor staurosporine, which lacks selectivity. BIS was found to be as

potent as staurosporine at inhibiting PKC, however it was not found to be strong

inhibitor of PKA unlike staurosporine.22

4.4 EXPERIMENTAL

4.4.1 Collection and Preparation of RBCs

Rabbits (male New Zealand whites, 2.0-2.5 kg) were anaesthetised with ketamine (8
ml/kg, i.m.) and xylazine (1 mg/kg, i.m.) followed by pentobarbital sodium (15 mg/kg
i.v.). A cannula was placed in the trachea and the animals were ventilated with room
air, having a ﬂow rate of 20 breaths per min (10 cc air). A catheter was placed into a
carotid artery for administration of heparin and for phlebotomy. After heparin (500
units, iv), the animals were exsanguinated. All procedures were approved by the
Animal Investigation Committee at Michigan State University. Blood was centrifuged
for 10 min at 500 g and 4°C. The plasma and buffy coat were discarded. The RBCs

were resuspended and washed three times in a physiological salt solution (PSS) (in

mM: 4.7 KCl, 2.0 CaClz, 140.5 NaCl, 12 MgSO4, 21.0

111

 

tris[hydroxymethyl]aminomethane, 11.1 dextrose with 5% w/v bovine serum albumin
[ﬁnal pH 7.4]).
4.4.2 Preparation of Reagents
Synthetic human C-peptide was commercially purchased (Genscript, Piscataway, NJ)
and puriﬁed in house using liquid chromatographic methods. Its content was veriﬁed
using electrospray mass spectrometry. A stock solution of 8.3 uM was prepared by
dissolving 0.25 mg of the puriﬁed peptide in an appropriate amount of puriﬁed water
(DDW).

A solution of zinc was prepared by dissolving 5.5 mg of zinc (II) chloride in
500 m1 of DDW yielding a stock concentration of 80 nM. From this stock, 31.8 pl
was diluted in 25 ml of DDW to yield a working solution with a concentration of 102
nM.
4.4.2.1 Preparation of Antagonists used to Inhibit ATP Release Pathway
PTX was prepared by diluting the 50 pg vial (Sigma-Aldrich, St. Louis, M0) to 50 ml
using DDW creating a 1 ug/ml stock solution. MAS-7 (Sigma-Aldrich, St. Louis,
MO) was prepared by dissolving 1 mg of MAS-7 in 10 ml of DDW creating a 70 iiM
stock solution. H-89 (Sigma-Aldrich, St. Louis, MO) was prepared by dissolving 5
mg of H-89 in 1 ml of phosphate buffered saline (PBS) creating a 9.6 mM stock
solution. MDL-12,33OA (Sigma-Aldrich, St. Louis, MO) was prepared by dissolving
2.5 mg of MDL-12,330A in 66.3 ml of DDW creating a 400 uM stock solution. BIS
(Sigma-Aldrich, St. Louis, MO) was prepared by dissolving 1 mg of BIS in 1 ml of

dimethylsulfoxide creating a 2.2 mM stock solution. Niﬂumic acid was prepared by

112

dissolving 0.29 g of niﬂumic acid in 10 ml of 100% ethanol, creating a 0.1 M stock

solution.

4.5 METHODS
4.5.1 ATP release from RBCs upon Incubation with Various Inhibitors

To determine the effect of PTX on ATP release of RBCs incubated with C-
peptide, 500 pl of the 1 pg/ml stock PTX was added to 4 ml of PSS, followed by the
addition of approximately 500 pl of RBCs creating a 100 ng/ml solution of PTX. The

RBC suspension incubated for a period of 2 hours, followed by centrifugation and the

addition of 600 p1 of C-peptide stock, 490 pl of Zn2+ stock previously mixed in 3410

pl of PSS. After the addition of C-peptide/Zn2+, the RBCs were incubated for an

additional 4 hours, followed by the measurement of ATP release. The ATP release
was determined using the non-ﬂow method as described in previous chapters.

