in vitro and in vivo studies on the antidiabetic and ... · 3.1.4 long-term effects of va extract...
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I
IN VITRO AND IN VIVO STUDIES ON THE ANTIDIABETIC AND
ANTILIPIDEMIC EFFECTS OF CHLOROGENIC ACID
ONG KHANG WEI
NATIONAL UNIVERSITY OF SINGAPORE
2013
II
IN VITRO AND IN VIVO STUDIES ON THE ANTIDIABETIC AND
ANTILIPIDEMIC EFFECTS OF CHLOROGENIC ACID
ONG KHANG WEI
[BSc. Biomedical Science (Hons.)]
A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF
PHILOSOPHY OF MEDICAL SCIENCE
DEPARTMENT OF PHARMACOLOGY
NATIONAL UNIVERSITY OF SINGAPORE
2013
III
DECLARATION
I hereby declare that this thesis is my original work and it has been written by me in its
entirety. I have duly acknowledged all the sources of information which have been used in the
thesis.
This thesis has also not been submitted for any degree in any university previously.
(ONG KHANG WEI)
07 Jan 2013
IV
ACKNOWLEDGEMENTS
I would like to express my sincere and greatest gratitude to Associate Professor
Benny Tan Kwong Huat. He has been a great and fantastic mentor who has always
guided me throughout my whole study. Without his guidance, I would not be able to
come out with this wonderful topic of study and complete the journey of research for
my PhD degree. I am greatly inspired by his dedication to academic and research
works. He has always been extraordinarily good in managing both academic and
research tasks which has in turn motivated me in equally handling my academic and
research assignments. As a supervisor, he shared his experiences and interesting
stories in his previous and current research lives. I would also like to take this
opportunity to thank him for his patience and words of encouragement when I was
once at the bottleneck of my study.
Next, I would like to thank Ms. Annie Hsu, our outstanding laboratory technician, for
her guidance and assistance throughout my study. As a mentor, her invaluable
experience in conducting experiments has tremendously facilitated the whole process
of my study. As a friend, she shared with me her life experience and gave me advices
when I was puzzled and stranded in predicament. Her positive attitude has helped me
sailed through every single unpleasant and undesirable moment.
I am greatly indebted to Associate Professor Huang DeJian and Ms. Song LiXia from
Department of Chemistry for their enormous assistance and support in aiding me to
identify and characterize the chemical composition of our herbal extract. Likewise, I
would like to express my very great appreciation to Mr. K.F. Leong and Mr. Chua
Keng Soon for their help in identifying the herb and specimen deposition in NUS
herbarium.
V
My grateful thanks are also extended to my fellow lab mates who make the life in the
laboratory more interesting and lively. I would like to offer my special thanks to one
of my lab mates, Ms. Chew Xin Yi for her assistance and guidance in performing the
immunoprecipitation experiments.
I also would like to express sincere appreciation to National University of Singapore
for supporting my full-time PhD research with scholarship.
Finally, I wish to thank my parents and family for their support and encouragement
throughout my study.
I
Contents
LIST OF PUBLICATIONS……………………………………………………………i
LIST OF ABBREVIATIONS………………………………………………………....ii
LIST OF FIGURES…………………………………………………………………...iv
LIST OF TABLES……………………………………………………………………vi
LIST OF APPENDICES………………………………………………………..........vii
SUMMARY…………………………………………………………………………viii
1 Chapter 1: Introduction ........................................................................................... 1
1.1 Diabetes Mellitus............................................................................................. 1
1.2 Classification of Diabetes Mellitus ................................................................. 2
1.3 Normal Glucose Homeostasis ......................................................................... 4
1.4 Insulin signaling vs AMPK-dependent pathway ............................................. 7
1.5 Pathogenesis of T2DM .................................................................................... 8
1.5.1 β-cell Dysfunction .................................................................................... 8
1.5.2 Insulin Resistance .................................................................................... 9
1.5.3 Fasting Hyperglycemia vs Postprandial Hyperglycemia ....................... 10
1.6 Management of T2DM .................................................................................. 11
1.7 Vernonia amygdalina and diabetes ............................................................... 12
1.8 Coffee and diabetes ....................................................................................... 18
1.9 CGA and diabetes.......................................................................................... 18
1.10 Objectives and Design of Study .................................................................... 23
1.10.1 Objectives of study ................................................................................ 23
1.10.2 Research design ..................................................................................... 24
2 Chapter 2: Materials and Methods ........................................................................ 26
II
2.1 Materials ........................................................................................................ 26
2.2 Studies of antidiabetic effects of VA in STZ-induced diabetic rats .............. 27
2.2.1 Plant materials ........................................................................................ 27
2.2.2 Preparation of plant extract .................................................................... 27
2.2.3 Experimental animals............................................................................. 28
2.2.4 Ethics statement ..................................................................................... 28
2.2.5 Induction of diabetes with STZ.............................................................. 28
2.2.6 Dose-response study in STZ-diabetic rats with VA .............................. 28
2.2.7 Chronic (28-day) study in STZ-diabetic rats ......................................... 29
2.2.8 Biochemical analyses ............................................................................. 29
2.2.9 Determination of G6Pase activity .......................................................... 30
2.2.10 Determination of muscle glycogen content ........................................... 30
2.2.11 Fractionation of rat skeletal muscle ....................................................... 30
2.2.12 Immunoblotting to detect GLUT 1 and GLUT 4 ................................... 31
2.2.13 HPLC analysis ....................................................................................... 31
2.2.14 LC-ESI-MS analysis .............................................................................. 32
2.3 Studies of antidiabetic and antilipidemic effects of CGA ............................. 32
2.3.1 Experimental animals............................................................................. 32
2.3.2 Ethic statement ....................................................................................... 33
2.3.3 Oral glucose tolerance test ..................................................................... 33
2.3.4 2-week CGA treatment in Leprdb/db
mice ............................................... 33
2.3.5 2DG transport in skeletal muscle isolated from Leprdb/db
mice .............. 34
2.3.6 Cell culture and differentiation of L6 skeletal muscle ........................... 34
2.3.7 Cell culture of HepG2 human hepatoma ............................................... 35
2.3.8 2DG transport in L6 skeletal muscle cells ............................................. 35
2.3.9 Myotube subcellular fractionation ......................................................... 36
III
2.3.10 siRNA transfection of myotubes and HepG2 ........................................ 36
2.3.11 Immunoprecipitation and detection of association between IRS-1 and
p85 subunit of PI3K .............................................................................................. 37
2.3.12 Glucose production assay ...................................................................... 38
2.3.13 AMPK activity assay ............................................................................. 38
2.3.14 ACC activity assay ................................................................................. 39
2.3.15 Fatty acid synthesis assay ...................................................................... 39
2.3.16 Fluo-4 direct calcium assay ................................................................... 40
2.3.17 Oil Red O staining ................................................................................. 40
2.3.18 Glucose and lipid profiles ...................................................................... 40
2.3.19 Hepatic G6Pase activity ......................................................................... 41
2.3.20 Fractionation of skeletal muscle ............................................................ 41
2.3.21 2DG transport in skeletal muscles ......................................................... 41
2.3.22 Liver histology or skeletal muscle immunohistochemistry ................... 41
2.3.23 Western blot analysis ............................................................................. 42
2.4 Statistical analysis ......................................................................................... 42
3 Results .................................................................................................................. 43
3.1 Studies of antidiabetic effects of VA in STZ-induced diabetic rats .............. 43
3.1.1 Acute effect of VA extract on fasting blood glucose in STZ-induced
diabetic rats ........................................................................................................... 43
3.1.2 Long-term effects of VA extract on body weight, food and water intakes
of STZ-induced diabetic rats ................................................................................ 44
3.1.3 Long-term effects of VA extract on fasting blood glucose, triglyceride
and total cholesterol levels ................................................................................... 45
3.1.4 Long-term effects of VA extract on pancreatic and serum insulin levels
................................................................................................................48
3.1.5 Long-term effects of VA extract on hepatic G6Pase activity ................ 48
3.1.6 Long-term effects of VA extract on hepatic GSH and antioxidant
enzymes ................................................................................................................48
IV
3.1.7 Long-term effects of VA extract on expression of GLUT 1/ GLUT 4 and
cellular distribution of GLUT 4 ............................................................................ 53
3.1.8 Long-term effects of VA extract on muscle glycogen synthesis ........... 57
3.1.9 Determination of main active constituents in VA extract ...................... 58
3.2 Studies of antidiabetic and antilipidemic effects of CGA ............................. 59
3.2.1 CGA lowers blood glucose levels in an OGTT on Leprdb/db
mice ......... 59
3.2.2 2-week treatment with CGA reduces body weight, water intake and
improves glucose and lipid profiles ...................................................................... 63
3.2.3 2-week treatment with CGA improves glucose tolerance and insulin
sensitivity in Leprdb/db
mice .................................................................................. 69
3.2.4 CGA inhibits gluconeogenesis in Leprdb/db
mice through downregulation
of gluconeogenic G6Pase ..................................................................................... 74
3.2.5 Suppression of glucose production and G6Pase expression in HepG2
hepatoma by CGA ................................................................................................ 78
3.2.6 CGA ameliorates hepatic lipid accumulation, triglyceride and total
cholesterol levels in Leprdb/db
mice ....................................................................... 78
3.2.7 CGA decreases oil droplets formation in HepG2 Cells ......................... 84
3.2.8 Amelioration of hepatic lipid accumulation by CGA is mediated through
inhibition of fatty acid synthesis ........................................................................... 84
3.2.9 Acute stimulation of glucose uptake by CGA in skeletal muscle isolated
from Leprdb/db
mice ............................................................................................... 87
3.2.10 Chronic treatment with CGA increases glucose uptake in skeletal
muscles by increasing GLUT 4 expression and translocation to plasma membrane
................................................................................................................88
3.2.11 Dose- and time-dependent stimulation of glucose transport by CGA in
L6 myotubes ......................................................................................................... 96
3.2.12 CGA stimulates GLUT 4 translocation to plasma membrane in L6
myotubes ............................................................................................................... 98
3.3 Studies of molecular pathways that mediate beneficial metabolic effects of
CGA .....................................................................................................................101
3.3.1 CGA increases AMPK and ACC phosphorylations in response to Ca2+
influx in HepG2 hepatoma cells ......................................................................... 101
V
3.3.2 Chronic treatment with CGA increases phosphorylations of AMPK and
ACC and expression of CAMKKβ in liver and skeletal muscles of Leprdb/db
mice
..............................................................................................................107
3.3.3 Inhibition and knockdown of AMPK abolished CGA-inhibited
gluconeogenesis and fatty acid synthesis in HepG2 cells .................................. 110
3.3.4 CGA stimulates phosphorylations of AMPK and ACC in L6 myotubes
..............................................................................................................110
3.3.5 Compound c diminishes glucose transport stimulated by CGA in L6
myotubes ............................................................................................................. 116
3.3.6 AMPK is necessary for the glucose transport stimulation by CGA in L6
myotubes ............................................................................................................. 119
3.3.7 CGA does not induce association of p85 subunit of PI3K to IRS-1 in L6
myotubes ............................................................................................................. 121
3.3.8 Effect of CGA on L6 myotubes viability and proliferation ................. 121
4 Discussion ........................................................................................................... 125
4.1 Studies on the antidiabetic effects of VA .................................................... 126
4.2 Studies on the antidiabetic effects of CGA ................................................. 130
4.3 Studies of antilipidemic effects of CGA ..................................................... 133
4.4 Studies of molecular targets that mediate beneficial metabolic changes by
CGA .....................................................................................................................134
4.5 Possible cytotoxic effect of CGA ................................................................ 137
4.6 VA vs CGA vs Met ..................................................................................... 138
5 Conclusions and Future Perspectives ................................................................. 140
6 References .......................................................................................................... 142
7 List of Appendices .............................................................................................. 168
i
LIST OF PUBLICATIONS
Journals
Ong KW, Hsu A, Tan BKH (2012) Chlorogenic acid stimulates glucose transport in
skeletal muscle via AMPK activation: A contributor to the beneficial effects of coffee
on diabetes. PLoS ONE 7.
Ong KW, Hsu A, Song L, Huang D, Tan BKH (2011) Polyphenols-rich Vernonia
amygdalina shows anti-diabetic effects in streptozotocin-induced diabetic rats.
Journal of Ethnopharmacology 133: 598-607.
Ong KW, Hsu A, Tan BKH (2013) Antidiabetic and antilipidemic effects of
chlorogenic acid are mediated by AMPK activation. Biochemical Pharmocology 85:
1341-1351.
Book Chapter
Tan BKH, Ong KW (2013) Influence of dietary polyphenols on carbohydrate
metabolism; Watson RR, Preedy VR, Zibadi S, editors. US: Elsevier.
ii
LIST OF ABBREVIATIONS
2DG 2-deoxyglucose
A1C Glycated hemoglobin
ACC Acetyl-CoA carboxylase
AICAR 5-amino-1-β-D-ribofuranosyl-imidazole-4-carboxamide
AMPK AMP-activated protein kinase
AS160 Akt substrate of 160 kDa
AUC Area under the curve
CAMKK Calcium/calmodulin-dependent protein kinase kinase
CAP Cbl-associated protein
CGA Chlorogenic acid
CGI Complete glucose intolerance
CQA Caffeoylquinic acid
DC Diabetic control
di-CQA Dicaffeoylquinic acid
DM Diabetes mellitus
DPP-4 Dipeptidyl peptidase-4
FBS Fasting blood sugar
FFA Free fatty acids
G6P Glucose-6-phosphate
G6Pase Glucose-6-phosphatase
GIP Gastric inhibitory polypeptide
GLP Glucagon-like peptide-1
GLUT 1 Glucose transporter 1
GLUT 4 Glucose transporter 4
GOT Glutamic oxaloacetic transaminase
GPT Glutamic pyruvic transaminase
iii
GPx Glutathione peroxidase
GSH Glutathione
HOMAIR Homeostatic model assessment index of insulin resistance
HRP Horse radish peroxidase
IAAs Insulin autoantibodies
IAPP Human islet amyloid polypeptide
ICAs Islet cell autoantibodies
IFG Impaired fasting glucose
IGT Impaired glucose tolerance
IKK IκB kinase
IRS Insulin receptor substrate
ITT Insulin tolerance test
KRBB Krebs-Ringer bicarbonate buffer
KRPH HEPES-buffered Krebs-Ringer phosphate
LKB-1 Liver kinase B1
NC Normal control
NF-κB Nuclear factor kappa-light-chain-enhancer of activated B cells
OGTT Oral glucose tolerance test
PEPCK Phosphoenolpyruvate carboxykinase
PI3K Phosphatidylinositol-3-kinase
PKC Protein kinase C
PM Plasma membrane
PPG Postprandial glucose
PTT Pyruvate tolerance test
SDS-PAGE Sodium dodecyl sulfate polyacrylamide gel electrophoresis
SOD Superoxide dismutase
STZ Streptozotocin
T1DM Type 1 diabetes mellitus
T2DM Type 2 diabetes mellitus
iv
TC Total cholesterol
TG Triglyceride
VA Vernonia amygdalina
v
LIST OF FIGURES
Figure 1.1 Fate of glucose………………………………………………………...4
Figure 1.2 Vernonia amygdalina………………………………………………...15
Figure 3.1.1 Acute effects of VA on glucose tolerance in STZ-induced diabetic
rats……………………………………………………………………44
Figure 3.1.2 Chronic effects of VA on fasting blood glucose of STZ-induced
diabetic rats…………………………………………………………..46
Figure 3.1.3 Chronic effects of VA on lipid profile of STZ-induced diabetic
rats……………………………………………………………………47
Figure 3.1.4 Chronic effects of VA on insulin levels of STZ-induced diabetic
rats……………………………………………………………………49
Figure 3.1.5 Chronic effects of VA on hepatic G6Pase levels of STZ-induced
diabetic rats………………………………………………………….50
Figure 3.16 Chronic effects of VA on hepatic antioxidant enzymes and GSH
activities of STZ-induced diabetic rats…….........................................51
Figure 3.1.7 Chronic effects of VA on skeletal muscle GLUT 4 expression and
translocation of STZ-induced diabetic rats……..................................54
Figure 3.1.8 Chronic effects VA on skeletal muscle glycogen levels in STZ-induced
diabetic rats……………………………..............................................57
Figure 3.1.9 Chemical profile of ethanolic VA extract…………………………...58
Figure 3.2.1 Acute effects of CGA on glucose tolerance in Leprdb/db
mice……….61
Figure 3.2.2 Decreased inhibitory effect of compound c in suppressing CGA-
mediated glucose lowering after 2-week treatment with CGA………62
Figure 3.2.3 Chronic effects of CGA on glucose and lipid profiles and insulin
sensitivity in Leprdb/db
mice…………………………………………..64
Figure 3.2.4 Chronic effects of CGA on glucose tolerance and insulin levels in
Leprdb/db
mice…………………………………………………………71
Figure 3.2.5 CGA decreases glucose production from gluconeogenic pyruvate in a
pyruvate tolerance test on Leprdb/db
mice…………………………….75
Figure 3.2.6 CGA inhibits expression and activity of hepatic G6Pase in Leprdb/db
mice………………………………………………………………….76
Figure 3.2.7 CGA suppresses glucose production and expression of G6Pase in
HepG2 hepatoma cells………………………………………….........79
vi
Figure 3.2.8 CGA ameliorates hepatic lipid accumulation in Leprdb/db
mice……...82
Figure 3.2.9 CGA lowers hepatic triglyceride and total cholesterol levels………..83
Figure 3.2.10 CGA decreases oil droplets formation in HepG2 cells………………85
Figure 3.2.11 CGA inhibits fatty acid synthesis in HepG2 cells……………………86
Figure 3.2.12 Acute stimulation of glucose uptake by in skeletal muscles isolated
from Leprdb/db
mice……………………………………………...........88
Figure 3.2.13 Chronic treatment with CGA increases glucose uptake in skeletal
muscles………………………………………………………….........90
Figure 3.2.14 Chronic CGA treatment increases GLUT 4 expression and
translocation to plasma membrane…………………………………...91
Figure 3.2.15 Dose- and time-dependent stimulation of glucose transport in L6
myotubes by CGA……………………………………………………97
Figure 3.2.16 CGA stimulates GLUT 4 translocation to plasma membrane in
myotubes…………………………………………………………….99
Figure 3.3.1 CGA increases AMPK and ACC phosphorylations in response to Ca2+
influx in HepG2 hepatocytes……………………………..................102
Figure 3.3.2 Chronic CGA administration phosphorylates AMPK and ACC in liver
and skeletal muscles of Leprdb/db
mice……………………………...108
Figure 3.3.3 Inhibition and knockdown of AMPK abolished CGA-inhibited
gluconeogenesis and fatty acid synthesis…………………………...111
Figure 3.3.4 Dose- and time- dependent phosphorylation of AMPK in L6 myotubes
by CGA……………………………………………………………..113
Figure 3.3.5 CGA increases AMPK activity in L6 myotubes……………………115
Figure 3.3.6 Effects of compound c on CGA-stimulated glucose transport in L6
myotubes……………………………………………………………117
Figure 3.3.7 Effects of gene silencing of AMPK on CGA-stimulated glucose
transport in L6 myotubes……………………………………………120
Figure 3.3.8 CGA phosphorylates Akt in the absence of PI3K in L6
myotubes……………………………………………………………122
Figure 3.3.9 Effect of CGA on cell viability and cell proliferation of L6
myotubes……………………………………………………………123
Figure 4.4 Cross-talk between insulin signalling & insulin-independent pathways
and schematic illustration of possible mechanism(s) of action of CGA
to cause beneficial metabolic outcomes……………………….........138
vii
LIST OF TABLES
Table 1.1 Summary of studies on antidiabetic effects of Vernonia
amygdalina…………………...………………………………………16
Table 1.2 Summary of studies on antidiabetic effects of CGA…………………21
Table 3.1.1 Effect s of 28-day treatment with VA on body weight, food intake and
water intake in STZ-induced diabetic rat…………………………….45
Table 3.1.2 Chemical profile of ethanolic extract of VA…………………………59
Table 3.2.1 Body weights, food and water intakes in Leprdb/db
mice following 2-
week treatment with CGA or metformin……………………………..69
viii
SUMMARY
Vernonia amygdalina (VA) is well-known for its medicinal importance and it is used
in Nigeria, Ghana, and South Africa for the treatment of diabetes. A dose-response
study was conducted to determine the optimum dose for the hypoglycemic effect of
VA in streptozotocin (STZ)-induced diabetic rats. The optimum dose (400 mg/kg)
was used throughout the 28-day chronic study. Body weight, food and water intakes
of the rats were monitored daily. Fasting blood serum, pancreas, liver and soleus
muscle were collected for biochemical analyses. Chemical composition of VA was
analysed using high-performance liquid chromatography (HPLC) and liquid
chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS). In an oral
glucose tolerance test, 400 mg/kg VA exhibited a significant improvement in glucose
tolerance of the STZ-induced diabetic rats. 28-day treatment with 400 mg/kg VA
resulted in decrease in fasting blood glucose compared to diabetic control. VA also
caused significant decrease in triglyceride and total cholesterol levels. Furthermore,
VA was found to increase expression of GLUT 4 in rat skeletal muscle. Further tissue
fractionation revealed that it can increase the GLUT 4 translocation to plasma
membrane as well, suggesting that VA may stimulate skeletal muscle’s glucose
uptake. This observation is in line with the restoration in skeletal muscle glycogenesis
of VA-treated group. In addition, VA also suppressed glucose-6-phosphatase
(G6Pase). Hence, VA possesses antihyperglycemic effect, most probably through
increasing GLUT 4 translocation and inhibiting hepatic G6Pase. 1,5-dicaffeoyl-quinic
acid, dicaffeoyl quinic acid, chlorogenic acid and luteolin-7-O-glucoside in the extract
may be the candidates that are responsible for the above-mentioned biological
activities.
ix
Besides VA, coffee also contains high levels of chlorogenic acid (CGA). Regular
consumption of coffee has been associated with a lower risk of Type 2 diabetes
mellitus (T2DM) but these beneficial effects cannot be explained by caffeine.
