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Michigan State
University

This is to certify that the
dissertation entitled

QUANTITATIVE DETERMINATION OF
GLUCOSE-G-PHOSPHATE DEHYDROGENASE
AND
ITS POTENTIAL IN DIABETIC COMPLICATIONS

presented by

CHINTHA WASANTHI SUBASINGHE

has been accepted towards fulfillment
of the requirements for the

Ph.D. degree in CHEMISTRY

 

 

@Em Professor’s Signature

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Date

MSU is an Affirmative Action/Equal Opportunity Employer

 

PLACE IN RETURN BOX to remove this checkout from your record.

TO AVOID FINES return on or before date due.
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5/08 KzlProlecc8-PreleIRCIDateDue.indd

QUANTITATIVE DETERMINATION OF GLUCOSE-6-
PHOSPHATE DEHYDROGENASE AND
ITS POTENTIAL IN DIABETIC COMPLICATIONS

BY

C. Wasanthi Subasinghe

A DISSERTATION

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY
Chemistry

2008

ABSTRACT

QUANTITATIVE DETERMINATION OF GLUCOSE-6-PHOSPHATE
DEHYDROGENASE AND
ITS POTENTIAL IN DIABETIC COMPLICATIONS

By

C. Wasanthi Subasinghe

Diabetes mellitus has become an epidemic during the last few decades without a

cure and with more than 170 million people diagnosed worldwide. In addition, oxidative
stress arising from hyperglycemia facilitates cardiovascular disease in diabetes,
increasing the risk factor by threefold in comparison to healthy controls. Appropriate
treatment of disease is dependent on the knowledge of the patho-physiology of the
disease, the mechanism underlying hyperglycemia and efficacy of exogenous insulin and
various pharmaceutical agents to improve postprandial hyperglycemia.

This work describes an attempt to evaluate the degree of damage caused by
oxidative stress and hyperglycemia to the red blood cells (RBCs) by investigating
molecular level changes in structure and certain metabolites within the RBC.
Deformation induced RBC-derived ATP release from erythrocytes is related to cellular
antioxidant status. Simultaneous monitoring of glucose-6-phosphate dehydrogenase
G6PD activity (via fluorescence determination of cellular NADPH) and ATP release
from erythrocytes in a microflow system is described. To confirm the relationship
between these analytes, a determination of NADPH and ATP release was performed

while inhibiting G6PD. Upon inhibition, NADPH concentrations decreased to 8.26 :t

0.29 uM from an original value of 12.73 :t 0.50 uM, while ATP release decreased from
0.21 :l: 0.07 uM to 0.06 at 0.02 uM. Results confirm that decreasing cellular NADPH
levels coincide with a decrease in ATP release from the RBCs. A similar trend was
observed using erythrocytes from type 2 diabetes patients.

In support of this hypothesis, the effect of proinsulin C-peptide (activated with
Zn2+) on G6PD-inhibited RBCs was investigated. G6PD inhibition leads to the inhibited
glucose transport into the RBC, lowered RBC-derived ATP release and lowered
incorporation of C-peptide in to the RBCs. In an attempt to investigate the mechanism of
metformin in type 2 diabetes, it was revealed that metformin (in combination with C-
peptide) exerts a “sensitizing” effect improving the ability of C-peptide to facilitate
glucose uptake into RBCs.

The sorbitol pathway was up-regulated under hyperglycemia. A further
investigation of G6PD inhibition on sorbitol pathway lead to inconclusive results.
However, despite the belief that hyperglycemia increases sorbitol levels, a relative
reduction in sorbitol level was observed upon changing the glucose concentration from
5.5 mM to 7.5 mM, although further increase in glucose concentration up to 17.5 mM
increased the sorbitol concentration.

In conclusion, G6PD enzyme activity is of extreme importance in maintaining
proper cellular antioxidant defense systems. Inhibition of G6PD resulted from an
exposure to hyperglycemia, leads to facilitate observed cardiovascular complications in

diabetes individuals.

~ To my Family ~

iv

ACKNOWLEDGEMENTS

First and foremost I offer my sincerest gratitude to my supervisor, Dr Dana
Spence, who has supported me throughout my PhD work with his knowledge and
guidance whilst allowing me the room to work in my own way. His enthusiasm, his
inspiration, and his great efforts to explain things clearly and simply gave me
determination and dedication needed to continue my work especially at times when the
things are not moving as they should. One simply could not wish for a better or friendlier

supervisor.

Also I would like to thank Dr. Garry Blanchard, Dr. John McCracken and Dr.
Gregory Baker for serving on my committee as well their helpful reviews on my
dissertation. Note of thanks goes to the Department of Chemistry, Michigan State
University for providing me this opportunity. Also my gratitude goes to NIH and various
funding agencies that funded my studies. I would like to thanks many people who have
taught me Chemistry: my high school teachers and professors at University of Colombo,

Sri Lanka and Wayne State University, Detroit, Michigan.

In my daily work I have been blessed with a friendly, fun and cheerful group of
fellow students. Special note of thanks goes to Luiza Genes and Madushi Raththagala for
being true companions who shared good and bad times from the fist day I met them
almost five years ago. Also I would like to thank Ajith Kamnarathne for his help with

labVIEW and other computer troubles which I seem to run in to more often than others.

My appreciation goes to Andrea, Chia, Jen and Nicole for their contribution in rabbit
surgery. Finally rest of the Spence group: wathsala (for good times in and out of the lab),
Teresa, Steve and Adam (may be I should say steve....?) need some recognition too. It

was a true pleasure to be a pan of Spence group.

I wish to thank all my friend specially Niro, Amila, Thilini, Aruni and Rhazes for
helping me get through the difficult times, and for all the emotional support, camaraderie,
entertainment, and caring they provided. Last but not least my special gratitude goes to
every single person who helped me even in a simplest possible way during last four and

half years.

Finally, I would like to thank my parents and my sister for their support,
encouragements and unconditional love and standing by me regardless of them approving

my decisions. To them I dedicate this thesis.

vi

TABLE OF CONTENTS

CHAPTER PAGE
LIST OF TABLES ............................................................. vii
LIST OF FIGURES ........................................................... viii

1. INTRODUCTION TO DIABETES MELLITUS ..................... 1
1.1 Diabetes mellitus ....................................................................... 1

1.1.1 Type 1 diabetes .................................................................. 4
1.1.2 Type 2 diabetes .................................................................. 4
1.1.3 Gestational Diabetes ............................................................ 6
1.1.4 Diabetes associated with contributed clinical states, diseases, drugs

and/or chemicals” . 7
1.1.5 Malnutrition— associated diabetes ........................................... 7

1.2 Glucose- 6-phosphate dehydrogenase and the pentose phosphate pathway. . . .. 8
1.3 Diabetes induced oxidative stress, antioxidant status of the red blood cell

and its effect on cardiovascular complications .................................... 12
1.4 Deformation induced, CFTR mediated ATP release from RBCs in

controlling vascular tone ............................................................. 16
1.5 Proinsulin C-peptide as a potential treatment regime for diabetes ............... 17
1.6 Channeling glucose via different pathways and polyol pathway up-

regulation in diabetes .................................................................. 21
List of references ............................................................................ 25

2. QUANTITATIVE ANALYSIS OF G6PD ACTIVITY AND
SIMULTANEOUS DETECTION OF NADPH LEVEL AND

RBC DERIVED ATP RELEASE ..................................................... 34
2.1 G6PD Structure and function” .. 37
2. 2NADPH asanindicator ofG6PD activity 38
2. 3 Dehydroepiandrosterone, as a G6PD inhibitor ..................................... 38
2.4 RBC deforrnability and CFTR mediated ATP release from RBCs .............. 40
2.5 Simultaneous detection of G6PD activity and its effect on ATP release ....... 41
2.6 Experimental ............................................................................ 41
2.6.1 Collection of RBCs ............................................................. 41
2.6.2 Preparation of RBCs ............................................................ 42
2.6.3 Reagents .......................................................................... 43
2.6.4 Instrumentation .................................................................. 44
2.7 Methods ................................................................................. 44
2.7.1 Optimization of NADPH detection parameters ......................................
44

vii

CHAPTER PAGE

2.7.1.1 Determination of the incubation time for resazurin ............... 44
2.7.1.2 Qualitative detection of NADPH in rabbit, control and type 2

diabetes RBCs ....................................................... 44

2.7.2 Chemiluminescence detection of RBC derived ATP release ............. 45

2.7.3 Flow through detection of NADPH levels in rabbit red blood cells. . . .. 47
2.7.4 Separation of RBCs into age-based fractions and measurement of

NADPH levels and ATP release .............................................. 48

2.7.5 Simultaneous detection Scheme .............................................. 50
2.7.6 Simultaneous detection of NADPH levels and deformation induced

RBC ATP release ............................................................... 50

2.8 Results and discussion ................................................................. 52

2.9 Conclusions ............................................................................. 69

List of references ........................................................................... 71

3. ROLE OF C-PEPTIDE AND EFFECT OF DIABETES
INDUCED OXIDATIVE STRESS ON C-PEPTIDE

ACTIVITY ............ . ............................. ..................... 78

3.1 C-peptide as a bioactive peptide ...................................................... 78
3.1.1 RBC membrane and effect of hyperglycemia on membrane structure. 84
3.1.2 Human C-peptide Enzyme Linked Immuno Sorbent Assay (ELISA)... 85
3.1.3 Insulin like activity of C-peptide; I4C-labeled glucose transport in to

the RBC .......................................................................... 88

3.2 Experimental ............................................................................ 91

3.2.1 Reagents .......................................................................... 91

3.2.2 Instrumentation .................................................................. 92
3.2.3 Effect of G6PD inhibition on ATP release from C-peptide treated red

blood cells... ... 92

3. 2. 4 Effect of G6PD 1nh1b1t10n onl C labeled glucose uptake in to RBCs.. 93
3.2.5 Effect of G6PD inhibition on C-peptide binding to red blood cell

membrane ........................................................................ 94

3.2.6 Effect of G6PD inhibition and elevated glucose levels on PS
translocation on RBC membrane ............................................. 95
3.3 Results and discussion .................................................................... 95
3.4 Conclusions ................................................................................. 109
List of references .............................................................................. 1 l 1

4. EFFECT OF POLYOL PATHWAY AND G6PD ACTIVITY IN

DIABETES RBCSH ...119
4.1 Channeling glucose via polyol pathway ............................................ 122
4. 2. Up- regulation of sorbitol pathway leads to complications 1n diabetes ........... 126
4.3. Monitoring the sorbitol level in RBCs ................................................ 128

viii

CHAPTER PAGE

4.4 Experimental .............................................................................. 130
4.4.1 Reagents ........................................................................... 130
4.4.2 Instrumentation .................................................................... 131
4.4.3 Detection of sorbitol levels in red blood cells upon G6PD enzyme
inhibition and C-peptide treatment ............................................. 131
4.4.4 Effect of elevated glucose levels on polyol pathway ........................ 132
4.5 Results and discussion ................................................................... 132
4.6 Conclusions ............................................................................... 139
List of references ............................................................................. 140
5. CONCLUSIONS AND FUTURE DIRECTIONS .......................... 144
5.1 Conclusions ............................................................................... 144
5.2 Future directions ......................................................................... 150
List of references ............................................................................. 154

ix

List of tables

TABLE PAGE

1.1 Different types of diabetes and their characteristics. . . . . . . . . . . . 3

FIGURE

1.1

1.2

1.3

1.4

1.5

LIST OF FIGURES

Schematic representation of the pentose phosphate pathway. During
oxidative phase G6P is converted to ribulose-5—phosphate. During the
nonoxidative phase pentose sugars are formed ................................

(a) Structure of NADPH, NADPH (b) protein structure of G6PD. G6PD
consists of two or four identical subunits each weighing about 59 kD (c)
structure of glutathione is a small tripeptide of cysteine, glutamate and
lysine with unusual peptide bond between the amine group of cysteine
and the carboxyl group of the glutamate side chain. GSH is the most
common non enzymatic antioxidant present in human RBCs ...............

Schematic representation of antioxidant defence mechanism in RBCs.
GSH undergoes oxidation to form GSSG protecting such important
components like spectrin. Glutathione reductase (GR) regenerates GSH
with the expense of NADPH. G6PD supply the required NADPH in the
process oxidizing G6P to 6GP. ATP released when subjected
mechanical deformation acts as no stimulator to the endothelial cells. . . ..

Proposed pathway for ATP release form RBCs. G-protein is activated as
a respond to external stimuli (such as mechanical deformation,
hypoxia). G protein will leads to the activate adinylyl cyclase,
increasing CAMP within RBCs. This increased CAMP will lead to the
activation of protein kinase-A, eventually activating CFTR, prompting
the ATP release. Whether ATP IS channeled through CFTR or other
channel 18 not very clear”

Schematic representation of the sorbitol pathway. Pathway consists of
two enzymes aldose reductase and sorbitol dehydrogenase. AR reduces
glucose in to sorbitol, while reducing NADPH. Then SDH oxidizes C2
of sorbitol, forming fructose. Free NADH is generated in the process. . . ..

xi

PAGE

11

15

18

23

FIGURE

2.1

2.2

2.3

2.4

2.5

2.6

(a) Fluorescence-based NADPH detection scheme. Enzyme diaphorase
converts non fluorescence resazurin to red fluorescence resorufm with
the expense of NADPH. (b) Dehydroepiandrosterone, a non competitive
steroid inhibitor of G6PD. DHEA is naturally produced from cholesterol
in a human body but the circulating concentration readily decreases by
the age of 20 ........................................................................

(a) Experimental setup used for ATP release measurements. (b)
Schematic representation of the PMT and microbore tubing connection
inside the light excluding box. X and Y represent the connecting “Tee”
and window with the PMT underneath for chemiluminescence detection
respectively. —+ indicates the direction of flow ................................

Pictorial representation of the separation of RBCs into fractions based
on the age. Two Percoll solutions with slightly different densities were
placed in a microcentrifuge tube and RBCs were introduced slowly to
the same tube. The content was centrifuged at X3000 g at 4°C for 15
min. RBCs were separated based on density (therefore age). The small
RBC layer between two Percoll layers represent the RBCs that were not
separated .............................................................................

Schematic representation of the flow-based system employed to
quantitatively and simultaneously determine the concentration of
NADPH and deformation induced ATP release from the RBCs obtained
from either rabbits or humans. The data acquisition program was
written using labVIEW8 (National Instrumentation, Austin, TX) .........

Kinetic study - optimization of the incubation time for resazurin.
Resultant fluorescence intensity was measured over a period of 70 min.
at 583 nm. Fluorescence intensity was increased linearly as a function
of time up to 70 min. period .....................................................

The effect of DHEA on G6PD activity (n = 6 rabbits). Gray bars
represent 30 min. incubation with DHEA whereas black bars represent
the 60 min. incubation. Diamide alone did not affect G6PD activity, nor
did it have any further effect on G6PD activity in the presence of
DHEA. The asterisks represent those values significantly different from
the control (p < 0.001) while the crosses indicate NADPH levels that are
statistically equivalent to the control (p > 0.02) ...............................

xii

PAGE

39

46

49

53

56

57

FIGURE PAGE

2.7

2.8

2.9

2.10

2.11

NADPH levels in rabbit, control human and diabetes RBCs measured

under non flow conditions. The NADPH level in the RBCs obtained

from the patients with diabetes was significantly different from the

healthy human controls and rabbit RBCs (p < 0.01). n = 5 samples ........ 58

Deformation induced ATP release from RBCs in the absence and
presence of a G6PD inhibitor is shown. The gray bars show the average
ATP release (n = 6 rabbits) at various time intervals measured from
RBCs that were subjected to 20 11M diamide alone. The black bars show
the ATP release from aliquots of the RBCs after the addition of
diamide; however, these RBCs were first incubated with 100 1.1M DHEA
for 30 min in order to inhibit the G6PD activity. The bars with lines
show ATP release from RBCs that were incubated with DHEA alone.
The asterisks represent those values significantly different from the
initial (t = 0 min, p < 0.001) while the crosses indicate ATP values that
are statistically equivalent to the initial value at t = 0 min (p > 0.02) ...... 60

NADPH standards measured using flow based fluorescence detection
scheme. Fluorescence intensity changes linearly within the NADPH
concentration range of 0 —- 20 11M (r 2 =0.998). n = 7 sample sets .......... 62

Quantitative measurement of cellular NADPH levels using flow-through
fluorescence detection. (a) An NADPH standard (lower trace) in

comparison with NADPH levels in 0.02% RBCs (top trace) and

chemically aged RBCs (middle trace). (b) Quantitative representation of
NADPH levels in 0.02% rabbit RBCs and DHEA inhibited rabbit RBCs

(n = 6 rabbits). There is a 39% decrease in NADPH concentration upon
incubation with the G6PD inhibitor DHEA for 30 min. The values in the
absence and presence of DHEA are significantly different (p < 0.01). . . .. 63

NADPH levels and deformation induced RBC derived ATP release
from young and old fractions of rabbit RBCs. The black bars represent
the fluorescence intensity corresponding to NADPH while the gray bars
represent the chemiluminescence intensity corresponding to ATP
release from the RBCs (n = 3 rabbits). The values between the two
groups are significantly different

(p<0.01) .................................................................... 64

xiii

FIGURE PAGE

2.12

2.13

2.14

3.1

3.2

Qualitative representation of simultaneous detection of NADPH levels

and deformation-induced ATP release from rabbit RBCs. NADPH

levels were measured using a fluorescence-based G6PD assay, while

ATP release was measured using chemiluminescence. The bottom two

traces represent ATP release from normal RBCs (higher of the pair) and
G6PD inhibited (lower of the pair) RBCs. The top two traces represent
NADPH levels in 0.02% RBCs in the absence (higher of the pair) and
presence (lower trace of the pair) of DHEA ................................... 66

Quantitative representation of simultaneously detected NADPH levels

and deformation-derived ATP release. The black bars represent NADPH
><10'l concentration (n = 5 rabbits), while the gray bars represent ATP

release (n = 5 rabbits). The values between the two groups are

significantly different (p < 0.01) ................................................ 66

Measurement of cellular NADPH levels and ATP release from RBCs
obtained from people with type 2 diabetes and healthy controls. The

black bars represent NADPH ><10'l (n = 5 individuals) and the gray bars
represent ATP release (n = 8 individuals). There is an approximate 50%
decrease in NADPH concentrations and a 78% decrease in ATP release

from the RBCs from diabetic patients as compared to healthy

individuals. The values between the two groups are significantly

different (p < 0.01) ................................................................ 68

(a) Structure of human proinsulin C-peptide (b) Schematic
representation of the human proinsulin structure with chains A and B of
insulin connected by C-peptide. (b) Amino acid sequence of human C-
peptide .............................................................................. 80

Figure 3.2: (a) Schematic representation of an antibody (b) Schematic
representation of C-peptide ELISA where C-peptide is sandwiched
between immobilized C-peptide-antibody and another biotinylated C-
pep-antibody. Then Streptavidin linked enzyme was allowed to
conjugate with detection antibody followed by addition of
substrate ............................................................................. 87

xiv

FIGURE

3.3

3.5

3.6

3.7

3.8

Effect on ATP release from rabbit RBCs upon C-peptide activated with
Cr3+. The gray bars represent the metal activated C-peptide treated
RBCs and black bars represent the RBCs with out C-peptide. DHEA
was used to inhibit the G6PD activity and diamide is a known oxidant. n
= 6 rabbits. * represent the values that are significantly different from
each other p < 0.001 ...............................................................

Effect on ATP release from rabbit RBCs upon C-peptide activated with
Zn2+. The gray bars represent the metal activated C-peptide treated
RBCs and black bars represent the RBCs with out C-peptide. DHEA
was used to inhibit the G6PD activity and diamide is a known oxidant. n
= 9 rabbits. * represent the values that are significantly different at p <
0.001 .................................................................................

Effect of G6PD inhibition on C-peptide mediated l4C labeled glucose
uptake in to the RBCs. The gray bars represent the metal activated C-
peptide treated RBCs and black bars represent the RBCs with out C-
peptide. DHEA was used to inhibit the G6PD activity and diamide is a
known oxidant. n = 8 rabbits. * represent the values that ate
significantly different p < 0.001 .................................................

Effect of G6PD inhibition and resulted oxidative stress on C-peptide
binding to the RBC. The gray bars represent the metal activated C-
peptide treated RBCs and black bars represent the RBCs with out C-
peptide. DHEA was used to inhibit the G6PD activity and diamide is a
known oxidant. n = 6 rabbits. * represent the values that ate
significantly different at p < 0.001. A picture of the ELISA assay (96-
well plate) is shown in upper left hand comer .................................
Annexin binding to the control, G6PD inhibited and RBCs incubated in
15 mM glucose. Row fluorescence intensity data of control RBCs and
RBCs incubated in 15 mM glucose. Lower trace represents the control
RBCs where as upper two traces represent RBCs incubated in DHEA
and 15 mM glucose..................

Quantitative representation of annexin binding to the control, G6PD
inhibited and RBCs incubated in 15 mM glucose. Upon inhibition of
G6PD (with DHEA) annexin binding increase by 28.2 % and incubation
with glucose increased the annexin binding by 41 %. n = 3 individuals.
* represent the values that are significantly different p < 0.001 ............

XV

PAGE

98

99

101

103

105

106

FIGURE

3.9

4.1

4.2

4.3

4.4

4.5

4.6

Annexin binding to the control and type 2 rat RBCs in the presence and
absence of diabetic treatment drug metformon (30 11M). Black bars
represent the control rats and the gray bars represent the type 2 diabetic
rats. Incubation with metformin seems to mask the overall negative
charge of PS facilitating C-peptide interaction with membrane. n = 5
ratts . * represent the values that are significantly diffatemt p < 0.001....

Structures of glucose (a) Sorbitol (b) and fructose (C). Sorbitol is sugar
alcohol of both glucose and fructose. Oxidation at C1 of glucose or C2
of fructose will result in sorbitol ................................................