The ATP release from RBCs incubated with MAS-7 was determined by taking
71 pl of the stock MAS-7 solution and diluting with 4.43 ml of PSS, followed by the
addition of approximately 500 pl of RBCs and allowing the RBC suspension to
incubate for 15 minutes prior to the measurement of ATP. The effect of PTX on
MAS-7- induced ATP release was determined by incubation of RBCs with PTX (as
described above), followed by the addition of 71 pl of MAS—7 and incubated for 15
minutes. The ATP release was determined as described above.

The effect of MDL-12,330A on ATP release was determined by adding 190 pl
of the 400 pM stock MDL-12,330A to 4.31 ml of PSS, followed by the addition of

500 pl of RBCs. This suspension was incubated for 20 minutes followed by

113

centrifugation and the addition of C-peptide/Zn2+/PSS as described above. The ATP

release was measured after 4 hours of incubation.
The ATP release after incubation with H-89 was determined by taking 15.5 pl
of the H-89 stock solution and diluting it in 4.5 ml of PSS, followed by the addition of

500 pl of RBCs. This suspension was incubated for 1.5 hours, followed by

centrifugation and the addition of C-peptide/Zn2+/PSS as described above. This

suspension was incubated for an additional 4 hours followed by the measurement of
ATP as described above.
Similarly, 22.7 pl of the BIS stock was diluted in 4.5 ml of PSS, followed by

the addition of 500 pl of RBCs and incubated for 15 minutes. The suspension was

centrifuged and the C-peptide/Zn2+/PSS (as described above) was added followed by

an incubation of 4 hours after which the ATP release was measured.

4.5.2 Fluorescence Determination of cyclic AMP (cAMP) Levels upon
Incubation of RBCs with C-peptide

RBCs were incubated with H-89 and/or C-peptide as described above. After

incubation, the RBC suspensions were centrifuged and the supernatant was removed.

The CAMP levels were determined using a ﬂuorescent-based competitive ELISA kit

(Catchpoint, Sunnyvale, CA).

4.5.3 Glucose Transport of RBCs Incubated with C-peptide and Inhibitors

Aliquots (10, 5, 2.5, and 1.25 pl) of the stock niﬂumic acid were added to the low

glucose PSS (550 pM, 300 pl) followed by the addition of 100 pl of RBCs and

. 2+
incubated for 15 minutes. After incubation, 120 pl of C-peptide and 98 pl of Zn

114

pre-mixed in low glucose PSS (containing 2 pCi of 14C-labeled glucose) were added

to the RBC suspension and allowed to incubate for an additional 4 hours. After

incubation, the RBC suspensions were centrifuged and washed twice with low glucose

PSS to remove any excess 14C-labeled glucose. The packed RBCs were lysed with 1

ml of bleach and the radioactivity was determined as described in previous chapters.
RBCs incubated with H-89 and BIS were prepared as described above,

however all volumes were reduced by 1/5 and the PSS that was added with the C-

peptide/Zn2+ contained 2 pCi of 14C-labeled glucose. The incubation times and

determination of radioactivity is as described above.

4.6 RESULTS
Using the established mechanism for ATP release from RBCs as a guide, we
hypothesized that C-peptide was resulting in an increase in ATP release by activating

a G-protein. As shown in ﬁgure 4.3, the ATP release from RBCs incubated with

Zn2+-activated C-peptide is 97.5 i 25.5% higher than that of RBCs alone.