Moreover, CGA has been shown to delay intestinal glucose absorption and thus
suppressing postprandial glucose levels. On the other hand, improvement in fasting
glucose and insulin cannot be explained by the delay in intestinal glucose absorption.
Therefore, the present author next studied its effect on other metabolic pathways and
likewise its effects after long-term consumption. He investigated the effects of CGA
on glucose tolerance, insulin sensitivity, hepatic gluconeogenesis, lipid metabolism
and skeletal muscle glucose uptake in Leprdb/db
mice. Hepatoma HepG2 was used to
investigate CGA’s effect on hepatic glucose production and fatty acid synthesis while
L6 myotubes was used to further strengthen our findings in animal skeletal muscles.
Subsequently, he attempted to evaluate whether these effects of CGA are associated
with the activation of AMPK. In Leprdb/db
mice, acute treatment with CGA lowered
AUCglucose in an OGTT. Chronic administration of CGA inhibited hepatic G6Pase
expression and activity, attenuated hepatic steatosis, improved lipid profiles and
skeletal muscle glucose uptake, which in turn improved fasting glucose level, glucose
tolerance, insulin sensitivity and dyslipidemia in Leprdb/db
mice. CGA activated
CAMKK and AMPK, leading to subsequent beneficial metabolic outcomes, such as
suppression of hepatic glucose production, fatty acid synthesis and glucose uptake in
skeletal muscles. Inhibition and knockdown of AMPK and CAMKK abrogated these
metabolic alterations. In conclusion, CGA improved glucose and lipid metabolism,
via the CAMKK-dependent activation of AMPK. All these suggest that CGA could
be the main component that contributes to the beneficial effects of VA and coffee and
also the paradoxical effect of coffee in T2DM.
1
1 Chapter 1: Introduction
1.1 Diabetes Mellitus
Diabetes mellitus is a metabolic disorder characterized by hyperglycemia resulting
from defects in insulin secretion, tissues sensitivity to insulin or both. Hyperglycemia
is usually accompanied by symptoms which include polyuria, polydypsia, polyphagia
and at times blurred vision. Chronic hyperglycemia results in complications such as
retinopathy with potential loss of vision, nephropathy which leads to renal failure,
neuropathy and other cardiovascular disorders. There are three criteria to make a
diagnosis of diabetes, which are elevated fasting blood glucose, abnormal oral glucose
tolerance test and symptoms of diabetes with hyperglycemia (Appendix 1).
Previously, the use of glycated-hemoglobin (A1C) for the diagnosis of diabetes was
not recommended due the lack of uniformity in the assays worldwide [1]. However,
A1C assays are now highly standardized so their results now can be uniformly applied
both temporally and across populations [2]. The A1C value of ≥6.5% is used as a
diagnostic threshold. However, the diagnostic test should be performed using a
method that is certified by the National Glycohemoglobin Standardization Program
(NGSP) and standardized or traceable to the Diabetes Control and Complications
Trial reference assay.
In 2000, the estimated prevalence of diabetes among adults was 2.8% or 171 million
people and it is expected to increase to 4.4% or 366 million people by the year of
2030 [3] (Appendix 2). This growing burden of diabetes will lead to global financial
2
burden and also indirect cost to society, which is the health status of human
population.
1.2 Classification of Diabetes Mellitus
Diabetes can be categorized into two major categories known as Type 1 diabetes
mellitus (T1DM) and T2DM. T1DM is often genetically-associated and immune-
mediated. Individuals with T1DM have an absolute deficiency in insulin secretion and
can be identified by serological evidence of autoimmune-mediated destruction of
pancreatic islets or by genetic markers. However, this form of diabetes only accounts
for 5-10% of those with diabetes. Also known as juvenile-onset diabetes, the rate of
β-cells destruction in this form of diabetes is usually rapid in infants and children.
However, it can occur at any age, even as late as eighties or nineties in life. Markers
responsible for this destruction include islet cell autoantibodies (ICAs), insulin
autoantibodies (IAAs), glutamic acid decarboxylase autoantibodies (GAD65), and
autoantibodies to tyrosine phosphatase IA-2 and IA-2α [4-7]. One and more of these
autoantibodies are present in 85-90% of individuals when fasting hyperglycemia is
initially detected. There is another form of T1DM where the pathogenicity is less well
understood and hence known as idiopathic diabetes. Individuals in this category
usually have permanent insulinopenia but lack signs of autoimmunity. This form of
diabetes is strongly inherited. Hormone replacement therapy is not absolutely
necessary for survival in this case as the degree of β-cell dysfunction varies among
individuals [8].
The most common type of diabetes, T2DM, accounts for 90-95% of those with
diabetes. Individuals in this category can either have predominant insulin resistance
with relative insulin deficiency or predominant insulin secretory defect with insulin
3
resistance. The etiology of this form of diabetes is wide and complicated, ranging
from abnormalities in lipoprotein metabolism, central or visceral obesity, to
cardiovascular risk factors such as hypertension. However, pancreatic islets
destruction does not occur in T2DM. On the contrary, insulin resistance may cause
patient to have normal or even higher level of insulin. This form of diabetes is always
associated with obesity. It’s becoming more common in developed and developing
countries, afflicting younger generations victimized by a global epidemic of
overweight and obesity [9].
There is another type of diabetes diagnosed during pregnancy named gestational
diabetes. Most of the cases resolve with delivery, but the condition may persist in
some cases as unrecognized glucose intolerance may have begun before the
pregnancy. Evaluation of gestational diabetes should be done early in the pregnancy
except for those in low risk group, who
Are less than 25 years old
Have a normal BMI
Have no family history of diabetes
Have no history of abnormal glucose metabolism
Have no history of poor obstetric outcome
Are not members of an ethnic/racial group with a high prevalence of diabetes
such as Hispanic Americanw, Native Americans, African-Americans, and
Pacific Islanders
4
Study has shown that gestational diabetes was associated with poor maternal and fetal
outcomes [10].
1.3 Normal Glucose Homeostasis
Plasma glucose is maintained at a rather consistent value of approximately 90 mg/dl
(5 mmol/l), with a maximal increase of not exceeding 165 mg/dl (9.2 mmol/l) after a
meal [11] or a decrease down to not lower than 55 mg/dl (3.1 mmol/l) after exercise
[12] or a moderate 60-hour fast [13]. Glucose can be from dietary source or is either
from the gluconeogenesis in liver and kidney or the breakdown of glycogen
(glycogenolysis) in liver. This glucose may be stored directly as glycogen through the
process of glycogenesis in liver or may undergo glycolysis, which can be non-
oxidative, producing pyruvate or oxidative, through oxidization of acetyl CoA to
carbon dioxide and water in the tricarboxylic acid cycle or commonly known as Krebs
cycle (Figure 1.1).
Figure 1.1 Fate of Glucose
5
They are several key regulators that regulate glucose homeostasis:
I. Insulin
This major regulator affects glucose metabolism both directly and indirectly.
Its receptors are available in insulin-sensitive organs such as liver, kidney,
muscle and adipose tissue. Activation of insulin signaling upon binding of
insulin to insulin receptors causes suppression of gluconeogenesis in liver and
kidney [14], translocation of glucose transporter-4 (GLUT 4) from inner
membranes to plasma membrane in liver, muscles and adipose tissue to
increase glucose uptake [15], and inhibition of free fatty acid release into
circulation [16]. As free fatty acid stimulates gluconeogenesis and reduce
glucose transport into cells, release of insulin also indirectly regulates
gluconeogenesis and glucose transport through free fatty acids. Besides,
insulin promotes glycogen synthesis by inhibiting glucose-6-phosphatase
(G6Pase) and glycogen phosphorylase while stimulating glycogen synthase
[17]. Increased plasma glucose results in increase in plasma insulin while
decrease in plasma glucose causes reduction in plasma insulin level as well.
II. Glucagon
Unlike insulin secreted from pancreatic β cells, glucagon is secreted from α-
cells of the pancreas. Glucagon secretion is stimulated by hypoglycemia
whereas hyperglycemia will inhibit its secretion. Glucagon acts exclusively on
liver by activating glycogen phosphorylase and results in immediate glucose
release [18]. Further action of glucagon will be through stimulation of
gluconeogenesis [19].
6
III. Catecholamines
Catecholamines are molecules that act as both hormone, in blood circulation
and neuromodulator, in central nervous system. During stress and
hypoglycemia catechoamines are released and they inhibit insulin secretion
and action. In the liver, through β2-adrenergic receptors, they activate
glycogen phosphorylase and augment gluconegenesis [20]. In the kidney, they
are potent stimulators of gluconegenesis. In skeletal muscle, they reduce
glucose uptake and stimulate glycogenolysis. They also activate lipase and
result in lypolysis in adipose tissue to increase release of free fatty acid [21].
IV. Growth Hormone and Cortisol
Both metabolic actions of growth hormone and cortisol are antagonistic to
those of insulin. These include increase secretion of gluconeogenic enzymes,
reduce glucose transport and inhibit lipolysis [22, 23]. In addition, cortisol also
impairs insulin secretion and therefore further debilitating insulin signaling.
V. Free Fatty Acids
As mentioned before, increased plasma free fatty acids will result in
stimulation of renal and hepatic gluconeogenesis, inhibition of glucose
transport in muscles and adipose tissue and competition with glucose as
metabolic fuel [24].
VI. Incretins
Incretins are hormones secreted by intestine in response to nutrients ingestion.
Their main effect is to stimulate pancreas to release insulin after meals intake.
7
Two incretin hormones were identified so far: gastric inhibitory polypeptide
(GIP) and glucagon-like peptide-1 (GLP1). Both of them have short half-life
due to rapid digestion by proteolytic enzyme known dipeptidyl peptidase-4
(DPP-4).
1.4 Insulin signaling vs AMPK-dependent pathway
The insulin signaling and AMP-activated protein kinase (AMPK)-dependent
pathways regulate glucose and fatty acid metabolism, cellular growth, differentiation
and survival in various eukaryotic tissues [25, 26]. Activation of insulin signaling is
an anabolic process while AMPK activates catabolic pathways to conserve energy.
Signal transduction from the stimulus to the regulation of various cellular processes
usually involves protein kinase signaling. Insulin signaling is initiated upon the
binding of the hormone to its receptor which triggers the conformational changes and
autophosphorylation of the tyrosine residues. Activated insulin receptor attracts
insulin receptor substrates and tyrosine phosphorylates them. Once activated, insulin
receptor substrates recruit downstream molecules such as phosphatidylinositol 3-
kinase (PI3K), Cbl-associated protein (CAP) and protein kinase C (PKC), which are
the major conduits for GLUT 4 recruitment and glucose regulation.
AMPK, a sensor of intracellular energy, is activated at low cellular energy levels and
regulates cellular processes accordingly. Physiological or pathophysiological stimuli
such as hypoxia, muscle contraction, oxidative stress and glucose deprivation cause
an increase in AMP/ATP ratio, which is crucial for AMPK activation [27]. The
activity of AMPK is also modulated by hormones and cytokines that affect whole-
body energy balance, and by insulin sensitizers like thiazolidinediones [28]. Also, the
effect of the widely used antidiabetic drug metformin have been shown to depend
8
largely on AMPK activation [29]. Other kinases such as liver kinase B1(LKB-1) and
calcium/calmodulin-dependent protein kinase kinase β (CAMKKβ) have been shown
to be the upstream kinases that phosphorylate AMPK. AMPK regulates glucose
homeostasis by increasing glucose uptake in peripheral tissues and glycolysis
independently of insulin [30]. In addition, AMPK regulates hepatic glucose output by
inhibiting expression and activity of hepatic gluconeogenic enzyme, G6Pase [31].
AMPK also enhances fatty acid transport and oxidation, while switching off fatty acid,
cholesterol and glycogen synthesis and therefore resulting in its insulin sensitizing
properties [32].
1.5 Pathogenesis of T2DM
Although T2DM makes up most cases of diabetes mellitus, its pathogenesis remains
unclear, most probably due to its heterogeneity. Two main factors account for the
development of T2DM, which are the genetic factors and the environmental
influences. Studies have shown that most patients have a positive family history and
the risk for developing T2DM is increased up to 40% by having a first-degree relative
with the disease [33]. Environmental factors such as physical inactivity, obesity and
dietary habits may interact with genetic factors and increase the risk of developing
diabetes.
1.5.1 β-cell Dysfunction
Compared to normoglycemic subjects, impaired glucose tolerance (IGT) subjects
secrete less insulin at any given glucose level [34]. Islet β-cells function declines
progressively from IGT to complete glucose intolerance (CGI) which appears to be
9
the reason why patients who are initially well controlled by a single oral
hypoglycemic agent require increasing dose or combined agents to maintain glycemic
indices [35]. There are several possible causes that result in β-cells dysfunction.
Glucotoxicity and lipotoxicity are conditions where islet β-cells are exposed to high
glucose or free fatty acids levels chronically. Long-term exposure to high glucose and
free fatty acid levels impair insulin secretion from β-cells [36, 37]. Human islet
amyloid polypeptide, IAPP or amylin, is normally co-localized within the same
secretory vesicles as insulin [38] and co-released with insulin in response to glucose
or non-glucose secretagogues [39]. Deceased IAPP release but increased islet amyloid
deposit have always been found in T2DM [40] and amyloid deposition has been
proposed to decrease β-cell mass [41].
1.5.2 Insulin Resistance
Instead of impaired insulin secretion, in some T2DM patients, hyperinsulinemia
coexists with hyperglycemia, which is commonly associated with obesity and insulin
resistance [42]. By using hyperinsulinemic-euglycemic clamp technique, obese and
diabetic patients have been correlated to decreased responsiveness or diminished
sensitivity in insulin-stimulated whole-body glucose disposal [43]. Reduced numbers
of insulin receptors, impairments in insulin receptor substrate and PI3K are primarily
related to insulin resistance. Besides genetic factor, acquired insulin resistance gains
much attention for the prevention and progression of the disease. Owing to
dyslipidemia in obese patients, elevated free fatty acid levels are associated with non-
alcoholic hepatic steatosis [44], insulin resistance [45], decrease in skeletal muscle
glucose disposal [46] and increased hepatic glucose production [47]. Apart from
decreasing β-cell function, chronic physiological increment in the plasma glucose
10
concentration also leads to progressive insulin resistance in T2DM [48]. However, to
date, it is still unclear with regard to the relative contributions of pancreatic β-cell
dysfunction and insulin resistance to the pathogenesis of T2DM.
1.5.3 Fasting Hyperglycemia vs Postprandial Hyperglycemia
In post-absorptive state, majority (65-70%) of glucose uptake occurs in insulin-
insensitive tissues such as brain, erythrocytes and splanchnic tissues and glucose
uptake is precisely matched by the rate of hepatic glucose production [49]. Therefore,
gluconeogenesis is the main source of fasting glucose elevation [50] and is regulated
primarily by insulin and glucagon [51]. In the condition of insulin resistance or
impaired insulin secretion, glucose uptake cannot increase appropriately in response
to hepatic glucose output. As a result, small increases in glucose production cause
proportional increase in fasting glucose level.
Following glucose ingestion, increase in plasma glucose stimulates insulin secretion,
which in turn suppresses hepatic glucose production and stimulates glucose uptake by
peripheral tissues (50-60% of glucose uptake is by skeletal muscles) to restore
normoglycemia [52]. Several factors contribute to postprandial hyperglycemia in
T2DM. First, the total amount of glucose entering the systemic circulation is
increased due to the insufficient suppression of hepatic glucose output during fasting
or post-absorptive state, as discussed above [53]. Second, the efficiency of glucose
disposal is reduced because of insulin resistance in peripherals, especially skeletal
muscles, and relative or absolute insulin deficiency [54]. Third, hepatic glucose
uptake is impaired, although this defect makes a relatively small contribution since
the liver normally takes up only 20-35% of a glucose load [55].
11
1.6 Management of T2DM
Management of diabetes and maintaining near-normal plasma glucose levels are of
utmost importance in order to prevent the development of diabetic complications such
as nephropathy, neuropathy, retinopathy, dyslipidemia and cardiovascular diseases,
which are comparatively more lethal. A number of therapeutic choices are available
for the management of the disease; however, none are free of disadvantages.
Pharmacological intervention remains the most effective way to control plasma
glucose level but it is always associated with unwanted side effects. Sulfonylureas
initiate insulin release even when glucose level is low [56] and therefore are more
likely to cause hypoglycemia [57]. Besides, as sulfonylureas stimulate insulin
secretion, their effective use requires significant residual β-cell function [58]. Efficacy
is better in patients shortly after diagnosis of T2DM when most β-cell function is still
preserved [59]. Thiazolidinediones often cause weight gain which will further
deteriorate insulin resistance [60] and increase cardiovascular mortality risk e.g.
pioglitazone [61, 62]. The use of biguanides, such as metformin, is always associated
with acidosis and severe gastrointestinal upset [63]. Over the past 30 to 40 years,
studies using approaches ranging from epidemiological to interventional and
molecular technologies have proven that regular exercise is effective in preventing
and delaying metabolic diseases and its complications [64]. Unfortunately, sustained
benefits are difficult to achieve due to incapability of human nature to adhere to
exercise regimen. Also, evolutionarily humans have been driven to minimize energy
expenditure and remain sedentary. As a result, dietary approach remains a crucial tool
to achieve the goal of cost-effective management with minimal complications but
maximal quality of life. Before the introduction of the therapeutic use of insulin, diet
12
is the main form of treatment of the disease, and dietary measures included the use of
traditional medicines which are mainly derived from plants [65]. Even now,
approximately 80% of the third-world population is still dependent on traditional
medicines. Metformin, the most prescribed and first-choice agent in T2DM
pharmacotherapy, was derived from Galega officinalis (also known as Goat’s rue or
French lilac), a herb known for relieving symptoms of diabetes since the Middle Ages
[66].
In addition, in recent years, a wealth of evidence has been obtained, correlating lower
consumption of carbohydrate, saturated fat, processed food and higher consumption
of fruits, vegetables, legumes, coffee, and tea with lower risk of diabetes and
improved glucose and lipid metabolism. It is evident that plant-based foods are rich in
phytochemicals known as polyphenols which include flavonoids, phenolic acids,
lignans and stilbenes, which have been shown to improve glucose homeostasis at
several organ sites, including the (1) gastrointestinal tract, which regulates
carbohydrate digestion and glucose absorption, (2) endocrine pancreatic system,
which secretes key regulatory hormones, insulin and glucagon, in response to
abnormal glucose levels, (3) liver, where glucose synthesis, glycogen storage and
breakdown are initiated, (4) insulin-sensitive peripheral tissues like skeletal muscle
and adipose tissue, where glucose is metabolized for energy or stored for future use.