Schematic representation of the polyol pathway. Glucose is oxidized to
its alcohol sorbitol by AR, while reducing NADPH. Then SDH oxidizes
C2 of sorbitol, forming a ketose, fructose. Free NADH is generated in
the process ..........................................................................

Fluorescent measurement for sorbitol standards. (a) Row fluorescence
intensity values for sorbitol standards 0, 20, 40 and 80 11M. (b)
Quantitative data for the same set of standards. Y= 10605.6 x + 10457.6
and r2 = 0.97 ........................................................................

Normalized sorbitol level in G6PD inhibited (by DHEA) RBCs. (a)
Normalized sorbitol decreased by 18.5 % when G6PD was inhibited. n=
8 (b) Normalized sorbitol level increased by 25.2 % upon G6PD
inhibition. n= 6. Date represent mean i SE. rabbits and * represent the
values that are significantly different (p<0.001) whereas + represent
(p<0.005) ...........................................................................

Sorbitol levels in type 1 diabetes and control RBCs. Upper trace
represent the Type 1 diabetes RBCs and lower trace represent the
control RBCs. Two values are significantly different (p < 0.005) ............

Normalized fluorescence intensity of RBCs incubated in PSS containing
increasing amounts of glucose. Normalized fluorescence intensity
decrease from 1.00 i 0.0 to 0.87 i 0.03 when glucose concentration
increase from 5.5 mM to 7.5 mM. However fluorescence intensity
increased up to 1.08 i005 when glucose concentration was increased up
to 10.5 mM. Date represent mean i SE. n= 4 rabbits and * represent the
values that are significantly different (p<0.005) ..............................

xvi

PAGE

108

120

125

133

135

137

138

FIGURE PAGE

5.1

5.2

5.3

Schematic representation of the result summary. (+) indicated the
increase in concentration or pathway activity where as (-) indicates
decrease ............................................................................. 146

Effect of estradiol on ATP release from RBCs. Upon incubation with
estradiol ATP release from RBCs decrease even in the presence of ATP
stimuli C- peptide (activated with an ). Black bars represent RBCs with

out C-peptide and gray bars represent gray bars with C-peptide. n=5

rabbits and I represent values that are significantly dirrerent (p < 0.001). 151

Effect of estradiol on RBC derived ATP induced NO production from
bPAEcells. (a) Qualitative representation and qualitative representation

(b) of NO formed in bPAEC when stimulated with ATP released from

RBCs incubated in estradiol 1n the presence and absence of C-peptide

using method and the device described by Genes et (11.22 NO production
decreased when cells are treated with estradiol even in the presence of
C-peptide. n — 1 rabbit. Represent values significantly different (p <

0. 05) .................. - 152

 

xvii

Chapter one

INTRODUCTION TO DIABETES MELLITUS

1.1 Diabetes mellitus
Diabetes mellitus is metabolic syndrome often characterized by disordered

metabolism, followed by increased blood sugar levels (hyperglycemia) resulting from
hereditary and environmental causes."2 Under normal conditions blood glucose level is
tightly regulated in the human body between about 4 and 6 mM (mmol/ L) with normal
average of about 5 mM.3 The homeostasis of blood glucose is achieved via the two
closely interacting, mutually antagonistic hormones, insulin and glucagon.4 Under normal
conditions the hormone insulin, secreted by pancreatic beta (0) cells, promotes glucose
uptake into the liver, muscle cells and peripheral tissues in order to maintain normal
fasting blood glucose 1evel.5‘6 Insulin is a small peptide hormone consists of the two
peptide chains (total 51 amino acids) linked together by disulfide linkages. In mammalian
species, insulin is synthesized in the pancreas within the B- cells of the islets of
Langerhans in the endocrine part of the pancreas.7 Upon dietary intake of carbohydrate,
resulting elevation of blood glucose triggers a series of signal transduction pathways that
trigger insulin secretion."1

Development of the diabetes mellitus can be characterized by impaired insulin
secretion from pancrease, insulin resistance and increased hepatic glucose production

resulting from both increased glyconeogenesis and glyconeolysis.8'10 Gluconeogenesis is

the process that synthesizes glycogen from dietary glucose in the liver and glyconeolysis
cleaves glycogen into glucose, addressing glucose demand of the body in-between the
meals.4’” Acute symptoms of diabetes include excessive urine production, resulting
compensatory thirst and increased fluid intake, blurred vision, unexplained weight loss,
lethargy, and changes in energy metabolism.”13

Over the last two decades diabetes mellitus has become an epidemic with about
180 million people diagnosed with the disease worldwide.l4 According to the American
Diabetes Association website, 23.6 million children and adults in the United States, alone
are diabetic. In other words 7-8 % of the American population are diabetic individuals.
Of the 23.6 million, 17.9 million are diagnosed with the disease while another 5.7 million

15"" Annual economic cost for direct and indirect diabetes

remains unaware of the disease.
care in 2007 was estimated at $174 billion in the United States alone.15"6 With the
increasing adult population due to advances in health care, and obesity among younger
generations due to high calorie diets and lack of exercise, the United. States has witnessed
an almost epidemic progression in diabetic cases.

The American Diabetes Association classifies five classes within the disorders
that represent diabetic symptoms. Those types are shown in the table 1.1 and discussed in
detail below. Out of those five classes, types 1, 2 and gestational diabetes are the most

commonly occurring forms and attempts to classify others remain somewhat

controversial. '7

 

Type

Characteristics

 

Type 1

No insulin production due to B-cell destruction
Developed during early years

Can not be cured but can be controlled by daily
lifelong insulin administration

If untreated, can be fetal due to brain damage

and coma

 

Type 2

Insulin resistance of the target cells/tissues
Eventually leads to reduced insulin secretion
Visible symptoms are mild during development
No ketoacedotic episodes

Leads to development of retinopathy,

neuropathy and cardiovascular diseases

 

Gestational diabetes

Resembles type 2 diabetes
Developed during 24-28 weeks of pregnancy

If untreated can be lethal to the infant

 

Diabetes associated with
contributed clinical
states, diseases, drugs

and/or chemicals

Resembles type 2
Arise due to other disease conditions, chemical

agents or drugs used

 

Malnutrition-associated

diabetes

 

 

Resembles type 2
Requires insulin

No ketoacidosis

 

Table 1.1: Different types of diabetes and their characteristics.

 

1.1.1 Type 1 diabetes

Type 1 diabetes, also commonly known as insulin dependent diabetes or juvenile
diabetes, is thought to be an autoimmune disease resulting from a genetic or
environmental trigger. As indicated by the name, type 1 diabetes usually develops during
childhood or the early stages of life even though the disease can develop during latter
stages as well.‘8"9 Type 1 patients demonstrate autoimmune destruction of pancreatic B-
cells which in turn, results in little to no insulin production in the body.20 As a result,
individuals suffering from type 1 diabetes are either totally or almost totally deprived of
insulin.“19 In addition, various antibodies to islet antigens such as insulin and glutamic
acid decarboxylase are found in these individualsls’19

Conditions associated with type 1 diabetes include hyperglycemia, hypoglycemia,
ketoacidosis which acidosis results from increased ketone body formation due to
deamination of amino acids and celiac disease, an autoimmune disorder of the small.
intestine.6 Current treatments for type 1 diabetes include lifelong intravenous insulin
administration upon intake of a carbohydrate rich meal. In fact, treatment does not disrupt
a normal life style if sufficient care awareness and timely testing and insulin
administration is taken care of.2]’22 But if untreated glucose depravation or hypoglycemia

of cells specially the brain cells can lead to the comma and decease.”22

1.1.2 Type 2 diabetes
Type 2 diabetes, also known as adult onset diabetes mellitus is insulin
independent and develops mostly during latter stages of life and contributes to 90 — 95 %

of the diagnosed patients in United States.15 In fact, according to the Center of Disease

Control, one fifth of the United States population over 60 years of age is diagnosed with
type 2 diabetes. Type 2 diabetes is often characterized by a resistance to insulin function
by target tissues followed by impaired insulin secretion.”23 Insulin resistance is the first
sign of the development of type 2 diabetes. Usually an insulin resistance develops 5 -10
years prior to elevation of fasting blood glucose levels up to a diabetic range of above 7
mM24. As a result of this resistance, the redistribution of the glucose transporter GLUT 4
into the muscle cell membrane fails, reducing the glucose uptake into the muscle cells.4'25
This insulin resistance can be characterized by abnormally high levels of insulin and C-
peptide in the circulatory system. In response to this insulin resistance, insulin synthesis
by pancreatic B-cells is up regulated, leading to extensive insulin production and eventual
B-cell death due to over exhaustion.25 Therefore as the disease progress replacement of
insulin become a necessity for survival.26

Although there are numerous explanations for the pathogenesis and disease
progression of type 2 diabetes, to date, an exact explanation is unavailable. Obesity is
believed to be one of the major contributors for the development of type 2 diabetes. In
fact statistics indicate 55 % of the type 2 diabetes patients in United States are obese. ln
facts there are reports suggesting the central obesity influence individuals to be insulin
resistance, by secreting group of hormones called adipokinases that could impair glucose
tolerance. Other major contributors include aging and family history.26

Treatment options to type 2 diabetes include an inclined physical activity,
decreased carbohydrate intake. Weight loss, especially from abdominal fat deposits also
improves the insulin sensitivity significantly.” The oral medications and insulin injection

will follow if the above options were unsuccessful in controlling the disease

progression.26 Though insulin resistance is one of the major causes of type 2 diabetes it is
not the only cause. Recent studies indicate C-peptide, a small peptide secreted during
insulin biosynthesis, may play a major role in blood glucose level control and
complications associated with diabetes specially in cardiovascular system. The effect of

C-peptide in diabetes pathogenesis will be discussed in-detail below and in chapter 5.

1.1.3 Gestational Diabetes

Gestational diabetes is usually developed during the pregnancy specifically
around 24‘’1 to 28th weeks.27 Occurrence rate for this disease is about 2 — 5 % among
pregnant women. Prevalence of the gestational diabetes is high among the older, obese,
women with family history of diabetes and women from diabetes prone ethnic
groups.”29 Symptoms of the disease are more or less similar to that of type 2 diabetes
and these women either fully recover from the disease or develop in to type 2 diabetes at
the end of their pregnancy.30 Statistics suggests that about 20 - 50 % of these women
develop types 2 diabetes later in life.'7

Even though gestational diabetes is temporary, if untreated it can damage both
mother and the fetus or even can be fetal to the fetus. Untreated elevated blood glucose
can cause macrosomia which is overweight of the fetus at the birth, congenital cardiac
and central nervous system anomalies, respiratory distress syndrome and

hyperbilirubinemia resulting from RBC destruction?l

1.1.4 Diabetes associated with contributed clinical states, diseases, drugs and/or
chemicals

This is a type 2 diabetes condition associated with other clinical states, drugs
administrated or chemical agents. Pancreatic diseases such as chronic pancreatitis, cystic
fibrosis, genetic syndromes such as muscular dystrophy and Huntington’s chorea can
cause development of type 2 diabetes. Also administration of certain drugs such as beta
blockers, glucocorticoids, some antidepressant medications and conditions that that
requires pancreatectomy leads to development of diabetes mellitus. Usually this can be
cured or controlled by either eliminating the drugs that cause the distress or treating the

clinical condition.

1.1.5 Malnutrition — associated diabetes

Malnutrition — associated diabetes is a condition somewhat similar to type 2
diabetes. In this case patients require insulin for glycemic control but they do not develop
diabetic ketoacidosis. Malnutrition — associated diabetes is usually developed in
individuals of age range between 10 and 40.

Both untreated or severe type 1 and type 2 diabetes can lead to the secondary
complications such as retinopathy, neuropathy, nephropathy and complications in the
cardiovascular system such as high blood pressure, angina and heart attack. Diabetic
retinopathy is retinopathy or damage to the retina of the eye, caused by elevated blood
glucose and resulting changes in microvasculature in diabetes mellitus, which can
eventually lead to blindness.32 In fact, there are reports suggesting that eight out of the 10

individuals diagnosed and liveing with diabetes, for 10 or more years develop diabetes

retinopathy.32 Diabetic neuropathy is a deranged function and structure of peripheral
nervous system resulting from injuries to the vasa nervorum (small blood vessel feeding
the nerves) and diabetic nephropathy is a progressive kidney disease caused by
angiopathy (disease) of capillaries in the kidney. It is the prime cause for dialysis in
diabetes patients.

In fact, the elevated blood glucose believed to be the major cause of structural and
rheological alternations in red blood cells (RBCs) and microvasculature which leads to
the complications. Therefore the firm understanding of the underlying glucose
metabolism mechanism, metabolic pathways involved and the energy status of the cells
specially, in RBCs is beneficial in disease control of diabetes and associated
cardiovascular complications. In-fact, there are reports suggesting the impact of altered
activity of different metabolic pathways such as pentose monophosphate shunt and
sorbitol pathway on development of the cardiovascular complications in diabetic

individuals.

1.2 Glucose-6-phosphate dehydrogenase and the pentose phosphate pathway

The pentose phosphate pathway (PPP) also known as the phosphogluconate
pathway or the hexose monophosphate shunt (HMP shunt) is an exclusively cytosolic
process that generates NADPH and synthesizes pentose (5-carbon sugars) during the
process (Figure 1.1).4‘33 There are two major phases in the PPP, namely the oxidative
phase and the non-oxidative phase.4 During the oxidative phase, reducing equivalents are

generated in the form of nicotinamide adenine dinucleotide phosphate (N ADPH) (Fig 1.2

Glucose-6-phosphate

NADP+ \

M 2+ Glucose-6-phosphatedehydrogenase
g
NADPH

 

V
6-phosphoglucono-d-lactone

 

H20
6-phosphoglucolactonase Oxidative
11+ phase

1
6-phosphogluconate
NADP+

6-phosphogluconate dehydrogenase

 

 

 

 

NADPH (:02
V
__ Ribulose—S-phosphate J
Ribulose-S' Ribulose-S-
phosphate-3 phosphate isomerase
-ep1merase
Ribose-S-phosphate Xylulose 5-phosphate

transketolase

l l

Glyceraldehyde 3-phosphate Sedoheptulose 7-phosphate
I |

 

 

 

 

transaldolase

 

 

Non-oxidative phase

Erythrose 4-phosphate Fructose 6-phosphate
|

l transketolase 1

fructose 6-phosphate

 

 

Glyceraldehyde 3-phosphate

J

Figure 1.1: Schematic representation of the pentose phosphate pathway. During oxidative

phase G6P is converted to ribulose-S-phosphate. During the nonoxidative phase pentose
sugars are formed.

a.) for reductive biosynthesis reactions within cells, and the synthesis of 5-carbon sugars,
which are essential for DNA and RNA synthesis, occur during the non-oxidative phase.4
The PPP is an alternative to the glycolysis, which is the primary method of glucose
oxidation in the body.4 Even though the PPP does involve in the oxidation of glucose, its
primary role is anabolic rather than catabolic.4

Upon entering the cell, glucose is converted to glucose-6-phosphate (G6P) by
enzyme hexokinase.4 This G6P can either be channeled to glycolysis for energy
production or the PPP. During the oxidative phase, the G6P molecule is subjected to
series of oxidative reactions resulting in two NADPH molecules and ribulose 5-
phosphate (Fig 1.1).4

Glucose-6-phosphate dehydrogenase (G6PD) is the first and the key enzyme of
the PPP.33 This X-linked enzyme show in figure 1.2.b, in its active enzyme form, is
made up of either two or four identical subunits, each having a molecular mass of about
59 kD; this is more than three times as large as the hemoglobin. G6PD catalyzes the
oxidation of G6P into S-glucono-l,5-lactone-6-phosphate, while reducing one NADP+
molecule to its reduced form (NADPH) transferring an electron from one molecule to
another. Being the rate determining step of the PPP, G6PD is responsible for maintaining
adequate levels of NADPH within the cell.33

NADPH is a required cofactor in many biosynthetic reactions and extremely
important in maintaining/regenerating glutathione (GSH), a small tri-peptide, in its
reduced form (Figure 1.2.C).34 Reduced GSH acts as a scavenger for dangerous oxidative
metabolites in the cell such as hydrogen peroxide and other free radical generating

species.34 Therefore, the function of G6PD is critical to human survival. The activity of

10

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G6PD has a profound effect on the stability of red blood cells (RBCs) because they are
especially sensitive to oxidative stresses and G6PD is the only NADPH-producing
enzyme present in RBCs. As a result, G6PD deficient individuals are not capable of
handling oxidative stress and consequently, hemolysis ensues.35

It is well established that G6PD deficient individuals are subjected to high
oxidative stress due to the lack of NADPH production, and hence a weakened antioxidant
defense system.”39 Though G6P levels appear to be normal in RBCs obtained from
people with diabetes, it is well known that G6PD activity in the RBCs from patients with

diabetes is deficient compared to healthy individuals40‘42

, thus resulting in decreased
NADPH levels in the diabetic.

In addition to its importance in the RBC as a major determinant in oxidative
stress, G6PD activity is also employed as an indicator of cell age.43 As the cell ages
G6PD activity decreases, making cell age inversely related to G6PD activity.44 Therefore
by understanding the behavior of PPP and G6PD in aged RBCs may exert beneficial

effect in understanding its activity under hyperglycemia and its contribution to the

development of cardiovascular complications.

1.3 Diabetes induced oxidative stress, antioxidant status of the red blood cell and its
effect on cardiovascular complications

Understanding the injuries at the cellular level induced by hyperglycemia is
critical in both damage control and treatment of diabetes. At the cellular level, various
metabolic pathways can be activated or up-regulated by either the elevated glucose levels

or metabolic derivatives of glucose. These pathways and products that are accelerated as

12

a result of hyperglycemia, includes increase in reactive oxidative species such as
peroxides, superoxides and other reactive free radicals, increase in formation of advanced
glycation end-products (AGE), activation of polyol pathway and enhanced aldose
reductase activity and activation of protein kinase C isoforms.

Activation of the above mentioned pathways/enzymes results in the accumulation
of products that lead to an oxidatively-stressed cellular environment, thereby damaging
important cellular components. In human RBCS that lack oxidative protection due to a
lowered G6PD activity and hence weakened anti-oxidant defense under hyperglycemia,
this effect seems profound.

RBCs are biconcave disk shape structures that lack nuclei and many other cellular
organelles such as mitochondria and golgi apparatus.45 RBCs that are synthesized in the
bone marrow contribute to about 45 % of the whole blood and are also the main cellular
component in blood. RBCs are about 6 — 10 pm in diameter with about 80 fL volume and
have a distinct red color as a result of the abundant oxygen carrier hemoglobin.”46
Hemoglobin is an iron-containing metalloprotein that contributes up to 35 % of the
volume of the mammalian RBCs.

Though RBCs major purpose is to transport oxygen and carbon dioxide in and out
of the tissues, recent findings suggest RBCs are not mere “sacks of hemoglobin” but they
actively participate in controlling vascular tone.45 Therefore it is important to understand
the effects of diabetes relative oxidative stress on RBCs structure and function and its
consequences in ensuing cardiovascular complications that arise from diabetes 40’47.

Commonly known as glutathione, 2—amino-5-{[2-[(carboxymethyl)amino]-l-(

mercaptomethyl)-2-oxoethyl]amino}-5-oxopentanoic acid is small tripeptide of cysteine,

13

glutamate and lysine (Figure 1.2.C). GSH is the most common non enzymatic antioxidant
present in human RBCs.48 It contains an unusual peptide linkage between the amine
group of cysteine and the carboxyl group of the glutamate side chain. In cells thiol groups
are kept in a reduced state and the reduced thiol group concentration is about 5 mM
within the cell.48 In its reduced form GSH, readily undergoes oxidation to the dimer
(GSSG) forming a disulfide linkage, to protect much important cellular components such
as proteins from oxidant insults (Fig 1.3)”.

Under normal conditions, enzyme glutathione reductase regenerates GSH at the
expense of the reducing cofactor NADPH. Therefore, a steady supply of NADPH is vital
for maintenance of healthy antioxidant defense in cells specially in RBCs where GSH is
the main non enzymatic antioxidant. Though other cells have several methods to produce
NADPH, RBCs rely solely on the enzyme G6PD for regeneration of NADPH.

In addition to having highly oxidative environment resulting from hyperglycemia,
having weakened antioxidant defense makes RBCs of diabetes individuals more prone to
oxidative damage. It is well established that spectrin a common membrane skeletal
protein of these diabetes RBCs are readily subjected to oxidation thus changing their
specific three dimensional structure, hence their function ensuing a stiffened RBC
membrane. 50 Stiffened RBC membrane will not only hinder the shape change they need
to undergo upon travelling through microvasculature but will result in decrease in degree
of deformation, resulting decrease in release of adenosine tripeptide (ATP) upon

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1.4 Deformation induced, CFTR mediated ATP release from RBCs in controlling
vascular tone

Originally it was believed that oxygen transport from blood vessel, within the
tissue occured by passive diffusion across the oxygen (0;) gradient maintained by the
uniform consumption of Oz by the target tissue itself.45‘51 However this model failed to
explain the observed sensitivity of the vasculature to changes in physiological 02 levels
inside the target tissues.”52 In 1993, studies conducted by Stein et al. reported that the
amount of the 02 present is more important in controlling oxygen supply than oxygen
tension, suggesting the role of RBCs as a oxygen sensor in controlling vascular
caliber.45‘52

RBCs contain millimolar amounts of adenosine 5-triphosphate (ATP) in their
cytosol. It has been shown using various experimental animal models and in human
RBCs, that when traveling through microvasculature, RBCs release nanomolar to low
micromolar amounts of ATP upon being subjected to mechanical deforrnation.45’53’54 This
ATP binds to the purinegic receptor (P2Y) on the vascular endothelium resulting in
synthesis and release of the vasodilator nitric oxide (NO) resulting in vasodilation
followed by an increase in blood flow to the target tissue. ATP is metabolized by
enzymes present in the circulation, therefore this effect is likely localized.45‘54‘55

The mechanism by which ATP is released remains somewhat controversial,
although several components of the signaling pathway have been identified over the fast

few years. However, it has been proven that ATP release from RBCs occurs only in the

presence of the cystic fibrosis transmembrane conductance regulator (CFTR). CFTR is a

16

cyclic adenosine monophosphate (CAMP) activated Chloride Channel expressed mainly in
epithelial cells. However, CFTR also regulates the transport of other components.