Additionally, without the metal present the ATP release is not statistically different

than RBCs. Moreover, when RBCs are incubated with PTX prior to the addition of

Zn2+-activated C-peptide the ATP release is not signiﬁcantly reduced or inhibited,

indicating that the mechanism for ATP release does not involve the G-proteins that are

essential to the established deformation-induced ATP release mechanism. However,

115

 

Normalized ATP Release

.N
o
l n

—l
01
J A

 

.5
O
l I

.0
01
I l n n I

 

 

.0
o

(9 19 § x Q :\ «+
Q90 \0 \§ Q‘tNél’ @09 (aﬁxQ

Q
\Q \QQ @‘b

Figure 4.3: Normalized ATP release from rabbit RBCs in the presence of zinc-
activated C-peptide (10P+IOZn). Resultant ATP release after incubation with
pertussis toxin prior to zinc-activated C-peptide (PTX+10P+IOZn). ATP
release using G-protein activator mastoparan-7 in combination with pertussis
toxin (mas-7+PTX). Error bars are i SEM (n=7), p<0.001.

116

as a positive control, RBCs were incubated with a known G-protein activator, MAS-7,
prior to the addition of PTX. It was determined that PTX is able to signiﬁcantly
reduce the ATP release from RBCs indicating that the pertussis toxin is functioning

properly. This ﬁnding is important because it demonstrates that the ATP release as a

. . . 2+ . . . . .
result of incubation With Zn -activated C-peptide 1S independent of the G-proteins

required for deformation induced ATP release. Summarily, these results suggest that
C-peptide may have an effect on RBCs that does not require G-protein activation.
Another requirement of the established deformation-induced ATP release

mechanism from RBCs includes the activation of adenylyl cyclase. In the proposed

pathway this occurs via activation of the GS protein, which is linked to the G-protein

coupled receptor. As indicated by the results above, Zn2+-activated C-peptide appears

to be resulting in an ATP release independent of G-proteins, and therefore may be
activating adenylyl cyclase by due to an increase in substrate (ATP). When RBCs

were incubated with an adenylyl cyclase inhibitor (MDL12,33OA) prior to the addition
of Zn2+-activated C-peptide, the ATP release detected was not reduced compared to
the ATP release of RBCs incubated with only Zn2+-activated C-peptide (ﬁgure 4.4).

cAMP is produced in RBCs upon the conversion of ATP by adenylyl cyclase.

cAMP activates PKA, which phosphorylates the cystic ﬁbrosis transmembrane

regulator (CFTR) protein. Due to Zn2+-activated C-peptide increase in ATP release

from the RBCs, it was hypothesized to increase cAMP levels. However, as shown in

117

 

 

 

 

     

2.5:
0
3 2.0 ~
2 1
G, .
n:
n. 1.5 -
I'-
<
'0
g 1.0—
g .
o 0.5:
2 4
0.03
"o \/
Q90 s"
1,0
XNQ

,9

Figure 4.4: Normalized ATP release of RBCs incubated with Zn2+-activated

C-peptide (10P+10Zn) and RBCs incubated with Zn2+-activated C-peptide
and the adenylyl cyclase inhibitor MDL12,330a (10P+10Zn+MDL). Error
bars are :1: SEM (n=7), p<0.001.

118

ﬁgure 4.5, the cAMP levels decreased (increase in ﬂuorescence intensity) in the

2+ . .
presence of Zn -activated C-peptide.

Interestingly, in the presence of H-89 and Zn2+-activated C-peptide the cAMP

levels increased back to levels that were near statistical equivalence to RBCs alone.

2+ . . . . .
These results suggest that Zn -act1vated C-peptide 1S somehow resulting in the

depletion of cAMP in the RBCs.

When RBCs were incubated with the PKA inhibitor H-89 prior to the addition

of Zn2+-activated C-peptide, the ATP release was approximately 120% greater than

that of RBCs incubated with Zn2+-activated C-peptide. These values suggest that

phosphorylation of CFTR may occur by another kinase resulting in an ATP release

that is greater than the ATP release from RBCs incubated with Zn2+-activated C-

peptide (ﬁgure 4.6). Furthermore, as shown in ﬁgure 4.7, the amount of glucose

transported in the presence of the PKA inhibitor did not increase signiﬁcantly
compared to Zn2+-activated C-peptide treated RBCs indicating that the inhibition of
PKA does not affect the ability of Zn2+-activated C-peptide to transport glucose.