1.7 Vernonia amygdalina and diabetes
Vernonia amygdalina (VA), belonging to the Asteraceae family, is a small shrub that
is native to South Africa (Figure 1.2). Its medicinal value was first reported in wild
chimpanzees who suffered from parasite-related disease. They were observed to
ingest and swallow only the highly bitter juice, spitting out the fibrous remains for
13
self-deparasitization [67, 68]. Since then, investigations were conducted and have
shown that this plant possesses antimalaria, antihelmintic and antimicobial properties
[69-72]. Its antioxidant and hepatoprotective activities have also been investigated
[73-75]. Recently, several reports on anticancer study especially against breast cancer,
indicate its antineoplastic property [76, 77]. VA is usually known as bitter leaf due to
its bitter taste. Bitter principles from plants have been associated with the
improvement in the symptoms of DM [78]. For instance, bitter materials present in
the decoction from the root of Coccinia indica and from the fruit of Momordica
charantia were found to be potent to different degrees in some experimental models
of diabetes [79]. The VA leaves are consumed locally either as vegetables (macerated
in soup) or aqueous extracts as a tonic for the treatment of various diseases [80]. It is
well-known for its medicinal importance and is prominently used in Nigeria, Ghana,
and South Africa for the treatment of diabetes [81, 82].
Akah et. al (1992) showed that acute treatment with aqueous leaf extract of VA
caused significant reductions of blood glucose levels in both normoglycemic and
alloxan-diabetic rabbits, comparable to the effect of tolbutamide [83]. The same effect
was repeated in another study on alloxan-induced diabetic Sprague-Dawley rats.
Aqueous VA extract (500mg/kg) produced significant reduction in fasting blood
glucose concentrations of normoglycemic and diabetic rats 1 to 12 hours after acute
treatment compared to vehicle-treated controls [84]. However, no data on serum or
pancreatic insulin levels or other parameters was reported in the studies on the acute
effects of VA. A 28-day study by Nwanjo (2005) [85] of streptozotocin (STZ)-
induced diabetic Wistar rats showed that aqueous VA extract (200mg/kg/twice a day)
significantly reduced fasting blood glucose levels by more than 50% compared to
14
diabetic controls[85]. Besides, the study provided some data on the hypolipidemic and
antioxidant activity of VA. Eteng et al. (2008) showed that 400mg/kg of daily
ethanolic VA extract was able to produce significant hypoglycemic activity in normal
and alloxan-induced diabetic Wistar albino rats in 21 days, compared to the vehicle-
treated controls [86]. In addition, the improvement in lipid profile is consistent with
Nwanjo’s findings [85]. Very recently, another chronic study has demonstrated that
VA significantly reduced fasting glucose levels in both normal and STZ-induced rats
over a period of 28 days. Histological assessment revealed that protective effect
against the oxidative effects of STZ was observed in the liver and pancreas treated
with VA [87]. Ebong et al. (2008) have conducted both acute and chronic studies of
VA on alloxan-induced diabetic albino Wistar rats. 400 mg/kg of ethanolic VA
extract caused significant reduction in peak blood glucose levels (47.31%) at 7 hours
after acute administration, compared to the diabetic controls. In the 24-day chronic
study, daily administration of 400mg/kg ethanolic VA extract resulted in a significant
decrease (>80%) in fasting blood glucose levels compared to levels 24 days before
[88]. Reduction in levels of Glutamic Pyruvic Transaminase (GPT) and Glutamic
Oxaloacetic Transaminase (GOT), which are markers of hepatotoxicity, are in
agreement with the hepatoprotective effect of VA [89, 90]. Another interesting study
showing the glucose-lowering effect of VA was conducted on normal broilers, which
were treated with VA leaf-meal for 28 days [91].
On the other hand, Uchenna et al. (2008) conducted a clinical study to investigate the
effect of VA on blood glucose concentrations of healthy human subjects by using
“squeeze wash” and “chew raw” methods of administration [92]. Significant
reductions were shown in levels of fasting and postprandial blood glucose, with
15
improved glucose tolerance, where the peak reduction occurred at 60 minutes post-
administration [92]. An in vitro study provided further evidence for antidiabetic effect
of VA by showing that it increased glucose utilization in C2C12 muscle cells and
Chang liver cells [93]. Another in vitro study also showed that VA extracted by
acetone or ethylacetate possessed inhibitory effect against digestive enzymes, α-
amylase and α-glucosidase [94].
From Table 1.1, it is evident that VA possesses hypoglycemic properties in various
animal models and human studies but the mechanisms of action are yet to be
elucidated. Furthermore, the active antidiabetic principle(s) from the extract has yet to
be isolated and identified.
Figure 1.2 Vernonia amygdalina
16
Table 1.1 Summary of studies on antidiabetic effects of Vernonia amygdalina
Experiment
al Model
(Normal/
Diabetic/ In
vitro)
Duration
Effective
Dose
Extraction
method
Results Suggested mechanism(s)
of action
References
In vivo studies
Normal/
Alloxan-induced
diabetic
Sprague-Dawley rat
Acute 500mg/kg aqueous FBS decreased 1 to
12 hrs after acute treatment in both
normal & diabetics.
Stimulate insulin
secretion. (Resembles the action of positive control
drug. No supportive data)
[84]
Normal/ Alloxan-
induced
diabetic rabbit
Acute 80mg/kg aqueous FBS decreased up to 50%
(normal), >40%
(diabetic) compared to
controls within 4-8
hrs.
Mechanisms other than insulin secretion
stimulation. (No
supportive data)
[83]
Streptozotoci
n-induced
diabetic Wistar rat
Chronic
(42 days)
400mg/kg ethanolic Glucose level was
normalized at the
end of week 3. Protective effects
against destructive
effects of STZ on pancreas.
NA [95]
Normal/
Streptozotoci
n-induced
diabetic Wistar rat
Chronic
(28 days)
200mg/kg ethanolic FBS decreased
over 28-day
treatments. Hepatic
antioxidant enzymes activities
were increased.
Protective effects against destructive
effects of STZ on
liver and pancreas.
Insulin mimetic and β-cell
regeneration.
[87]
Normal/ Alloxan-
induced
diabetic albino Wistar
rat
Chronic (21 days)
400mg/kg ethanolic FBS decreased by 29% (normal), 42%
(diabetic). Reduced
triglyceride & total cholesterol level in
both normal &
diabetcs.
Increase insulin secreation from β-cells.
(Resembles the action of
positive control drug. No supportive data)
[86]
Streptozotoci
n-induced diabetic
albino Wistar
rat
Chronic
(28 days)
200mg/kg aqueous FBS
decreased >45% after 28-day
treatment.
Improved in antioxidant and
lipid profile.
NA [85]
Normal
broiler
Chronic
(28days)
15% VA leaf
meal in feed
NA The percentage
reductions of
glucose level were 14.30%, 22.90%
and 28.60% for
5%, 10% and 15% VA-feed
respectively.
NA. [91]
17
Alloxan-
induced diabetic
albino Wistar
rat
Acute;
Chronic (24 days)
400mg/kg
for both acute and
chronic
studies
ethanolic FBS decreased by
29% an hour after treatment. FBS
reduced by 88% for
24-day treatment relative to initial
FBS. Decreased in
GPT & GOT indicate its
hepatoprotective
effect.
Increase insulin
production/ peripheral carbohydrate
mechanisms. (Resembles
the action of positive control drug. No
supportive data)
[88]
Experiment
al Model
(Normal/
Diabetic/ In
vitro)
Duration
Effective
Dose
Extract
method
Results Suggested mechanism(s)
of action
References
In vitro studies
In vitro (C2C12
muscle cells
and Chang liver)
NA 50µg/ml Acetone, Methanol,
Water,
N-hexane/ isopropanol
Water & n-hexane/isopropanol
fractions increased
glucose utilization by 78% & 95% (C2C12
muscle); 66% & 60% (Chang liver). No
effect on 3T3-L1
adipocytes.
Increase peripheral tissues glucose uptake.
[93]
In vitro (non-
cell based
enzymatic assay)
NA 6.8-
10.62µg/ml
Free
phenols:
Acetone
Bound
phenols:
Ethylacetate
Free phenols: α-
amylase
(IC50=8.44µg/ml); α-glucosidase
(IC50=7.12µg/ml)
Bound phenols: α-
amylase
(IC50=10.62µg/ml); α-glucosidase
(IC50=6.8µg/ml)
Inhibition of digestive
enzyme activities.
[94]
Clinical study
Normoglycemic human
Acute 50g Squeeze-wash-drink/
chew raw
FBS, GTT & PPG decreased for both
squeeze-wash-
drink and chew raw methods.
Possess insulin-like effect/ stimulate insulin
secretion. (Resembles the
action of positive control drug. No supportive data)
[92]
18
1.8 Coffee and diabetes
The present study has greatly been inspired by the studies of the correlation between
coffee and diabetes. To date, a total of 13 cohort studies involving 1,247,387
participants and 9,473 incident cases of T2DM from various populations groups of the
United States (American [96], African Americans [97]), Europe (England [98],
Sweden [99]) and Asia (Japan [100], Singapore [101]), have demonstrated an inverse
correlation between habitual coffee consumption and the development of T2DM.
However, Battram et al. (2006) showed that the area under the curve (AUC) of
glucose was significantly lowered during an oral glucose tolerance test (OGTT)
following consumption of decaffeinated coffee compared with caffeinated coffee and
a placebo [102]. By using both OGTT [102-105] and euglycemic-hyperinsulinemic
clamp [105-108] techniques, the acute administration of caffeine had been shown to
impair insulin sensitivity. Likewise, a 5-day consumption of high doses of caffeine
induced glucose intolerance [109] while a 7-day consumption of caffeine induced the
development of insulin resistance [110]. Besides caffeine, coffee contains numerous
compounds like phenols, diterpenes, trigonelline and minerals such as potassium and
magnesium. Among them, chlorogenic acid [111-114], trigonelline [111], quinides
[115] and magnesium [116] have been shown to affect glucose metabolism. As a
result, attention has been diverted to other components in coffee, in particular the
major phenolic compound, CGA, which was also found out to be a major constituent
of VA extract later in our studies.
1.9 CGA and diabetes
CGA is a type of hydroxycinnamic acids, which is found in many types of fruits and
in high concentration in coffee [117]. It is an ester formed from cinnamic acids and
19
quinic acid and is also known as 5-O-caffeoylquinic acid (5-CQA) (IUPAC
numbering) or 3-CQA (pre-IUPAC numbering) [118]. CGA and its derivatives have
been shown to inhibit G6P translocase in microsomes of rat livers, suggesting its
inhibitory role in gluconeogenesis for the first time [119, 120]. It was later found out
that G6P translocase 1 facilitates the flux of G6P into endoplasmic reticulum in
enterocytes [121] and a patient genetically deficient in this transporter had been
shown to experience carbohydrate malabsorption [122]. This reveals the possibility
that CGA might be able to inhibit and delay intestinal glucose absorption. Another
study supported this hypothesis by showing that CGA inhibited sodium-dependent
glucose uptake in rat intestinal brush border membrane vesicles[123]. Besides, at IC50
of 0.07mM, CGA has also been shown to inhibit porcine pancreatic α-amylase [124].
Consistent with this, in an animal study using obese and insulin-resistant Sprague-
Dawley Zucker (fa/fa) rats, CGA was found to lower postprandial hyperglycemia,
plasma triacylglycerol and cholesterol [125]. Bassoli et al. (2008) also observed a
significant reduction in plasma glucose peak caused by CGA in an OGTT [114]. In a
human cross-over trial in obese men, ingestion of 1g CGA caused a significant
reduction in glucose and insulin levels 15mins after oral glucose load in an OGTT
[111]. However, no effect was observed on fasting glucose and insulin levels,
contradicting the previous finding in normaglycemic rats that CGA reduced fasting
blood glucose level over a period of 5 hours [120]. Karthikesan et al. (2010) showed
that in STZ-nicotinamide-induced diabetic rats, CGA lowered fasting blood glucose
levels and restored STZ-diminished insulin levels. Also in the same study, CGA was
shown to positively modulate several gluconeogenic and glycolytic enzymes, such as
G6Pase, frutose-1,6-biphosphatase, hexokinase and glucokinase [126]. Svetol, a
decaffeinated green coffee extract that has a high CGA content, has been shown to
20
inhibit G6Pase in human liver microsomes [127], in line with previous study
demonstrating that CGA could be a specific inhibitor of G6Pase [112]. Besides, there
are studies showing that CGA stimulated glucose uptake in L6 myocytes [128] and
3T3-F442A adipocytes [129], indicating that CGA might be able to regulate fasting
glucose levels as well.
21
Table 1.2 Summary of studies on antidiabetic effects of CGA
Experimental
Model (Normal/
Diabetic/ In-
vitro)
Duration of
treatment
Effective
Dose
Results Suggested
mechanism(s) of
action
References
In vivo studies
Normal Wistar
rats
Acute 50mg/kg i.v. S4048, a CGA derivative,
decreased blood glucose levels
in a dose-dependent manner over 5 hours.
Inhibition of
hepatic
gluconeogenesis (No supportive
data).
[120]
SD Zucker (fa/fa)
rats
Chronic
(3 weeks)
5mg/kg i.v. CGA lowered postprandial
glucose, plasma triacyglycerol
and cholesterol levels. CGA did not affect fasting glucose and
insulin levels.
Improved insulin
sensitivity (No
supportive data).
[125]
Normal Wistar rats
Acute 3.5mg/kg oral
CGA reduced plasma glucose peak in an OGTT. 1mM CGA
inhibited 40% G6Pase activity
in hepatic microsomes but failed to reduce glucose production in
isolated perfused liver.
Inhibition of intestinal glucose
absorption.
[114]
STZ-
nicotinamide-
induced diabetic rats
Chronic
(45days)
5mg/kg CGA reduced plasma glucose
levels and restored STZ-
diminished insulin levels. CGA also positively modulated
gluconeogenic and glycolytic
enzyme activities.
Inhibition of
gluconeogenesis
an glycogenolysis. Protection against
STZ-induced
damage on
pancreatic β-cells.
[126]
In vitro studies
In vitro (Isolated
hepatic microsomes and
perfused liver
from rats)
NA 230µM CGA inhibited G6P hydrolysis
by inhibiting G6P translocase in hepatic microsomes. CGA also
inhibited hepatic glucose output
in perfused liver.
Inhibition of
hepatic gluconeogenesis.
[119]
In vitro (rat
intestinal brush border membrane
vesicles)
NA 1mM 1mM CGA caused up to 80%
inhibition in glucose transport in the intestinal membrane
vesicles.
Inhibition of
intestinal glucose absorption.
[123]
In vitro (3T3-
F442A
adipocytes)
60mins 100µM CGA caused a dose-dependent
increase in glucose uptake of
adipocytes. CGA also acted
synergistically with insulin to stimulate glucose uptake. CGA
also restored glucose-uptake in
TNFα-induced insulin-resistant adipocytes.
Stimulation of
glucose uptake in
adipocytes.
[129]
In vitro (non-cell based enzymatic
assay)
NA 0.07mM CGA inhibited porcine pancreatic α-amylase activity.
NA [124]
In vitro (human liver microsomes)
NA 160µM CGAs from Svetol inhibited G6P hydrolysis in human liver
microsomes.
Inhibition of gluconeogenesis.
[127]
In vitro (L6
myotubes)
NA 25µM CGA inceased glucose uptake in
L6 myotubes, possibly via
increased expression of GLUT 4 and PPARγ.
Stimulation of
glucose uptake in
skeletal muscle.
[128]
22
Experimental
Model (Normal/
Diabetic/ In-
vitro)
Duration of
treatment
Effective
Dose
Results Suggested
mechanism(s) of
action
References
Clinical study
Overweight men Acute 1g CGA significantly lowered glucose and insulin
concentrations 15mins after
glucose load.
NA [111]
23
1.10 Objectives and Design of Study
1.10.1 Objectives of study
Although there are a number of studies investigating the antidiabetic effects of VA, its
mechanism(s) of action is/are yet unclear. Hence, the present study aims to elucidate
the possible mechanisms of the hypoglycemic effect of VA, using STZ-induced
diabetic animal model. Besides, the present study aims to identify active constituents
in the extract in order to determine possible chemical compounds that may be
responsible for the hypoglycemic effects.
On the basis of evidence that (i) VA exerts hypoglycemic effects through inhibition of
gluconeogenic enzyme activity and increased expression and translocation of GLUT 4
in skeletal muscle, (ii) VA is rich in CGA and its derivatives, (iii) beneficial
antidiabetic effects of coffee are not attributable to caffeine, and (iv) CGA possesses
hypoglycemic effects in human, animal and in vitro models, the present author's next
aim is to investigate the hypoglycemic effects of CGA in both in vivo and in vitro
models and to identify its possible mechanism(s) of action. Also, he will examine the
effect of long-term consumption of CGA as the beneficial metabolic effects of coffee
on T2DM are associated mainly with the long-term consumption of the beverage. By
using several pharmacological and molecular inhibitors, he will elucidate whether the
hypoglycemic effects of CGA are insulin-dependent or non-insulin dependent. Last
but not least, owing to the fact that T2DM is closely related to obesity and there have
been a few studies reporting that CGA enhanced fat metabolism in the liver [125, 130],
he will therefore investigate the effect of CGA on lipid metabolism in both in vivo and
in vitro models.
24
1.10.2 Research design
Briefly, 6 to 8 week-old male Wistar rats were treated with intraperitoneal STZ
(65mg/kg) to induce diabetes. After five days, those with fasting blood glucose of
250mg/dL and above were considered diabetic and selected for the study. They were
randomly divided into six groups (n=6). The hypoglycemic activity of VA was
screened using an incremental dose study in an OGTT. The rats were then allowed to
rest for two weeks to clear away the treatment effects prior to the initiation of the
long-term (28-day) study. All treatments were given twice daily orally for 28 days.
Body weight, food and water intakes of the rats were monitored daily. Fasting blood
glucose was assessed before the initiation of chronic treatment and at the end of the
study. Serum, pancreas, liver and soleus muscle were collected at the end of the study
and frozen at -80°C till time of use.
The author will next use a total T2DM diabetic model, which is the genetically
modified Leprdb/db
mouse for the studies involving CGA. Leprdb/db
mouse is a
spontaneously mutated diabetic model which becomes obese at approximately three to
four weeks of age. Elevations of plasma insulin begin at 10 to 14 days and elevations
of blood sugar at four to eight weeks. Homozygous mutant mice are polyphagic,
polydipsic, and polyuric. The severity of disease with this genetic background leads to
an uncontrolled rise in blood sugar for which exogenous insulin fails to control blood
glucose levels. Both metabolic efficiency and gluconeogenic enzyme activities are
increased in this model (Source: The Jackson Laboratory Database
http://jaxmice.jax.org/strain/000642.html).
He will investigate the effect of CGA on glucose tolerance before and after 2-week
treatment in Leprdb/db
mice as the beneficial metabolic effects of coffee on T2DM
25
have been shown to result mainly from the long-term consumption of the beverage.
He will performe insulin tolerance test to assess effect of CGA on insulin sensitivity.
He will also examine the effect of 2-week treatment with CGA on various organs
involved in glucose metabolism, namely the liver and skeletal muscle. HepG2 human
hepatoma cell line was used to study CGA’s effect on hepatic glucose production.
The present author will also investigate the effect of CGA on lipid metabolism in both
in vivo and in vitro models as previous studies [125, 130] have shown that CGA
enhances fat metabolism in the liver. He will also investigate the effect of CGA on 2-
deoxyglucose (2DG) transport in L6 myotubes. He will subsequently evaluate
whether these effects of CGA are associated with the activation of AMP-activated
protein kinase (AMPK) and try to determine the possible upstream target(s) that
activate(s) CGA-mediated AMPK activation.
26
2 Chapter 2: Materials and Methods
2.1 Materials
CGA, DMEM, Krebs-Ringer bicarbonate buffer (KRBB), antibiotic/antimycotic,
insulin, wortmannin, cytochalasin B, Fluoroshield with DAPI, Oil Red O, STO-609,
amyloglucosidase, Light Green SF Yellowish, Pararosaniline hydrochloride and AMP
were obtained from Sigma (St. Louis, MO, USA). HepG2 hepatocytes were obtained
from ATCC (Manassas, VA, USA). FBS was from Hyclone (Cramlington, UK).
DMSO was purchased from MP Biomedicals (Illkirch, France). Hematoxylin and
Eosin (H&E) stain, glucose oxidase kits, InfinityTM
Tryglyceride and Total
Cholesterol reagent kits were obtained from Thermo Scientific (Waltham, MA, USA).