The currently accepted mechanism suggests that ATP release occurs through a G-
protein coupled signal transduction pathway (F igure1.4).45 ‘5 6 Receptor mediated
activation of hetero-trimeric G protein will lead to the activate adinylyl cyclase enzyme,
increasing CAMP within RBCs. This increased CAMP will lead to the activation of
protein kinase-A, eventually activating CFTR, prompting the ATP release.

ATP release is mediated by pathway that involves various membrane bound
proteins and enzymes, thus, oxidative damage to the RBC membrane can lead to
decreased ATP release, which will eventually lead to impaired 0; supply to the target

tissues.

1.5 Proinsulin C-peptide as a potential treatment regime for diabetes

Until recently, microvascular complications arising from diabetes were attributed
to elevated glucose levels and its consequences such as oxidative stress and activation of
the polyol pathway. But recently with new findings in diabetic pathogenesis, it is
believed that a deficiency of circulating C-peptide in the blood stream has a significant
role, especially in type 1 diabetic complications.”59 Earlier, it was believed that
microvascular complications are much more aggressive and rapidly developed in type 1
individuals in comparison to that of type 2.60 Only in light of recent findings has it
become clear that an essential lack of circulating C- peptide has contributed to these

accelerated disease progressions and secondary damages in type 1 individuals.57‘60'61

17

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In fact, currently, there are studies suggesting acute administration of C-peptide can
improve vascular, kidney and neural dysfunction in type 1 diabetes.59 It is now
increasingly evident that C-peptide, in combination with insulin, is successful in

controlling diabetic complications rather than insulin itself.

Although C-peptide was discovered in 1967, up until the 1990’s, little or no
biological significance was attributed to its function.62 In fact, as indicated by the name
“Connecting peptide”, C-peptide was thought to be physiologically inert beside.
providing the spatial arrangement necessary for the secondary and tertiary folding of the

A and B chains of insulin.62

C-peptide is a single chain small peptide consisting of 31 amino acids connecting
the N-terminal B-Chain and the C-terminal A-Chain.63 It is synthesized during insulin
biosynthesis in pancreatic B cells as a part of proinsulin.63 Later, it is cleaved from
proinsulin, stored in secondary secretory granules and released into the blood stream in

. . . . 64,5
equlmolar amounts W1th 1nsu11n. 6

Even though the half life of insulin in the
bloodstream falls in the range of a few minutes, C-peptide remains for hours. As a result
of this, C-peptide is utilized as a biomarker for insulin.66'69 Also, this longer half life of
C-peptide in the bloodstream reinforces the claim that suggest at high hormone
concentration, C-peptide suppresses the non-hypoglycemic effect of insulin and at lower

- - . - 5770-72
concentratlon 1t enhances the 1nsu11n effect. ’

Despite these new findings that suggest bioactivity of C-peptide, unequivocal lack

of knowledge concerning the mechanism of C-peptide activity (as well as its inability to

19

diminish complications in a certain percentage of the treated individuals during studies
conducted), limits the use of C-peptide as a potential treatment option in diabetes. In fact,
there are major drawbacks that stand against the notion of C-peptide as a bioactive

peptide.

In a conventional way of thinking, a bioactive peptide exerts its effect by binding
as a ligand to a specific molecule known as “receptor”.73 In the case of ligand receptor
binding, there is a specific moiety in the ligand, as well as a receptor, that act as an active
site for binding.73 However, there is no known receptor for C-peptide in any of the target
cells such as RBCs. Also, studies indicating not only the full-length native C-peptide, but
also its C-terminal pentapeptide fragment is reported to exert physiological effects

confirm the lack of the receptor based interaction for proinsulin C-peptide.73

Recently, non receptor mediated membrane interactions in C-peptide activity has
been suggested. In fact, in 1999 Rigler et al. demonstrated that C-peptide binds
specifically in nanomolar concentration to cell membranes, possibly to a G-protein-
coupled receptor, resulting in internalization of the peptide.59 Clinical studies conducted
with type 1 diabetes patients who lack endogenous C-peptide production show that
administration of C-peptide could increase regional blood flow in several tissues.58'69’74 In
addition to the binding data, they have shown that C-peptide involves the activation of
Ca2+ dependent signaling pathway, stimulation of Na+/K+ ATP ase and endothelial nitric
oxide synthase (eNOS) activation. In fact, all of these factors are related to the

improvement of blood flow through the microvasculature, indicating the importance of

C-peptide in glycemic control.75’76

20

Also, it has shown by our group that upon interacting with transition metal ions
such as Cry, Fey, C-peptide increases both glucose uptake in to RBCs and ATP release
from the RBCs, indicating a novel possible role of C-peptide in controlling blood glucose

levels.58

1.6 Channeling glucose via different pathways and polyol pathway up-regulation in

diabetes.

Upon entering the digestive track, food is broken down in to nutrition components
such as proteins, carbohydrates and fats and then eventually to the buildings blocks of
these macromolecules such as amino acids, triglycerols, monosaccharides and
oligosaccharides, prior to absorption in to the bloodstream.4 Glucose, a monosaccharide
of vast importance, is the ubiquitous source of energy and important metabolic
intermediate in living cells.4’” Also, glucose is the primary source of energy for the brain,

and hence its availability influences psychological processes.4

Glucose is a simple six carbon aldohexose with two stereoisomers (L and D) with
only the D form being bioactive, and hence available in the dietary intake.4 In aqueous
solutions, the glucose molecule exhibits an equilibrium between in an open-Chain
(acyclic) form and a ring (cyclic) form.4 The cyclic form, a result of a covalent bond
between the aldehyde C atom and the C-5 hydroxyl group, form a six-membered cyclic

hemiacetal and it is the predominant species at physiological pH.4

Glucose is taken up into the circulation and distributed in the body in a manner

dependant upon the energy demand of the cells.4 In healthy subjects, a portion of

21

absorbed glucose is stored as glycogen in the liver and the remaining fraction is then
distributed among various cells based on their bio-energy needs.4 Glucose, once entered
into the target organ via facilitated diffusion, is immediately converted in to G6P to
prevent reverse diffusion.4 The majority of the G6P will undergo oxidation via glycolysis
to enter anaerobic or aerobic respiration depending on the type of the cell, producing the
much needed ATP energy.4 Some fraction of the G6P will be Channeled through the

pentose phosphate pathway to generate a redox equivalent and five carbon sugars.77

Under hyperglycemic conditions, activation of polyol/sorbitol pathway occurs to
open another route to channel the excess glucose entering the cell.78 The polyol pathway

is a non insulin dependent metabolic alternation for glucose.4

The polyol pathway consists of two enzymatic reactions catalyzed by aldose
reductase (AR) and sorbitol dehydrogenase (Figure 1.5).“79‘80 AR catalyzes the rate
determining step converting glucose to sorbitol, utilizing the coenzyme NADPH.“82
Then sorbitol dehydrogenase (SDH) oxidizes generated sorbitol in to fructose reducing

nicotinamide adenine dinucleotide (NADH ) in the process.83

Although the polyol pathway is less functional to nonfunctional in normal cells, it
is known that non insulin dependant diabetes (NIDDM) patients show increased polyol
pathway activity. 80’“ It has been reported that the high glucose level increases activity of

both aldose reductase and sorbitol dehydrogenase.85

In RBCs, where the PPP is the sole NADPH producer, activation of AR that

consumes NADPH, will further escalate oxidative stress. (Fig 1.3) In fact, this is

22

Glucose
NADPH

Aldose
reductase

 

V NADP+
Sorbitol

A NAD+

Sorbitol
dehydrogenase

 

 

Y NADH

D-fructose

Figure 1.5: Schematic representation of the sorbitol pathway. Pathway consists of two
enzymes aldose reductase and sorbitol dehydrogenase. AR reduces glucose in to sorbitol,
while reducing NADPH. Then SDH oxidizes C2 of sorbitol, forming fructose. Free
NADH is generated in the process.

23

believed to be the main cause of such diabetic complications as retinopathy, neurOpathy
and nephropathy and cataracts in diabetic individuals.85 Therefore, the sorbitol level in
RBCs is an indication of the sorbitol accumulated in the retina of an eye and static

nerve.”87

Collectively, as a result, early detection and development ofan effective treatment
plan that includes life style changes such as diet and exercise, and new findings from
conducted research, suggest that control of the pathogenesis of diabetes mellitus can be
successful. Thus, despite the vast amount of money and research dedicated toward
finding a cure for diabetes, diabetes still remains somewhat of an unsolved mystery with

an ever-increasing occurrence world wide.

Therefore, understanding the molecular basis of RBC derived vascular regulation,
contribution of various metabolic pathways to the oxidative status of RBCs and finally a
factor that enables regulation of glucose in non insulin dependent targets will enable the
improvement of the diagnosis and treatment options in diabetes associated vascular

complications.

The work described here attempts to quantitatively determine the activity of the
' PPP enzyme G6PD by monitoring multiple parameters such as NADPH and ATP
simultaneously in the presence of hyperglycemia. Also, the relation between proinsulin

C-peptide, G6PD activity and the sorbitol pathway was investigated.

24

List of references

(1)

(2)

(3)

(4)

(5)

(6)

(7)

(8)

(9)

(10)

Brownlee, M., Biochemistry and molecular cell biology of diabetic complications.
Nature (London, U. K.) 2001, 414, (6865), 813-820.

Maritim, A. C.; Sanders, R. A.; Watkins, J. B., 111, Diabetes, oxidative stress, and
antioxidants: A review. J. Biochem. M01. Toxicol. 2003, 17, (1), 24-38.

Ito, C., Reference value of fasting blood sugar level. Bunshi T onyobyogaku no
Shinpo 2008, 95-99.

Lehninger, A. L.; Nelson, D. L.; Cox, M. M., Principles of Biochemistry, Pt. 1.
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Lestradet, H., The discovery of insulin. Bull. Acad. Natl. Med. (Paris) 1996, 180,
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Schade, D. S.; Eaton, R. P., Pathogenesis of diabetic ketoacidosis: a reappraisal.
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Brange, J .; Langkjoer, L., Insulin structure and stability. Pharm Biotechnol 1993,
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Ehrmann, D. A.; Sturis, J.; Byme, M. M., Karrison, T.; Rosenfield, R. L.;
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Masaki, Y.; Tanigawa, K.; Ohguni, 8.; Note, 8., Rat fetal islets as a useful model
for the study of insulin release failure. Pancreas 1987, 2, (6), 632-7.

Pollock, A. S.; Christakos, S.; Reddy, D. Method for restoring glucose
responsiveness to insulin secretion. 96-USl6736

9714441, 19961017.,1997.

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Stryer, L.; Editor, Biochemistry, 4th Revised Edition. 1996; p 1 100 pp.

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Singh, R.; Pearson, E. R., The importance of making a genetic diagnosis of
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from erythrocytes. Med. Sci. Monit. 2001, 7, (4), 669-674.

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Meyer, J. A.; Froelich, J. M.; Reid, G. E.; Karunarathne, W. K. A.; Spence, D. M.,
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Rigler, R.; Pramanik, A.; Jonasson, P.; Kratz, G.; Jansson, O. T.; Nygren, P. A.;
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improves neuropathy in type 1 diabetic BB/Wor-rats. Diabetes/Metabolism
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Sima, A. A. F.; Editor, Special Issue: Physiological and Pathophysiological
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97 pp.

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B. L.; Rigler, R.; Jomvall, H., Role of C-peptide in human physiology. Am J
Physiol Endocrinol Metab 2000, 278, (5), E759-68.

Wahren, J.; Ekberg, K.; Jomvall, H., C-peptide is a bioactive peptide.
Diabetologia 2007, 50, (3), 503-9.

Sima, A. A. F.; Kamiya, H., Is C-peptide replacement the missing link for
successful treatment of neurological complications in type 1 diabetes? Current
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type 1 diabetic polyneuropathies and encephalopathy in the BB/Wor-rat.
Experimental Diabetes Research 2008, No pp given.

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peptide and insulin administration on renal function and metabolic control in
diabetes type 1. J Clin Endocrinol Metab 1993, 77, (4), 976-81.

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Diabetologia 2007, 50, (3), 500-2.

Wahren, J.; Joemvall, H., C-peptide makes a comeback. Diabetes/Metabolism
Research and Reviews 2003, 19, (5), 345-347.

Wallerath, T.; Kunt, T.; Forst, T.; Closs, E. 1.; Lehmann, R.; Flohr, T.; Gabriel,
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(2), 95-102.

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Effects of C-peptide on expression of eNOS and iNOS in human cavemosal
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Clinical Biochemistry 2001, 38, (6), 701-707.

Chung, S. S. M.; Ho, E. C. M.; Lam, K. S. L.; Chung, S. K., Contribution of
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Society of Nephrology 2003, 14, (Suppl. 3), 8233-8236.

Mishra, J .; Pandeya, S. N., Aldose reductase inhibitors in diabetic complications.
Oriental Journal of Chemistry 2006, 22, (3), 571-592.

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Progress in Drug Research 1993, 40, 99-161.

Demiot, C.; Tartas, M.; Fromy, 8.; Abraham, P.; Saumet, J. L.; Sigaudo-Roussel,
D., Aldose reductase pathway inhibition improved vascular and C-fiber functions,
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neuropathy. Diabetes 2006, 55, (5), 1478-1483.

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Journal Title: The Journal of diabetic complications 1989, 3, (2), 70-6.

33

Chapter Two

QUANTITATIVE ANALYSIS OF G6PD ACTIVITY AND
SIMULTANEOUS DETECTION OF NADPH LEVEL AND RBC
DERIVED ATP RELEASE

Since the late 19th century, cardiovascular diseases (CVD) were thought to be

associated with diabetes."2 These speculations were further strengthened with the
discovery of insulin in 1922 3'6 followed by improvements in complications such as renal
failure, diabetes induced infections, and a marked increase in mortality and morbidity
resulting from CVD in people with diabetes.1 In fact, some scientists suggest that
research and development during the past two decades has fueled a view of diabetes as an
unusually severe form of cardiovascular disease (CVD) with a poor prognosis.7’8
According to the 2007 diabetes statistics released by Center for Disease Control
and Prevention, diabetic patients shows up to a 3-fold increase in coronary heart disease
events, independent of other traditional CVD risk factors, in comparison to their non-
diabetic counterparts.7 There are reports indicating that the physical Characteristics of red
blood cells (RBCs) such as deformability, plays a role in CVD pathogenesis in diabetic
individuals};10 Also there are reports indicating that damage to the oxidative defense
system lead to the less deformable RBCs.ll Therefore, understanding the effect of

hyperglycemia on major contributors to the oxidative defense such as glutathione,

34

NADPH levels and G6PD activity will provide beneficial effects for treatments and

prevention of CVD in diabetes.

2.1 G6PD Structure and function

Human glucose-6-phosphate dehydrogenase (G6PD, EC 1.1.1.49, B-D-glucose-6-
phosphate; NADP oxido-reductase) is an X — linked (Gene is located on X-Chromosome)
enzyme that is classified as a NADP+-dependant dehydrogenasem3 Also, G6PD is the
enzyme that catalyses the first and rate determining step of the hexose monophosphate
shunt or pentose phosphate pathway (PPP) and is located in the cytosol of animal, plant
and microbacterial cellsn’M'18 In addition, G6PD is found in the mitochondria of animal
cells and the Chloroplast of metaphytes or green plantsg’15

In solutions, G6PD exerts a rapid equilibrium between a dimeric and tetrameric
form and this equilibrium is affected by pH and ionic strength.‘5 ’19‘20 Even though there is
evidence suggesting that only the dimeric form is biologically active, this issue remains
controversial to date because little is known about the biological significance of the

'2’” However, it is widely accepted that the monomer of G6PD is

interconversion.
biologically inactive, indicating the importance of the subunit contact for it’s catalytic
function and Michaelis-Menten kinetic properties.'2 Both the amino acid sequence and
DNA sequence of the G6PD gene for almost all G6PD variants are known, and the
crystal structure of the human Canton G6PD variant (most common G6PD variant) has
been solved with up to 2.5 A resolution. ”"5

Being an X-linked enzyme, deficiencies in G6PD activity are more prominent in

males compared to femalesn’z' G6PD deficiency is the most commonly occurring

35

enzymopathy in humans, but remains somewhat benign unless triggered by the intake of

15,17,21

such oxidative agents as premaquine or foods like fava beans. Also, the occurrence

of the G6PD deficiency disease remains somewhat geographical with high frequent

outbreaks in malaria-endemic regions.21

This, combined with the high frequency
polymorphism (over 150 different human varieties) of the G6PD gene among different
populations, has made G6PD very interesting to biochemists, geneticists and medical
researchers. ' 7‘7"

G6PD is recognized as an important antioxidant enzyme, especially in RBCs and
in the microvasculaturew’22 G6PD activity is the main source of NADPH in the body
and in RBCs; in fact in RBCs, G6PD is the sole producer of NADPH.”23 NADPH is an
important cofactor utilized by many antioxidant defense enzymes, therefore signifying
the importance of G6PD activity in oxidant defense.‘9 It has been shown in isolated adult
cardiomyocytes that G6PD translocation to the cell membrane and G6PD activity
increase with increasing oxidative stress.22

Disturbing the redox balance of the cell, caused by deficiency in G6PD activity,
can lead to dysregulation of cell growth and signaling, abnormal embryonic deve10pment,
altered susceptibility to viral infection as well as increased susceptibility to degenerative
diseases.24 In-fact there are reports suggesting that G6PD activity is inhibited in diabetic
individuals.20 Though the mechanism by which inhibition occurs remains unclear to date,
there are two suggested pathways. One such possible mechanism was glycation induced
inactivation of G6PD.20 The other proposed mechanism suggests that high circulating

glucose levels result in the activation of protein kinase A and subsequent phosphorylation

leads to G6PD inhibition.'°’23

36

It is well known G6PD activity is an indicator of cell age.25 In 1986 Gross et a!
separated RBCs into four different age fractions and reported that G6PD activity
decreased as the cell age.25 Therefore understanding the Changes in oxidant defense as
cell age may lead to understanding the hyperglycemia induced decrease in G6PD activity
and its effect on physiological functions of the cell.

Also, diabetes patients, and those individuals who are G6PD deficient, exhibit a
high prevalence of hypertension and other cardiovascular complications.”26
Understanding the underlying effects of reduced G6PD activity, and hence lowered
NADPH levels, in the RBCs of diabetic individuals may be beneficial in explaining its
resultant effects on the microvasculature and complications in the cardiovascular

9
system. 1

2.2 NADPH as an indicator of G6PD activity

The conunon method for determining G6PD activity is by monitoring the increase
in NADPH absorbance at 340 nm.'9‘20 In this study, a fluorescence based detection
scheme was used to monitor cellular NADPH levels.
Fluorescence based detection scheme

NADPH was determined using the Vybrant Cytotoxicity Assay Kit (Invitrogen
Corp., Carlsbad, CA). In this assay, NADPH produced by G6PD is utilized by the
enzyme diaphorase to reduce resazurin to the fluorescent product resorufin as shown in

figure 2.1 (a). The resultant emission was measured at 587 nm.

37

2.3 Dehydroepiandrosterone, as a G6PD inhibitor

Commonly known as the “fountain of youth” or “the mother of all the hormones”,
dehydroepiandrosterone (DHEA) (Figure 2.1 (b)) is a naturally occurring steroid that is

, 8
27 2 Even

produced in the adrenal glands, gonads, adipose tissue, brain and the skin.
though it is produced in different parts of the body, more than 90 % of the Circulating
DHEA in human body is produced by the adrenal glands.29 Though the exact function of
DHEA is not well defined, it acts as a precursor for androgens and sex hormones such as

7 2
ICSIOSIBI‘OI’IB and estrogen. 2 ’ 9

DHEA seems to exert contrasting effects on individuals with diabetes. There are
reports suggesting that administration of DHEA exerts beneficial effects on type 2
diabetes subjects.29'3' Coleman and coworkers reported that dietary administration of
DHEA to type 2 diabetic mice induces remission of hyperglycemia and insulin
sensitivity.”'32 Interestingly, DHEA acts as a non-competitive steroid inhibitor of G6PD
and therefore will increase the oxidative stress on the cells by decreasing cellular

NADPH, hence weakening antioxidant defense.'6'33'38

There are also reports indicating that G6PD deficient/inhibited Chinese hamster
ovary cells are more sensitive to effects of ionizing radiation and oxidizing agents such as
diamide.l6 Diamide is a yellow, non-hygroscopic small diazene dicarbonyl oxidant that
reacts rapidly with GSH, oxidizing it to the dimeric form GSSG.”40 Also decrease in
deforrnation-induced RBC-derived ATP release upon treatment of RBCs with DHEA was

reported by our group.34

38

(a)

 

06

ll; NADPH NADP+ N\

e\ V

p
HO O O Diaphorase HO O O
Resazurin Resorufin
(hex 563, item 587 nm)
(b)
O

 
   
 

I'l'ullc

HO

Figure 2.1: (a) Fluorescence-based NADPH detection scheme. Enzyme diaphorase
converts non fluorescence resazurin to red fluorescence resorufin with the expense of
NADPH. (b) Dehydroepiandrosterone, a non competitive steroid inhibitor of G6PD.
DHEA is naturally produced from cholesterol in a human body but the circulating
concentration readily decreases by the age of 20.