There have been recent reports suggesting that C-peptide activates PKC in

various cell lines including Swiss 3T3 ﬁbroblasts,23 and. human renal tubular cells.6’7

PKC has not been shown to phosphorylate CFTR in RBCs, however, PKC has been

shown to phosphorylate CFTR in other cell types.24 When RBCs were incubated with

BIS prior to the addition of Zn2+-activated C-peptide, the ATP release increased

119

 

1.8 3
1.65
1.45
1.2%
1.05
0.85
0.65
0.45
0.2%
0.0 5

 

Fluorescence Intensity

 

 

Figure 4.5: Normalized ﬂuorescence intensity obtained using competitive
ELISA for cAMP levels and RBCs containing H-89 (RBCs+H-89), Zn2+-

activated C-peptide (10P+IOZn), and Zn2+-activated C-peptide and H-89
(10P+10Zn+H-89). Error bars are i SEM (n=6), p<0.001.

120

3.0 1
2.5 5;
2.0 f

1.5{

 

1.05

Normalized ATP Release

0.5 {

 

   

0.0 -'
r9 0 q
8’0 ’56" 0,23%
s (a
X

.98

Figure 4.6: Normalized ATP release of RBCs incubated with Zn2+-activated C-

peptide (10P+10Zn) and Zn2+-activated C-peptide and H-89 (10P+IOZn+H89).
Error bars are :1: SEM (n=4), p<0.001.

121

 

1.6

1.4

1.2

 

1.0
0.8
0.6

Normalized CPM

0.4
0.2
0.0

 

e
e0 6‘9
Q‘ xN 0x
62 (a
X

 

\
\QQ \QQ

Figure 4.7: The normalized counts per minute (CPM) of RBCs incubated with
Zn2+-activated C-peptide (10P+10Zn). The resultant CPM aﬁer inhibition with
inhibitors (10P+10P+H89, 10P+IOZn+BIS). Error bars are :t SEM (n=3), p<0.005.

122

approximately 75% compared to RBCs incubated with only Zn2+-activated C-peptide

(ﬁgure 4.8). Moreover, the incubation of BIS with RBCs alone did not result in an

increase in ATP release above RBCs indicating that PKC is only active in the presence

+
of an activator such as an -activated C-peptide (or perhaps when ATP levels are

increased). Additionally, the glucose transport observed after incubation with BIS in

2+ . . . .
the presence of Zn -activated C-peptide was Similar to the glucose transport observed

after incubation with the PKA inhibitor H-89 (ﬁgure 4.7).

While it is still uncertain which kinase plays the most important role in the

release of ATP from RBCs incubated with Zn2+-activated C-peptide, ﬁgure 4.9

demonstrates that CF TR is still required for glucose transport into the RBC. Using
various concentrations of the CFTR inhibitor niﬂumic acid, the glucose transport into
the RBC decreased With increasing concentration of niﬂumic acid. These results
suggest that when CFTR is inhibited and ATP release is therefore inhibited, the
amount of glucose entering the RBC is reduced, indicating the ability of the RBC to
self-regulate the amount of glucose required based on the amount of ATP contained

within the RBC.

4.7 DISCUSSION
Since its discovery in 1967, the mechanism by which C-peptide is exerting biological
effects has not been completely determined. A receptor has not been identiﬁed, and

most literature indicates that C-peptide activates a G-protein, implying the

6,23,25-27

involvement of a G-protein coupled receptor. However, this has not been

123

3.0 -_
2.5 5

2.0 {

 

Normalized ATP Release
01

 

   

0.5 f
0.0 -'
e o «a
so (a ,9 a9
Q‘ Qx 1’0
*9 ,{9
.98

Figure 4.8: Normalized ATP release of RBCs incubated with Zn2+-activated C-
peptide (10P+10Zn), Zn2+-activated C-peptide and BIS (10P+10Zn+BIS) and RBCs
with BIS alone (BIS). Error bars are :t SEM (n=4), p<0.001.