Compound c, Orange G stain and NP 40 were obtained from Merck (Darmstadt,
Germany). [γ-32
P]-ATP, NaH14
CO and 14
C-sodium acetate were purchased from
PerkinElmer (Waltham, MA, USA). Protease inhibitor cocktail was purchased from
Abcam (Cambridge, UK). AMPK α1/2 siRNA and an unrelated siRNA (control
siRNA-A) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).
The antibodies (anti-GLUT 4, anti-GLUT 1, anti-Na+K
+ATPase, anti-CAMKKβ, anti-
phospho-AMPK α1/2, anti-AMPK α1/2, anti-ACC, anti-PEPCK, anti-e-cadherin,
anti-G6Pase and FITC-conjugated rabbit IgG) were also obtained from Santa Cruz
Biotechnology (Santa Cruz, CA, USA). Anti-GAPDH and anti-phospho-ACC were
from Cell Signaling Technology (Danvers, MA, USA) and Millipore (Billerica, MA,
USA) respectively. Oligofectamine, Alexa Fluor 555-conjugated mouse IgG, Fluo-4
direct calcium assay kit and OPTI-MEM were purchased from Invitrogen (Carlsbad,
CA, USA). Bradford protein estimation kit was from Bio-Rad (Hercules, CA, USA).
Ultra-sensitive Rat Insulin and Leptin ELISA kits were obtained from Crystal Chem
27
Inc. (Downers Grove, IL, USA) while adiponectin ELISA kit was obtained from
Bertin Pharma (Artigues Pres Bordeaux, France). Free fatty acids detection kits were
purchased from Wako Diagnostics (Richmond, VA, USA). G-Sepharose beads and
ECL detection kit were obtained from GE Healthcare (Piscataway, NJ, USA). SAMS
peptide was purchased from Tocris Bioscience (Minneapolis, MN, USA).
2.2 Studies of antidiabetic effects of VA in STZ-induced diabetic
rats
2.2.1 Plant materials
Fresh leaves of VA were collected from Penang, Malaysia in January, 2009. Its
botanical identity, Vernonia amygdalina Delile (Asteraceae) was confirmed by Mr.
K.F. Leong, a research officer in Singapore Botanic Gardens, National Parks Board. A
voucher specimen (specimen voucher number: 2007018334) has been deposited in
SINU Herbarium, National University of Singapore (NUS).
2.2.2 Preparation of plant extract
Harvested fresh leaves (3 kg) were rinsed with distilled water and homogenized with
80% ethanol. The homogenate was extracted over 24 hours at room temperature. The
extracted mixture was then filtered. The filtrate was concentrated in a rotary
evaporator at 50°C. Plant residues were again extracted with 80% ethanol for another
24 hours until exhaustion, followed by filtration and concentration. Accumulated
filtrate was subjected to freeze drying and the resulting solid residues (62.4 g
28
yellowish green powder) are referred to as “extract” in this paper. The extract yield
(w/w) from 3 kg of fresh VA leaves was approximately 2.1%.
2.2.3 Experimental animals
Sixty male Wistar rats were obtained from Centre for Animal Resources (CARE),
NUS. They were allowed to acclimatize to conditions in the Animal Holding Unit
(AHU), NUS, where they were housed throughout the experiment on a 12-hour
light/dark cycle. Water and feeds were available to the animals ad libitum. The
Principles of Laboratory Animal Care (NIH, 1985) were followed throughout the
duration of experiment. The experimental protocol for animal study was approved by
NUS Institutional Animal Care and Use Committee (IACUC).
2.2.4 Ethics statement
The Principles of Laboratory Animal Care (NIH, 1985) were followed throughout the
duration of the experiment. The experimental protocol for animal study was approved
by NUS IACUC [Protocol No: 085/07(A3)10].
2.2.5 Induction of diabetes with STZ
Overnight-fasted rats (n=50) were injected with a single intraperitoneal dose of STZ
(65 mg/kg), which was freshly dissolved in sodium citrate buffer (pH 4.5) [131].
Their fasting blood glucose levels were measured five days after the injection, using
glucose oxidase method. Rats with fasting blood glucose of 250 mg/dL and above
were considered diabetic and selected for the study.
2.2.6 Dose-response study in STZ-diabetic rats with VA
Thirty diabetic rats and six normal rats used in this study were randomly divided into
six groups (n=6). The anti-hyperglycemic activity of VA was screened by using a
29
modified oral glucose tolerance test. Briefly, blood samples were collected from the
normal/diabetic rat, followed by oral gavaging of treatments (vehicle, 500 mg/kg
metformin, 200/400/600 mg/kg VA) for each group of animals. Diabetic rats treated
with vehicle were assigned as diabetic control (DC) while normal rats treated with
vehicle were assigned as normal control (NC). Thirty minutes after the treatments,
blood samples were collected for glucose estimation, followed by oral administration
of 2 g/kg of glucose. Blood samples were further collected 1 hour and 2 hours post-
glucose loading for glucose estimation.
2.2.7 Chronic (28-day) study in STZ-diabetic rats
Two weeks after the dose-response study, the rats were randomly re-grouped for
chronic treatment. Six rats without STZ treatment were taken as NC and treated with
vehicle. Eighteen diabetic rats were randomly selected and divided into three groups
(vehicle, 500 mg/kg metformin and 400 mg/kg VA). All treatments were given twice
daily orally for 28 days. Body weight, food and water intakes of the rats were
monitored daily. Fasting blood glucose was assessed before the initiation of chornic
treatment and at the end of the study. Blood sample, pancreas, liver and soleus muscle
were collected at the end of the study and frozen at -80°C till the time of use.
2.2.8 Biochemical analyses
Pancreatic insulin extraction was done as described previously [132]. Briefly, 0.1 g of
pancreas tissue was homogenized in 5ml of acetic acid for 1 min by ultrasonic
disintegration at 4°C. It was left to stand overnight at -20°C. After which it was
centrifuged at 1000 g for 10 minutes. The supernatant was used for insulin
measurement. Both serum and pancreatic insulin levels were measured using Ultra-
Sensitive Rat Insulin ELISA kit. InfinityTM
Tryglyceride and Total Cholesterol
30
reagents kits were used for the measurement of serum triglyceride and total
cholesterol levels. Kits for antioxidant enzyme assays (Superoxide Dismutase,
Catalase and Glutathione Peroxidase) and glutathione assay were purchased from
Cayman Chemicals (Ann Arbor, MI, USA).
2.2.9 Determination of G6Pase activity
Hepatic G6Pase activity was measured in microsomes isolated from liver as described
previously [133]. Liver was homogenized in sucrose buffer and centrifuged at 10 000
g for 10 minutes at 4°C. Supernatant was taken and re-centrifuged at 100 000 g for 45
minutes at 4°C. The pellets (microsomes) were re-suspended with sucrose buffer for
assay. The activity of the enzyme was expressed as amount of phosphate liberated
from G6P in a minute, adjusted to the amount of protein extracted.
2.2.10 Determination of muscle glycogen content
Muscle glycogen content was measured according to a protocol described previously
[134]. Muscle was homogenized in citrate buffer (pH 4.5) and 10 µl of homogenate
was aliquoted to determine free glucose in the tissue by using glucose oxidase reagent.
Amyloglucosidase (1mg) was added to 1ml of homogenate and incubated for 4 hours
at 37°C. Then, 10µl was taken to measure the total glucose content. Glycogen content
was calculated by subtracting initial free glucose from total glucose and expressed as
mg/g tissue.
2.2.11 Fractionation of rat skeletal muscle
Fractionation of skeletal muscle was done as described previously [135]. The soleus
muscle was isolated and homogenized in KCl buffer with protease inhibitors at low
speed. Homogenate was treated with Triton X-100 for 30 minutes at 4°C. It was then
31
centrifuged at 14 000 g for 15 minutes at 4°C. Supernatant was collected as total cell
lysates and stored for immunoblotting. A solution of KCl was added to the remaining
homogenate to a final concentration of 0.65 M. The mixture was left on ice for 15
minutes, followed by centrifugation at 2000 g for 10 minutes at 4°C. Supernatant was
collected and put on ice. Pellet was re-suspended in KCl buffer and steps were
repeated. Two supernatants were pooled and centrifuged at 190 000 g for 60 minutes
at 4°C. Resulting pellet was crude membranes. It was re-suspended in sucrose buffer.
The re-suspended pellet was then loaded onto the top of a discontinuous sucrose
gradient (25%, 30% and 35%). The sucrose gradient columns were then subjected to
ultracentrifugation at 150, 000 g for 16 hours at 4°C. Plasma membrane was collected
at the top of 25% sucrose while microsomal fraction was collected in between 30-35%
sucrose. They were re-suspended in KCl buffer and stored for immunoblotting.
2.2.12 Immunoblotting to detect GLUT 1 and GLUT 4
Protein extracted was separated with SDS-PAGE and electro-transferred to
nitrocellulose membranes. Blotted protein was then probed with primary antibodies.
They were then probed with HRP-conjugated secondary anti-rabbit antibodies. Probed
proteins were visualized with advanced ECL kit.
2.2.13 HPLC analysis
Chemical composition of the extract was analysed using Waters HPLC equipped with
a photodiode array detector, a Shimadzu ODS-VP column (4.6x250 mm, 5 μm i.d.),
and an auto-sampler. It was set to scan from 200-400 nm at 25°C. The binary mobile
phase consists of (A) water-acetic acid (99.9: 0.1, v/v) and (B) CH3CN. The gradient
elution program was set to 5–13% B (0–4 min), 13–26% B (4–16 min), 26–28% B
32
(16–24 min), 28–36% B (24–32 min), 36–90% B (32–36 min), 90–5% B (36–40 min)
and 5% B (40–44 min). The injection volume of each sample was 20 µl. The flow rate
of the mobile phase was set to 1 ml/min and peaks were monitored at 330 nm.
2.2.14 LC-ESI-MS analysis
LC-ESI-MS analysis was conducted on Finnigan/MAT LCQ ion trap mass
spectrometer fitted with an electrospray interface (ESI) and a TSP 4000 HPLC system.
The LC conditions were identical to those used for HPLC analysis as above. The
injection volume of each sample was 20 µl. Both positive ion and negative ion modes
were used for further characterization of the compounds. The heated capillary and
spray voltage were maintained at 250°C and 4.5 kV, respectively. Nitrogen was
operated at 80 psi for sheath gas flow rate and 20 psi for auxiliary gas flow rate. Full
scan mass spectra from m/z 50 to 2000 were recorded with a scan speed of one scan
per second. MS/MS was operated with the collision energy of 30–40%. Identities of
the compounds were obtained by matching their molecular ions (m/z) recorded by
LC-ESI-MS and LC-ESI-MS/MS with existing literature data [136].
2.3 Studies of antidiabetic and antilipidemic effects of CGA
2.3.1 Experimental animals
Leprdb/db
mice homozygous for diabetes spontaneous mutation were obtained from
The Jackson Laboratory (Sacramento, CA, USA). C57BL/6 mice were purchased
from CARE, NUS. They were allowed to acclimatize to conditions in the AHU, NUS.
They were housed throughout the experiment on a 12-hour light/dark cycle. Water
and feeds were available to the animals ad libitum.
33
2.3.2 Ethic statement
Refer to Section 2.2.4.
2.3.3 Oral glucose tolerance test
Leprdb/db
mice were randomly assigned into four groups (n=4) and four C57BL/6 mice
were assigned as lean control group. They were fasted for six hours before the test.
For inhibitor study with compound c (AMPK inhibitor), mice were pre-treated with
50 mg/kg compound c. Blood samples were collected from the tail vein for fasting
glucose measurement using glucose oxidase method before treatments (vehicle, i.p.
250 mg/kg CGA, oral 250 mg/kg metformin). Ten minutes after the treatments, blood
samples were collected again followed by oral gavaging of 2 g/kg glucose. Blood
samples were collected 15, 30, 60 and 120 minutes after the glucose challenge.
2.3.4 2-week CGA treatment in Leprdb/db
mice
After the oral glucose tolerance test, the mice were given a one-week rest to clear the
acute effects of various treatments. They were randomly re-assigned (n=4) and treated
with vehicle, 250 mg/kg oral metformin or 250 mg/kg CGA i.p. daily. On Day 0, oral
glucose tolerance test was done followed by initiation of chronic treatment.
Measurements of body weight, food and water intakes were started and monitored in
the subsequent days. At the end of the treatment, besides OGTT, insulin tolerance test
(ITT) and pyruvate tolerance test (PTT) were also performed. For PTT and ITT, 2
g/kg sodium pyruvate or 0.75U/kg insulin or 3U/kg insulin was injected
intraperitoneally and blood was sampled as described in OGTT. The mice were then
sacrificed for collection of whole blood, skeletal muscles (soleus and gastrocnemius)
and liver. They were frozen immediately in liquid nitrogen and stored at -80ºC till
time for assays. The score for homeostatic model assessment (HOMAIR) index of
34
insulin resistance was determined from: fasting blood glucose (mg/dL) X fasting
serum insulin (µU/ml)/ 405 [137].
2.3.5 2DG transport in skeletal muscle isolated from Leprdb/db
mice
Skeletal muscles were isolated from Leprdb/db
mice as described previously [138]. It
was then incubated with treatments (vehicle, metformin or CGA) in KRBB for 30
minutes at 37ºC. Treated muscle strips were subsequently incubated with 0.5 ml
KRBB containing 1 µCi/ ml 2-Deoxy-[3H]D-glucose for 30 minutes at 37ºC. Reaction
was terminated by immediately blotting the tissues and dissolving them in 0.5N
NaOH for an hour followed by neutralization with equal amount of 0.5N HCL. After
centrifugation, supernatant was collected for quatitation of 2DG taken up by the tissue
using liquid scintillation counter (Beckman Coulter LS6500 Multi-Purpose
Scintillation Counter, Fullerton, CA, USA). Non-specific uptake was measured in the
presence of 10 µmol/l cytochalasin B and subtracted from the total uptake. 2DG
uptake was expressed as a percentage of the basal uptake of cells incubated with
KRBB buffer only.
2.3.6 Cell culture and differentiation of L6 skeletal muscle
The culture was maintained in DMEM containing 10% FBS and 1%
antibiotic/antimycotic in a humidified atmosphere of 5% CO2 at 37ºC. Differentiation
of myoblasts into myotubes was carried out as described by Klip et al. [139]. L6
myoblast was seeded in 10% FBS-DMEM until it reached 80-90% confluence; the
FBS content was reduced to 2% for a further 5-7 days to induce myotube formation.
The degree of differentiation was determined as the percentage of nuclei present in the
multinucleated myotubes under a phase-contrast microscope. Before all experimental
35
manipulations, L6 myotubes were deprived of serum for 4 hours to render the cells
quiescent.
2.3.7 Cell culture of HepG2 human hepatoma
HepG2 human hepatoma was cultured in Dulbecco’s Modified Eagle’s Medium
(DMEM) supplemented with 10% FBS under 5% CO2 at 37ºC. Confluent cells were
serum-starved for four hours before treatment with CGA, metformin and inhibitors at
indicated concentrations and incubation periods. For inhibitor study, cells were pre-
treated with compound c or STO-609 for 30 minutes before addition of drugs for
another 4-hour incubation.
2.3.8 2DG transport in L6 skeletal muscle cells
The cells were grown and differentiated in 96-well plates as described in section 2.3.6.
After the indicated periods of incubation with different treatments, the cells were
rinsed with KRPH (HEPES-buffered Krebs-Ringer phosphate) buffer, consisting of
118 mmol/l NaCl, 5 mmol/l KCl, 1.3 mmol/l CaCl2, 1.2 mmol/l MgSO4, 1.2 mmol/l
KH2PO4 and 30 mmol/l HEPES (pH 7.4). CGA was prepared in 5% DMSO and
diluted with appropriate amount of DMEM to obtain different concentrations. 5%
DMSO was used for all drug preparations. For the study with CGA + insulin,
myotubes were treated with 2 mmol/l CGA for 24 hours and stimulated with 100
nmol/l insulin for 30 mins before 2DG uptake measurement. For the study involving
inhibitors, myotubes were pre-incubated with 100 nmol/l wortmannin or 10 µmol/l
compound c for 30 minutes before adding CGA. Washed cells were incubated with 10
µmol/l 2-Deoxy-[3H]D-glucose (1µCi/ml) in KRPH buffer for 30 minutes at 37ºC.
The 2-DG uptake was terminated immediately by aspirating the radioactive
36
incubation solution and washing three times by ice-cold phosphate-buffered saline
(PBS). The cells were then lysed with 0.5 N NaOH, followed by 0.5 N HCL for
neutralization. The quantity of 2DG taken up by the cells was measured with liquid
scintillation counter. Non-specific uptake was measured in the presence of 10 µmol/l
cytochalasin B and subtracted from the total uptake. 2-DG uptake was expressed as a
percentage of the basal uptake of cells incubated with KRPH buffer only.
2.3.9 Myotube subcellular fractionation
The subcellular fractionation of myotubes was done as previously described [140].
Treated cells were scraped gently from 10 cm dishes and centrifuged at 700 g, 4ºC for
10 mins. The pellet was then resuspended in sucrose buffer, consisting of 250 mmol/l
sucrose, 5 mmol/l sodium azide, 2 mmol/l EGTA, 20 mmlol/l HEPES (pH 7.4) and
protease inhibitor cocktail, and homogenized with 20 strokes using a Dounce
homogenizer. The homogenate was centrifuged at 760 g, at 4ºC for 5 minutes to
remove nuclei and cell debris. The supernatant was collected as total cell lysate. For
plasma membrane isolation, the supernatant was removed and centrifuged at 31,000 g,
4ºC for 60 minutes to pellet the crude plasma membrane (PM). PM fraction was
resuspended in sucrose buffer and stored at -80ºC.
2.3.10 siRNA transfection of myotubes and HepG2
Transfection of siRNA into myotubes was done as described previously with a minute
modification [141]. Cells were seeded and grown in 6-well plates as described earlier
(see section on cell culture and differentiation). After cells were treated with 2% FBS
for 3 days, siRNAs were transfected with oligofectamine, according to the instructions
of the manufacturer. Briefly, the complex mixture in serum- and antibiotic-free OPTI-
37
MEM was layered onto the cells and incubated for 4 hours at 37ºC. The serum content
was then brought up to 2% by adding half volume of antibiotic-free DMEM
supplemented with 6% FBS and incubated for an additional 20 hours. Another volume
of 2%-FBS DMEM with antibiotic was then added and incubated for further 24 hours
to allow the siRNA to remain on the cells for a total of 48 hours. For HepG2 cells,
Confluent cells were transfected with siRNAs in oligofectamine according to the
instructions of the manufacturer. Briefly, the complex mixture in serum- and
antibiotic-free OPTI-MEM was layered onto the cells and incubated for 8 hours at
37ºC. The medium was then replaced with DMEM containing 10% FBS and
incubated for an additional 48 hours. Efficiency of transfection was optimized using
FITC-tagged siRNA.
2.3.11 Immunoprecipitation and detection of association between IRS-1 and p85
subunit of PI3K
Treated cells were scraped gently from 6-well plate and pelleted with ice-cold PBS at
3,000 rpm, 4ºC for 5 minutes. Cell pellet was then lysed in lysis buffer (50 mmol/l
Tris [pH 8], 170 mmol/l NaCl, 1 mmol/l DTT, 0.5% NP40 and protease inhibitor
cocktail for 30 minutes at 4ºC. Cell lysate was then centrifuged at 13,000 rpm for 10
minutes to remove cell debris. Cell lysate (2 µl) was used for Bradford protein
estimation. Total cellular protein (1 mg) was immunoprecipitated with 1 µg of anti-
IRS-1 antibody coupled to 40 µl of 100 mg/ml protein G-Sepharose beads.
Immunoprecipitated proteins, together with the beads, were separated with SDS-
PAGE and blotted as mentioned in the western blot analysis below. Blotted proteins
were probed with anti-PI3K p85α antibody.
38
2.3.12 Glucose production assay
Treated cells were washed with PBS. Glucose-free glucose production buffer (phenol
red-free DMEM supplemented with 2 mmol/l sodium pyruvate, 20 mmol/l sodium
lactate and 44 mmol/l sodium bicarbonate) was added and incubated for four hours.
After that, medium was collected for the measurement of glucose, using the glucose
oxidase kit. The readings were then normalized to the total protein content.
2.3.13 AMPK activity assay
AMPK activity was measured as described previously [142]. HepG2 cells were
treated with CGA at different concentrations for indicated periods of time. Treated
cells were scraped and pelleted with ice-cold PBS at 3,000 rpm, 4ºC for 5 minutes.