39

2.4 RBC deformability and CFTR mediated ATP release from RBCs

Adenosine triphosphate (ATP), commonly known as a “universal energy carrier”
or “molecular currency” is a multifunctional nucleotide generated and consumed within
the body in a multitude of anabolic and catabolic reactions. ATP is mainly generated
during respiration in non-photosynthetic organisms and during photosynthesis in
photosynthetic organisms, and is readily consumed by many anabolic and catabolic
enzymes.“42 It is involved in signal transduction pathways and acts as a substrate for
kinases, a group of enzymes that catalyze the transfer of a phosphate group from high
energy donors, to specific target molecules.”42

It is well known that RBCs contain millimolar concentrations of ATP, and upon
being subjected to a stimuli such as mechanical deformation, 0; tension (hypoxia) or low
pH, these RBCs release ATP into the lumen of the vessel.”46 It was reported that RBCs
typically release nanomolar to low micromolar amounts of ATP.”4547 This ATP can
activate the purinergenic receptors on the vascular endothelial cells that will stimulate the
synthesis of nitric oxide (NO), a known vasodialator.34‘43'47 This NO induces the
production of cyclic guanosine monophosphate (CGMP) that will eventually lead to
smooth muscle relaxation and an increase in blood flow to the target tissue. 46

Previously, our group has shown that the amount of ATP released from the RBCs
upon mechanical deformation is significantly decreased if the RBCs are subjected to
oxidant stress prior to the deformation.34 Based on these results it was hypothesized that
in type 2 diabetic individuals, where RBCs are under constant oxidative stress and

lowered G6PD activity and hence lowered NADPH levels, reduction of ATP release

upon deformation is pronounced.33‘34 Our results suggested that when RBCs were

40

subjected to an oxidative stress with 20 11M diamide (a known oxidant), ATP release
from these RBCs was decreased by 63 %.34 This decreased level of ATP release may
impair blood distribution and increase the risk of developing cardiovascular

. . . . q 4
complicatrons 1n vrvo.33 3

2.5 Simultaneous detection of G6PD activity and its effect on ATP release

The G6PD enzyme is a determinant in the antioxidant status of the RBC and is
also used as an indicator of cell age.48'50 Also, deformation-induced RBC-derived ATP
release depends upon the deformability of the cell membrane and, hence, the antioxidant
defense system. However, it is unknown if the relationship between the antioxidant status
and RBC-derived ATP release occurs immediately or over a period of time. Therefore, it
is important to be able to detect both G6PD activity (via the determination of NADPH)
and deformation-induced ATP release from RBCs. In order to demonstrate a direct
relationship between G6PD activity and deformation-induced ATP release from RBCs, a

simultaneous microflow detection scheme was developed.

2.6 Experimental

2.6.1 Collection of RBCs

All surgical procedures involving animals used in this study were performed
under protocols approved by the appropriate Animal Investigation Committee or
Institutional Review Board. Rabbit RBCs were obtained from male New Zealand White

rabbits (2.0-2.5 kg). Rabbits were anesthetized with ketamine (8.0 mg kg", im) and

41

xylazine (1.0 mg kg", im) followed by pentobarbital sodium (15 mg kg'l iv). After
tracheotomy, the rabbits were mechanically ventilated (tidal volume 20 mL kg", rate 20
breaths min"; Harvard ventilator). A catheter was placed into a carotid artery, heparin
(500 units, iv) was given, and after 10 min, animals were exsanguinated. Human blood
was obtained by venipuncture and collected in to a heparinized syringe. Human diabetes
subjects are adults, mostly African Americans with hemoglobin Alc value of 7 % or

greater.33

2.6.2 Preparation of RBCs

To prepare the RBCs from the collected blood sample, whole blood was
centrifuged at x 500 g at 4°C for 10 min. The plasma and buffy coat were removed and
stored for other experiments within the laboratory. RBCs were resuspended and washed 3
times in a physiological salt solution (PSS). The PSS was prepared by combining TRIS
buffer (25 mL, prepared by mixing 50.9 g of TRIS in 1 L of distilled and deionized water
(DDW)) and Ringer’s buffer ( 25 mL, prepared by mixing 164.2 g NaCl, 7.0 g KCl, 5.9 g

CaC12.2HzO, and 2.5 g MgS04 in 1 L DDW). After the addition of dextrose (0.50 g) and

albumin bovine fraction 1V (fatty acid free, 2.5 g) to the above mixture, the solution was
diluted to 500 mL with DDW and the pH was adjusted to 7.35- 7.45. The PSS was then
triple filtered using a filter with 0.45 pm pores (Corning, Fischer Scientific). Cells were
prepared on the day of use and studied within 8 h of removal from the animal or human

subject.

42

2.6.3 Reagents
All reagents were prepared in DDW or PSS unless otherwise noted.

Resazurin stock was prepared by dissolving the content of the vial in 75 uL of
analytical grade dimethyl sulfoxide (DMSO) to make a 4 mM resazurin stock solution.
Next, the resazurin-reaction mixture was prepared by diluting 75 11L of the resazurin
stock solution to 10.00 mL in the solution prepared by using the component C
(lyophilized mixture of diaphorase, G6P and NADP+) and D (0.5 M tris, pH = 7.5)
according to the instructions provided with the Vibrant Cytotoxicity assay kit.34

A 200 11M NADPH stock solution was prepared by dissolving NADPH (0.0043 g)
in 25 mL of DDW. NADPH standards (0.0- 20.0 pM) were prepared by diluting the stock
solution of NADPH in DDW, accordingly.33

20 uL of 1000 U mL'l G6PD stock solution was diluted with DDW to 4.00 mL to
make a 5 U mL'l G6PD solution.

A 100 11M ATP stock solution was prepared by dissolving ATP (0.0619 g) in
DDW and diluting to 1.00 L in DDW. ATP standards (0.0 - 1.5 11M) were prepared by
diluting appropriate aliquots of the stock ATP solution in PSS.34

To prepare the luciferin/luciferase mixture required for chemiluminescence
determination of ATP, luciferin (2 mg, Sigma) was dissolved in 20 mL of DDW. A 5 mL
aliquot of the luciferin solution was added to a vial containing luciferin/luciferase (FLE-
50, Sigma).34

A 2 mM stock solution of diamide was prepared by adding 0.0344 g of diamide to
100 mL of DDW. A 1 mL aliquot of the diamide stock solution was added to 9 mL of

wash buffer to create a buffered 200 11M solution of diamide.34

43

2.6.4 Instrumentation

All non — flow fluorescence measurements were performed with a Shimadzu
R5301 scanning spectrofluorometer. All Chemiluminescence-based ATP measurements
were conducted using a photomultiplier tube (PMT; Hamamatsu Corporation,
Hamamatsu, Japan) placed in a light excluding box (Figure 2.2.). Flow based
fluorescence measurements were carried out using a Jasco FP-2020 intelligent flow-

through fluorescence detector with 16 11L flow cell.

2.7 Methods

2.7. 1 Optimization of NADPH detection parameters
Fluorescence detection of NADPH was optimized under non-flow based

conditions prior to the application to flow-based system.

2.7.1.1 Determination of the incubation time for resazurin
50 pL of G6PD (5 U mL") was diluted with PSS to 100 1.1L. The resazurin -
reaction mixture (100 uL) was added and resulted fluorescence intensity was measured

(ex. 563 nm, cm. 587 nm) as a function of time for 65 minutes.

2.7.1.2 Qualitative detection of NADPH in rabbit, control and type 2 diabetes RBCs

A 0.04 % rabbit RBC sample was prepared by diluting the appropriate volume of
RBCs in PSS. The resazurin reaction mixture (100 pL) was added to a solution of RBCs

(0.04% HCT, 100 uL) and the content was mixed thoroughly and incubated at 37 °C for

44

30 min. The fluorescence intensity of the resorufin was measured (ex. 563 nm, cm. 587
nm) using a scanning spectrofluorometer under static conditions.

To verify the Change in fluorescence emission was the result of G6PD activity,
RBCs were incubated with 100 11M DHEA, a known inhibitor of G6PD, for 30 min prior .
to addition to the reaction mixture. Finally, to identify the involvement of G6PD in ATP
release from RBCs upon deformation, RBCs were incubated with 2 11M diamide (i 100

11M DHEA) for 20 min and monitored for G6PD activity as described above.

2.7.2 Chemiluminescence detection of RBC derived ATP release

To quantitatively determine ATP release, the luciferin/luciferase mixture was
placed in a 500 11L syringe. ATP standards, or RBCs, were placed in the second syringe
and both solutions were pumped through 30 cm sections of microbore tubing having an
internal diameter of 50 pm at a rate of 6.7 11L min'1 using a dual syringe pump (Harvard
Apparatus, Holliston, MA). The streams containing the luciferin/luciferase mixture and
ATP standard/RBCs were combined (figure 2.2 (b)) at a mixing T-junction with an
internal volume of 560 nL (Upchurch Scientific, Oak Harbor, WA). The combined
stream flowed through a segment of microbore tubing having an internal diameter of 75
um, allowing the detection of the resultant Chemiluminescence from the reaction of the
ATP (either in standard form or that released from RBCs) using a PMT ( Hamamatsu
Corporation, Hamamatsu, Japan) placed in a light excluding box. The polyimide coating
was removed from the microbore tubing on the segment over the PMT to facilitate light

transport through the tubing and to the PMT. (fig 2.2.)

45

(a)

 

    

Computer

  

 

 

(b)

 

Microbore tubing

 

 

 

 

 

— Dark box

 

Figure 2.2 (a) Experimental setup used for ATP release measurements. (b) Schematic
representation of the PMT and microbore tubing connection inside the light excluding
box. X and Y represent the connecting “Tee” and window with the PMT underneath for
chemiluminescence detection respectively. —> indicates the direction of flow.

For studies involving DHEA, 7% RBCs (10.0 mL) were prepared by adding 1
mM DHEA (1.0 mL) to make 7% RBCs in 100 11M DHEA and this solution was
incubated at 37 °C for 30 minutes. For diamide studies, an appropriate volume of RBCs
were placed in a 5.0 mL volumetric flask containing 0.5 mL of the 200 11M diamide
creating a 7% RBC solution in 20 11M diamide. This mixture was then placed in a 500 uL
syringe and ATP release was measured at 5 minute intervals. The syringe was rotated

periodically to ensure a homogeneous mixing of red blood cells.

2.7.3 Flow through detection of NADPH levels in rabbit red blood cells

A 0.02 % hematocrit of RBCs was used in all the experiments where NADPH
was quantitatively measured. To determine NADPH, an aliquot of the resazurin reaction
mixture was placed in a 500 11L syringe (Hamilton, Fisher Scientific). NADPH standards,
or the RBCs, were placed in another syringe and both solutions were pumped through 30
cm sections of microbore tubing, having an internal diameter of 50 um (Polymicro
Technologies, Phoenix, AZ), at a rate of 1.0 pL min'1 using a dual syringe pump
(Harvard Apparatus, Boston, MA). The two streams were combined at a mixing T-
junction having an internal volume of 560 nL (Upchurch Scientific, Oak Harbor, WA).
The now combined content was allowed to flow through a 90 cm long segment of
microbore tubing having an internal diameter of 150 pm. The larger bore tubing resulted
in a slower linear rate of the stream, thus enabling the required incubation period prior to
detection of the resultant fluorescence using a flow-through fluorescence detector (Jasco,
FP-2020 fluorescence detector). The total time taken for a single measurement was 30

min. The fluorescence intensity was measured at 587 nm (excitation at 563 nm). The

47

same instrumental setup and experimental procedure described above was used to

quantitate the cellular NADPH in diabetic and human control RBCs.

2.7.4 Separation of RBCs into age-based fractions and measurement of NADPH
levels and ATP release

RBCs were separated into fractions based on cell density centrifugation.51 In a 15
mL tube, 2 mL of a solution of Percoll (Sigma Chemical Co, St. Louis, MO) having a
density of 1.115 g mL" were added to the bottom of the tube. Next, 2 mL of a similar
solution of Percoll having a density of 1.105 g mL'1 were slowly added on to the top of
the first 2 mL of the higher density solution. Finally, 1 mL of RBCs (70% hematocrit)
was added to the top of the Percoll layers. The tube containing the Percoll and RBCs was
then centrifuged at 3000 x g at 4 °C for 15 min. After centrifugation, two layers of RBCs
are present; those having a density of less than the 1.105 g mL Percoll solution will
appear at the top of the tube and represent those RBCs having the lowest density; these
RBCs are considered the “younger” of the RBCs in the original RBC sample3’7‘5'52
(Figure 2.3). Those below the higher density Percoll solution are the denser, or aged,
RBCs. These separated RBC fractions were removed by pipette and diluted to a
hematocrit of 7% using the PSS described above. ATP release and NADPH levels

measurements were made with 7% and 0.02% hematocrit RBCs, respectively, using the

methods described above.

48

 

Centrifuged at X3000 g at
4 °C for 15 min

“-

 

 

 

 

RBC - low density younger
fraction

Percoll, 1.105 g mL"

RBC, 1 mL, 70 %

Percoll, 2 mL, 1.105 g mL"

 

Percoll, 2 mL, 1.115 g mL" I Percoll, 1.115 g mL"

RBC - high density older
fraction

 

Figure 2.3: Pictorial representation of the separation of RBCs into fractions based on the
age. Two Percoll solutions with slightly different densities were placed in a
microcentrifuge tube and RBCs were introduced slowly to the same tube. The content
was centrifuged at X3000 g at 4°C for 15 min. RBCs were separated based on density
(therefore age). The small RBC layer between two Percoll layers represents the RBCs
that were not separated.

49

2.7.5 Simultaneous detection Scheme

In order to determine both cellular NADPH levels (hence, G6PD activity) and
deformation-induced RBC-derived ATP release simultaneously, under continuous flow
conditions, the experimental setup shown in Figure 2.4 was employed. Here, NADPH
was measured using the previously described resazurin/resorufin fluorescence based
assay while ATP was measured with the firefly luciferin/luciferase Chemiluminescence.
This setup mainly consists of dual syringe pumps, PMT, fluorescence detector and
various lengths of fused silica microbore tubing (internal diameters of 50, 75 and 150 um
and an external diameter 363 um, Polymicro Technologies, Phoenix, AZ) and connectors.
(U pchurch Scientific Inc., Oak Harbor, WA)

Deformation was achieved by using 50 1.1M internal diameter microbore tubing
whereas longer incubation times needed for fluorescence detection was achieved by
application of microbore tubing with larger inner diameter (150 um). Microbore tubing
carrying different reacting agents were connected with a mixing tee (see figure 2.2 (b))
and the exciting stream from mixing tee was flowed in to a segment of 75 um i.d.
microbore tubing, for chemiluminescence measurements using the PMT setup explained

above.

2.7.6 Simultaneous detection of NADPH levels and deformation induced RBC ATP
release

A required volume of RBCs was placed in a 10 mL volumetric flask and was

diluted with PSS to prepare a 7 % hematocrit of RBCs. Next, approximately 285 uL of

50

this solution were diluted up to 100 mL in DDW to create a solution of RBCs with
hematocrit of 0.02 %.

For the simultaneous determination of ATP and NADPH, two dual syringe pumps
were employed as shown in figure 2.4. NADPH standards or the 0.02 % RBCs were
placed in one syringe and the resazurin reaction mixture was placed in another and both
were pumped at a rate of 1.0 11L min'l through 50 um internal diameter tubing. The
content was combined at a mixing-T and incubated for 30 min by passing through the 90
cm long, 150 pm internal diameter microbore tubing. The resultant fluorescence was
detected online with the flow-through fluorescence detector mentioned above.

Simultaneous to the NADPH measurements, the luciferin/luciferase mixture was
placed in a 500 11L syringe. ATP standards, or RBCs, were placed in the second syringe
and both solutions were pumped through 30 cm sections of microbore tubing having an
internal diameter of 50 pm at a rate of 6.7 uL min—1 using the dual syringe pump
mentioned above. The streams containing the luciferin/luciferase mixture and ATP
standard/RBCs were combined at the mixing T-junction. The combined stream flowed
through a segment of microbore tubing having an internal diameter of 75 um, allowing
the detection of the resultant chemiluminescence from the reaction of the ATP (either in
standard form or that released from RBCs) using a PMT setup described in section 2.5.5.
These measurements were repeated with the RBCs in the presence of the DHEA. The
identical experimental setup and procedure were occupied to monitor control and human

RBCs

51

2.8 Results and discussion

Reactive oxygen species (ROS) such as superoxides, peroxides and hydroxyl
radicals are generated during various cellular filnctions.22 Under normal biological
conditions these generated ROS are transformed into non-harmful species by the cellular
antioxidant defense mechanism.22 However, it is well known that the antioxidant defense
system of the RBCs obtained from people with type 2 diabetes is deficient in comparison
to those RBCs obtained from healthy individuals.53'55 This weakened antioxidant status is
believed to be the result of decreased levels of the cofactor NADPH due to the reduced
activity of G6PD.43‘56‘57 In fact, several studies have confirmed that RBCs having a
down-regulated G6PD gene, show lowered basal NADPH concentrations.15

NADPH acts as a substrate for GSHrcd, an enzyme that maintains GSH in its
reduced, antioxidant form.58 Upon oxidant insult, GSH is oxidized to its dimeric form
GSSG, thereby protecting important cellular components and membrane proteins from
being subjected to oxidant stress.59 Upon decrease of NADPH, GSHrcd activity decreases,
thereby resulting in lower levels of reduced GSH and subsequent weakening of the
cellular antioxidant defense. Also, it is well established that hyperglycemic conditions
up-regulate metabolism and activate various biochemical pathways such as the sorbitol
pathway that leads to overproduction of ROS.”60 This overproduction of ROS can
diminish the already weakened antioxidant defense system and permanently damage
important cellular components.60

When subjected to oxidation by ROS, membrane protein such as spectrin undergo

structural modifications and Change its specific three-dimensional structure, hence its

function.”“ "6' In fact, the cytoskeleton formed by spectrin is of extreme importance to

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53

RBCs, allowing them to undergo shape changes when traveling through the
microvasculature.

Recently, our group and several other groups have shown that RBCs release
nanomolar to low micromolar amounts of ATP when subjected to a mechanical
deformation while traveling through the microvasculature.34’62 This ATP can bind to the
P2Y receptor on the endothelial cell of the vessel wall and stimulate endothelial NO
synthase (e-NOS), stimulating synthesis of NO, a known vasodilator, thereby
participating in the control of vascular caliber.34‘45‘62

We hypothesized that a decrease in the PPP, an increase in ROS and weakened
antioxidant defense would lead to a more stiffened RBC membrane. These stiffened
RBCs will undergo reduced deformation and an accompanying decrease in ATP release,
facilitating complications in the microvasculature and the importance of understanding
G6PD activity and its effect on RBC derived ATP release. But neither we, nor others,
have studied whether this effect is a consequence of a long time event or an immediate
result of the G6PD inhibition.

As the first step towards establishing the relationship between G6PD activity and
RBC-derived ATP release, a scheme to monitor G6PD activity under continuous flow
conditions, an essential element of the circulation was designed. Though UV absorption
of NADPH is the most commonly utilized technique for measuring G6PD activity, this
method carries certain drawbacks such as interference from NADH and reduced

detection limits in comparison to fluorescence. Therefore, a fluorescence based

resazurin/resorufin assay was used for the NADPH measurements.

54

In order to optimize the experimental conditions used to measure NADPH in
RBCs, resazurin was incubated with G6PD in the presence of NADP+, G6P and
diaphorase. A linear increase in the fluorescence intensity as a function of a time was
observed over a 70 min. period and then stabilized due to resazurin becoming the limiting
reagent (Figure 2.5). A 30 min. incubation period was selected for further studies as it
provides adequate intensity changes, as well as a shorter incubation time.

Upon optimization of the experimental conditions, the next step was to apply
these optimized conditions to RBCs. To further Clarify that the changes are due to G6PD
activity, the RBCs were incubated with DHEA (a known G6PD inhibitor), and then
treated with diamide (a known oxidant) prior to the NADPH measurements (Figure 2.6).
These data confirm the reduction of cellular NADPH levels upon G6PD inhibition.
However diamide did not have a significant effect (p > 0.02) on NADPH levels in
comparison to control RBCs nor did it have any effect on G6PD inhibited RBCs. Also,
the effect of DHEA after a 60 min. incubation was not significantly different from effect
at 30 min. Therefore a 30 min. incubation time was used in further studies.

Biaglow et al. has shown that cell growth and bio-reduction of thiol species
decrease in Chinese hamster ovary cells on a time and concentration dependant manner
upon incubation with DHEA due to decreased NADPH levels.l6 Also, there are reports
indicating that overexpression of G6PD results in an increase in NADPH and a decrease
of the damaging ROS.22 Therefore, as expected, the effect of DHEA was profound in
RBCs where G6PD is the sole NADPH producer.

NADPH levels were also measured in human control and type 2 diabetes RBCs

under non-flow-based conditions. As shown in the data in Fig. 2.7, the NADPH level in

55

 

Fluorescence intensity (a.u)

A
O
l

 

 

0 E I l l T l l
o 10 20 30 40 50 6O 70

Time (min)

 

Figure 2.5: Kinetic study - optimization of the incubation time for resazurin. Resultant
fluorescence intensity was measured over a period of 70 min. at 583 nm. Fluorescence
intensity was increased linearly as a function of time up to 70 min. period.

56

 

 

 

 

 

 

 

 

 

 

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.35 1 l
v . +
E - l
100 —
g .
‘5 .
2 .
LL. 4
50 4
0 i 1 I .
Control Diamide DHEA Diamide + DHEA

Figure 2.6: The effect of DHEA on G6PD activity (n = 6 rabbits). Gray bars represent 30
min. incubation with DHEA whereas black bars represent the 60 min. incubation.
Diamide alone did not affect G6PD activity, nor did it have any further effect on G6PD
activity in the presence of DHEA. The asterisks represent those values significantly
different from the control (p < 0.001) while the crosses indicate NADPH levels that are
statistically equivalent to the control (p > 0.02)

57

300 .

 

 

250

 

200 l 1

 

 

H

 

150?

 

100

Fluorescence intensity (a.u.)

50%

 

 

 

 

 

 

 

 

0 i . . ,
Control(Human) Diabetes Control(Rabbit)

 

Figure 2.7: NADPH levels in rabbit, control human and diabetes RBCs measured under
non flow conditions. The NADPH level in the RBCs obtained from the patients with
diabetes was significantly different from the healthy human controls and rabbit RBCs (p
< 0.01). n = 5 samples.