124

12:

 

Glucose Transported (g)
0)

 

 

0 1 0.5 0.25 0.125
Concentration (mM)

Figure 4.9: The grams (g) of glucose transported into RBCs using 14C-labeled
glucose and various amounts of the CFTR inhibitor niﬂumic acid in the presence

of Zn2+-activated C-peptide. Error bars are i SEM (n=5), p<0.01.

125

shown in RBCs. As reported here, the ATP release from RBCs incubated with Zn2+-

activated C-peptide is not affected by incubation with PTX indicating that C-peptide’s

mechanism of action does not involve G-proteins. This, coupled with a report

suggesting that mechanically induced ATP release involves G-proteins,8 indicates that

2+ . . . . .
Zn -activated C-peptide does not result in an ATP release through activation of G—

proteins.
Once determined that the increase in ATP release was independent of G-

proteins, the mechanism by which C-peptide is able to elicit an ATP release was

. . 28 . . . .
reexammed. As preViously reported, incubation of RBCs With metal-activated C-

peptide results in an increase in glucose transport into the RBC. Once glucose enters
the RBC, approximately 90% of it undergoes glycolysis where ATP is produced.
Glycolysis is the only metabolic pathway that can produce ATP in RBCs. The

previously established mechanism for ATP release involved the conversion of ATP to

cAMP by adenylyl cyclase.29 With the increase in glucose transport, it was

hypothesized that there would be more ATP produced, which would lead to more
substrate available for conversion to cAMP by adenylyl cyclase.

However, as shown in ﬁgure 4.4, the amount of ATP released by the RBCs in

the presence of Zn2+-activated C-peptide and MDL12,330A, an inhibitor of adenylyl

. . . . 2+ .
cyclase, is increased compared to RBCs 1ncubated With only Zn -activated C-

peptide. These results indicate that even with the inhibition of adenylyl cyclase it is

possible for RBCs that had been incubated with Zn2+-activated C-peptide to release

126

 

ATP. This data does not suggest that adenylyl cylcase is not involved in the signal

transduction pathway; it merely suggests that the accumulation of ATP resulting from

. . . 2+ . . .
incubation With Zn -activated C-peptide is able to be released by some other

mechanism.

As previously reported, the mechanism of ATP release from RBCs incubated

with Zn2+-activated C-peptide involved CFTR.28 It is well established that CFTR

requires phosphorylation by PKA, a cAMP-dependent kinase.3O However, as shown

in ﬁgures 4.5 and 4.6, inhibition of PKA normalizes the amount of cAMP produced in
the RBC and increases the amount of ATP released from the RBCs. Taken
collectively, these results indicate that While an inhibition of PKA results in higher

cAMP levels, the amount of ATP that is being released is still considerably higher

than that of RBCs incubated with Zn2+-activated C-peptide, providing further

evidence of an alternative mechanism for the release of ATP from the RBC.
A possible alternative route for ATP release from RBCs could involve PKC.

As previously discussed, C-peptide has been shown to activate PKC in various cell

3.6’7’23’3l While not part of the established mechanism for ATP release, PKC has

type
been reported to directly affect the phosphorylation of CFTR,32 a protein already

. . . 2+
established to be a requirement for ATP release from RBCs 1ncubated With Zn -

activated C-peptide. As shown in ﬁgure 4.8, the ATP release of RBCs incubated with

. 2+
Zn2+-activated C-peptide and BIS was increased compared to RBCs With Zn -

. . 2+ . . . .
activated C-peptide alone. However, Without Zn -activated C-peptide, the addition

127

of BIS did not have an affect on the ATP release of RBCs providing evidence that the
inhibition of PKC affects the ability of RBCs incubated with Zn2+-activated C-peptide
to release ATP. Despite data demonstrating the involvement of both PKA and PKC in

the ATP release mechanism for RBCs incubated with Zn2+-activated C—peptide, there

does not appear to be an increase in the amount of glucose transported in the presence

of either inhibitor. A possible explanation for this may be an increased release of the
stored ATP Within the RBC in the presence of Zn2+-activated C-peptide. RBCs alone
contain low mM amounts of ATP, however only high nM to low pM concentrations

are released. It is possible that Zn2+-activated C-peptide is causing a release of the

stored ATP, which may not require an increase in glucose transport to compensate for
the ATP that is lost upon release.