Cell pellets were then lysed in lysis buffer containing 150 mmol/l NaCl, 50mmol/l
Tris (pH 8.0), 10 mmol/l sodium fluoride, 1 mmol/l sodium pyrophosphate, 1 mmol/l
sodium orthovanadate, 1% NP-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl
sulphate and protease inhibitor cocktail. 2 µl of the cell lysate was used for Bradford
protein estimation. 1 mg of total cellular protein was immunoprecipitated with 1 µg of
anti-AMPK α1/2 antibody coupled to 40 µl of 100 mg/ml protein G-Sepharose beads.
Kinase reaction was carried out on washed immunoprecipitate in 40 mmol/l HEPES
(pH 7.0), 0.2 mmol/l AMP, 80 mmol/l NaCl, 0.8 mmol/l DTT, 5mmol/l MgCl2, 0.2
mmol/l ATP (2mCi [γ-ATP]) and 0.1 mmol/l SAMS peptide for 20 minutes at 37ºC.
Reaction mixture was then spotted on P81 Whatman filter paper and washed
extensively using phosphoric acid and acetone. Radioactivity on the filter paper was
measured by liquid scintillation. Kinase activity was expressed as incorporated
ATP/mg protein/minute.
39
2.3.14 ACC activity assay
ACC activity was assayed by measuring the incorporation of (14
C)HCO3- into malonyl
CoA [143]. Treated HepG2 cells were lysed in lysis buffer containing 150 mmol/l
NaCl, 50mmol/l Tris (pH 8.0), 10 mmol/l sodium fluoride, 1 mmol/l sodium
pyrophosphate, 1 mmol/l sodium orthovanadate, 1% NP-40, 0.5% sodium
deoxycholate, 0.1% sodium dodecyl sulphate and proteases inhibitors cocktail. Cell
lysate (2 µl) was used for Bradford protein estimation. ACC activity was measured by
incubating lysates with reaction mixture consisting of 50 mmol/l HEPES (pH7.5), 10
mmol/l potassium citrate, 10 mmol/l MgCl2, 4 mmol/l potassium carbonate, 0.5
mmol/l adenosine triphosphate, 0.4 mmol/l NaH14
CO3, 0.25 mmol/l acetyl CoA and
0.075% bovine serum albumin for 40 minutes at room temperature. The reaction was
stopped by adding in half volume of 1N HCL and allowing to stand for 30 minutes at
room temperature. Total 14
C incorporated was measured using a liquid scintillation
counter (Beckman Coulter LS6500 Multi-Purpose Scintillation Counter, Fullerton,
CA, USA). ACC activity was expressed as incorporated 14
C /mg protein/minute.
2.3.15 Fatty acid synthesis assay
Fatty acid synthesis was assayed as described previously with slight modification
[143]. After the appropriated treatment, medium was removed and replaced with
serum-free DMEM containing 14
C-sodium acetate (2µCi/ml). Cells were then
incubated with the radiolabelled acetate for 4 hours at 37ºC. Radioactive medium was
then removed and washed immediately with ice-cold PBS for three times. 0.5N NaOH
was added to lyse the cells followed by neutralization with equal volume of 0.5N
HCL. Radioactivity incorporated into fatty acid was monitored using scintillation
40
counter. Readings were expressed as percent inhibition of fatty acid synthesis
compared to control.
2.3.16 Fluo-4 direct calcium assay
Measurement of free Ca2+
influx was performed on HepG2 hepatoma cells according
to the instructions of the manufacturer. Briefly, treated cells were incubated with
equal volume of 2X Fluo-4 Direct calcium reagent for one hour at 37˚C. Fluorescence
was measured at 494 nm (excitation) and 516 nm (emission) with a microplate reader
(Tecan Infinite m200, Mannedorf, Switzerland).
2.3.17 Oil Red O staining
Treated cells were washed with ice-cold PBS. The cells were then fixed with 10%-
buffered formalin for 24 hours at room temperature. Fixed cells were stained with Oil
Red O in 60% isopropanol for ten minutes at room temperature. Stained cells were
washed with deionized water and images were acquired for analysis. Quantitation was
done by eluting the stain with 100% isopropanol. OD was measured at 500nm.
2.3.18 Glucose and lipid profiles
Following manufacturers’ protocols, different enzymatic or ELISA kits were used for
the measurement of serum glucose, insulin, triglyceride, total cholesterol, adiponectin
and free fatty acid levels. For hepatic lipids measurement, tissue was homogenized in
10% w/v isopropanol and kept at 4ºC for two days. Homogenate was centrifuged at
4ºC and 1000g for 10 minutes. Supernatant was collected for the measurements of
triglyceride, cholesterol and free fatty acids.
41
2.3.19 Hepatic G6Pase activity
Refer to Section 2.2.9.
2.3.20 Fractionation of skeletal muscle
Skeletal muscles (soleus and gastrocnemius) isolated from Leprdb/db
mice after 2
weeks of treatments. Please refer to Section 2.2.12 for details in skeletal muscle
fractionation.
2.3.21 2DG transport in skeletal muscles
Refer to Section 2.3.5.
2.3.22 Liver histology or skeletal muscle immunohistochemistry
Isolated skeletal muscle and liver samples were fixed in 10% buffered formalin for 24
hours at 4ºC. Fixed tissues were then processed using standard tissue processing
protocol. Processed tissues were embedded in paraffin and sectioned into 4µm-thick
slices. Tissue sections were dewaxed and hydrated in descending concentrations of
ethanol. Liver sections were stained with H&E for normal histological assessment,
where cytoplasm stained pink, nuclei stained deep purple and pancreatic islets
appeared pale in color. Antigen retrieval was then performed on skeletal muscle by
boiling tissue sections in Tris-EDTA buffer containing 10 mmol/l Tris (pH 9), 1
mmol/l EDTA and 0.05% Tween 20 for 20 minutes. The tissuee were then blocked
with 1% bovine serum albumin in TBS supplemented with 0.1% Tween 20. After that
tissues were incubated in a mixture of primary antibodies (anti-GLUT 4 and anti-e-
cadherin) overnight at 4ºC. Tissue sections were washed and incubated in a mixture of
FITC-conjugated rabbit IgG and Alexa Fluor 555-conjugated mouse IgG secondary
antibodies. They were then mounted with Fluroshield with DAPI.
42
2.3.23 Western blot analysis
Treated HepG2 cells were scraped from 6-well plates and pelleted with ice-cold PBS
at 3,000 rpm, 4ºC for 5 minutes. Cell pellets were then lysed in lysis buffer containing
150 mmol/l NaCl, 50mmol/l Tris (pH 8.0), 10 mmol/l sodium fluoride, 1 mmol/l
sodium pyrophosphate, 1 mmol/l sodium orthovanadate, 1% NP-40, 0.5% sodium
deoxycholate, 0.1% sodium dodecyl sulphate and protease inhibitor cocktail. To
extract protein from liver, liver tissue was first homogenized in lysis buffer. The
lysate was then centrifuged at 14,000rpm for 10 minutes. Supernatant was collected
and separated via SDS-PAGE and the separated proteins were then blotted onto
nitrocellulose membrane. Membranes were probed with anti-GAPDH, anti-e-cadherin,
anti-GLUT 4, anti-PEPCK, anti-G6Pase, anti-CAMKKβ, anti-phospho-AMPK α1/2,
and anti-phospho-ACC. They were then developed using the ECL detection kit.
2.4 Statistical analysis
Experiments were repeated three times, each time in triplicate. Values are expressed
as mean ± SE. One-way ANOVA followed by Tukey’s t test was used to determine
significant differences between groups. P values <0.05 were interpreted as statistically
significant.
43
3 Results
3.1 Studies of antidiabetic effects of VA in STZ-induced diabetic
rats
3.1.1 Acute effect of VA extract on fasting blood glucose in STZ-induced
diabetic rats
Figure 3.1.1 shows the acute hypoglycemic effect of various treatments on blood
glucose level measured at various time intervals. The values are percent change in
blood glucose level at time t, relative to blood glucose level at time -0.5 hour (%
Change = At/ A-0.5 X 100). Thirty minutes after various treatments at time -0.5 hour,
metformin caused 4.4% decrease of blood glucose whereas 400 mg/kg of VA, which
was found to be the most potent dose, resulted in a slight increase of 1.3%. After
glucose loading at time 0 hour, a surge of 36.6% was observed in DC compared to
15.2% in NC at time 1 hour, which indicates an impaired glucose tolerance in DC.
Metformin and VA continued to cause a 54.5% and 25.4% suppression on glucose
elevation for the first hour after glucose loading if compared to DC, respectively,
indicating an improvement of glucose tolerance. For the second hour post-glucose
loading, metformin-treated group showed 74.3% suppression in blood glucose while
VA showed a 35.2% suppression on increased blood glucose. Metformin suppressed
the rise in glucose levels, with glucose levels significantly lower than those of NC; on
the other hand, VA suppressed elevation of glucose levels to near-normal levels.
44
Figure 3.1.1 Acute effects of VA on glucose tolerance in STZ-induced diabetic rats
Treatments were given at time -0.5 hour and glucose was loaded at 0 hour. Blood
samples were collected at -0.5 hour, 0 hour, 1 hour and 2 hour for blood glucose
assessment.
Values were expressed as mean ± S.E.M. (n=6). NC=Normal Control, DC=Diabetic
Control, Metformin=metformin-treated diabetic, VA=Vernonia amygdalina-treated
diabetic. * P<0.05.
3.1.2 Long-term effects of VA extract on body weight, food and water intakes
of STZ-induced diabetic rats
Table 3.1.1 shows the effects of various treatments on body weight, food and water
intakes of the rats. After 28 days of treatment, both metformin and VA (400mg/kg)
showed significant decreases in body weight compared to DC. However, there was no
significant difference in food intake for all the experimental groups. On the other hand,
there were significant reductions in water intake for metformin- and VA-treated
groups, compared to DC.
45
Table 3.1.1. Long-term effect s of VA on body weight, food intake and water
intake in STZ-induced diabetic rat.
Treatments Body Weights (g)a
Food Intake (g/kg)a
Water Intake (g/kg)a
Day 1 Day 28 Day 1 Day 28 Day 1 Day 28
NC 281.5 ±
18.5
386.0 ±
17.0
143.5 ±
5.7
114.7 ±
4.2
188.3 ±
9.5
175.6 ±
1.7
DC 301.3 ±
7.2
338.7 ±
5.9
153.7 ±
2.4
136.3 ±
5.9
657.7 ±
19.8
644.0 ±
19.6
Metformin 283.3 ±
9.2
311.2 ±
10.3*
133.7 ±
8.8
127.0 ±
8.9
637.7 ±
49.8
367.7 ±
26.2*
VA 288.8 ±
9.3
303.4 ±
2.7*
143.7 ±
9.8
137.7 ±
4.5
641.3 ±
25.3
346.7 ±
24.3*
a Values were expressed as mean ± S.E.M. (n=6); *P<0.05 compared to DC.
3.1.3 Long-term effects of VA extract on fasting blood glucose, triglyceride and
total cholesterol levels
One week after STZ injection, an increase of 140% in fasting blood glucose was
observed compared to NC. After 28-day treatments, fasting blood glucose of DC
group continued to rise for another 9% while significant decreases of 36.1% and
26.5% were observed in metformin-treated and VA-treated groups respectively.
Compared to DC, there were decreases of 35.3% and 32.1% respectively in fasting
blood glucose of metformin- and VA-treated groups (Figure 3.1.2). Triglyceride level
of DC was observed to be 140% higher than NC. There was a significant 18.2%
reduction in VA-treated group compared to vehicle in DC while no significant change
was observed in metformin-treated group (Figure 3.1.3A). Total cholesterol level was
increased in DC by 120% compared to NC. However, VA treatment lowered total
46
cholesterol level by 41%, compared to vehicle in DC. Metformin showed no
significant effect on total cholesterol level (Figure 3.1.3B).
Figure 3.1.2. Long-term effects of VA on fasting blood glucose of STZ-induced
diabetic rats.
Treatments (500mg/kg metformin, 400mg/kg VA and vehicles for both NC and DC)
were given twice daily through gavage for 28 days. Blood samples were collected at
the end of first and last week for glucose level determination.
Values were expressed as mean ± S.E.M. (n=6). NC=Normal Control, DC=Diabetic
Control, Metformin=metformin-treated diabetic, VA=Vernonia amygdalina-treated
diabetic. * P<0.05 compared to DC.
47
Figure 3.1.3. Long-term effects of VA on lipid profile of STZ-induced diabetic rats
Treatments (500mg/kg metformin, 400mg/kg VA and vehicles for both NC and DC)
were given twice daily through gavage for 28 days. Blood samples were collected at
the end of day 28 for the determination of A: triglyceride and B: total cholesterol.
Values were expressed as mean ± S.E.M. (n=6). NC=Normal Control, DC=Diabetic
Control, Metformin=metformin-treated diabetic, VA=Vernonia amygdalina-treated
diabetic. * P<0.05 compared to DC.
48
3.1.4 Long-term effects of VA extract on pancreatic and serum insulin levels
STZ depleted pancreatic and serum insulin levels by up to 95%, compared to NC.
After the treatments for 28 days, there were significant increases of 54% and 58% in
pancreatic and serum insulin levels respectively, caused by VA administration,
compared to vehicle in DC. However, there was no significant change caused by
metformin in both pancreatic and serum insulin levels (Figure 3.1.4A & B).
Nonetheless, pancreatic and serum insulin levels from all the diabetic rats are
negligible if compared to NC.
3.1.5 Long-term effects of VA extract on hepatic G6Pase activity
STZ caused an increase of hepatic G6Pase activity compared to NC. Metformin, a
gluconeogenesis suppressor, restored the G6Pase activity to near-normal, by an
approximate reduction of 76% compared to DC. On the other hand, 400 mg/kg VA
was found to decrease G6Pase activity by 40% compared to vehicle in DC (Figure
3.1.5).
3.1.6 Long-term effects of VA extract on hepatic GSH and antioxidant enzymes
STZ-induced diabetic rats were observed to have decreased hepatic GSH levels and
antioxidant enzyme activities (SOD, catalase, and GPx) compared to NC (Figures
3.1.6). Metformin treatment did not produce significant changes in both hepatic GSH
and antioxidant enzymes. VA significantly increased SOD (Figure 3.1.6A) and
catalase activities (Figure 3.1.6B) by 129% and 39.2% respectively compared to
vehicle in DC. VA enhanced GPx activity significantly compared to vehicles in both
DC and NC (Figure 3.1.6C). In addition, VA restored the glutathione level to near-
normal (Figure 3.1.6D).
49
Figure 3.1.4. Long-term effects of VA on insulin levels of STZ-induced diabetic rats
Treatments (500mg/kg metformin, 400mg/kg VA and vehicles for both NC and DC)
were given twice daily through gavaging for 28 days.
A: Pancreas was isolated at the end of day 28 for the determination of pancreatic
insulin level. B: Blood samples were collected at the end of day 28 for the
determination of serum insulin level. Values were expressed as mean ± S.E.M. (n=6).
NC=Normal Control, DC=Diabetic Control, Metformin=metformin-treated diabetic,
VA=Vernonia amygdalina-treated diabetic. * P<0.05 compared to DC.
50
Figure 3.1.5. Long-term effects of VA on hepatic G6Pase levels of STZ-induced
diabetic rats
Treatments (500mg/kg metformin, 400mg/kg VA and vehicles for both NC and DC)
were given twice daily through gavaging for 28 days.
Liver was isolated at the end of day 28 for the determination of G6P level. Values
were expressed as mean ± S.E.M. (n=6). NC=Normal Control, DC=Diabetic Control,
Metformin=metformin-treated diabetic, VA=Vernonia amygdalina-treated diabetic.
* P<0.05, **P<0.01 compared to DC.
51
Figure 3.16. Long-term effects of VA on hepatic antioxidant enzymes and GSH
activities of STZ-induced diabetic rats
Treatments (500mg/kg metformin, 400mg/kg VA and vehicles for both NC and DC)
were given twice daily through gavaging for 28 days. Liver was isolated at the end of
day 28 for the determination of A: SOD and B: catalase activities.
Values were expressed as mean ± S.E.M. (n=6). NC=Normal Control, DC=Diabetic
Control, Metformin=metformin-treated diabetic, VA=Vernonia amygdalina-treated
diabetic. * P<0.05 compared to DC.
52
Treatments (500mg/kg metformin, 400mg/kg VA and vehicles for both NC and DC)
were given twice daily through gavaging for 28 days. Liver was isolated at the end of
day 28 for the determination of C: GPx activity and D: glutathione level.
Values were expressed as mean ± S.E.M. (n=6). NC=Normal Control, DC=Diabetic
Control, Metformin=metformin-treated diabetic, VA=Vernonia amygdalina-treated
diabetic. * P<0.05 compared to DC.
C
D
53
3.1.7 Long-term effects of VA extract on expression of GLUT 1/ GLUT 4 and
cellular distribution of GLUT 4
There was a decrease in total expression of GLUT 4 (~40%) but no change in total
expression of GLUT 1 in DC, compared to NC. Metformin almost restored GLUT 4
expression to the normal state while VA caused a significant increase of GLUT 4
expression by 24% compared to vehicle in DC. Neither metformin nor VA affected
the expression of GLUT 1 (Figure 3.1.7A). Translocation of GLUT 4 to plasma
membrane was reduced by 75% after treatment with STZ. Chronic administration of
metformin or VA significantly increased translocation of GLUT 4 to the plasma
membrane (Figure 3.1.7B&D). Na+-K
+-ATPase was used as plasma membrane
marker [140]. No significant changes were observed in the expression of Na+-K
+-
ATPase in different treatment groups (Figure 3.1.7b). A higher portion of GLUT 4
was observed to be retained in the light microsomal pool in DC compared to NC.
Metformin and VA treatments decreased the light microsomal GLUT 4 pools by
56.8% and 35.7% respectively, compared to vehicle in DC. The expression of Na+-
K+-ATPase in the light microsomal fraction was negligible (Figure 3.1.7c).
54
Figure 3.1.7. Long-term effects of VA on skeletal muscle GLUT 4 expression and
translocation in STZ-induced diabetic rats
A
GLUT 4
GLUT 1
NC DC Metformin VA
GAPDH
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
2
NC DC Metformin VA
Pro
tein
/ G
AP
DH
Den
sity
(A
rbit
rary
Un
it)
Treatments
GLUT 4
GLUT 1
**
*
Treatments (500mg/kg metformin, 400mg/kg VA and vehicles for both NC and DC)
were given twice daily through gavaging for 28 days. Soleus muscle was isolated for
the determination of A: total GLUT 4 expression through SDS-PAGE and
immunoblotting. Images shown are representative of the corresponding treatment
group.
Values were expressed as mean ± S.E.M. (n=6). NC=Normal Control, DC=Diabetic
Control, Metformin=metformin-treated diabetic, VA=Vernonia amygdalina-treated
diabetic. * P<0.05, ** P<0.01 compared to DC.
55
B
NC DC Metformin VA
GLUT 4
Na+-K+-ATPase
GAPDH
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
2
NC DC Metformin VA
Pro
tein
/ G
AP
DH
(A
rbit
rary
Un
it)
Treatment
GLUT 4
Na+-K+-
ATPase
***
Treatments (500mg/kg metformin, 400mg/kg VA and vehicles for both NC and DC)
were given twice daily through gavaging for 28 days. Soleus muscle was isolated for
the determination of B: amount of GLUT 4 on plasma membrane through SDS-PAGE
and immunoblotting. Na+-K
+-ATPase was used as a plasma membrane marker.
Images shown are representative of the corresponding treatment group.
Values were expressed as mean ± S.E.M. (n=6). NC=Normal Control, DC=Diabetic
Control, Metformin=metformin-treated diabetic, VA=Vernonia amygdalina-treated
diabetic. * P<0.05, ** P<0.01 compared to DC.
56
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
NC DC Metformin VA
Pro
tein
/ G
AP
DH
Den
sit
y (
Arb
itra
ry
Un
it)
Treatments
GLUT 4
Na+-K+-
ATPase
CNC DC Metformin VA
GLUT 4
Na+-K+-ATPase
GAPDH
**
*
D
0
1
2
3
4
5
6
NC DC Metformin VA
PM
/LM
GL
UT
4
(Arb
itra
ry
Un
it)
Treatments
*
*
Treatments (500mg/kg metformin, 400mg/kg VA and vehicles for both NC and DC)
were given twice daily through gavaging for 28 days. Soleus muscle was isolated for
the determination of C: amount of GLUT 4 remains on intracellular membranes
through SDS-PAGE and immunoblotting. Na+-K
+-ATPase was used as a plasma
membrane marker. D: Ratio of plasma membrane/inner membranes GLUT4. Images
shown are representative of the corresponding treatment group.