58

the RBCs obtained from patients with diabetes was about 30% less than healthy human
controls and rabbit RBCs. These results follow the same trend as previously published
ATP release data from RBCs of people with type 2 diabetes}4 To provide further
evidence that the G6PD activity may be a major determinant of RBC-derived ATP
release, ATP release from the G6PD-inhibited and diamide-treated rabbit RBCs were
measured. As shown in Fig 2.8, RBCs that were subjected to diamide alone were able to
recover from the oxidant insult (diamide addition) as evident by the recovery of ATP
release seen in measurements taken after 20 min.34 The ability to recover from the
diamide insult is similar to previous data generated by our group.34 However RBCs
incubated in DHEA did not recover in the same amount of time as RBCs incubated in
diamide alone. In fact, the ATP release never recovered, even after a 40 min incubation
period (Figure 2.8). These data, combined with the NADPH measurements in the
presence of diamide incubated RBCs (Figure 2.6), suggest that inhibition of the G6PD
enzyme, a major component of the pentose phosphate pathway, may be a determinant in
the ability of RBCs to release ATP upon deformation.

Upon successful monitoring of the cellular NADPH levels and the resulting
G6PD activity, it is essential to evaluate the relationship between the G6PD activity,
deformation induced RBC-derived ATP release, in order to understand the relation
between elevated cardiovascular complications and diabetes. The first step toward
establishing such a correlation would be to assess the NADPH levels hence G6PD
activity under continuous flow, an essential element of microcirculation.

We successfully extended our fluorescence detection scheme to a flow based

measurement scheme. For calibration and quantification purposes, NADPH standards (0

59

 

Normalized ATP release
.9 .o .0
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O

30
Time (min)

A

Figure 2.8: Deformation induced ATP release from RBCS in the absence and presence of
a G6PD inhibitor is shown. The gray bars show the average ATP release (n = 6 rabbits) at
various time intervals measured from RBCs that were subjected to 20 uM diamide alone.
The black bars show the ATP release from aliquots of the RBCs after the addition of
diamide; however, these RBCs were first incubated with 100 uM DHEA for 30 min in
order to inhibit the G6PD activity. The bars with lines show ATP release from RBCs that
were incubated with DHEA alone. The asterisks represent those values significantly
different from the initial (t = 0 min, p < 0.001) while the crosses indicate ATP values that
are statistically equivalent to the initial value at t = 0 min (p > 0.02).

60

— 20 mM) (Fig 2.9) were measured prior to measuring NADPH levels in RBCS. Once the
method was established using NADPH standards, NADPH levels in normal rabbit RBCs
and chemically aged RBCs (by inhibiting G6PD with DHEA) were measured. As
summarized in Figure 2.10 (b), the DHEA-induced aging (via inhibition of the G6PD and
the pentose phosphate pathway) resulted in a decrease in NADPH concentration from
13.2 i 1.8 uM (normal RBCs) to 8.0 :tl.1 uM (RBCs incubated in DHEA) in a 0.02%
solution of RBCs, a 39% decrease in NADPH concentrations.

It is well established that as cells age, the G6PD activity within the cell decreases
resulting in a smaller RBC that is more dense and, importantly, less deformable.
Therefore, to further verify the relationship between G6PD activity and ATP release,
RBCs from healthy rabbits were separated based on cell density centrifugation as
described above. The cellular NADPH levels and deformation induced ATP release were
measured in these young and old fractions obtained from the whole sample of RBCS. As
shown in figure 2.9, both NADPH concentration in, and deformation-induced ATP
release from, the RBCs are significantly different in the younger and older fractions. As
expected, both NADPH levels and ATP release from RBCs obtained from the less dense,
“younger” fraction are greater than more dense RBCs with less G6PD activity. In the
younger fraction, the NADPH level is 11 % greater compared to the older fraction, while
the ATP release was 56 % greater in the younger RBC fraction compared to the older
portion of the cells. See Figure 2.1 1.

Although there are reports suggesting a relation between decreased G6PD activity
and increased RBC stiffness, this is the first attempt to monitor the consequences of these

characteristics simultaneously and quantitatively while permitting the forces required

61

 

0.030 p

0.025

l 1 1 1

0.020

0.015

L +4 #1 l 1

0.010 a

Fluorescence intensity (a.u.)

114

0.005

1

0.000 G 1 1 l I
0 5 10 15 20 25

 

 

 

NADPH (0M)

Figure 2.9: NADPH standards measured using flow based fluorescence detection scheme.
Fluorescence intensity changes linearly within the NADPH concentration range of 0 —- 20
uM (r 2 =0.998). n = 7 sample sets.

62

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Figure 2.11: NADPH levels and deformation induced RBC derived ATP release from
young and old fractions of rabbit RBCS. The black bars represent the fluorescence
intensity corresponding to NADPH while the gray bars represent the chemiluminescence
intensity corresponding to ATP release from the RBCs (n = 3 rabbits). The values
between the two groups are significantly different (p < 0.01).

64

(flow) to induce the ATP release. Both NADPH and ATP were measured simultaneously,
using the experimental setup shown in figure 2.4. As expected, a direct relationship
between decreased NADPH levels and the decreased values of deformation-induced
RBC-derived ATP is observed. Results showed in figures 2.12 and 2.13 indicate that the
decrease in ATP release is proportional to the decrease in the cellular NADPH levels.
Upon inhibition with DHEA, the ATP release decreased to 0.06 d: 0.02 uM, down from
an initial value of 0.21 d: 0.07 uM in 7% RBCS. In a similar manner, the initial
concentration of NADPH (12.73 i: 0.50 uM) decreased to 8.62 :t 0.29 uM upon addition
of DHEA.

As the last step towards understanding the relationship between oxidative stress,
G6PD activity and ATP release in diabetic subjects, we monitored these parameters in
RBCs obtained from diabetic and healthy individuals. There is an approximate 50%
decrease in NADPH concentrations and a 78% decrease in ATP release from the RBCs

from diabetic patients as compared to healthy individuals (Figure 2.14)

65

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0.010‘ . . . . . . r
6 8 10 12 14 l6 18 20
Time (min)

Figure 2. 12: Qualitative representation of simultaneous detection of NADPH levels and
deformation-induced ATP release from rabbit RBCS. NADPH levels were measured
using a fluorescence-based G6PD assay, while ATP release was measured using
chemiluminescence. The bottom two traces represent ATP release from normal RBCs
(higher of the pair) and G6PD inhibited (lower of the pair) RBCS. The top two traces
represent NADPH levels in 0.02% RBCs in the absence (higher of the pair) and presence
(lower trace of the pair) of DHEA.

66

 

0.8 1

Concentration (uM)

0.4 a

 

0.2 -

,T

0.0 ‘ T 9%
control RBC+DHEA

 

 

 

 

 

 

 

 

Figure 2.13: Quantitative representation of simultaneously detected NADPH levels and
deformation-derived ATP release. The black bars represent NADPH ><10’l concentration
(n= 5 rabbits), while the gray bars represent ATP release (n = 5 rabbits). The values
between the two groups are significantly different (p < 0.01).

67

 

 

 

 

 

 

 

 

 

 

1.4 J
1.2 f
A 1.0 {
E :
3 .
r: 0.8 -
.2 ‘
‘53 I
S 0.6 3
o .4
c: .
C q
U .
0.4 1
i ..T
0.2 1 l
0.0 I “'1'“
Diabetes Human control

Figure 2.14: Measurement of cellular NADPH levels and ATP release from RBCs
obtained from people with type 2 diabetes and healthy controls. The black bars represent
NADPH XIO‘ (n= 5 individuals) and the gray bars represent ATP release (n= 8
individuals). There is an approximate 50% decrease in NADPH concentrations and a
78% decrease in ATP release from the RBCs from diabetic patients as compared to
healthy individuals. The values between the two groups are significantly different
(p < 0.01).

68

2.9 Conclusions

In type 2 diabetes, RBCs are under high oxidative stress and are believed to be
less deformable, leading to lowered levels of deformation-induced ATP release. In an
attempt to understand simultaneous changes in G6PD activity and deformation induced
RBC derived ATP release, we successfully developed and extended an NADPH
measurement scheme to continues flow based system, which is an essential condition in
microcirculation, with the expectation of application of simultaneous detection. As
expected cellular NADPH levels decrease by 39 % upon inhibition of enzyme G6PD
signifying its importance in antioxidant defense.

When subjected to oxidative stress, if unprotected, oxidation of important
membrane proteins will leads to a stiffening of the cell membrane. This effect is
pronounced in RBCs where G6PD is the sole producer of NADPH, an essential cofactor
in the antioxidant defense mechanism. In type 2 diabetes, RBCs are under high oxidative
stress and are believed to be less deformable, leading to lowered levels of deformation-
induced ATP release. Also G6PD activity is an indication of cell aging and upon aging
cell membranes become more stiffened due to a weakened oxidative defense system.

Here we were able to measure cellular NADPH levels in the RBC (via a
fluorescence-based assay) and deformation-derived ATP release from the RBC (via
chemiluminescence assay) simultaneously, under continuous flow conditions. Results are
consistent with the expected pattern of a direct relationship between NADPH
concentrations in the RBC and deformation-derived ATP release from these cells. With a
decrease in NADPH concentration, there is a decrease in deformation-derived ATP

release from the RBC. A similar trend was observed with chemically aged (via inhibition

69

of G6PD) RBCs, as well as young and old fractions of RBCs separated from whole
blood. RBCs obtained from people with type 2 diabetes showed the same trend
confirming that oxidative stress leads to a decrease in deformation-derived ATP release.
Due to the ability of ATP to stimulate NO production in other cells (e.g., endothelial cells
and platelets), the RBC thus becomes a potential determinant of blood flow in the
diabetic circulation. Perhaps more evident from the work reported here, however, is the

potential use of NADPH and ATP as biomarkers of oxidative stress in the RBC.33

70

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77

Chapter 3

ROLE OF C-PEPTIDE AND EFFECT OF DIABETES
INDUCED OXIDATIVE STRESS ON C-PEPTIDE

ACTIVITY

3.1 C-peptide as a bioactive peptide
Proinsulin connecting-peptide, commonly known as a proinsulin C- peptide is a

small 31 amino acid peptide synthesized and secreted together with the hormone insulin
from the islets of Langerhans.1 Its name was also inspired by the fact that insulin is
composed of two peptide chains named as chain "A" and "B".I

The discovery of proinsulin by Steiner and his coworkers in 1965 is not only the
milestone in the elucidation of the biosynthesis of proteins, but also leads to the complete
understanding of insulin biosynthesis followed by the discovery of C-peptide." 2 In 1968,
Chance and coworkers isolated proinsulin from crystalline porcine insulin." 3 They
evaluated the amino acid sequence of a 33-amino acid peptide, linking the A and B
chains of insulin and described it as a “connecting peptide”." 3‘ 4 During the same year
Steiner and Clark successfully demonstrated that the primary role of proinsulin appeared
to be to facilitate efficient formation of the disulfide bonds of insulin, leading to further

1.4.5

investigations of C-peptide. Subsequent research followed, leading to the isolation

and analysis of human proinsulin and eventual preparation and sequence determination of

78

human C-peptide." 4’ 6'”) Figure 3.1 depicts the structure and amino acid sequence of

human C-peptide.
Upon investigating the role of C-peptide, studies conducted on rats indicated the kidney
as the main organ responsible for the degradation of proinsulin and C-peptide.”’ '2
However extended studies on the possible physiological function and activity of C-
peptide under various conditions were concluded to be negative by most researchers,
despite some reports stating otherwise.“ '3 Subsequently, it was concluded that C-peptide
is devoid of any biological activity.‘ '2‘ '3
During the following years, it was discovered that equivalent amounts of insulin
and C-peptide were secreted from the pancreas. Therefore measuring C-peptide in the
circulation became an important diagnostic tool for various diseases associated with
either pancreatic B cells or insulin." ‘4 Between 1973 to 1976 there was a considerable
increase in immunological studies involving C-peptide, exploring the possibility of using
C-peptide as diagnostic tool." 15'” Currently, C-peptide assays are used in a large variety
of studies, such as liver metabolism, ketoacidosis, insulin-induced hypoglycemia, oral
glucose tolerance test, comparing portal and peripheral blood, diagnosing patients with
islet cell tumors and examining children with juvenile diabetes." '7‘ '8
Despite its successful passive role as a diagnostic tool, C-peptide was thought to
be biologically inert for most of its 40 years of existence since its discovery in 1967.” 20
In fact, except for the importance of using C-peptide as a unique biomarker for insulin
biosynthesis, C-peptide was considered to have little or no importance beyond its role as

a mere integral part of proinsulin.” 22

79

(a) (b)

 

10
GIu-Ala-Glu-Asp-Leu-Gln-Val-Gly-Gln-Val_Glu-Leu-Gly-

20
Gly-Gly-Pro-Gly-Ala-Gly-Ser-Leu-Gln-Pro-Leu-Ala-Leu-

30
Glu-Gly-Ser-Leu-Gln

Figure 3.1: (a) Structure of human proinsulin C-peptide (b) Schematic representation of
the human proinsulin structure with chains A and B of insulin connected by C-peptide.
(c) Amino acid sequence of human C-peptide

80

During the late 1990’s, interest in C-peptide research was renewed." ”'25 New
studies reported by Wahren et al concluded that C-peptide has properties of an intracrine
peptide hormone." 26 In fact, a clinical study with 139 patients, conducted by Ekberg and
coworkers has shown that C-peptide treatment for 6 months improves sensory nerve
function in early-stage type 1 diabetic neuropathy.“ 26 During the 1990s, reports surfaced
indicating that C-peptide was able to ameliorate complications associated with diabetes in
rat models, including vascular and neural dysfunction, glucose utilization and renal
function.” 28 Other reports followed, demonstrating the ability of C-peptide to improve
blood flow in the skin muscles and microvasculaturezg‘ 30 Also, it was reported that C-
peptide was capable of improving the deformability of RBCS.3 I C-peptide has also been
reported to increase endothelial nitric oxide synthase (eNOS) activity.” 33

As increasing number of studies were directed towards the biological role of C-
peptide, more beneficial effects were observed. Beneficial effects observed with both
pancreas transplants and the combined replacement of insulin and C-peptide in treatment
of type 1 diabetes are such examples.24 The present stand on C-peptide has been
summarized by Wahren et al as a “C-peptide is a bioactive peptide.”26

Recently, our group has shown that RBCs treated with metal activated C-peptide
release increased levels of ATP, even in the absence of flow (mechanical deformation) or
hypoxia.“ 35 RBCs were able to release increased concentrations of ATP for a period of
about 24 h when incubated with C-peptide bound to Fe“, whereas binding of C-peptide
to Cr3+ results in sustained ATP release over a period of 3 to 5 days.34‘ 35 Further studies
indicated that the ATP release is related to an increase in glucose transport across the

RBC membrane.“ 35

81

C-peptide, when activated with metal ions such as Cr3+, Zn2+ and F e2+ stimulates
glucose uptake in to rabbit RBCS.:M’ 3'5 There are reports suggesting that there are
milimolar levels of Zn2+ in the islets of Langerhans where proinsulin C-peptide is
synthesized and secreted.3640 Therefore it is highly likely that C-peptide is associated
with Zn2+ upon secretion from the pancreas. Thus, C-peptide, when complexed to a
metal, has properties similar to insulin, facilitating glucose transport in to RBCs.3'4
However, insulin acts upon the glucose transporter GLUT 4 and RBCs do not have
GLUT 4 transporters. Therefore, metal-activated C-peptide may contribute to the proper
blood glucose maintenance through activation of GLUT 1, the major glucose transporter
on the RBC.34

Beyond the experimental level, no current studies were successfully extended to
the therapeutic level of C-peptide replacement in preventing and ameliorating diabetic
complications.l Also, despite recent findings there are debates about the biological
activity of the C-peptide. These arguments appear to be based on three major facts that C-
peptide lack relative to other bioactive peptides.

One such negative concern with C-peptide is that despite elevated concentrations
of circulating C-peptide, people with type 2 diabetes develops complications associated
with the disease. Recently, we reported that the ATP released from the RBCs of patients
with type 2 diabetes was approximately 50% less in comparison to the ATP released from
healthy controls.“ 42 Further investigations in to this issue indicated a decrease in G6PD
activity, a common trait in the diabetic RBC, leads to a less deformable cell eventually
decreasing ATP release, and this effect is indeed acute or immediate rather than

chronic.42 Interestingly, there were reports suggesting that ATP release was decreased in

82

the RBCs of patients with type 2 diabetes due to improper accumulation of cyclic AMP
levels in the RBCs of these patients.43 Therefore, under such circumstance C-peptide
activation of glucose transport and subsequent ATP release from the RBCs may be
impaired or less effective due to other complications within the diabetic red cells itself.34
Another negative concept of C-peptide is despite recent efforts, a receptor for C-
peptide has not been reported.44 C-peptide does not assume a stable, defined
conformation in solutions. '9‘ 45’ 46 This lack of a defined tertiary structure also facilitates
the arguments of a non-receptor based interaction.44 However, it is highly probable that a
receptor for C-peptide binding has yet to be discovered.46 A possible explanation to the
absence of tertiary structure is that coordination of Zn” to negatively charged amino acid

4‘ 6' '0 and/or C-terminal of the

side chains (4 glutamic acid sites and 1 aspartic acid site)
C-peptide molecule may result in the formation of a stable secondary structure that
facilitates intracellular signal transduction via conformation-dependent interactions
between the C-peptide—metal complex and a cell membrane or unidentified cell-surface
receptor.34

Due to lack of a receptor, C-peptide upon activation with Zn2+, binds to RBC
membrane to exert its biological insulin-like activity. Here it is hypothesized that RBCs
are subjected to oxidative stress for long periods (e.g. type 2 diabetes individuals)
changes occur at the cell membrane surface that affect C-peptide binding to the RBC
membrane. In order to further evaluate this hypothesis, it is necessary to understand the

fundamentals of the RBC membrane structure and effect of hyperglycemia on that

structure.

83

3.1.1 RBC membrane and effect of hyperglycemia on membrane structure

Like any other mammalian cell membrane, the RBC membrane is a selectively
permeable lipid bilayer, outlining the cell. The cell membrane is mainly composed of an
asymmetric bilayer of phospholipids and different types of associated proteins such as
integral proteins providing passage for various ions and hydrophilic compounds,
membrane bound enzymes involved in catalytic processes, various receptors both
extracellular and intracellular involving signal transduction, and finally they also provide
attachment points to spectrin forming the cytoskeleton.

Amphipathic phospholipids, which are the main constituent of cell bilayer,
spontaneously arrange so that the hydrophobic "tail" regions are shielded from the
surrounding polar fluid, causing the more hydrophilic "head" regions to associate with
the cytosolic and extracellular faces of the resulting bilayer. The arrangement of
hydrophilic heads and hydrophobic tails of the lipid bilayer act as a barrier preventing
diffusion of polar solutes such as amino acids, nucleic acids, carbohydrates, proteins, and
ions) across the membrane, but generally allow for the passive diffusion of hydrophobic
molecules.47

In bilayers, the lipids are organized non-randomly.47 In general, the outer leaflet
of the eukaryotic cell membrane and other internal organelles are enriched with
phosphatidylcholines where as in inner leaflet, flippases and scramblases concentrate
anionic lipids such as phosphatidyl serine (PS), phosphatidic acids and
phosphatidylethanolamine creating an extra barrier to passage of charged species

4
across.“ 8

84

Lipid asymmetry in the membrane originated from the vectoral biosynthesis in
which most eukaryotic glycerophospholipids are synthesized on the cytoplasmic side of
the endoplasmic reticulum (ER), whereas sphingolipids are either synthesized or modified
on the lumenal side of the ER and Golgi apperatus.47‘ 49 In oder to maintain bilayer
balance, some newly synthesized lipids are translocated to the side of the membrane
opposite to that of the site of synthesis.” 49 This translocation process is facilitated either
by slow movement of lipids from one layer to the other or by activation of flippases, a
family of transmembrane phospholipid transporter enzymes.” 49’ 50 In RBCs, both
binding of cytoskeleton to the anionic inner leaflet and PS flippase activity helps to
maintain PS in the inner membrane.50

Under normal conditions cells are capable of withstanding changes in membrane
polarity, but reports suggest that certain pathological conditions such as exposure to high
glucose levels results in the translocation of PS from the inner leaflet of the membrane to
the outer leaflet resulting in changes in cell membrane polarity.”52 Alterations in
membrane polarity has profound effects on cell function.” 52 In fact, if C-peptide exerts
its activity by interacting/intemalization with the cell membrane, then increases in the
negative charge on the outer layer of phospholipids will exert repulsive effects on C-

peptide. Therefore it is important to understand the C-peptide’s ability to bind to the RBC

membrane under hyperglycemic conditions as well as under high oxidative stress.
3.1.2 Human C-peptide Enzyme Linked Immuno Sorbent Assay (ELISA)

By 1969, a differential immunoassay with proinsulin and insulin antibodies was

developed allowing the detection of C-peptide in human serum and plasma.”’ 54 Enzyme

85

linked immuno sorbant assay (ELISA) is a commonly used test in biological experiments
that requires the measurement of the concentration of a substance in a biological sample
using the binding of an antibody, or antibodies, to its antigen.” 55 The immunoassay has
the advantage of highly specific binding of an antibody to its antigen.“ 55
Antibodies, also known as immunoglobulins (lg) are globulin proteins that are found in
the bloodstream of vertebrates, and are used by the immune system to identify and
neutralize foreign particles such as infectious bacteria and viruses.55 The typical structure
of an antibody consists of Y shaped structural unit made with two heavy chains and two
light chains (Figure 3.2 a). Though all the antibodies posses structural similarity of the Y
shape and four chains, there is a small region at the tip of the protein known as the
“hypervariable region” that is extremely variable, allowing for millions of possible
various antibodies.56 Each of these variants binds to a different and specific target antigen
allowing the immune system to recognize wide diversity of antigens.56

Antigens are substances that induced the generation of antibody and are usually
proteins or polysaccharides. The unique part of the antigen recognized by an antibody is
called an epitope and these epitopes bind with their antibody in a highly specific
interaction allowing antibodies to identify and bind only their unique antigen in the midst
of the different molecules that are present in the bloodstream.