As previously discussed, CFTR is required for ATP release from RBCs

incubated with Zn2+-activated C-peptide. However, as shown in ﬁgure 4.9, the

amount of glucose transported into the RBC decreases with increasing concentration

. . . . 2+ . . . . . .
of niﬂum1c ac1d, even in the presence of Zn -activated C-peptide. The inhibition of

CFTR does not allow for the release of ATP, so the ATP that is being produced within

. . . 2+ . . .
the RBC as a result of incubation With Zn -act1vated C-peptide continuously

increases. As reported by Carruthers et a1,33 ATP has the ability to inhibit GLUTl,

which may explain why there is a decrease in glucose transport upon incubation with

niﬂurnic acid.

128

'1'

While the exact mechanism for ATP release from RBCs incubated with Zn2+-

activated C-peptide has not been determined yet, some important questions about the

mechanism by which C-peptide exerts its effects have been answered. Contrary to

previous studies, Zn2+-activated C-peptide appears to behave independently of G-

proteins in the RBC and also appears to be affected by kinases that are not present in
the previously established mechanism for ATP release. While a receptor has not been

discovered, it is clear that C-peptide exerts biological effects.

129

 

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133

CHAPTER 5- CONCLUSIONS AND FUTURE DIRECTIONS

5.1 OVERALL CONCLUSIONS

Since its discovery in 1967, C-peptide has generally been considered to be a
biologically inert peptide, its primary role to maintain the A- and B- chains of insulin.
However, within the past 15 years, C-peptide has been shown to ameliorate several
complications associated with diabetes such as neuropathy, nephropathy, retinopathy,
and microvascular circulation problems.

More speciﬁcally, C-peptide has been shown to improve microvascular blood

. . . . . . . . . 1,2 . .
ﬂow in several tissues in diabetic patients including muscle and skin. In addition to

these reports, a report demonstrating the ability of C-peptide to improve red blood cell

(RBC) deformability in patients with type 1 diabetes has also been published.3 These

reports were inspirational to research discussed in this dissertation because it has been

. 4
reported that a more deformable RBC releases increased amounts of ATP.

As described in previous chapters, when ATP is released from RBCs it has the

ability to bind to the sz receptor of endothelial cells, which is the beginning of a

signal transduction pathway ultimately resulting in vasorelaxation of the smooth
muscle cells. Based on this information, we chose to investigate the ability of C-
peptide to induce an ATP release from RBCs as a function of time and concentration.
As presented in chapter 2, C-peptide has the ability to increase ATP release
from RBCs as determined using a ﬂow-based assay. In fact, the amount of ATP

released from RBCs upon incubation with C-peptide increased nearly 300% over 8

134

hours compared to RBCs without C-peptide. While not quantiﬁed, this increase is

signiﬁcant because previous reports have shown that RBCs generally release around

200 nM ATP upon mechanical deformation,5 and Sprague has shown that as little as

100 nM ATP can result in a signiﬁcant vasorelaxation.6

However, it was noted that after 24 hours of preparation, C-peptide did not
induce ATP release from RBCs subjected to mechanical deformation. Initially, it was
hypothesized that the C-peptide may be modiﬁed in some way such as a
phosphorylation or glycosylation. However, mass spectrometry could not conﬁrm
this. Interestingly, it was determined that upon initial preparation C-peptide

(purchased commercially and reported to be >98% pure) contained trace metal

. . . 2+ . .
contamination forming a Fe -peptide adduct as veriﬁed by mass spectrometry.