Values were expressed as mean ± S.E.M. (n=6). NC=Normal Control, DC=Diabetic
Control, Metformin=metformin-treated diabetic, VA=Vernonia amygdalina-treated
diabetic. * P<0.05, ** P<0.01 compared to DC.
57
3.1.8 Long-term effects of VA extract on muscle glycogen synthesis
STZ caused a decrease of 77% in muscle glycogen content compared to NC (Figure
3.1.8). Metformin significantly increased the glycogen level, compared to both DC
and NC, whereas VA only restored the glycogen level to normal.
Figure 3.1.8. Long-term effects of VA on skeletal muscle glycogen levels in STZ-
induced diabetic rats.
Treatments (500mg/kg metformin, 400mg/kg VA and vehicles for both NC and DC)
were given twice daily through gavaging for 28 days. Soleus muscle was isolated for
the determination of glycogen level.
Values were expressed as mean ± S.E.M. (n=6). NC=Normal Control, DC=Diabetic
Control, Metformin=metformin-treated diabetic, VA=Vernonia amygdalina-treated
diabetic. ** P<0.01 compared to DC.
58
3.1.9 Determination of main active constituents in VA extract
Figure 3.1.9 shows HPLC profile of ethanolic VA extract at 330 nm. 4 main peaks
were observed and further characterized by LC-ESI-MS analysis. Comparison
between previous literature data and LC-ESI-MS profile (peak # refers to those in
Figure 3.1.9) [Table 3.1.2] suggested that the 4 main components are 1,5-dicaffeoyl-
quinic acid, dicaffeoyl-quinic acid, chlorogenic acid and luteolin 7-O-glucoside.
Figure 3.1.9. Chemical profile of ethanolic VA extract.
HPLC analysis was performed at a mobile phase flow rate of 1mL/min, and the peaks
were monitored at 330 nm.
59
Table 3.1.2. Chemical profile of ethanolic extract of VA.
3.2 Studies of antidiabetic and antilipidemic effects of CGA
3.2.1 CGA lowers blood glucose levels in an OGTT on Leprdb/db
mice
Compared to lean control, glucose levels of DC group are significantly higher at all
time points during the OGTT, with a significant 127.7% higher in the total AUC. As a
positive control, metformin reduced fasting glucose levels in a near-linear manner
especially after glucose loading, with a suppression of 48.8% total AUC, compared to
DC. Intraperitoneal 250 mg/kg CGA decreased fasting blood glucose levels of
Leprdb/db
mice by 29.8±5% in the first 10 minutes before glucose challenge. After
glucose loading, the glucose-lowering effect continued to increase up to 35.7±4% for
30 minutes. At 2 hours post-glucose loading, this effect diminished gradually and the
fasting glucose levels returned to that observed in -10-minute (Figure 3.2.1). The
AUC of CGA-mediated glucose lowering decreased by 49.6%, compared to DC
(Figure 3.2.1B). However, this hypoglycemic effect of CGA was abated by pre-
intraperitoneal administration of compound c, an AMPK inhibitor (Figure 3.2.1A).
Nevertheless, the AUC of CGA-stimulated fasting glucose after the pre-treatment of
Peak
#
Rt
(min)
max (nm) MS
[M–H]－
MS/MS Tentative
ID
Quantitative
Estimation
(%Area)
1 12.729 242, 328 353 191,
179
Chlorogenic
acid
26.13
2 17.950 254, 348 447 285 Luteolin 7-
O-glucoside
6.93
3 20.684 244, 330 515 353,
191,179
1,5-
Dicaffeoyl-
quinic acid
18.81
4 21.501 244, 330 515 353,
191,179
Dicaffeoyl-
quinic acid
42.82
60
compound c was still lower compared to DC (Figure 3.2.1B). Interestingly, we
observed a decrease in the efficiency of compound c in inhibiting the glucose-
lowering effect of CGA after 15-day treatment with CGA. Instead, a higher dose of
compound c (100mg/kg) was required to achieve similar inhibitory effect on the
OGTT curve (Figure 3.2.2A). Figure 3.2.2B shows AUC of OGTT after
administration of compound c at Day 0 and Day 15. Efficiency of compound c
(50mg/kg) in inhibiting glucose-lowering effect of CGA was decreased by 21.5%
after 15-day treatment with CGA (Figure 3.2.2B).
61
Figure 3.2.1. Acute effects of CGA on glucose tolerance in Leprdb/db
mice and effect
of compound c on CGA-mediated glucose lowering
A: Oral glucose tolerance test was performed in 6-hour fasted Leprdb/db
mice. For the
inhibitor study, mice were pre-treated with compound c for 10 minutes. Blood
samples were collected from the tail vein for fasting glucose measurement before
treatments (vehicle, i.p. 250 mg/kg CGA, oral 250 mg/kg metformin). Ten minutes
after the treatments, blood samples were collected again followed by oral gavaging of
2 g/kg glucose. Blood samples were collected 15, 30, 60 and 120 minutes after the
glucose challenge. B: AUC of fasting glucose levels in OGTT (A).
Data were expressed as the means ± SE of three independent experiments.
DC=Diabetic Control, Met=Metformin. *P<0.05 compared to diabetic control,
*P<0.05 compared to CGA only-treated mice, #P<0.05 compared to lean control.
62
Figure 3.2.2. Decreased inhibitory effect of compound c in suppressing CGA-
mediated glucose lowering after 2-week treatment with CGA
A: Efficiency of different concentrations of compound c in inhibiting CGA-mediated
glucose lowering was measured before and after 2-week treatment with CGA. Pre-
treatment of compound c was performed intreaperitoneally 10 mins before
administration of CGA. B: AUC of fasting glucose levels in OGTT (C).
Data were expressed as the means ± SE of three independent experiments.
DC=Diabetic Control, Met=Metformin. *P<0.05 compared to mice treated with
compound c at Day 0.
63
3.2.2 2-week treatment with CGA reduces body weight, water intake and
improves glucose and lipid profiles
Leprdb/db
mice demonstrated apparent phenotypes of metabolic syndromes including
glucose intolerance, hyperinsulinemia, obesity, polyphagia, polydipsia and
dyslipidemia as compared to lean C57BL/6 mice (Table 3.2.1 and Figure 3.2.3A-F).
Leprdb/db
mice showed significantly higher food and water intakes, body weight,
fasting glucose and insulin levels, serum triglyceride (TG), total cholesterol (TC), and
free fatty acids (FFA). However, serum adiponectin was significantly lower in
Leprdb/db
mice, compared to lean animals. Leprdb/db
mice treated with CGA or
metformin showed similar food intakes which were significantly lower compared to
vehicle-treated DC. Together with improved lipid profiles (reduced serum TG, TC
and FFA) (Figure 3.2.3C-E), CGA- or metformin-treated animals demonstrated a
significant reduction in body weights. A day after treatments with CGA or metformin,
water intakes of the animals were found to be significantly decreased, indicating
improved blood glucose level which will otherwise cause excessive urination and
thirst, owing to the excretion of excessive glucose in urine (glucosuria). This
condition continued to improve over the 2-week treatment with CGA or metformin
(Table 3.2.1). Consistent with this, fasting glucose (Figure 3.2.3A) and insulin (Figure
3.2.3B) levels in the CGA- or metformin-treated groups were significantly lower
compared to vehicle-treated DC.
64
Figure 3.2.3. Chronic effects of CGA on glucose and lipid profiles and insulin
sensitivity in Leprdb/db
mice.
Serum was collected after the chronic treatment for the measurement of A: fasting
glucose and B: fasting insulin levels.
Data were expressed as the means ± SE of three independent experiments.
DC=Diabetic Control, Met=Metformin. *P<0.05 compared to diabetic control,
#P<0.05 compared to lean control.
65
Serum was collected after the chronic treatment for the measurement of C: total
cholesterol and D: triglyceride levels.
Data were expressed as the means ± SE of three independent experiments.
DC=Diabetic Control, Met=Metformin. *P<0.05 compared to diabetic control,
#P<0.05 compared to lean control.
66
Serum was collected after the chronic treatment for the measurement of E: free fatty
acids and F: adiponectin levels.
Data were expressed as the means ± SE of three independent experiments.
DC=Diabetic Control, Met=Metformin. *P<0.05 compared to diabetic control,
#P<0.05 compared to lean control.
67
G: At the end of the study, ITT was performed on the animals. Blood samples were
collected from tail vein for fasting glucose measurement 15, 30, 60 and 120 minutes
after insulin injection. H: AUC of fasting glucose levels in ITT (G).
Data were expressed as the means ± SE of three independent experiments.
DC=Diabetic Control, Met=Metformin. *P<0.05 compared to diabetic control
injected with 0.75U/kg insulin.
68
I: HOMAIR index of insulin resistance was determined as: fasting blood glucose
(mg/dL) X fasting serum insulin (µU/ml)/ 405.
Data were expressed as the means ± SE of three independent experiments.
DC=Diabetic Control, Met=Metformin. *P<0.05 compared to diabetic control,
#P<0.05 compared to lean control.
69
Table 3.2.1. Body weights, food and water intakes in Leprdb/db
mice following 2-
week treatment with CGA or metformin
Treatment Body Weight (g) Food Intake (g/kg) Water Intake (g/kg)
Day 1 Day 14 Day 1 Day 14 Day 1 Day 14
Lean 21.5 ± 1.8 22.5 ± 2.3 0.55 ±
0.05
0.60 ±
0.11
0.70 ±
0.08
0.80 ±
0.09
DC 51.5 ± 4.4 #
53.5 ± 5.4 #
0.66 ±
0.09 #
0.81 ±
0.11 #
1.51 ±
0.17 #
1.89 ±
0.29 #
Metformin 53.25 ±
4.5
50.25 ±
6.0 *
0.63 ±
0.14
0.74 ±
0.15
0.69 ±
0.22 *
0.45 ±
0.18 *
CGA 50.75 ±
3.9
44.75 ±
5.8 *
0.69 ±
0.09
0.73 ±
0.17
0.97 ±
0.25 *
0.59 ±
0.22 *
*P<0.05 compared to DC, #P<0.05 compared to lean control
3.2.3 2-week treatment with CGA improves glucose tolerance and insulin
sensitivity in Leprdb/db
mice
Insulin resistance is a major concomitant phenotype associated with obesity in
Leprdb/db
mice. Compared to lean control, they showed an approximate five-fold
incease in HOMAIR index. However, CGA or metformin treatment significantly
attenuated insulin resistance by 60% and 53% respectively in this diabetic model
(Figure 3.2.3I), consistent with increased insulin sensitivity in an ITT after 2-week
treatment with CGA (Figure 3.23G-H). In the ITT, exogenous insulin (0.75U/kg) did
not cause significant lowering of glucose levels in DC, compared to NC. The effect
can only be seen by injecting 4X the normal concentration of insulin (3U/kg).
However, after 2-week treatment with CGA, 0.75 U/kg insulin significantly decreased
blood glucose levels, comparable to those achieved by 3 U/kg insulin in DC,
indicating attenuated insulin resistance. This was further supported by the insulin area
under the curve (AUCinsulin) in an OGTT. AUCinsulin was higher in Leprdb/db
mice
compared to lean C57BL/6 and it was significantly elevated after two weeks,
suggesting diminished insulin sensitivity (3.2.4B&D). Treatment with CGA or
70
metformin for 15 days significantly lowered AUCinsulin in the OGTT, suggesting
improved insulin sensitivity (Figure 3.2.4B&D). In addition, glucose area under curve
(AUCglucose) was also significantlyly reduced after 15-day treatment with CGA or
metformin, compared to DC (Figure 3.2.4A&C). This indicates that both CGA and
metformin treatments ameliorated glucose intolerance.
71
Figure 3.2.4. Chronic effects of CGA on glucose tolerance and insulin levels in
Leprdb/db
mice.
A: Oral glucose tolerance test was performed in 6-hour fasted Leprdb/db
mice. Blood
samples were collected from the tail vein for glucose levels measurement before
treatments (vehicle, i.p. 250 mg/kg CGA, oral 250 mg/kg metformin). Ten minutes
after the treatments, blood samples were collected again followed by oral gavaging of
2 g/kg glucose. Blood samples were collected 15, 30, 60 and 120 minutes after
glucose challenge for the measurement of glucose levels. Results were compared to
those obtained from the acute study at Day 0.
Data were expressed as the means ± SE of three independent experiments.
DC=Diabetic Control, Met=Metformin. *P<0.05, **P<0.01 compared to respective
groups at Day 0.
72
B: Oral glucose tolerance test was performed in 6-hour fasted Leprdb/db
mice. Blood
samples were collected from the tail vein for insulin levels measurement before
treatments (vehicle, i.p. 250 mg/kg CGA, oral 250 mg/kg metformin). Ten minutes
after the treatments, blood samples were collected again followed by oral gavaging of
2 g/kg glucose. Blood samples were collected 15, 30, 60 and 120 minutes after the
glucose challenge for the measurement of insulin levels. Results were compared to
those obtained from the acute study at Day 0.
Data were expressed as the means ± SE of three independent experiments.
DC=Diabetic Control, Met=Metformin. *P<0.05, **P<0.01 compared to respective
groups at Day 0.
73
C: AUC of fasting glucose levels in OGTT (A). D: AUC of fasting insulin levels in
OGTT (B). Results were compared to those obtained from the acute study at Day 0.
Data were expressed as the means ± SE of three independent experiments.
DC=Diabetic Control, Met=Metformin. *P<0.05, **P<0.01 compared to respective
groups at Day 0.
74
3.2.4 CGA inhibits gluconeogenesis in Leprdb/db
mice through downregulation of
gluconeogenic G6Pase
To assess the effect of CGA on gluconeogenesis in vivo, the present author performed
a pyruvate tolerance test (PTT), as administration of the gluconeogenic substrate
(pyruvate) increases blood glucose level by promoting gluconeogenesis in the liver.
Pyruvate intolerance indicated hepatic insulin resistance in DC (>100% increase in
AUC of pyruvate tolerance test, compared to normal lean control) [Figure 3.2.5A-B].
Treatment with CGA or metformin significantly ameliorated pyruvate intolerance
(28% and 35% reduction in AUC, respectively, compared to Leprdb/db
mice). Also,
elevation of glucose levels induced by sodium pyruvate was effectively lowered by
CGA at all time points of the test. To further investigate the effect of CGA on hepatic
insulin resistance in Leprdb/db
mice, he examined its effect on hepatic gluconeogenic
enzymes, G6Pase and PEPCK. As expected, under the condition of the hepatic insulin
resistance, the expressions of G6Pase and PEPCK were significantly increased in
Leprdb/db
mice (Figure 3.2.6A). Both CGA and metformin decreased the expression of
G6Pase but they did not affect the expression of PEPCK. Consistent with this, the
activity of G6Pase was lower in both CGA- and metformin-treated Leprdb/db
mice
(Figure 3.2.6B).
75
Figure 3.2.5. CGA decreases glucose production from gluconeogenic pyruvate in a
pyruvate tolerance test on Leprdb/db
mice.
A: Pyruvate tolerance test was performed on 6-hour fasted Leprdb/db
mice. Mice were
treated with vehicle, ip 250 mg/kg CGA, oral 250 mg/kg metformin. Ten minutes
after the treatment, blood samples were collected for the measurement of blood
glucose levels, followed by ip administration of 2 g/kg sodium pyruvate. Blood
samples were collected 15, 30, 60 and 120 minutes after the pyruvate challenge. B:
AUC fasting glucose levels in PTT (A).
Data were expressed as the means ± SE of three independent experiments.
DC=Diabetic Control, Met=Metformin. #P<0.05 compared to lean control, *P<0.05
compared to diabetic control.
76
Figure 3.2.6. CGA inhibits expression and activity of hepatic G6Pase in Leprdb/db
mice
After 2-week treatment, liver was collected and homogenized for A: Quantitation of
gluconeogenic genes expression, using immunoblotting.
Data were expressed as the means ± SE of three independent experiments.
DC=Diabetic Control, Met=Metformin. *P<0.05 compared to diabetic control,
#P<0.05 compared to lean control.
77
After 2-week treatment, liver was collected and homogenized for B: microsomes
isolation and measurement of G6Pase activity.
Data were expressed as the means ± SE of three independent experiments.
DC=Diabetic Control, Met=Metformin. *P<0.05 compared to diabetic control,
#P<0.05 compared to lean control.
78
3.2.5 Suppression of glucose production and G6Pase expression in HepG2
hepatoma by CGA
To further evaluate the role of CGA on gluconeogenesis, the present author studied
the effect of CGA on glucose production in HepG2 hepatoma cells. As shown in
figure 3.2.7A & B, CGA effectively inhibited glucose synthesis in vitro, in dose- and
time- dependent manners, for up to 50%, as compared to vehicle. In addition,
expression of G6Pase in HepG2 was correspondingly inhibited by CGA, suggesting
an evident role of CGA on gluconeogenesis inhibition through the regulation of
G6Pase (Figure 3.2.7C & D). Again, he did not detect any changes in the expression
of PEPCK by CGA or metformin in HepG2 cell line.
3.2.6 CGA ameliorates hepatic lipid accumulation, triglyceride and total
cholesterol levels in Leprdb/db
mice
Non-alcoholic fatty liver or non-alcoholic hepatic steatosis is one of the metabolic
syndromes caused by impaired leptin signaling [144]. Due to the disruption of leptin
signaling in this animal model, histological examination of the liver in Leprdb/db
mice
showed lipid accumulation in the vesicles of hepatocytes and eventually fatty
degeneration of hepatocytes (arrows) [Figure 3.2.8]. Treatment with CGA or
metformin significantlyly attenuated the vacuolar degeneration, as shown by the
significant decrease in the formation of fat vacuoles in the liver sections. Likewise,
triglyceride and total cholesterol levels in the liver of Leprdb/db
mice were higher
(more than 2 times), if compared to lean control. Treatment with metformin or CGA
significantly improved the elevated lipids content in the liver (Figure 3.2.9 A-B).
79
Figure 3.2.7. CGA suppresses glucose production and expression of G6Pase in
HepG2 hepatoma cells.
HepG2 hepatoma cells treated in various A: doses of CGA and B: incubation periods
were washed and incubated with glucose production buffer for four hours. Medium
was collected for the measurement of glucose produced. Readings were then
normalized to the total protein content.
Data were expressed as the means ± SE of three independent experiments.
DC=Diabetic Control, Met=Metformin, Veh=vehicle-treated control. *P<0.05,
**P<0.01 compared to vehicle-treated control.
80
C: HepG2 cells treated in various doses of CGA were lysed for the quantitation of
gluconeogenic genes expression, using immunoblotting.
Data were expressed as the means ± SE of three independent experiments.
DC=Diabetic Control, Met=Metformin, Veh=vehicle-treated control. *P<0.05,
**P<0.01 compared to vehicle-treated control.
81
D: HepG2 cells treated in various incubation periods were lysed for the quantitation of
gluconeogenic genes expression, using immunoblotting.
Data were expressed as the means ± SE of three independent experiments.
DC=Diabetic Control, Met=Metformin, Veh=vehicle-treated control. *P<0.05,
**P<0.01 compared to vehicle-treated control.
82
Figure 3.2.8. CGA ameliorates hepatic lipid accumulation in Leprdb/db
mice.
Isolated livers were fixed in 10% buffered formalin for 24 hours at 4ºC. Fixed tissues
were processed and sectioned into 4µm-thick slices. Tissue sections were stained with
H&E for normal histological assessment, where cytoplasm stained pink and nuclei
stained deep purple.
Arrows indicate vacuoles formed as a result of lipid accumulation. Images shown
(scale bar = 50 µm) are representatives of the corresponding treatment group.
DC=Diabetic Control, Met=Metformin.
83
Figure 3.2.9. CGA lowers hepatic triglyceride and total cholesterol levels.
Liver was homogenized in 10% w/v isopropanol and kept at 4ºC for two days.
Homogenate was centrifuged at 4ºC and 1 000g for 10 minutes. Supernatant was
collected for the measurements of A: total cholesterol and B: triglyceride levels.
Data were expressed as the means ± SE of three independent experiments.
DC=Diabetic Control, Met=Metformin. *P<0.05 compared to diabetic control,
#P<0.05 compared to lean control.