In the sandwich ELISA used for the detection of C-peptide, a monoclonal C-
peptide antibody is affixed to a surface of a plate or ELISA well, and then a specific
antigen (e.g.: C-peptide) is added (Figure 3.2.b).54’ 55 Once the antigen is immobilized, a

biotinylated detection antibody is added and forms complex with antigen. Between each

step, the plate is typically washed with a mild detergent solution to remove any proteins

86

 

(b)

 
            
  

®U°

ago/D

9Q

 

0c. Ow.
{Q} Q}

 

QBiotin
streptavidin
link

Immobilized
antibody

Surface

Figure 3.2: (a) Schematic representation of an antibody (b) Schematic representation of
C-peptide ELISA where C-peptide is sandwiched between immobilized C-peptide-
antibody and another biotinylated C—pep-antibody. Then Streptavidin linked enzyme was
allowed to conjugate with detection antibody followed by addition of substrate.

87

or antibodies that are not specifically bound.54 After the washings, bioconjugation of
streptavidin linked horseradish peroxidase to the detection antibody is enabled. After the
final wash step, the plate is developed by adding an enzymatic substrate that is converted
in to some detectable signal (usually fluorescence or absorbance) by enzymatic reaction,
which indicates the quantity of antigen present in the sample.54 Use of a C-peptide
ELISA provides the accounts of C-peptide interaction with RBC membrane under
various oxidative stress conditions but provides no understanding of the it’s effect on

glucose transport in to cells.

3.1.3 Insulin like activity of C-peptide; I‘lC-labeled glucose transport in to the RBC
Glucose, being the primary energy substrate for anaerobic glycolysis, is an
essential component in metabolism. Being a polar molecule, free passage of glucose
across cell membranes is restricted and requires the assistance of a specific transporter
protein. In mammalian cells, glucose transport is facilitated by two major families of
structurally related glucose transporters.57 These are the GLUT and SGLT family of
transporters.57 The GLUT family, or the facilitative glucose transporter family, allows
energy-independent, bidirectional, glucose transport (facilitated diffusion) in most tissues
and cells.57 The Na+/ glucose cotransporter family, or the SGLT, mediates an active, Na+-
linked transport process against an electrochemical gradient.57 SGLT transporters are
mostly seen in intestine and kidney epithelial cells. The GLUT family is the more
important glucose transporter and consists of thirteen members divided into three major

classes based on their protein structure.57

88

GLUT 4 is the major insulin-dependant glucose transporter and is commonly
found in fat storage adipose tissues and skeletal and cardiac muscle cells.58 Moreover,
GLUT 4 is the most studied glucose transporter in diabetic pathogenesis.58 Even though
GLUT 4 is an insulin dependant glucose transporter, it is not the major glucose
transporter in the RBCs or blood brain barrier, a pivotal component in glucose
consumption in the human body.58

In fact, GLUT 1, the first and the most characterized glucose transporter,
equilibrates the glucose across the RBC membrane.58’ 59 In addition, GLUT l is
commonly found in fetal tissues and in the blood brain barrier.59 The structure of GLUT 1
consists of 12 membranespanning a-helical proteins, each with 20 amino acids.59 There
are reports suggesting that glucose transport rates in to the RBCs of diabetic individuals

are significantly less compared to that of healthy individuals. 60’ 6'

Also, there are reports
indicating that the decrease in glucose entry in the RBC membranes of diabetic patients
was due to a GLUTl change in structure — mostly the outer domain of the glucose
transporter.61 These findings indicate that reduction in non-insulin-mediated glucose
uptake plays an important pathogenic role in disorders of carbohydrate metabolism and
associated complications in both the macro-and microvasculature in diabetic patients.” 61

In fact, our group has previously shown using l4C labeled glucose, that in the
presence of metal activated C-peptide, glucose transport in to RBCs is up-regulated,
indicating that metal activated C-peptide has an insulin like effect on the glucose uptake

1.34 As indicated

by RBCS, possibly by activating the glucose transporter GLUT
previously, one problem with this finding is that the effect of C-peptide seems to

diminish in the case of type 2 diabetic individuals where there is a elevated C-peptide

89

levels during disease development.34 In an effort to further investigate the possible
method of C-peptide action and drawbacks in use of C-peptide as a diabetes treatment
option, we further evaluated the 14C labeled glucose transport under increased oxidative
stress, a common environmental effect in diabetes.

Finally, to support our hypothesis, we further evaluated C-peptide binding to RBC
membranes using C-peptide ELISA and changes in membrane PS content in the presence
of oxidative stress and hyperglycemia and its effect on C-peptide activity and binding. In
order to extent C-peptide therapy as a treatment option in diabetes, it is important to
understand the possible ways of overcoming the factors hindering the insulin like C-
peptide activity, allowing C-peptide to interact with the RBC membrane and exert its
effect. Therefore, we further investigated the effect of certain pharmaceutical drugs used
in treatment for diabetes, namely, metformin.

Metformin is the most popular oral anti-diabetic prescription drug in the United
States.62 Also, it is one of only two oral anti-diabetic drugs approved by World Health
Organization as a treatment for diabetes. Metformin belongs to the biguanide class of
anti-diabetic drugs and was discovered in 1957.62‘ 63 Though it’s potential for anti-
diabetic effect was known for almost 30 years, it was not used in diabetic treatment in the
United States until the late 19905. Currently, it is the only biguanide currently in use,
attributing to the reduced side effects on gastrointestinal complications compared to other
drugs of the same class .62

There are reports suggesting metformin acts as an insulin sensitizer.64'66 Also
there are reports suggesting metformin, when used in type 2 diabetes individuals with

. . . . . . . . 62, 67
cardiovascular complications, seems to allevrate their cardlovascular complrcatrons.

90

3.2 Experimental

Rabbit and Human RBCs were collected and prepared using the same techniques
applied in chapter two. Rat blood was obtained from S-month type 2 diabetic rats
(BB/ZDB, blood glucose 24.0 :t 1.9 mM) and matching controls (blood glucose 5.0 i 0.4
mM) according to the procedures approved by Animal Investigation Committee at
Wayne State University (Detroit, MI) and prepared according to the same procedure

described in chapter 2.

3.2.1 Reagents

Reagents were prepared in DDW or PSS unless otherwise noted. For 14C labeled glucose
transport studies, low glucose (0.55 mM glucose) PSS was prepared by adjusting the
amount of glucose added appropriately.

C-peptide stock (8.3><10'6 M) solution was prepared by dissolving 99.9 % purified human
C-peptide (0.25 mg) in 10.0 mL of DDW. This solution was stored in the 4 °C and used
over a period of two weeks. From the C-peptide Stock, 250 uL was diluted up to 25.00
mL to prepare 8.3><10'8 M C-peptide solution.

Cr3+ stock (8.0x10'5 M) solution was prepared by dissolving CrCl3 (0.0107 g) up to
500.00 mL in DDW. Then 31.9 mL of the Zn2+ stock was diluted up to 25.00 mL to make
1.02x10'7 M Zn2+ solution.

Zn2+ (8.0xlO'5 M) stock solution was prepared by dissolving ZnClz (0.0055 g) up to
500.00 mL in DDW. Then 31.9 mL of the Zn2+ stock was diluted up to 25.00 mL to make

1.02x10'7 M Zn2+ solution.

91

Metformin (0.0207 g) were dissolved in DDW (10.00 mL) to make 0.0125 M metformin

solution.

3.2.2 Instrumentation

All the fluorescence measurements were performed with a Horiba-Jobinyvon
F 1uromax-4 spectrofluorometer. All chemiluminescence-based ATP measurements were
conducted using a photomultiplier tube (PMT; Hamamatsu Corporation, Hamamatsu,
Japan) placed in a light excluding box. Absorption measurements for the C-peptide
immunoassay were conducted using a Molecular Devices Spectra Max M5c platereader.
l4C labeled glucose transport measurements were taken using a Wallac 1.450 microbeta

plus liquid scintillation counter.

3.2.3 Effect of G6PD inhibition on ATP release from C-peptide treated red blood
cells

5.00 mL of 14% hematocrit RBCs in 200 uM DHEA was prepared by diluting an
appropriate volume of RBCs in PSS containing DHEA. This mixture was incubated for
30 min. Meanwhile in a second microcentrifuge tube, C-peptide (600 pl) was mixed with
the Zn2+ (490 or.) or Cr3+ (490 pL) and the content was diluted up to 2.50 ml with PSS.
At the end of a 30 min. incubation period, 2.50 mL of 14 % RBCs in 200 mM DHEA
solution was transferred to the second centrifuge tube containing metal activated C-
peptide to make 5 .00 ml of 7% G6PD-inhibited RBCs in metal activated C-peptide. The
remaining 2.50 mL of the 14% RBCs were diluted up to 5.00 ml with PSS to make a

control of G6PD-inhibited RBCS. Control RBC samples and RBCs in metal activated C-

92

peptide were prepared in a similar manner. Next, the samples were allowed to incubate
for 6 hr. at room temperature. The resultant ATP release was measured using the

chemiluminescence assay as described above.” 67

At the end of the incubation period, 100 uL of RBCs were placed in a standard
disposable fluorimetric cuvette. Then, 100 uL of a luciferin] luciferase (L/L) solution was
added. The content of the cuvette was mixed and the chemiluminescence intensity was
measured 15 s after the UL addition. The resultant space current from the PMT, which is
proportional to the ATP-induced chemiluminescence, was recorded as a potential by a
data acquisition board controlled by a program written with labVIEW8 (National

Instruments, Austin, TX, USA).

3.2.4 Effect of G6PD inhibition on '4 C labeled glucose uptake in to RBCs
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. Additionally, 1.554 Bq l4C-labeled glucose
was added to the low glucose PSS. This created a ratio of 14C labeled glucoseznon-radio
labeled glucose of 1:10. RBCs were added, creating a 7% hematocrit solution and
allowed to incubate for a period of 4 hrs. The RBCs were then centrifuged at 500>< g for
10 min. The resultant cells were resuspended in low glucose PSS. After another
centrifugation (at 500><g), the RBCs were lysed with bleach and combined with
scintillation cocktail and the radioactivity was measured using a scintillation counter over

an integrations period of 1min. for each sample. G6PD activity was inhibited by

93

incubating RBCs with DHEA for 30 min. prior to the incubation with l4C labeled

glucose.

3.2.5 Effect of G6PD inhibition on C-peptide binding to red blood cell membrane

A 14 % solution of G6PD inhibited RBCs was prepared by incubating RBCs in
appropriate volume of DHEA diluted in PSS. This solution incubated at room
temperature for 30 min. 20 nM C-peptide and Zn2+ solutions were prepared by diluting
250.0 and 63.8 uL of C-peptide stock (8.3x10'6 M) and Zn2+ stock (80.40:5 M)
solutions, respectively, in DDW. C-peptide (600 uL) and Zn2+ (490 uL) were placed in a
14 mL microcentrifuge tube and diluted up to 2.50 mL with PSS. Next, 2.50 mL of 14 %
G6PD inhibited RBCs was added to this immediately to make a 7 % solution of G6PD
inhibited RBCs in 20 nM metal activated C-peptide and the content of the tube was
incubated for 4 hrs. A solution of 7 % RBCs in 20 nM metal activated C-peptide, 7 %
RBCs and 7 % G6PD inhibited RBCs were prepared in the same manner.

At the end of the 4 hr incubation period, the content of the vials were centrifuged
(>< 500 g) for 10 min. and the cells were resuspended in PSS. After another centrifugation
(at 500><g), the RBCs were lysed and the C-peptide ELISA was performed using a
Human C-peptide ELISA kit (LINCO research, St. Charles, MO). The resulting
absorbance was measured at 450 nm using the Molecular Devices Spectra Max M5e

platereader.

94

3.2.6 Effect of G6PD inhibition and elevated glucose levels on PS translocation on
RBC membrane

Experiments were performed using RBCs that had been incubated in a high
glucose concentration (15 mM) PSS, in addition to the normal PSS. The 7 % hematocrit
solutions of RBCs, G6PD inhibited (with DHEA) RBCs and RBCs in high glucose (15
mM) were prepared by diluting appropriate volumes of RBCs in appropriate PSS
containing the required inhibitors. These solutions were then allowed to incubate at room
temperature for 4 hrs. In order to evaluate the effect of metformin, each sample was
divided in to two equal portions and an appropriate volume of metformin was added to
one to make a RBCs suspension in 30 uM metformin while adding PSS to the set of
controls. After 2 hrs of incubation time, 2 uL of the 7 % RBCs were diluted in 498 uL of
IX binding buffer (100 mM HEPES/ NaOH, pH 7.5 containing 1.4 M NaCl and 25 mM
CaClz) provided with the Annexin V-FITC apoptosis detection kit (Sigma) to prepare a
cell suspension of approximately 1X 106 cells/ mL . To this, 5 uL of annexin labeled with
the fluorescence dye fluorescein isothiocyanate (FITC) was added. After a 10 min
incubation, the samples were centrifuged (X 500 g) for 5 min and the cells were rewashed
and resuspended prior to measuring the fluorescence emission using the Horiba-

Jobinyvon Fluromax-4 spectrofluorometer. (Excitation 487 nm and emission 515 nm)

3.3 Results and discussion
Despite the fact that it is one of the most studied pathological conditions, diabetes
still remains dangerously epidemic with no cure and well over 171 million people

suffering worldwide.68'7' As scientists and researchers struggle to understand and explore

95

the secrets behind the pathology and resultant associated complications, interest has
focused on a wide variety of factors other than insulin. C-peptide, despite its original
proclaim as a biologically inert compound, has been revived with a renewed interest
almost 40 years after its discovery in 1967.20 Despite the ever-increasing reports
suggesting the possible insulin like activity of C-peptide in ameliorating the
complications in type 1 diabetes, the issue still remains some what controversial.34

While groups of researchers strongly suggest that the C-peptide is bioactive other
groups argue otherwise.26 However during the last decade, an increasing number of
reports have appeared describing the beneficial effects of C-peptide in diabetic
complications especially, in type 1 diabetesn‘ 73 In fact, there are reports that when
administrated in replacement doses, C-peptide seems restore the diabetes induced

14. 20. 72 1

changes in both kidney and peripheral nerves system in type 1 animal models. n

addition, there are reports including clinical studies indicating C-peptides ability to
improve blood flow in skin, skeletal muscle tissues, myocardium and nerves tissues.20

Recently our group reported that C-peptide, in the presence of transition metal
ions such as Cr3+ and Fe”, could increase the amount of ATP released from RBCs and

4 5 -
3 ’3 In fact, we reported a 70 % increase

also increase the glucose uptake in to the RBCS.
in ATP release and a 31 % increase of glucose transport in comparison to control RBCs
and purified C-peptide (no metal added), after incubation of the RBCs for 6 h in a C-
peptide activated with Fe2+ or Cr3+. These findings clearly identify C-peptide is bioactive
and its activity depends upon metal binding.34

Even though C-peptide exerts beneficial effects on ameliorating type 1 diabetic

complications, similar effects were not observed with type 2 diabetes making annotations

96

of biological activity as somewhat doubtful.72 In this study, we attempt to address the
particular issue by investigating the effect of hyperglycemia induced oxidative stress and
resulted structural modifications and their effect on C-peptide activity.

Previously we have shown that in type 2 diabetes individuals, the glucose-6-
phosphate dehydrogenase (G6PD) enzyme was deficient and resulted in the immediate
decrease in RBC derived ATP release.“42 As the first step of understanding C-peptide’s
mode of action, as well as its inability to ameliorate complications in type 2 patients, we
inhibited the G6PD enzyme activity with DHEA and then these G6PD inhibited RBCs
were subjected to 6 hr incubation with Cr3+ activated C-peptide. As expected, upon
incubation with C-peptide, the ATP release from control RBCs increased by 58 %
(Figure 3.3). However, the ATP release from G6PD inhibited cells only increased by 27
% indicating that effect of C-peptide is diminished when the RBCs were subjected to
oxidative stress. This results were further confirmed by an observed 32 % increase of
ATP release from RBCs incubated with diamide a know oxidant 4' prior to the C-peptide
treatment.

Though our original studies with C-peptide were conducted with Cr3+, it is well
known that there are milimolar levels of Zn2+ in pancreatic islets where C- peptide is
synthesized and therefore it is logical to assume that Zn2+ has a higher chance to bind to
C-peptide compared to Cr“. Therefore Zn2+ was selected for further studies and 10 nM
C-peptide incubated in 10 nM Zn2+ was used unless otherwise noted. C-peptide
concentrations used were in accordance with the physiological limits.

Similar trends of ATP release were observed upon treating RBCs with C-peptide

activated using Zn”. As shown in figure 3.4. ATP release increased by 57 % in control

97

0.30

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

’2? 0.25 r r
(6 .
.E‘ 3 T
g 0.20 n
:9. .
a) .
8 0.15 -
0 .
0
8 . ..
.E . :5
E 0.10:
u -.
J: l
0 0.05 -
- 2
1 iiiiiiii P
0.00 . 1
RBC DHEA Diamide

Figure 3.3: Effect on ATP release from rabbit RBCs upon C-peptide activated with Cry".
The gray bars represent the metal activated C-peptide treated RBCs and black bars
represent the RBCs with out C-peptide. DHEA was used to inhibit the G6PD activity and
diamide is a known oxidant. n = 6 rabbits. * represent the values that are significantly
different from each other p < 0.001.

98

 

0.3 a

 

 

0.2 -

 

 

 

0.l -

Chemiluminescence Intensity (a.u.)

 

 

 

 

 

0.0

RBCs DHEA Diamide

Figure 3.4: Effect on ATP release from rabbit RBCs upon C-peptide activated with Zn”.
The gray bars represent the metal activated C-peptide treated RBCs and black bars
represent the RBCs with out C-peptide. DHEA was used to inhibit the G6PD activity and

diamide is a known oxidant. n = 9 rabbits. * represent the values that are significantly
different at p < 0.001.

99

RBCs in comparison to 30 % and 32% increase in DHEA and diamide treated RBCs
respectively further confirming oxidative stress indeed affects the C- peptide induced
ATP release . However, it is inconclusive whether the decrease in ATP release is a result
of a direct inhibition of C-peptide activity or false positive results occurred due to
reduced ATP release from RBCs subjected to the oxidative stress.4|

Previously, we reported C-peptide increased glucose uptake in to RBCS.34 To
provide further evidence that the exposure to high glucose concentration and resultant
oxidative stress inhibits metal-activated C-peptide facilitated glucose transport into the
RBC, l4C-labelled glucose was included in the PSS at a 1:10 ratio with unlabelled
glucose creating competition between the different glucose isotopes. As shown in Figure
3.5 the amount of glucose entering the RBC did not increase with comparison to the
control C-peptide treated sample. In fact when G6PD activity was inhibited glucose
transport increased only by less than 10 % compared to the 35 % increase in control RBC
sample treated with C-peptide. In fact, there is no significance difference (P < 0.001) of
ATP release in C-peptide treated G6PD inhibited RBCs from RBCs without C-peptide.

While this increase in glucose transport was not as high as the ATP released from
the RBCs, it should be noted that the ratio of radio-labeled glucose to unlabelled glucose
was not stoichiometrically equivalent. In addition, we have previously shown that RBCs
incubated with the metal ions in the absence of C-peptide did not result in any increase in
glucose transport across the RBCs membrane or RBC-derived ATP release.34 This result
indeed confirms C-peptide activity is hindered by increased oxidative stress. We propose
that C-peptide excerts its bioactivity via membrane mediated interaction followed by

activation of GLUT 1; therefore, it is important to understand the structural changes that

100

2.0

 

 

 

 

 

 

 

 

 

 

A 1.8 f

3' -.

5i, : l

.5 J l

E 1.4 -

q) '1

o. 1

fl -

5 1.2 j l

O . ~ iii-5.71:}:

O J 42‘...

.E 1.0 “j

g 3

2 0.8 1
0.6

 

 

 

 

 

   

Figure 3.5: Effect of G6PD inhibition on C-peptide mediated l4C labeled glucose uptake
in to the RBCS. The gray bars represent the metal activated C-peptide treated RBCs and
black bars represent the RBCs with out C-peptide. DHEA was used to inhibit the G6PD
activity and diamide is a known oxidant. n = 8 rabbits. * represent the values that ate
significantly different p < 0.001.

101

the RBC membrane undergoes under oxidative stress, and its effect on C-peptide’s
possible mode of action.

Conventional methods by which bioactive peptides exert their effect is via
binding to its specific receptor at the target tissue or organ.'4 Proinsulin C-peptide has
neither conserved amino acid sequences through the various species, nor any known
receptor, supporting the claims that it is biologically inert.l4

The first evidence suggesting a possible C-peptide interaction with cell
membranes was reported in 1986 using radioactive ”51 labeled rat C-peptide.74 Recently,
Wharen and co-workers have shown that C-peptide in nanomolar concentrations binds to
the cell surface, suggesting the possibility of a G-protein coupled membrane receptor.l4
Our studies also indicated the possible membrane mediated interaction followed by
activation of the GLUT 1 transporter.34
The ability of exogenously added C-peptide (activated with Zn”) to bind to RBC
membrane; under normal and oxidative stress C-peptide ELISA was performed. Our
group has evidence suggesting that metal activation is required for C-peptide to bind to
the RBC membrane. After incubation of normal and G6PD inhibited (by incubating with
DHEA) RBCs with 20 nM peptide for 4 hrs, cells were washed, centrifuged and lysed
and ELISA was performed. As shown in figure 3.6, the amount of C-peptide present in
the centrifuged RBCs decreased from 73 % in regular RBCs to 25 °/o in RBCs incubated
with DHEA, further suggesting the inability of proper C-peptide membrane interaction
under type 2 diabetic conditions.

In summary, all the studies we have conducted so far suggest that not only

does C-peptide bioactivity occur via activation of GLUT 1 by interacting with RBC

102

 

Absorbance (a.u.)