Moreover, after more than 24 hours of being in solution, the adduct dissociated, likely

. . 2+ . . . . .
due to ox1dation of the Fe rendering the C-peptide inactive. Once this was

. 2+ 3+ 2+ . . .
determined, a metal (Fe , Cr , or Zn ) was routinely used to activate the peptide.

It was subsequently discovered that mechanical deformation was not necessary
to induce ATP release from RBCs incubated with C-peptide. Furthermore, it was
hypothesized that the increase in ATP release was due to an increase in glucose
transport into the RBC. Indeed, using liquid scintillation counting it was determined
that incubation of RBCs with metal-activated C-peptide was resulting in an increase in
glucose transported into the cell. Using various inhibitors, it was determined that the
glucose was entering the cell using the GLUTl transporter, a non-insulin dependent

glucose transporter. These results are signiﬁcant because it indicates that metal-

135

activated C-peptide can aid in glucose clearance in the bloodstream without the
presence of insulin. Interestingly, almost all previous studies involving success with
C-peptide required co administration of insulin.

Collectively, these results suggest a solution to the lack of reproducibility
previously experienced working with C-peptide. It can be hypothesized that the
success experienced in the past with C-peptide may be due to a metal impurity present
within the commercially available C-peptide. As discussed above, successful C-
peptide replacement therapy was always accompanied by the co-administration of
insulin. When administered intravenously, insulin is often in its hexameric form,
which contains 2 atoms of zinc. It can be deduced that upon the meeting of C-peptide
and insulin in the injection device prior to reaching the body, C-peptide was able to
dehexamerize insulin by causing dissociation between zinc and the insulin hexamer. It

has been reported that C-peptide has the ability to disaggregate insulin, similar to

EDTA (a metal chelator).7 The dehexamerization of insulin by C-peptide would result

in an activated form of C-peptide, which would most likely produce positive results
for the patient. However, if insulin did not contain any zinc, the meeting of C-peptide
and insulin prior to injection would not be beneﬁcial and would not result in biological
activity. These results offer an explanation as to why there have been reproducibility
issues with C-peptide in the past.

Work presented here also provides an explanation to another common
argiunent against C-peptide being a biologically active peptide. Patients with type 2
diabetes have an abundance of C-peptide (when initially diagnosed); yet still suffer the

same complications as people with type 1 diabetes who are without C-peptide. Using

136

 

various techniques including chemiluminescence detection of ATP, liquid scintillation
counting, ELISAs, and ﬂuorescence it was determined that the RBCs of type 2
diabetic rats appear to be resistant to the effects of C-peptide.

As discussed above, C-peptide requires metal-activation to elicit a biological
response from RBCs. It is hypothesized that this is due to the ability of the positively
charged metal ion to help neutralize the highly negatively charged C-peptide (4
glutamic acid and 1 aspartic acid residues). By neutralizing the negativity of the

peptide, the peptide would be able to interact easier with the RBC membrane, which

. . . 8 . . .
also has negative characteristics. The diabetic RBC membrane, however, is even

more negative in character due to the increased extemalization of phosphatidylserine
9
(PS).

A potentially important clinical ﬁnding, is the decreased ability of RBCs from
type 2 rats to release ATP, intake glucose, and interact with the RBC membrane in the
presence of C-peptide compared to healthy controls. It was determined that this
decrease in activity was most likely due to an increase in the PS exposure on the
membrane of the diabetic RBC. However, this lowered activity was normalized when

RBCs from type 2 diabetic rats were incubated with metforrnin prior to the addition of

Zn2+-activated C-peptide. It is hypothesized that metforrnin, which would likely

display positive characteristics when exposed to a physiological pH, is able to mask

the negatively charged PS groups on the RBC membrane surface therefore allowing

the Zn2+-activated C-peptide to interact with the RBC more effectively. Collectively,

137

 

these results suggest that C-peptide may be effective in diabetic patients if given in
conjunction with an oral antidiabetic medication such as metformin.