84
3.2.7 CGA decreases oil droplets formation in HepG2 Cells
In line with the in vivo findings, CGA or metformin reduced the formation of oil
droplets in HepG2 cells. In the vehicle-treated cells, the oil droplets were big and
obvious (arrow) while in CGA- or metformin-treated cells, the oil droplets were
smaller and less dense (Figure 3.2.10 A). There was a decrease of 37.8% in the OD of
eluted dye from CGA-treated cells, compared to vehicle-treated cells (Figure 3.2.10
B).
3.2.8 Amelioration of hepatic lipid accumulation by CGA is mediated through
inhibition of fatty acid synthesis
All of the above data suggested that at least one of the intermediary building-blocks
for the synthesis of a more complex lipid molecule in the liver was reduced by the
treatment with CGA or metformin. Therefore, the present authors examined the effect
of CGA on fatty acid synthesis in HepG2 cells. Inhibition of fatty acid synthesis as
measured by the incorporation of 14
C-acetate into fatty acids was observed in CGA- or
metformin-treated hepatocytes. CGA inhibited fatty acid synthesis in a dose- (Figure
3.2.11 A) and time-dependent (Figure 3.2.11 B) manner.
85
Figure 3.2.10. CGA decreases oil droplets formation in HepG2 cells
A: Fixed HepG2 cells were stained with Oil Red O in 60% isopropanol for ten
minutes at room temperature. Accumulation of fat droplets was examined under
inverted light microscope (scale bar = 25µm). B: Quantitation was done by eluting the
stain with 100% isopropanol. OD was measured at 500nm
Images shown are representative of the corresponding treatment group. DC=Diabetic
Control, Met=Metformin.**P<0.01, compared to vehicle-treated control
86
Figure 3.2.11. CGA inhibits fatty acid synthesis in HepG2 cells
Treated HepG2 cells were incubated with 14
C-sodium acetate (2µCi/ml) for 4 hours at
37ºC. Cells were lysed and radioactivity incorporated into fatty acid was monitored
using scintillation counter. Readings were expressed as percent inhibition of fatty acid
synthesis compared to control.
Data were expressed as the means ± SE of three independent experiments.
DC=Diabetic Control, Met=Metformin, Veh=vehicle-treated control. *P<0.05,
**P<0.01 compared to vehicle-treated control.
87
3.2.9 Acute stimulation of glucose uptake by CGA in skeletal muscle isolated
from Leprdb/db
mice
Skeletal muscles isolated from Leprdb/db
mice showed significant increase in glucose
transport after treated with CGA (Figure 3.2.12). CGA (2 mmol/l) increased the
glucose transport of myotubes by 48±13% and the stimulation continued to increase
and achieve stability at higher concentrations. No significant difference in glucose
transport was observed between lean mice and diabetic mice. However, a significant
decrease of 54±3% in insulin-stimulated glucose transport was shown in diabetic mice
compared to lean mice. CGA treatment further enhanced the insulin-mediated glucose
transport for up to 145±13% at concentrations of 4, 8 and 10 mmol/l, compared to
diabetic control.
88
Figure 3.2.12. Acute stimulation of glucose uptake by in skeletal muscles isolated
from Leprdb/db
mice
Skeletal muscles were isolated from Leprdb/db
mice and treated with CGA or/ and 100
nmol/l insulin or 2 mmol/l metformin for 30 minutes. 2-deoxyglucose uptake was
measure over a 30-minute period, using liquid scintillation counter.
Data were expressed as the mean ± SE of three independent experiments.
DC=Diabetic Control, DMet=Diabetic treated with metformin, D(0.5, 1, 2, 4, 8,
10)=Diabetic treated with various concentrations of CGA. *P<0.05, **P<0.01
compared with controls. DC=Diabetic Control.
3.2.10 Chronic treatment with CGA increases glucose uptake in skeletal muscles
by increasing GLUT 4 expression and translocation to plasma membrane
GLUT is a superfamily of genes, encoding homologous proteins with different
functional properties and tissue-specific expressions [145]. Glucose transport is
mediated by the members of GLUT protein family, which consists of 12
transmembrane transporters [146]. GLUT 4 is exclusively expressed in peripheral
insulin-sensitive tissues like fat, skeletal and cardiac muscles. It is the only insulin-
responsive GLUT identified so far, with the observation that its level of expression in
89
various muscles and fat cells generally corresponded to the magnitude of insulin-
stimulated glucose disposal in the tissues [147]. Later on, it was established that this
insulin-stimulated glucose transport was mediated through the redistribution of GLUT
4 from the intracellular membrane compartment to the cell surface [148].
Expression of GLUT 4 and translocation of GLUT 4 to plasma membrane of skeletal
muscles were dramatically decreased in Leprdb/db
mice, as shown by
immunohistochemistry staining, compared to normal lean mice (Figure 3.2.14A-D). 2
weeks of treatments with CGA or metformin sinificantly elevated the expression and
translocation of GLUT 4, as depicted by the increase in stained GLUT 4 in both
intracellular and plasma membrane compartments of the skeletal muscles. To further
corroborate these findings, the present authors quantitated the amount of GLUT 4
protein from both whole tissue lysate and fractionated plasma membrane of the
skeletal muscles using immunoblotting. Consistently, GLUT 4 content was lower in
both whole tissue lysate and plasma membrane fraction in vehicle-treated Leprdb/db
mice, suggesting insulin resistance in skeletal muscle. However, treatment with CGA
or metformin significantly increased the expression and translocation of GLUT 4
(Figure 3.2.14E). We next examined the effect of CGA on glucose transport in the
skeletal muscles to provide further confirmatory evidence for the stimulatory effect of
CGA on GLUT 4 expression and translocation. Consistent with decreased plasma-
membrane GLUT 4 available for glucose transport into skeletal muscles of Leprdb/db
mice, glucose uptake by the tissues were significantly lower, compared to lean control
(Figure 3.2.13). In addition, exogenous insulin failed to stimulate glucose transport
into these tissues, indicating insulin resistance. As expected, increased plasma-
membrane GLUT 4 by CGA caused a rise of 67.9% in glucose transport of skeletal
90
muscles, compared to vehicle-treated skeletal muscles. In the presence of insulin,
CGA caused a more than 2.5-fold increase in glucose transport in skeletal muscles,
compared to vehicle-treated control, suggesting an additive effect of CGA treatment
to insulin action (Figure 3.2.13).
Figure 3.2.13. Chronic treatment with CGA increases glucose uptake in skeletal
muscles
Skeletal muscles isolated from Leprdb/db
mice after 2 weeks of treatments were
incubated with 0.5 ml KRBB containing 1 µCi/ ml 2-Deoxy-[3H]D-glucose for 30
minutes at 37ºC. Quatitation of 2DG taken up by the tissue was performed using
liquid scintillation counter. Readings were expressed as nmol 2DG uptake per mg
tissue in one minute.
Data were expressed as the means ± SE of three independent experiments.
DC=Diabetic Control, Met=Metformin, Veh=vehicle-treated control. *P<0.05,
**P<0.01 compared to diabetic control, #P<0.05 compared to lean control.
91
Figure 3.2.14. Chronic CGA treatment increases GLUT 4 expression and
translocation to plasma membrane.
A: Skeletal muscle sections were stained with DAPI for nuclear staining. Images were
captured using fluorescence microscope (scale bar = 50µm).
Images shown are representatives of the corresponding treatment group from three
independent experiments. DC=Diabetic Control, Met=Metformin.
92
B: Skeletal muscle sections were incubated with and anti-e-cadherin (plasma
membrane marker) overnight at 4ºC before staining with Alexa Fluor 555-conjugated
mouse IgG secondary antibody.
Images were captured using fluorescence microscope (scale bar = 50µm). Images
shown are representatives of the corresponding treatment group from three
independent experiments. DC=Diabetic Control, Met=Metformin.
93
C: Skeletal muscle sections were incubated with anti-GLUT 4 overnight at 4ºC before
staining with FITC-conjugated rabbit IgG secondary antibody. Images were captured
using fluorescence microscope (scale bar = 50µm).
Images shown are representatives of the corresponding treatment group from three
independent experiments. DC=Diabetic Control, Met=Metformin.
94
D: Merged images of (B) and (C). DC=Diabetic Control, Met=Metformin.
95
E: GLUT 4 was quantitated in total cell lysate and plasma membranes fractionated
from skeletal muscles of Leprdb/db
after treatments for 2 weeks, using immunoblotting.
Data were expressed as the means ± SE of three independent experiments.
DC=Diabetic Control, Met=Metformin, Veh=vehicle-treated control. *P<0.05,
**P<0.01 compared to diabetic control, #P<0.05 compared to lean control.
96
3.2.11 Dose- and time-dependent stimulation of glucose transport by CGA in L6
myotubes
In order to strengthen our in vivo findings, the present authors next investigated the
effects of CGA on glucose transport in L6 myotubes. Treatment of myotubes with
increasing concentrations of CGA for 24 hours led to a significant dose-dependent
increase in glucose transport, which was first observed at 1 mmol/l (55±8%). The
highest increase was observed at 2 mmol/l (63±6%) and the stimulation was
maintained up to the highest concentration tested, which was 10 mmol/l (Figure
3.2.15A). Besides, a time-dependent stimulation of glucose transport was also
observed when myotubes were incubated with 2 mmol/l CGA at different incubation
periods. Significant increase was first observed after 1-hour incubation (14±1%), and
continued to increase to 60±5% after 24 hours of incubation (Figure 3.2.15B). In all
subsequent experiments, the effects of CGA were determined at a concentration of 2
mmol/l and incubation period of 24 hours.
\
97
Figure 3.2.15. Dose- and time-dependent stimulation of glucose transport in L6
myotubes by CGA.
A: L6 myotubes were incubated with incremental concentrations of CGA for 24 hours.
B: L6 myotubes were incubated with 2 mmol/l CGA at different incubation periods up
to 24 hours. 2-deoxyglucose uptake was measure over a 30-minute period, using
liquid scintillation counter.
Data were expressed as percentage increase over basal uptake of cells incubated with
vehicle. Data were expressed as the mean ± SE of three independent experiments.
*P<0.05, **P<0.01 compared with vehicle-treated control.
98
3.2.12 CGA stimulates GLUT 4 translocation to plasma membrane in L6
myotubes
From our in vivo study, the present author knows that simulation of glucose transport
in skeletal muscles by CGA was associated with increased GLUT 4 expression and
translocation to plasma membrane. He therefore investigated whether the glucose
transport in response to CGA in L6 myotubes was accompanied by an increase in
expression and translocation of the GLUT 4 to the plasma membrane of myotubes.
Also, in this in vitro model, he investigated the effect of CGA on another GLUT
member, GLUT 1. The broad distribution of GLUT 1makes it an important
transporter regulating the basal glucose disposal. Its expression has been shown to be
altered by sulfonylureas [149], insulin [150], hypoxia [151], insulin-like growth
factor-1[152] and a number of other factors. Treatment of myotubes with CGA for
different time periods augmented GLUT 4 content on plasma membrane of myotubes,
with the significant increase first observed at 1-hour incubation, increasing in a time-
dependent manner for up to 24 hours (Fig 3.2.16B). Metformin demonstrated a similar
increase in GLUT 4 on plasma membrane of myotubes. However, neither metformin
nor CGA caused significant changes in total GLUT 4 expression or plasma
membrane-GLUT 1 (Figure 3.2.16A-B).
99
Figure 3.2.16. CGA stimulates GLUT 4 translocation to plasma membrane in
myotubes
Myotubes were treated with 2 mmol/l CGA for various incubation periods up to 24
hours. A: Whole cell lysate was used to detect for GLUT 4 and GLUT 1 expression
through immunoblotting. Illustrated are the representative images of three
independent experiments.
Data were expressed as the mean ± SE of three independent experiments. *P<0.05,
**P<0.01 compared with controls.
100
Myotubes were treated with 2 mmol/l CGA for various incubation periods up to 24
hours. B: isolated plasma membranes were used to detect for amount of GLUT 4 or
GLUT 1 translocated through immunoblotting. Illustrated are the representative
images of three independent experiments.
Data were expressed as the mean ± SE of three independent experiments. *P<0.05,
**P<0.01 compared with controls.
101
3.3 Studies of molecular pathways that mediate beneficial metabolic
effects of CGA
3.3.1 CGA increases AMPK and ACC phosphorylations in response to Ca2+
influx in HepG2 hepatoma cells
In HepG2 cells, CGA induced AMPK phosphorylation in a time- (Figure 3.3.1A) and
dose-dependent (Figure 3.3.1B) manner. AMPK immnunoprecipitated from treated
cells were evaluated for its kinase activity against SAMS peptide and it showed that
CGA activated and increased AMPK activity in a similar manner (Figure 3.3.1C-D).
To further elucidate its activation by CGA, the present author investigated the effect
of CGA on its downstream substrate, ACC. Increased ACC phosphorylation
correlated with AMPK activation was observed in both treatments with CGA or
metformin (Figure 3.3.1A-B). Since AMPK activation inhibits ACC activity, we
observed decreases in ACC activities in response to CGA or metformin treatments
(Figure 3.3.1E-F), and therefore it is evident that CGA or metformin induced
activation of AMPK but inhibited ACC. Several upstream signaling were known to
phosphorylate AMPK such as cellular stress that causes a fall in ATP: AMP ratio
[153], tumor suppressor kinase LKB-1 [154] and CaMKK [155]. He showed that
activation of AMPK by CGA was mediated by CAMKKβ in HepG2 hepatoma
cells(Figure 3.3.1A-B). In addition, we further demonstrated that treatment of HepG2
hepatoma cells with CGA resulted in an increase in the intracellular Ca2+
concentration measured by Fluo-4 (Figure 3.3.1G-H), suggesting that CGA caused
influx of extracellular Ca2+
into hepatoma cells, thereby activating CAMKK which in
turn phosphorylated AMPK.
102
Figure 3.3.1. CGA increases AMPK and ACC phosphorylations in response to Ca2+
influx in HepG2 hepatocytes.
A: Treated HepG2 cells in various incubation periods were lysed for the quantitation
of pAMPK, total AMPK, pACC, total ACC and CAMKK using immunoblotting.
Phosphorylated proteins were adjusted to total proteins while CAMKK was adjusted
to GAPDH.
Data were expressed as the means ± SE of three independent experiments.
DC=Diabetic Control, Met=Metformin, Veh=vehicle-treated control. *P<0.05,
**P<0.01 compared to vehicle-treated control.
103
B: Treated HepG2 cells with various CGA concentrations were lysed for the
quantitation of pAMPK, total AMPK, pACC, total ACC and CAMKK using
immunoblotting. Phosphorylated proteins were adjusted to total proteins while
CAMKK was adjusted to GAPDH.
Data were expressed as the means ± SE of three independent experiments.
DC=Diabetic Control, Met=Metformin, Veh=vehicle-treated control. *P<0.05,
**P<0.01 compared to vehicle-treated control.
104
C-D: Treated cells were lysed and immunoprecipitated with anti-AMPK α1/2
antibody. Kinase reaction was initiated against SAMS peptides in the presence of γ-
ATP. Radioactivity was measured using liquid scintillation and kinase activity was
expressed as incorporated ATP/mg protein/minute.
Data were expressed as the means ± SE of three independent experiments.
DC=Diabetic Control, Met=Metformin, Veh=vehicle-treated control. *P<0.05,
**P<0.01 compared to vehicle-treated control.
105
E-F: Treated cells were lysed and assayed against NaH14
CO3 for 40 minutes at room
temperature. Total 14
C incorporated was measured using liquid scintillation counter
and ACC activity was expressed as incorporated 14
C /mg protein/minute. G-H:
Treated cells were incubated with equal volume of 2 X Fluo-4 Direct calcium reagents
for one hour at 37˚C. Fluorescence was measured at 494 nm (excitation) and 516 nm
(emission).
Readings were expressed as % increase in free calcium influx compared to vehicle-
treated cells. Data were expressed as the means ± SE of three independent
experiments. DC=Diabetic Control, Met=Metformin, Veh=vehicle-treated control.
*P<0.05, **P<0.01 compared to vehicle-treated control.
106
G-H: Treated cells were incubated with equal volume of 2 X Fluo-4 Direct calcium
reagents for one hour at 37˚C. Fluorescence was measured at 494 nm (excitation) and
516 nm (emission). Readings were expressed as % increase in free calcium influx
compared to vehicle-treated cells.
Data were expressed as the means ± SE of three independent experiments.
DC=Diabetic Control, Met=Metformin, Veh=vehicle-treated control. *P<0.05,
**P<0.01 compared to vehicle-treated control.
107
3.3.2 Chronic treatment with CGA increases phosphorylations of AMPK and
ACC and expression of CAMKKβ in liver and skeletal muscles of Leprdb/db
mice
Leptin has been shown to stimulate phosphorylation of AMPK in skeletal muscles
through elevation of AMP levels in the muscles [156]. The present author's data
showed that in this leptin-signaling deficient model, phosphorylation of AMPK was
significantly lowered in both liver and skeletal muscles, compared to NC (Figure
3.3.2), consistent with previous proposal that there is a close association between
AMPK activation and leptin sensitivity [157]. Two-week treatment with CGA or
metformin increased phosphorylation of AMPK in the liver and skeletal muscles of
the animals, which in turn resulted in increased phosphorylation of its downstream
substrated, ACC. Consistently, CGA was found to increase expression of CAMKKβ
in the animal but metformin did not significantly increase its expression, indicating
CGA and metformin may activate AMPK via two different upstream regulators.
Interestingly, for the first time, he observed a decreased CAMKKβ expression in this
leptin receptor-deficient model, creating a question whether disruption in leptin
signalling will lead to the lower expression of CAMKKβ.
108
Figure 3.3.2. Chronic CGA administration phosphorylates AMPK and ACC in liver
and skeletal muscles of Leprdb/db
mice.
A: At the end of the treatment, liver were collected and homogenized to quantitate for
pAMPK, total AMPK, pACC, total ACC and CAMKK using immunoblotting.
Phosphorylated proteins were adjusted to total proteins while CAMKK was adjusted
to GAPDH.
Data were expressed as the means ± SE of three independent experiments.
DC=Diabetic Control, Met=Metformin, Veh=vehicle-treated control. *P<0.05,
**P<0.01 compared to vehicle-treated control.
109
B: At the end of the treatment, isolated skeletal muscles were collected and
homogenized to quantitate for pAMPK, total AMPK, pACC, total ACC and CAMKK
using immunoblotting. Phosphorylated proteins were adjusted to total proteins while
CAMKK was adjusted to GAPDH.
Data were expressed as the means ± SE of three independent experiments.
DC=Diabetic Control, Met=Metformin, Veh=vehicle-treated control. *P<0.05,
**P<0.01 compared to vehicle-treated control.
110
3.3.3 Inhibition and knockdown of AMPK abolished CGA-inhibited
gluconeogenesis and fatty acid synthesis in HepG2 cells
The present author next examined whether AMPK is responsible for the inhibition of
hepatic gluconeogenesis and fatty acid synthesis by CGA. As predicted, inhibitory
effects of CGA on glucose production and fatty acid synthesis were abolished by pre-
incubation with the AMPK inhibitor, compound c (Figure 3.3.3B&C). Also, AMPK
knockdown by using siRNA almost completely abrogated CGA-mediated inhibition
of glucose production and fatty acid synthesis in HepG2 hepatoma. Besides, CAMKK
selective inhibitor, STO-609, resulted in similar effect on the inhibition of glucose
production and fatty acid synthesis by CGA. Taken together, in vivo or in vitro, he
provided evidence that AMPK is vital for the action of CGA on hepatic glucose and
lipid metabolism.
3.3.4 CGA stimulates phosphorylations of AMPK and ACC in L6 myotubes
AMPK activation has been associated with increased GLUT 4 translocation to plasma
membrane and enhanced glucose transport [158, 159]. The present author has shown
that AMPK is vital for CGA to inhibit hepatic glucose production and fatty acid
synthesis in HepG2 hepatoma. Likewise, long-term administration of CGA in
Leprdb/db
mice resulted in phosphorylations of AMPK and ACC in animal skeletal
muscles. Thus, to further show that AMPK plays an important role in CGA-mediated
glucose transport in skeletal muscle, he evaluated the effect of CGA on AMPK and
ACC phosphorylations in L6 myotubes. Like metformin, CGA increased AMPK
phosphorylation and also ACC phosphorylation in a time- (Figure 3.3.4A) and dose-
dependent (Figure 3.3.4B) manners. In addition, AMPK activity was increased time-
and dose-dependently also by CGA and therefore explaining its phosphorylation
111
stimulated by CGA (Figure 3.3.5A&B). In line with our findings in vivo, activation of
AMPK by CGA was also mediated by CaMKK in L6 myotubes (Figure 3.3.4A & 5B).