 

 

 

 

 

 

 

0.00
RBC DHEA

Figure 3.6: Effect of G6PD inhibition and resulted oxidative stress on C-peptide binding
to the RBC. The gray bars represent the metal activated C-peptide treated RBCs and
black bars represent the RBCs with out C-peptide. DHEA was used to inhibit the G6PD
activity and diamide is a known oxidant. n = 6 rabbits. * represent the values that ate
significantly different at p < 0.001. A picture of the ELISA assay (96-well plate) is shown
in upper left hand corner.

103

membrane but also changes in the RBC itself under hyperglycemia and oxidative stress
will cause a decrease in expected C-peptide bioactivity.

Under normal conditions, the RBC maintains asymmetry of membrane lipids by
transporting anionic phospholipids such as PS into the inner leaflet. There are reports
suggesting certain pathological conditions such as hyperglycemia promote the
translocation of PS in to the outer membrane, disturbing regular lipid asymmetry.75 Also,
it is known that as a cell ages the PS content of the outer leaflet increases.75 We believe
this change in phospholipid composition exerts repulsive effects on C-peptide bioactivity.

Annexin V is a cellular protein belonging to the annexin protein group found in
all the living organisms including fungi. Though the specific function of the protein is
unknown, Annexin V selectively binds to the anionic phosphatidyleserine. Therefore
Annexin V is used as a probe in the annexin A5 affinity assay to detect cells that have
expressed PS on the cell surface, as commonly found in apoptosis as well as other forms
of cell death. Here we investigated the PS content in the outer membrane of regular,
G6PD inhibited and RBCs incubated in high glucose using FITC labeled annexin V.
FITC is commonly used fluorescence dye with 494 nm excitation and 521 nm emission.
(Figure 3.7)

As shown in figure 3.8 both G6PD inhibition as well as incubation with high glucose for
5 hrs resulted in a marked increase in annexin binding to the cell membrane of human
RBCS. In fact incubation with 15 mM glucose for 5 hrs increased annexin binding to

membrane by 41 % whereas G6PD inhibition increased that value by 28 % (p < 0.001).

104

 

 

 

 

 

 

 

490 500 510 520 530 540
wave lenght nm

Figure 3.7: Annexin binding to the control, G6PD inhibited and RBCs incubated in 15
mM glucose. Row fluorescence intensity data of control RBCs and RBCs incubated in 15
mM glucose. Lower trace represents the control RBCs where as upper two traces
represent RBCs incubated in DHEA and 15 mM glucose.

105

2.0

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

;

*7 1.8 a
z, .
e 3 t
a; 1.6-j I
8 : 1"
.‘é . l -.-
,, 1.4 - ...
U
5 . 45.4
0 .,
2’3 1.2 -
‘5 l
:3 .
C .
E 1.0 ‘2
'5 :
E 0.8 -
O 4
Z 1

0.6 {

RBC DHEA 15mMGLU

Figure 3.8: Quantitative representation of annexin binding to the control, G6PD inhibited
and RBCs incubated in 15 mM glucose. Upon inhibition of G6PD (with DHEA) annexin
binding increase by 28.2 % and incubation with glucose increased the annexin binding by
41 %. n = 3 individuals. * represent the values that are significantly different p < 0.001.

106

As mentioned above metformin is one of only two oral anti-diabetic drugs
approved by the World Health Organization as a treatment for diabetes, and there are
reports suggesting metformin acts as an insulin sensitizer.64'66 Also, there are reports
suggesting that metformin, when used in type 2 diabetes individuals with cardiovascular
complications, seems to alleviate their cardiovascular complications.62’ 67

Here we evaluated the ability of the prescription drug metformin to enhance the
C-peptide effect on type 2 diabetic rat RBCS. As shown in figure 3.9 type 2 rats have 62
% higher annexin binding in comparison to the RBCs obtained from control rats. Upon
incubation with 30 uM metformin for 2 hrs the amount of annexin binding to RBC
membrane decreased by 28 %. In a separate study carried out in our laboratory, we
measured the ATP lease from these RBCs after incubating them with C-peptide
(activated with Zn2+) for 4 hrs and then incubated with 30 uM metformin for 2 hrs. The
increase in normalized ATP release from type 2 rat RBCs (31.2 %) was significantly less
than that of the control rats (78.4 %) with p < 0.01 Upon treatment with 30 uM
metformin ATP release from both control and type 2 rat RBCs increased by almost 100
% and the two values are not significantly different from each other. Similar trends were
observed with C-peptide binding to the RBCs as well as C-peptide stimulated glucose
transport in to the RBCs confirming the fact that metformin indeed enhances the effect of

C-peptide activity in type 2 diabetes RBCS.

107

 

 

2000
1:
g 1500 .
3 .
>\
.‘z.’ *
g A
8 4
5 1000 «
8 1
C
8 -4
«‘3
o .
:3
a: 500+
,
0

 

 

 

 

 

 

RBC+MET

Figure 3.9: Annexin binding to the control and type 2 rat RBCs in the presence and
absence of diabetic treatment drug metformon (30 uM). Black bars represent the control
rats and the gray bars represent the type 2 diabetic rats. Incubation with metformin seems
to mask the overall negative charge of PS facilitating C-peptide interaction with
membrane. 11 = 5 ratts . * represent the values that are significantly diffatemt p < 0.001.

108

3.4 Conclusions

Proinsulin C-peptide, when activated with transition metals such as Zn2+ increases
the RBC derived ATP release by 50 % above its original value. However, when RBCs
were subjected to hyperglycemia or oxidative stress, the C-peptide effect was decreased.
This increase in ATP release can lead to an increase in endothelial NO synthase activity
resulting in increased NO levels leading to enhanced vasodilation and improved blood
flow in to the target organs.

We propose that C-peptide upon activating with Zn2+ interacts with the RBC
membrane, leading to insulin like affects by up-regulating the glucose uptake through
GLUT 1. This effect was decreased when the RBCs were subjected to oxidative stress
prior to C-peptide treatment. In fact, when the RBCs were subjected to a type 2 diabetic
like environment, either by incubating with high glucose or by inhibiting G6PD activity,
the effect of C-peptide on the RBC derived ATP release, l4C labeled glucose transport
and C-peptide binding to the RBCs was decreased.

It is known that hyperglycemia induces the PS translocation from the inner leaflet
to the outer leaflet of cell membrane creating a net negative charge.“ 76 Similar effect
was observed in cells that are exposed to hyperglycemia or oxidative stress, decreasing
the interaction between proinsulin C-peptide and RBC membrane. However, when these
cells were incubated with prescription drug metformin, amount of the annexin V binding
to the membrane decreased indicating the ability of metformin to masks the charge
repulsion between C-peptide and RBC membrane.

Collectively we can state that C-peptide is indeed bioactive when interact with

metal ions such as Zn”, and the method by which it exerts its bioactivity seems to be via

109

some membrane based interaction and this effect seems to diminish when cells are
subjected to an oxidative stress. Use of prescription drug in combination with metal
activated C-peptide replacement may improve C-peptides ability to ameliorate

complications in diabetes therefore acting as a “sensitizer” for C-peptide.

110

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118

Chapter 4
EFFECT OF POLYOL PATHWAY AND G6PD ACTIVITY

IN DIABETES RBCs

Glucose, derived from the Greek word glykys, meaning "sweet", is a simple

monosaccharide that holds the central position of metabolism of living organisms
including plants, animals and microorganisms.1 This relatively energy rich
monosaccharide is the main product of photosynthesis and the starting material for
respiration in both eukaryotes and prokaryotes, thus serving as a life sustaining fuel
source." 2 Glucose contains the six carbon sugar (hexose) with an aldehyde group making
it an aldohexose (figure 4.1).I Glucose has two stereoisomers L and D, but only D-
glucose is biologically active." 2 D-glucose is often referred to as dextrose monohydrate
or simply as dextrose, especially in the food industry. The mirror-image of the D-glucose
molecule, L-glucose, cannot be metabolized by living organisms/cells in the biochemical
process known as glycolysis.“ 2

Under normal conditions, upon entering to the blood stream via absorption from
the digestive system, the fate of glucose will vary on the energy status of the
cells/organism.l There are three major possible pathways that the glucose can take.l A
portion of the absorbed glucose will be stored as high molecular weight polymers such as

glycogen in animals (or starch in plants) without altering the cytosolic osmolarity, for

rapid utilization at a later time. l‘ 2 Another portion of absorbed glucose will be transported

119

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in to every tissue and cell within organism to be oxidized to pyruvate via glycolysis,
which may undergo further oxidation in the citric acid cycle of aerobic respiration
depending on the location and organism, producing ATP energy required for cellular
functions.

Glucose is such an efficient fuel that it will generate more ATP per 0; molecule in
comparison to other fuel sources such as fat.1 Glucose is not only a fuel molecule, but
also is a remarkably versatile precursor that supplies array of metabolic intermediates for
various biochemical reactions. Therefore, a third portion of absorbed glucose will enter
the pentose phosphate pathway (PPP), to be converted in to other sugars and
intermediates essential for other important biosynthetic and/or metabolic pathways (e. g.,
synthesis of DNA and RNA and phospholipid synthesis).l

In healthy individuals, all circulating sugars with the exception of glucose are
controlled by the liver, whereas glucose concentrations are controlled mainly by insulin
and several other factors.3 In other words, glucose homeostasis in the human body is
primarily maintained through peripheral glucose uptake and utilization with the activation
and regulation of insulin, regulation of glycogen break down in the liver via glucagon,
regulation of hepatic glucose production and the PPP via glucose-6—phosphate
dehydrogenase. '

In diabetic individuals, this tight regulation of blood glucose levels is disrupted as
a result of impaired insulin action. In the pathogenesis of diabetes, either the pancreas
does not produce insulin or the cells are resistant to the insulin activity, thereby hindering
the uptake and storage of glucose by tissues that depend upon insulin activity for glucose

uptake such as muscle cells. This insulin resistance results in hyperglycemia or increased

121

blood glucose levels.4 The abnormally high blood glucose levels will lead to the

activation and up-regulation of another metabolic fate of glucose named polyol pathway.5

4.1 Channeling glucose via polyol pathway

Glucose is transported in to the cells via a facilitated diffusion by two major
families of structurally related glucose transporters known as the glucose transporter
(GLUT) and sodium-dependent glucose cotransporters (SGLT).6 GLUT4 is the insulin-
dependant glucose transporter, found in adipose tissues and striated muscle such as
skeletal and cardiac muscles.7 In diabetes, due to insulin deficiency or resistance, glucose
utilization by these is tissues diminished exerting severe glucose strain on non-insulin
dependant glucose users such as RBCs and neurons.4’ 7'9 As a result, these cells will
experience high glucose influx and disturb cellular homeostasis.

Upon entering the cells, glucose is phosphorylated by the enzyme hexokinase
(with the expense of ATP) in to glucose-6-phosphate (G6P), and depending on the energy
states of the cells, G6P will enter either glycolysis or PPP." ‘0 Glycolysis utilization is
saturated under hyperglycemic condition, thereby, exerting excessive glucose strain on
cell and activating polyol pathway. Disruption of the osmotic regulation of cells, resulting
from glucose accumulation, will also act as catalytic forces driving more and more
glucose to be metabolized via the polyol pathway.3‘ H

The polyol pathway, also known as the sorbitol pathway, consists of two
reactions, one reduction step and the other an oxidation step (Figure 4.2).4‘ '2 In the first
step, glucose is reduced in to the alcohol sorbitol (Figure 4.1.b.) by the NADPH-

dependant enzyme aldose reductase (AR).4’ H During the second step this sorbitol is

122

oxidized to fructose (Figure 4.1.c.) by the NAD+-dependant enzyme sorbitol
dehydrogenase (SDH), reducing NAD+ in to NADH. There are reports suggesting that
hyperglycemic conditions in cells, leads to the activation of both aldose reductase and
SDH.4‘ "

The first enzyme of the polyol pathway is aldose reductase (EC: 1.1.1.21;
alcoholzNADPH oxidoreductase) which belongs to the aldo-keto reductase enzyme
family.l3 Most of the enzymes belonging to this family are involved in detoxification
processes as they catalyze the reduction of a wide variety of substrates such as aliphatic
and aromatic aldehydes, monosaccharides, steroids and polycyclic aromatic
hydrocarbons.” Human AR is a monomeric protein of 315 amino acids with a molecular
mass of about 36 kD.'4

Human AR is a cytosolic enzyme widely distributed among the retina and the lens
of the eye, kidney and various reproductive organs.'4 In the presence of NADPH, AR
catalyses rate determining step of the polyol pathway reducing glucose to its
corresponding alcohol sorbitol while oxidizing NADPH to NADP+.4’ 1" '2’ ”'16 Under
normal physiological conditions, AR is capable of converting a minor fraction of glucose
(< 3 %) in to sorbitol before glucose is phosphorylated, but does not occur to an extent

that can be harmful to the tissue.4‘ M

This is due to the fact that glucose, at normal
physiological conditions, is not the preferred substrate for AR. The specificity constant
(kw, / K,,,), a measure of how efficiently an enzyme converts a substrate into product is

only 2.8 X 102 M'lmin", which is considerably low value in comparison to the normal

range of 1010 M454.l4

123

Sorbitol dehydrogenase (EC. 1.1.1.14) is the second enzyme of the polyol
pathway.‘7 It oxidizes sorbitol back to fructose, reducing NAD+ to NADH to complete
the pathway. Human SDH (hSDH) is a tetramer of four identical catalytically active

18‘ l9 and one

subunits with each subunit consisting of 356 amino acid residues (38 kD)
catalytic zinc atom.20 According to the X-ray crystal structure of hSDH (3.0 A
resolution), coenzyme NAD(H) binding reversibly to the each subunit and single zinc
atom housed at the large catalytic domain is essential for the hSDH activity.18

Once sorbitol is formed, SDH readily and reversibly catalyses the oxidation of the
C2 of sorbitol producing fructose (Figure 4.2) with the reduction of NAD+ to NADH.l8
In-fact SDH is capable catalyzing the reaction in the reverse direction too ( K equilibrium
sheep liver SDH = 3.7 nM).5 It is reported that high pH facilitates the forward reaction
whereas low pH facilitates the reverse reaction.5

As mentioned earlier, glucose is not the preferred substrate for AR, making the
significance of the polyol pathway limited under normal glycemic conditions.” But there
are reports suggesting some beneficial effects observed with the sorbitol pathway.2| One
such report is that sorbitol acts as a neutral organic osmolyte, regulating osmotic pressure
of the cells hence maintaining the whole body homeostasis and correct cell volumes.3

Also there are reports suggesting AR plays an important role in cellular

detoxification by removing reactive aldehyde species.”24

Though the antioxidant system
is capable of handling reactive oxidant species (ROS) in the case of excessive ROS
formation, it would cause increased lipid peroxidation followed by lipid aldehyde

formation.24 While glutathione (GSH) scavenges these aldehydes forming GSH-lipid

aldehydes molecules, AR readily oxidizes these aldehydes forming GSH-lipid alcohol

124

Glucose

N DPH
Aldose

reductase

NADP+

 

Sorbitol
t NAD+

Sorbitol
dehydrogenase

 

 

‘L NADH
Fructose

Figure 4.2: Schematic representation of the polyol pathway. Glucose is oxidized to its
alcohol sorbitol by AR, while reducing NADPH. Then SDH oxidizes C2 of sorbitol,
forming a ketose, fructose. Free NADH is generated in the process.

125

and actively eliminates then from cells.” 24

In addition, fructose produced by SDH acts
as energy source for sperm and sorbitol is involved in controlling absorption in renal
medulla by regulating the osmosis. '4

Under hyperglycemic conditions, the excess glucose is removed from the body
through increased urine production. This process will lead to dehydration in vivo and
cells are forced to maintain their volume by accumulating organic osmolytes,
predominantly sorbitol. In addition to the overloaded glycolysis and impaired PPP, this

process will also leads to the up-regulation of the polyol pathway under hyperglycemia.25

4.2. Up-regulation of sorbitol pathway leads to complications in diabetes

This up-regulation of polyol pathway has been linked to the onset of progressive
microvascular complications leading to the retinopathy, nephropathy and neuropathy,
observed in a portion of patients with diabetes especially type 1 diabetes.3 Diabetic
retinopathy is a vascular disorder affecting the microvasculature of retina and it was
estimated that 75 % of more diabetes patients eventually develop retinopathy.l3 Though
the mechanism by which up-regulation of polyol pathway increase hyperglycemia related
injuries is not well understood, some of these complications are attributed to the physical
accumulation of sorbitol and fructose within the cells, changes is osmosis and oxidative
stress resulted from sorbitol and NADH accumulation and NADPH depletion.3‘ 4‘ H‘ ‘3

Cells, that are living under normal physiological conditions maintains optimum
(N AD+/NADH):(N ADPVN ADPH) ratio. One negative effect of this up-regulated activity
of both AR and SDH enzymes is consumption of NADPH and production of NADH,

1011
l.”

impairing the regular redox balance of cel 26 In fact both accumulation of NADP+

126

and NADH in the cells will leads to a state call “Pseudohypoxia”, increasing the
oxidative potential of the cells, and therefore increasing the oxidative damage to such
important cellular components. '0’ H’ 26
Glutathione in its reduced state is a major form of antioxidant defense and
cofactor NADPH is required for regeneration of GSH from its oxidized dimmer
(GSSG).27'29 This reduction of NADPH levels will cause both weakening of the
antioxidant defense and increase in the oxidative stress,26 and this situation is far worse in
RBCs where glutathione is the most abundant nonenzymatic antioxidant and G6PD, the
only NADPH producer is also impaired due to increased blood glucose levels.25,28'30
These suggestion were further backed up by the extensive evidence provided in
the literature suggesting that the inhibition of AR leads to amelioration of diabetic
neuropathy ,retinopathy and nephropathy.4 Bravi et al. reported that upon inhibition of
AR with inhibitor tolrestat both sorbitol and GSH/GSSG were resoted to near normal
value in RBCs obtained from type 2 diabetes individuals.26 However these reports on
reduced NADPH levels due to up-regulated sorbitol pathway seem contradictory.I0
Unlike glucose, sorbitol does not bypass cell membrane therefore accumulating
inside the cells.ll In-fact there are repots suggesting elevated D-sorbitol levels in lens and
peripheral nervous tissues in patients with diabetes suggesting its contribution to diabetic
retinopathy and neuropathy.‘6
Nonenzymatic glycation is a post translational modification of protein structure
by binding of a sugar such as glucose or fructose with out enzyme involvement and the

extent of modification has been found increased in a variety of proteins such as the lens

crystalline, the red cell membrane proteins, the peripheral nerve proteins, serum albumin

127

in diabetic patients.3 I In most tissues where polyol pathway is active, the concentration of
fructose is similar to that of glucose. ln-fact in diabetes subjects, the level of fructose in
the lens of an eye may rise 23-fold becoming twice as concentrated as glucose.3 '

Fructose is known to be ten fold higher glycation agent compare to the glucose.I "
3 I This is attributed to the fact that Schiff base formation rate by fructose is about 8-fold
faster comparison to the that of glucose.3 I Therefore accumulation of fructose within the
cell can leads to glycation of important proteins causing sever damage in comparison to
elevated glucose levels hence disrupting regular functions of these proteins and is a major
contributor to the diabetic retinopathy.” 3‘

All these findings collectively suggest that it is important to be able to monitor

sorbitol levels in cells to assets the diabetic related damages especially in the cells in

which sorbitol pathway is active.

4.3. Monitoring the sorbitol level in RBCs

Since human RBCs are metabolically capable of g1ucose-sorbitol-fructose
conversion (polyol pathway), and RBCs are more prone to oxidative stress compared to
other cells RBC sorbitol levels can be use as an indicative of diabetic control.” 17 There
are reports suggesting that RBC sorbitol level reflect the sorbitol level in lens and sciatic
nerve, the longest and widest nerve that supplies nerves to most of lower half of the
human body.32 In addition to that sorbitol levels in RBCs may be useful too in assessing
the damages caused to the tissues that are less accessible in comparison to RBCs, in
clinical level especially in developing countries where numbers of patients are maximum

. 4
and resourced and technology rs scarce.” 3

In-fact many different analytical techniques are being used to detect the sorbitol
levels in RBCS. Among these methods are liquid chromatography and gas-liquid
chromatography with refractive index detector, enzymatic techniques coupled to
absorption or fluorescence detection schemeslé’ 32

As shown in figure 4.2, sorbitol dehydrogenase converts sorbitol in to fructose,
while reducing non fluorescence NAD+ to fluorescence NADH. Nicotinamide adenine
dinucleotide (NADVNADH), discovered by British biochemists Arthur Harden and
William Youndin in 1906 is a dinucleotide, coenzyme /cofactor found in most living
cells. NADH is mainly involved in catabolic reaction undergoing oxidation, but also
contributes as a substrate/co-factor for certain enzymes.35

Both NAD+ and NADH strongly absorb in the UV region of the spectrum, due to
presence of adenine base. (NAD+ an NADH absorb respectively at 295/339 nm).5' 36‘ 37
This difference in the ultraviolet absorption spectra makes them easily distinguishable in
a simple absorption measurement couple to an enzyme assay.36

Also NADH in solution has an emission peak at 460 nm and a fluorescence
lifetime of 0.4 us, while the oxidized form of the coenzyme does not fluoresce.37 These
changes in fluorescence are also used to measure changes in the redox state of living
cells, through fluorescence spectroscopy and microscopy.36 During this study increase in
NADH fluorescence was used to monitor RBC sorbitol levels.

Here we hypothesize that oxidative stress induced G6PD inhibition, coupled with
elevation in polyol pathway activity will increase the oxidative stress on diabetes RBCs

even further. There are reports suggesting that sorbitol activity increase with increasing

blood glucose levels.38 Also there are reports suggesting that G6PD activity is inhibited

129

under hyperglycemic conditions.25 But in RBCs G6PD is the only producer of NADPH.
Therefore investigation of the effect of G6PD inhibition on sorbitol pathway activity was

conducted and also the effect of metal activated C-peptide on sorbitol levels of regular

RBCs and G6PD inhibited RBCs were also investigated.39

4.4 Experimental
Rabbit and Human RBCs were collected and prepared using the same techniques applied

in chapter two.