The ﬁnal and most common argument against C-peptide being a biologically
relevant peptide is the lack of a known receptor. Nearly all peptide hormones (i.e.
insulin) have a known receptor. C-peptide has previously been shown to operate

through activation of G proteins, with its receptor hypothesized to be a G-protein

9

l . . . .
coupled receptor. However, other reports suggest that C-peptide 1S activating a

. . . . 12 . .
tyrosme kinase-coupled receptor of insulin. Other reports suggest that C-peptide is

interacting through nonspeciﬁc membrane interactions, implying the lack of a

l 3
receptor.

It was determined here that the ATP release from RBCs incubated with Zn2+-

activated C-peptide does not follow the proposed mechanism of deformation-induced

ATP release as presented by Spraguem-20 While it is apparent by some of the data

obtained that Zn2+-activated C-peptide involves some of the feature of the proposed

pathway (CFTR), it appears to behave independently of Cr in conjunction with protein
kinase a (PKA) and protein kinase c (PKC), respectively. Inhibition of either of these
kinases does not inhibit ATP release from the RBCs as would be the case if the

mechanism of ATP release was that proposed by Sprague. In fact, data presented in

. . . . . . . 2+
chapter 4 indicates an increase in the ATP release upon incubation With Zn -

2+ .
activated C-peptide and the kinase inhibitors. These results suggest Zn -activated C-

peptide activates a different signal transduction pathway within the RBC while still

138

 

utilizing CF TR. However, it is still unknown if there is a receptor, but work
presented here has been successful in making C-peptide work reproducibly.
Collectively, data presented here offer rebuttals to the arguments against C-
peptide as a biologically relevant peptide. Work presented here has been able to
correct reproducibility issues facing C-peptide replacement therapy and offer a
suggestion as to why C-peptide is ineffective in patients with type 2 diabetes.
Additionally, the mechanistic data presented here provides evidence that C-peptide
may act independently of a receptor, but is still able to elicit biological effects in

RBCs.

5.2 FUTURE DIRECTIONS
The ultimate goal of this project was to provide a better biological understanding of C-
peptide’s effects and active form in the body. However, more work still needs to be

done to determine the exact mechanism of C-peptide in vivo. While it has been

determined that the ATP release from RBCs incubated with Zn2+-activated C-peptide

does not follow the previously established mechanism for ATP release from RBCs, the
exact signal transduction pathway has not been determined.

Additionally, it has been hypothesized that C-peptide is activated by zinc in
vivo due to the relatively high concentrations of zinc in the granules, which are at a pH
of ~ 5. On going research in the group by Wathsala Medawala indicates that C-
peptide cannot bind to zinc at such a low pH, possibly due to the protonation of the
negatively charged R groups. However, once released from the B-cell, the pH is

increased, which could allow for C-peptide to bind to zinc. Preliminary studies, as

139

 

veriﬁed through CD spectroscopy, indicate that C-peptide has the ability to
dehexamarize insulin, much in the same way as EDTA. Determining exactly how C-
peptide becomes activated in vivo would help the scientiﬁc community to better
understand how to implement C-peptide replacement therapy.

Moreover, all of the studies performed here have been in vitro. To be
clinically relevant, it would be important to carry these studies out in vivo. By using
animal models, it would be possible to determine the best way to administer the C-
peptide in conjunction with zinc. Experiments performed in vivo would allow for the
determination of improved blood ﬂow resulting from an increase in ATP release and
subsequent increase in nitric oxide production.

Finally, diabetes occurs in direct conjunction with other diseases such as cystic

ﬁbrosis, Where as many as 65% of people with cystic ﬁbrosis also have diabetes.21

The cause for diabetes in these patients varies among patients, with some appearing to
have an autoimmune destruction of the B-cells, and others behaving more like type 2
patients. Based on knowledge gained through the studies performed here, it may be
possible to apply these results to obtaining new information about cystic ﬁbrosis

related diabetes.

140

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