4.4.1 Reagents
Reagents were prepared in DDW or PSS unless otherwise noted.

A Sorbitol stock solution 0.1 M Sorbitol was prepared by dissolving D-sorbitol (0.18217
g) in DDW up to 10.00 mL. Then 10.0 uL of this solution was diluted to make 10.00 mL
of 100 uM sorbitol solution.

A 150 Units/ mL sorbitol dehydrogenase enzyme solution was prepared by diluting the
content of the vial in an appropriate volume of cold DDW. Stock solution was divided in
to aliquots of 0.133 mL and frozen till the day of use.

A 0.03 M ZnSO4 solution was prepared by dissolving ZnSO4.7H 20 (0.2628 g) up to 5.00
mL with DDW.

Then a 10.00 mL solution of 0.475 M NaOH was prepared by dissolving NaOH pellets
(0.19 g) in DDW.

A 100.00 mL solution of 0.15 M TRIS-HCI buffer (pH=8.6) containing 4.5 mM

ethylenediaminetetraacetic acid (EDTA) was prepared by dissolving TRIS (1.882 g) and

130

EDTA (0.1675 g) in DDW and adjusting pH up to 8.6 with 2 M HCl and diluting up to
mark with DDW. Then NAD+ (0.0199 g) and 0.133 mL of 150 Units/ mL sorbitol
dehydrogenase enzyme was diluted up to 10.00 mL using the above mentioned TRIS-
HCl-EDTA buffer to prepare a sorbitol dehydrogenase reaction mixture (SDH ram).

Low glucose (55 uM) PSS was prepared by using the same procedure described in
chapter 2, by replacing 0.5 g of glucose with 0.005 g of glucose. Then 10.00 mL of each
5.5, 7.5, 8.5 and 10.5 mM glucose PSS solutions were prepared by dissolving

appropriated weight of glucose in PSS.

4.4.2 Instrumentation
All the fluorescence measurements were made using Horiba-jobinyvon Fluromax-

4 spectrofluorometer.

4.4.3 Detection of sorbitol levels in red blood cells upon G6PD enzyme inhibition

and C-peptide treatment

5.0 mL of 14% hematocrit RBCs in 200 uM DHEA was prepared by diluting
appropriate volume of RBCs in a PSS containing DHEA. This mixture was incubated for
30 min. Meanwhile in a second microcentrifuge tube C-peptide (600 uL) was mixed
with the Zn2+ (490 uL) and content was diluted up to 2.50 mL with PSS. At the end of 30
min. incubation period 2.50 mL of the 14 % RBCs in 200 uM DHEA solution was
transferred to the second centrifuge tube containing metal activated C-peptide to make a
5.00 mL of 7% G6PD inhibited RBCs in metal activated C-peptide. Remaining 2.50 mL

of the 14% RBCs were diluted up to 5.00 mL with PSS to make control G6PD inhibited

131

RBCS. Control RBC sample, RBCs in 1 nM Zn were prepared in a similar manner. Then
the samples were allowed to incubate for 6hr. at room temperature.

At the end of incubation period, RBCs were lysed by diluting up to 7.00 mL with
DDW then the proteins were precipitated by adding 1.00 mL of 0.475 M NaOH followed
by 1.00 mL of 0.3 M ZnSO4, Then the content of the tube was thoroughly mixed by
vortexing and was centrifuged for 10 min. at X2000 g. Then 1.00 mL of the resulted
supernatant was placed in a vial and then 500 uL of SDH remix was added to it and mixed
thoroughly and was incubated at 37 °C for 30 minutes. Then the resulted fluorescence for

NADH was measured (Ex 366 nm, Em 474 nm).

4.4.4 Effect of elevated glucose levels on polyol pathway

7 0/o RBC sample in PSS containing X mM glucose was prepared by diluting
appropriate volume of RBCs in corresponding PSS solution. (X= 5.5, 7.5, 8.5 and 10.5)
Contents were incubated at room temperature for 5 hrs. Then the cellular sorbitol levels

were measured using the same procedure described above.

4.5 Results and discussion

In order to monitor sorbitol levels in RBCs, method described by Shinohara at el
was occupied due to the convenience of the method in comparison to the available other
method such as chromatographic techniques.33‘ 34 In this method sorbitol accumulated
within the cell was allowed to react with sorbitol dehydrogenase enzyme in the presence
of exogenously introduced NDA+ ensuring the occurrence of the forward reaction.

(Figure 4.2)

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Fluorescence intensity changes in sorbitol standards 0-80 uM were conducted
daily prior to the measurements in for quantification and error elimination purposes. As
expected linear increase in fluorescence intensity ((r2 = 0.97) with increasing sorbitol
concentration was observed (Figure 4.3). However, any conclusive data on sorbitol levels
upon G6PD enzyme inhibition was not observed. In-fact these studies were conducted on
RBC samples obtained from 14 different rabbits, and it appears that sorbitol level
increased by 18.5 % upon G6PD inhibition in 6 samples and it decreased by 25.3 % in 8
samples (Figure 4.4). Despite the C-peptide’s ability to increase glucose uptake to the
RBCS39, similar trend was seen with the RBCs treated with metal activated C-peptide
treated RBCS. Here normalized sorbitol was increases by 47 % (0.58 :t 0.01) and was
decreased by 17 % (1.22 :1: 0.08).

In order to further clarify the method used here is indeed precise RBCs were
spiked with sorbitol standards and resulted fluorescence intensity was measured. Values
obtained from this method were slightly greater than the sorbitol standards alone,
nonetheless not significantly different (p<0.005). Successful measurement of sorbitol
levels in type 1 diabetes sample was performed in an attempt to further verify the method
used here indeed accurate and executed with out any experimental errors (Figure 4.5).

As the next step change in sorbitol level in RBCs incubated in PSS containing varying
levels of glucose was studied. Here RBCs were incubated with PSS containing 5.5, 7.5,
8.5 and 10.5 mM glucose (See figure 4.6) with the expectation of observing increase in
sorbitol levels with increasing glucose concentration. However, very interesting
observation was made upon analysis of the data acquired. As shown in figure 4.6

normalized fluorescence intensity corresponding to the NADH levels in the RBCs,

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hence normalizes RBC sorbitol level was decreased from 1.00 :t 0.0 to 0.87 i 0.03, when
glucose concentration was increased from 5.5 mM (regular blood glucose value) to 7.5
mM. Then when the concentration of glucose was further increased up to 10.5 mM
normalized fluorescence intensity increased up to 1.08 i005. These values were in fact
significantly different (p<0.005) from each other indicating that with increase in blood
glucose level, sorbitol levels show a slight decrease followed by increase larger than
original value.

This observed trend is similar to the change in glutathione levels in rabbit RBCs
upon oxidant insult from diamide, a known oxidant.” 30 Also similar trend was observed
with NO levels derived from vascular endothelial cells upon stimulation with ATP in a
different study conducted in our laboratory.

Previously more than one group has reported that upon incubation of RBCs in

high glucose, increase in sorbitol levels were observed.” 34

At a glance this results seems
to contradict the results observed during this study. But upon through investigation it was
clear most of these studies were conducted at glucose levels that are higher than 11.5 mM
up to 27.8 mM which are two to tree folds increase in comparison to blood glucose in
sever diabetise.33‘ 34 But our study was conducted in range of 5.5 mM to 10.5 mM. In—fact
during this study, sorbitol level in RBCs incubated in 17.5 mM glucose was measured by
us and increase in normalizes sorbitol levels were observed. There for it is possible that
these previous studies were conducted overlooking the possible decrease in sorbitol
levels upon slight increase in blood glucose. However further through investigation upon

this observation is required prior to apply these results in any attempts to evaluate

changes in sorbitol pathway activity in diabetes.

136

 

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Figure 4. 5: Sorbitol levels in type 1 diabetes and control RBCS. Upper trace represent the
Type 1 diabetes RBCs and lower trace represent the control RBCS. Two values are
significantly different (p < 0.005).

137

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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Figure 4.6: Normalized fluorescence intensity of RBCs incubated in PSS containing
increasing amounts of glucose. Normalized fluorescence intensity decrease from 1.00 i
0.0 to 0.87 i 0.03 when glucose concentration increase from 5.5 mM to 7.5 mM.
However fluorescence intensity increased up to 1.08 i005 when glucose concentration
was increased up to 10.5 mM. Date represent mean 1‘ SE. n= 4 rabbits and * represent the
values that are significantly different (p<0.005)

138

4.6 Conclusions

Effects of G6PD inhibition on cellular sorbitol levels in rabbit RBCs were some
what inconclusive. ln-fact upon G6PD inhibition sorbitol levels were increased in 43 %
of the samples and it was deceased in other 57 %. During this study sorbitol
dehydrogenase based NADH fluorescence method was occupied for the detection of
cellular sorbitol.” 34 Though this method is widely used technique, sorbitol standards and
sorbitol levels in type 1 human diabetes RBCs were measured in order to eliminate the
experimental errors associated and further verification of the method. Very conclusive,
expected increase in sorbitol levels were observed with both standards and human type 1
RBCS. Similar results were observed with RBCs incubated with metal activated C-
peptide. Therefore it is necessary to further investigate on this aspect.

Despite the common beliefe that hyperglycemia increase the cellular sorbitol
levels, interesting observation was made with the RBCs incubated with increasing
glucose concentration PSS. When glucose levels were changed from 5.5 mM to 7.5 mM,
decrease in sorbitol levels were observed. But when changing from 7.5 mM up to 17.5
mM increase in sorbitol level is increased. This observed trend coincide with the changes
in cellular glutathione levels upon introduction of diamide30 and also change in platelet

derived nitric oxide levels upon addition of ATP treatment.

139

 

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Harden, A.; Young, W. J., Alcoholic ferment of yeast juice. Proceedings of the
Royal Society of London 1906, 77, (B), 405-20.

Lakowicz, J. R.; Szmacinski, H.; Nowaczyk, K.; Johnson, M. L., Fluorescence
lifetime imaging of free and protein-bound NADH. Proc. Natl. Acad. Sci. U. S. A.
1992, 89, (4), 1271-5.

Dawson, R. M. C., Data for Biochemical Research. 3rd Ed. 1986; p 580 pp.

Greene, D. A.; Arezzo, J. C.; Brown, M. B., Effect of aldose reductase inhibition
on nerve conduction and morphometry in diabetic neuropathy. Zenarestat Study

Group. Neurology 1999, 53, (3), 580-91.

Meyer, J. A.; Froelich, J. M.; Reid, G. E.; Karunarathne, W. K. A.; Spence, D. M.,
Metal-activated C-peptide facilitates glucose clearance and the release of a nitric
oxide stimulus via the GLUTl transporter. Diabetologia 2008, 51, (1), 175-82.

143

Chapter Five

CONCLUSIONS AND FUTURE DIRECTIONS

5.1 Conclusions

Although the etiology of the disease is not well defined, diabetic mellitus can be
defined as a disordered metabolism followed by hyperglycemia resulting from either
insufficient secretion of endogenous insulin or resistance of the target tissues to the
insulin action, triggered by autoimmune response, viral infection or genetic cause."2
Based on the pathogenesis of the disease, diabetes is subdivided in to various types, with
type 1 and 2 being the most commonly occurring types. While exogenously administrated
insulin and other currently approved medications address some symptoms and
complications of the disease, numerous other complications affecting cardiovascular
system, nerves or kidney and lens and retina of an eye are yet to be understood.3‘4

In type 2 diabetes, RBCs are under high oxidative stress and are believed to be
less deformable, leading to lowered levels of deformation-induced ATP release.5'7 In an
attempt to understand simultaneous changes in G6PD activity and deformation induced
RBC derived ATP release, as NADPH measurement scheme was successfully developed
and extended to a continuous flow based system, which is an essential element in the
microcirculation.7 As expected, cellular NADPH levels decreased by 39 % upon
inhibition of G6PD signifying its importance in antioxidant defense.7

When subjected to oxidative stress, if unprotected, oxidation of important

membrane proteins will lead to a stiffening of the cell membrane.6‘8‘9 This effect is

144

pronounced in RBCs where G6PD is the sole producer of NADPH, an essential cofactor
in the antioxidant defense mechanism.9'l2 In type 2 diabetes, RBCs are under high
oxidative stress and are believed to be less deformable, leading to the lowered levels of
deformation-induced ATP releasef”7 Also G6PD activity is an indication of the cell age
and upon aging, cell membranes are believed to become more stiffened."l3

Here cellular NADPH levels in the RBC (via a fluorescence-based assay) and
deformation-derived ATP release from the RBC (via chemiluminescence assay) were
measured simultaneously, under continuous flow conditions.7 Results were consistent
with the expected pattern indicating a direct relationship between NADPH concentrations
in the RBC and deformation-derived ATP release from these cells.7 With a decrease in
NADPH concentration, there is a decrease in deformation-derived ATP release from the
RBC and vice versa (Figure 5.1).7

A similar trend was observed with chemically aged (via inhibition of G6PD)
RBCs, as well as young and old fractions of RBCs separated from whole blood.7 RBCs
obtained from people with type 2 diabetes showed the same trend confirming that
oxidative stress leads to a decrease in deformation-derived ATP releasef"7 Due to the
ability of ATP to stimulate NO production in endothelial cells and even platelets, the
RBC thus becomes a potential determinant of blood flow in the diabetic circulation.
Perhaps more evident from the work reported here, however, is the potential use of
NADPH and ATP as biomarkers of oxidative stress in the RBC.7

Since its discovery in 1967 proinsulin C-peptide was thought to be devoid of any

biological activity up until late 903’. '4 However, with the recent developments in

145

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146

research involving C-peptide, new light has been shed on its possible biological role.15
In-fact, proinsulin C-peptide, when activated with transition metals such as Zn2+,
increases the RBC derived ATP release by about 50 % over its original value. However,
when RBCs were subjected to hyperglycemia and resulted in oxidative stress, the C-
peptide effect was decreased. As mentioned above, an increase in ATP release can lead to
an increase in endothelial NO synthase activity and increased NO levels resulting in
vasodilation and improved blood flow in to the target organs.16 Unfortunately, our data
suggests C-peptide activity is decreased that the RBCs obtained from people with
diabetes.

We propose that upon interacting with Zn2+, C-peptide forms a secondary
structure that interacts with RBC membrane, leading to insulin like effect by up-
regulating the glucose uptake by GLUT 1 and this effect is decreased if RBCs are
subjected to oxidative stress prior to C-peptide treatment. In-fact, when RBCs were
subjected to chemically induced type 2 diabetic like environment (either by incubating
with high glucose, diamide or by inhibiting G6PD activity) the effect of C-peptide on
RBC derived ATP release, l4C labeled glucose transport and C-peptide binding to the
RBCs were decreased.

It is known that hyperglycemia induces the phosphatidyleserine (PS) translocation
from the inner leaflet to the outer leaflet of the cell membrane creating a net negative
charge in the cells that are exposed to hyperglycemia. This amount of PS present in the
cell membrane was detected using annexin V (tagged with FITC) binding to the PS
present in outer membrane.l7 It appears this overall charge alteration diffuses the

interaction between proinsulin C-peptide and RBC membrane. Upon incubation of these

147

cells with prescription metformin, the one of the two drugs approved by the world health
organization (glybacamdie is the other one) for diabetes, binding of annexin V to
membrane was reduced indicating its ability to masks the charge repulsion between C-
peptide and RBC membrane.

Therefore, collectively it can be stated that C-peptide is indeed bioactive when
interact with metal ions such as Zn”, and the method which it exert its bioactivity seems
to be via some membrane based interaction and this effect seems to diminish when cells
were under oxidative stress. Use of prescription drug metformin in combination with
metal activated C-peptide replacement may improve C-peptides ability to ameliorate
complications in diabetes therefore acting as a “snsitizer” for C-peptide.

However, results observed here with the metformin is rather indirect. Though
annexin binding to PS is a specific interaction and increase in ATP release was observed
with the combined C-peptide and annexin treatment, direct evidence for such interaction
between C-peptide, metformin and annexin is needed.

It is well know that the polyol pathway is up-regulated in diabetes individuals
leading to the accumulation of glucose-alcohol sorbitol in cells.18 These elevated sorbitol
levels were capable of triggering series of biological complications leading to
retinopathy, neuropathy and nephropathy in diabetes.18 However, for sorbitol pathway
activation, a continuous supply of NADPH is required. In the RBCs, G6PD is the sole
producer of NADPH and in the case of diabetes RBCs, G6PD activity is inhibited,
resulting in lowered NADPH levels. Here, the effects of down-regulated G6PD activity

on RBC sorbitol levels were evaluated.

148

Effects of the G6PD inhibition on cellular sorbitol levels in rabbit RBCs were
some what inconclusive. In-fact, upon G6PD inhibition sorbitol levels were increased in
43 % of the samples analyzed and it was deceased in other 57 %. During this study
sorbitol dehydrogenase based NADH fluorescence method was employed for the
detection of cellular sorbitol.19‘20 Though this method is a widely used technique, sorbitol
standards and sorbitol levels in human type 1 diabetes RBCs were measured in order to
eliminate the experimental errors associated and further verification of the method and
expected trend was observed with the both standards and the human type 1 RBCs.
However, ambiguity was observed with both G6PD inhibited RBCs and RBCs incubated
with metal activated C-peptide providing inconclusive results. However, since sorbitol is
a food additive, dietary intake can affect the overall assay results. Therefore it is
necessary to further investigate this aspect.20

Despite the common beliefe that hyperglycemia increases the cellular sorbitol
levels, an interesting observation was made with the RBCs incubated with increasing
glucose concentration PSS. When glucose levels were changed from 5.5 mM to 7.5 mM,
a decrease in sorbitol levels were observed. But when changing from 7.5 mM up to 17.5
mM increase in sorbitol level is increased. This observed trend coincide with the changes
in cellular glutathione levels upon introduction of diamide6 and also change in platelet
derived nitric oxide levels upon addition of ATP treatment.

Finally we can conclude this study by stating that hyperglycemia and oxidative
stress induced by hyperglycemia caused both structural and physiological changes in
RBCS. Thereby monitoring changes in various metabolites enables the predictions, early

detection and treatment in diabetes mellitus.

149

5.2 Future directions

Ultimate goal of the all the above studies conducted are to use the knowledge of

behavior of the various metabolites in the RBCs in prediction, diagnosis and treatment of
diabetes and other cardiovascular complications. In-fact, detecting multiple components
from a single RBC sample, with out the requirement of sample separation within a flow-
based system will enable improved in vitro determinations of drug efficacy and cellular
response to administered drugs.
The first step towards such measurement scheme was taken in above described work with
development of dual detection scheme. Therefore, incorporation of measurement
schemes of metabolites such as ATP, NADPH, NADH and also antioxidant GSH2| in to a
single measurement scheme on a PDMS (polydimethylsiloxane) microchip will be an
interesting future work. This incorporation of microfluidic chip will proved the ability to
better mimic the in vivo circulation, while monitoring in real time, changes in multiple
metabolites present within the RBC.

Multiple sclerosis (MS) is an autoimmune disease in which the immune system
attacks the central nervous system leading to the destruction of myelin sheaths
(demyelination). Myelin sheaths are cells that wrap around the axon of the neuron,
electrically insulating and protecting axon (Figure 5.2 and 5.3). There are reports
indicating that demyelination in MS was caused by increased NO levels. Also, there are
reports indicating that administration of steroids such as estrogens ameliorate the pain

episodes in MS patients.

150

 

2.0 a

 

 

1.5 .

 

 

 

0.5

l 1 1 n

Normalized Chemiluminescence Intensity
C
l

 

 

 

 

 

 

 

 

 

 

0.0

 

Figure 5. 2: Effect of estradiol on ATP release from RBCS. Upon incubation with
estradiol ATP release from RBCs decrease even in the presence of ATP stimuli C-peptide
(activated with Zn2+). Black bars represent RBCs with out C-peptide and gray bars
represent gray bars with C-peptide. n=5 rabbits and I represent values that are
significantly dirrerent (p < 0.001).

151

 

* p<0.05

15 -
10 1

Fluorescence intensity (a.u.)

 

 

 

control C-pep+Zn C-pep+Zn+estradiol

RBC 7%

RBC + C-pep /Zn

RBC + C-pep /Zn
+ estradiol

 

Figure 5. 3: Effect of estradiol on RBC derived ATP induced NO production from
bPAEcells. (a) Qualitative representation and qualitative representation (b) of NO formed
in bPAEC when stimulated with ATP released from RBCs incubated in estradiol in the
presence and absence of C-peptide using method and the device described by Genes et
al.22 NO production decreased when cells are treated with estradiol even in the presence
of C-peptide. n = 1 rabbit. Represent values significantly different (p < 0.05)

152

In a separate study conducted in our group it was discovered that RBCs of MS
patients shows increased levels of ATP release upon mechanical deformation. In a closer
infection, it was very clear that estrogens are structural analogs of DHEA. Therefore it is
possible that these estrogens exerts DHEA like effect on RBCs hence lowering the
amounts of ATP release which will leads to decrease levels on NO production
ameliorating the autoimmune destruction and pain relapses in MS.

In fact, preliminary studies on effect of estradiol (a component of estrogen) on
ATP release from RBCs were conducted. Here RBCs and RBCs treated with metal
activated (Zn2+) C-peptide was treated with estradiol. C-peptide was used as an ATP
stimulator and RBCs incubated in DHEA was used as a control experiment. Both ATP
release from RBCs7 and NO22 release from bovine pulmonary artery endothelial cells
stimulated with RBC derived ATP was measured (Figure 5.1). Interestingly, decrease in
both ATP release and NO was observed supporting our hypothesis. Therefore
investigation of effects of DHEA, G6PD activity and effect of estrogen on G6PD activity
and RBC derived ATP release will be an interesting future study that can be conducted

using the duel detection system developed here.

153

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