Figure 3.3.3. Inhibition and knockdown of AMPK abolished CGA-inhibited
gluconeogenesis and fatty acid synthesis
A: HepG2 cells were transfected with AMPK α1/2 siRNA using oligofectamine. B:
Cells pre-treated with inhibitors or siRNA were incubated with CGA to study possible
mediator(s) for CGA-inhibited glucose production.
Data were expressed as the means ± SE of three independent experiments.
DC=Diabetic Control, Met=Metformin, Veh=vehicle-treated control. **P<0.01
compared to CGA-treated control.
112
C: Cells pre-treated with inhibitors or siRNA were incubated with CGA to study
possible mediator(s) for CGA-inhibited fatty acid synthesis.
Data were expressed as the means ± SE of three independent experiments.
DC=Diabetic Control, Met=Metformin, Veh=vehicle-treated control. **P<0.01
compared to CGA-treated control.
113
Figure 3.3.4. Dose- and time- dependent phosphorylation of AMPK in L6 myotubes
by CGA
A: Myotubes were treated with CGA for various incubation periods. Whole cell lysate
was used for the detection of p-AMPK, AMPK, p-ACC, ACC and CaMKKβ.
Phosphorylated proteins were adjusted to respective total protein while CAMKK was
adjusted to GAPDH.
Illustrated are the representative images of three independent experiments. Data were
expressed as the mean ± SE of three independent experiments. *P<0.05, **P<0.01,
compared with controls.
114
B: Myotubes were treated with CGA in various concentrations. Whole cell lysate was
used for the detection of p-AMPK, AMPK, p-ACC, ACC and CaMKKβ.
Phosphorylated proteins were adjusted to respective total protein while CAMKK was
adjusted to GAPDH.
Illustrated are the representative images of three independent experiments. Data were
expressed as the mean ± SE of three independent experiments. *P<0.05, **P<0.01
compared with controls.
115
Figure 3.3.5. CGA increases AMPK activity in L6 myotubes
A: Myotubes were treated with 2 mmol/l CGA for various incubation periods up to 24
hours. B: Myotubes were treated with 2 mmol/l CGA for various incubation periods
up to 24 hours. Whole cell lysate was immunoprecipitated with anti-AMPK α1/2.
Immunoprecipitate was assayed against SAMS peptide in the presence of [γ-32
P]ATP.
kinase activity was expressed as incorporated ATP/mg protein/minute.
Data were expressed as the mean ± SE of three independent experiments. *P<0.05,
**P<0.01, compared with controls.
116
3.3.5 Compound c diminishes glucose transport stimulated by CGA in L6
myotubes
Previously, the present author has observed inhibitory effects of compound c on
CGA-mediated inhibition of hepatic glucose and fatty acid synthesis (Section 3.3.3).
To explain the mechanism underlying the glucose transport stimulated by CGA, he
examined the effect of several molecules which are capable of mediating the
stimulation of glucose transport. Wortmannin is a well-known selective inhibitor for
PI3K, a key regulator in insulin signaling [160]. Pretreatment of myotubes with 100
nmol/l wortmannin for 30 minutes abolished the stimulatory effect of insulin on
glucose transport (Fig. 3.3.6A). However, no effect was observed with the
wortmannin pretreatment on CGA-stimulated glucose transport. CGA further
increased insulin-stimulated glucose transport its glucose transport up to 100±2%,
suggesting that CGA may not work on the insulin-dependent pathway. Moreover,
pretreatment of CGA and insulin-stimulated transport with wortmannin only partially
inhibited the stimulation but not to the extent of that obtained in insulin-only-
stimulated transport. Hence, to investigate whether CGA acts through the insulin-
independent pathway, he examined the effect of compound c, a selective inhibitor of
AMPK [161, 162], on the CGA-stimulated glucose transport. Pretreatment with 10
µmol/l compound c for 30 minutes significantly abated the glucose transport
stimulated by both metformin and CGA (Fig. 3.3.6B).
117
Figure 3.3.6. Effects of compound c on CGA-stimulated glucose transport in L6
myotubes
A: L6 myotubes were incubated with 2 mmol/l CGA for 24 hours. A: Myotubes were
preincubated with 100 nmol/l wormannin for 30 minutes before incubated with CGA
or insulin. Myotubes were then incubated with 100 nmol/l insulin (30 minutes) or 2
mmol/l metformin (2 hours) or 2mmol/l CGA (4 hours) before 2DG uptake
measurement. 2DG uptake was measured over a 30-minute period using liquid
scintillation counter. Readings are expressed as percentage increase over basal uptake
of cells incubated with vehicle.
Data were expressed as the mean ± SE of three independent experiments. *P<0.05,
**P<0.01, compared with controls.
118
B: L6 myotubes were incubated with 2 mmol/l CGA for 24 hours. A: Myotubes were
pre-incubated with 10 μmol/l compound c for 30 minutes before incubated with CGA
or insulin. Myotubes were then incubated with 100 nmol/l insulin (30 minutes) or 2
mmol/l metformin (2 hours) or 2mmol/l CGA (4 hours) before 2DG uptake
measurement. 2DG uptake was measured over a 30-minute period using liquid
scintillation counter. Readings are expressed as percentage increase over basal uptake
of cells incubated with vehicle.
Data were expressed as the mean ± SE of three independent experiments. *P<0.05,
**P<0.01, compared with controls.
119
3.3.6 AMPK is necessary for the glucose transport stimulation by CGA in L6
myotubes
Besides using inhibitor compound c, AMPKα content was reduced with RNA
silencing. AMPKα 1/2 siRNA nucleotides reduced the total expression of AMPKα 1/2
by 74±7% compared with transfection with equal concentration of unrelated control
siRNA sequence (Fig 3.3.7A). Transfection with AMPKα 1/2 or unrelated siRNAs
alone did not affect glucose transport. However, reduction in total expression of
AMPKα 1/2 significantly reduced glucose transport stimulated by CGA by 58±5%
(Fig 3.3.7B). Unrelated control siRNA did not significantly affect CGA-stimulated
glucose transport, though a slight increase was detected. Taken together, the data
suggested that AMPK may have a major contribution to CGA-induced stimulation of
glucose transport.
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Figure 3.3.7. Effects of gene silencing of AMPK on CGA-stimulated glucose
transport in L6 myotubes
L6 myotubes were transfected with vehicle, unrelated siRNA or AMPKα1/2. A:
Expression of AMPKα1/2 after transfection with or without unrelated siRNA or
AMPKα1/2 siRNA. B: Transfected or non-transfected myotubes were incubated with
2 mmol/l CGA for 24 hours. 2DG uptake was measure over a 30-minute period using
liquid scintillation method. Readings were expressed as percentage increase over
basal uptake that was obtained from non-transfeted cells incubated with vehicle.
Data were expressed as the mean ± SE of three independent experiments. **P<0.01
compared with non-transfected-control treated with CGA.
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3.3.7 CGA does not induce association of p85 subunit of PI3K to IRS-1 in L6
myotubes
Activation of PI3K requires association of its p85 subunit to IRS-1 as shown by
insulin in Fig 3.3.8C. However, CGA did not produce any significant effect on the
association of p85 to IRS-1 immunoprecipitates of myotubes compared to the
association observed in vehicle-treated myotubes. This again suggests that the
stimulatory effect of CGA on glucose transport may not be attributable to PI3K
activation. To further examine whether CGA activates the PI3K-Akt pathway, the
present author investigated the effect of CGA on Akt phorphorylation. Surprisingly,
he observed that Akt was phosphorylated at Thr 308 by insulin as well as CGA (Fig
3.3.8D).
3.3.8 Effect of CGA on L6 myotubes viability and proliferation
To discard the possibility that increases in glucose transport in response to CGA
might be due to the changes in myotube numbers, the present author assessed the
effect of increasing doses of CGA on cell viability and cell proliferation. No
significant changes were observed up to a concentration of 4 mmol/l (Fig 3.3.9A).
However, significant increases in cell death were observed at 6 mmol/l (17.4±5%), 8
mmol/l (19.9±4%) and 10 mmol/l (17.6±4%) compared to vehicle-treated myotubes.
On the other hand, myotubes treated with different concentrations of CGA did not
produce any significant changes in all three cell-cycle phases, G0/G1, S and G2/M,
compared to the vehicle-treated myotubes (Fig 3.3.9B). Taken together, at the
concentration of 2 mmol/l, CGA did not cause significant cell death or cell-cycle
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arrest, thus eliminating the possibility that cell numbers may affect the study of
glucose transport.
Figure 3.3.8. CGA phosphorylates Akt in the absence of PI3K in L6 myotubes
A: Myotubes were treated with vehicle, 100 nmol/l insulin or 2 mmol/l CGA. Whole
cell lysate was immunoprecipitated with IRS-1 and immunoblotted for IRS-1 and p85
subunit of PI3K. B: Myotubes were treated with vehicle, 100 nmol/l insulin or 2
mmol/l CGA. Whole cell lysate was detected for p-Akt through immunoblotting.
123
Data were expressed as the mean ± SE of three independent experiments. *P<0.05,
**P<0.01 compared with controls.
Figure 3.3.9. Effects of CGA on cell viability and cell proliferation of L6 myotubes
A: Myotubes were incubated with incremental concentrations of CGA for 24 hours.
Viability of myotubes was measured using MTT staining. Readings are expressed as a
percentage of non-viable cells compared to vehicle-treated myotubes.
Data were expressed as the mean ± SE of three independent experiments. *P<0.05
compared with vehicle-treated control.
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B: Myotubes were incubated with incremental concentrations of CGA for 24 hours.
Numbers of cells in cell-cycle phases were examined using propidium iodide staining
and FACS analysis. Readings are expressed as percentage of cells stained by
propidium iodide at different phases.
Data were expressed as the mean ± SE of three independent experiments. *P<0.05
compared with vehicle-treated control.
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4 Discussion
The global prevalence of diabetes mellitus has been increasing dramatically due to the
fact that globalization has led to social impacts such as urbanization and lifestyle
changes [163]. Diabetes, especially T2DM, previously thought to be prevalent in
developed countries [164], has now emerged as a potential epidemic in developing
countries like China and India [165]. T2DM was once believed to be a metabolic
syndrome exclusive to adults, but has now risen as a plague in adolescents and also
children [166]. In 2010, an estimated 285 million cases of diabetes were reported; this
figure is expected to increase by 54% to 439 million by 2030 [165]. Despite the
availability of a variety of pharmacological agents to control blood glucose level
(from insulins, sulfonylureas, biguanides, thiazoledinediones to the most-recent DPP
IV), lifestyle changes involving exercise and dietary intervention are pivotal to
achieving the goal of cost-effective management with minimal complications but
maximal quality of life. This is because pharmacological management of diabetes is
always associated with unwanted side effects and incapability of human nature in
adhering to sustaining exercise regimen as discussed in Section 1.5. Therefore, dietary
intervention becomes the most powerful tool to combat and keep the disease at bay.
Polyphenol-rich coffee and other plant-based foods like Vernonia amygdalina are
always associated with improved glycemic responses (Section 1.6 & 1.71). It is thus
imperative to learn what is/are the component(s) in these foods that cause this
beneficial metabolic changes and how they work in order to manage the disease in a
more efficient and cost-effective manner.
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4.1 Studies on the antidiabetic effects of VA
In the beginning of the present study, the author started with the investigation on the
anti-diabetic effects of VA and its possible mechanisms of action, at the same time
identifying the bio-constituents that are responsible for the biological activities. The
potential health benefits of VA remain anecdotal until Kupchan et al. demonstrated
that chloroform fraction of VA contains compounds which are toxic to cancerous cells
in vitro[167]. Since then, several active molecules, belonging to the family of
sesquiterpene lactones, such as vernomygdaline, vernolide, vernodaline and
vernolepin, were identified as potential anticancer agents [168]. The present study
focused on identifying possible active constituents from more polar fraction, in the
hope that they would be less toxic but possessing beneficial health effects. Four main
polyphenols were identified in the ethanolic VA extract. They are dicaffeoyl quinic
acid, 1,5-dicaffeoyl-quinic acid, chlorogenic acid and luteolin-7-O-glucoside.
Dicaffeoyl quinic acid and its isomers appear to be the most abundant compounds
present in the extract. These findings are consistent with the study by Ola et al. (2009)
[136]. In an acute glucose tolerance test, 400 mg/kg of VA exerted the most effective
anti-hyperglycemic activity. This effective dose is similar to that reported in previous
studies [86, 88]. A 28-day chronic study revealed that VA significantly decreased
fasting blood glucose. Both of these designs demonstrated the anti-diabetic efficiency
of VA and supported the findings of previous studies [84-86, 88, 95].
STZ (2-deoxy-2(3-methyl-3-nitrosoureido)-D-glucopyranose) is a nitrosourea widely
used to induce experimental diabetic animal model. Its glucose moiety is responsible
for STZ to be transported into insulin-secreting β-cells via GLUT 2 [169] while the
nitrosoamide moiety is accounted for the toxicity of this compound [170]. The
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mechanism of cytotoxicity of STZ on β-cells were extensively reviewed by
Szkudelski et al. [131]. STZ-induced diabetes is characterized by hypoinsulinemia,
polydipsia, polyuria and decreased body weight [171]. Several other abnormalities
like increased activities of alkaline phosphatase, aspartate transaminase, alanine
transaminase and changes in the activities of several enzymes in carbohydrate
metabolism were also observed in this model [172, 173]. Impairment in the
antioxidant defence was also reported in this model. Lipid peroxides, hydroperoxides
and protein carbonyls are significantly higher in plasma, pancreatic tissue and kidney
of these animals; activities of antioxidant enzymes such as superoxide dismutase,
catalase, glutathione peroxidase and glutathione-S-transferase were found to be lower
in plasma, pancreas and kidney [174, 175].
Treatment with metformin or VA greatly improved polyuria and polydipsia (Table
3.1.1), indicating improvement in the diabetic conditions. Currently available oral
hypoglycemic drugs such as sulfonylureas and thiazolidinediones often lead to weight
gain, which is an unwanted side effect that will further promote insulin resistance [56].
In this study, both metformin and VA caused significant improvements in body
weight, suggesting that they may be better candidates for combating diabetes,
especially T2DM. Moreover, there were neither significant changes in food intake nor
any sign of toxicity. VA possessed anti-lipidemic activity, in accord with
Adaramoye’s study which also showed its lipid-lowering effect [176]. Several
previous studies proposed that VA possibly acted through stimulating insulin
secretion from pancreatic β-cells [84, 86, 88, 92]. In spite of the fact that we observed
significant increase in insulin level in VA-treated animals, the insulin levels in STZ-
diabetic rats are negligible if compared to normal control. This might be due to
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immense loss of pancreatic β-cells as a result of toxicity from a single high-dose STZ.
Also, another study has shown that β-cells isolated from STZ-treated animal are
almost completely unresponsive to stimulation by glucose or sulfonylureas [177]. In
some studies, co-administration of nicotinamide was performed in STZ-induced
diabetic animal in order to prevent excessive depletion of insulin secretion [178, 179].
Further investigations are needed to clarify whether VA stimulates insulin secretion.
Physiological levels of reactive oxygen species (ROS) are essential for cell
proliferation and regulation of cell signaling. Oxidative stress occurs when our body
loses its ability to maintain redox homeostasis such that the generation of ROS
exceeds their utilization, neutralization and elimination. In diabetes, the generation of
ROS is greatly increased due to glucose auto-oxidation and lipid peroxidation.
Excessive ROS results in tissue damage and subsequent diabetic complications
against which our conventional hypoglycemic drugs appear to be ineffective. Our
body’s first-line anti-oxidative defense consists of enzymatic (SOD, catalase, GPx)
and non-enzymatic (glutathione) antioxidants. SOD accelerates dismutation of O2- to
H2O2, while catalase and GPx catalyze the conversion of H2O2 to H2O. VA resulted in
up-regulation of antioxidant enzymes, especially GPx and glutathione. These effects
may all be due to the high polyphenolic content of the extract. Several studies showed
that polyphenols modulate promoter activities of several antioxidant enzymes and this
may be due to the activation of antioxidant/electrophile responsive elements
(AREs/EpREs) [180]. Further study is required to determine how VA polyphenols,
especially dicaffeoyl-quinic acids, regulate the activities of antioxidant enzymes.
As discussed before, GLUT 1 (non insulin-responsive) and GLUT 4 (insulin-
responsive) are two major glucose transporters that regulate glucose uptake into
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various tissues. GLUT 1 is expressed ubiquitously while GLUT 4 is mainly expressed
in skeletal muscles and adipocytes. STZ was reported to affect expression of GLUT 4
[181-184] but not GLUT 1 [182]. Our results showed a similar effect. We further
assessed GLUT 4 translocation to plasma membrane and found that rats treated with
STZ have most of their GLUT 4 retained in the light microsomal membrane while the
functional GLUT 4 on plasma membrane was significantly decreased. Treatment with
metformin or VA increased both total GLUT 4 expression and likewise the functional
plasma membrane GLUT 4. This observation is further strengthened by the finding of
increase in muscle glycogen synthesis which is consistent with the finding of
increased muscle glucose uptake regulated by GLUT 4. Metformin has been shown to
modulate GLUT 4 translocation through the AMPK pathway [29, 185]. Further
investigations are needed to characterize the pathway by which VA increased both
GLUT 4 expression and translocation.
G6Pase is one of the rate-limiting gluconeogenic enzymes that regulate glucose
synthesis. Due to insulin deficiency from β-cell loss caused by STZ, activation of
G6Pase was observed [186]. Metformin is well-known for its antigluconeogenic
property [187] and our results again showed its strong suppression of G6Pase activity.
On the other hand, VA also exerted some degree of inhibition of G6Pase activity. The
limitation of this study is that variations in insulin level could be a confounding factor.
VA caused a slight increase in insulin which can, in turn, increase translocation of
GLUT 4 and glycogenesis while inhibiting gluconeogenic enzymes such as G6Pase. It
appears that other diabetic models in which insulin levels are constant should be
employed to further confirm these findings.
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4.2 Studies on the antidiabetic effects of CGA
As discussed in Section 1.7.2, most of the studies involving CGA and its antidiabetic
effects were associated with regulation of fasting and postprandial glucose levels,
which were attributable to the inhibition of gluconeogenesis, increased peripheral
glucose uptake and reduced intestinal glucose absorption. None of these studies has
suggested that CGA may stimulate insulin secretion from pancreatic β-cells. Hence,
we strongly believe that CGA may have a greater impact on T2DM rather than T1DM.
As a result, the animal model which the present author next used for the study of CGA
is a genetically-induced T2DM model, known as Leprdb/db
mice. Leptin is the primary
adipose hormone that conveys adiposity signal to the brain. The brain, particularly the
hypothalamus, integrates leptin and various other metabolic signals to regulate energy
homeostasis and body weight by controlling both behavioral and metabolic responses
[188]. Leptin decreases body weight by suppressing appetite and by increasing energy
expenditure [189-191]. This mutant species is leptin receptor-deficient. This genetic
deficiency of leptin receptors results in obesity and obesity-associated metabolic
changes (detailed metabolic changes was discussed previously in Section 1.7.4) which
closely resembles human T2DM [192, 193].
Acute administration of CGA significantly lowered fasting blood glucose in a genetic
T2DM model, Leprdb/db
mice. However, reduction in fasting glucose level cannot be
explained by the delay in intestinal glucose absorption. Moreover, it has been shown
that CGA suppressed G6Pase activity [114, 119], suggesting a possible role of CGA
on gluconeogenesis. Yet, no study has been conducted to investigate its direct effect
on hepatic glucose production. Thus, for the first time, the present author
demonstrated its inhibitory effect on glucose production in HepG2 cells. Concurrent
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inhibition of the expression of G6Pase was observed with the decreased glucose
production by CGA. We believe that although its duration of acute action in
suppressing glucose levels during the OGTT is relatively short, compared to
metformin, long-term consumption of CGA may be beneficial as with coffee
consumption. As predicted, 2-week treatment with CGA decreased fasting blood
glucose and insulin levels. In addition, glucose and insulin tolerance of the animals
were also significantly improved. He further assessed its long-term effect on
gluconeogenesis in vivo, via PTT, as administration of gluconeogenic substrate
pvruvate increased blood glucose by stimulating gluconeogenesis in the liver. CGA
attenuated pyruvate-induced hyperglycemia, indicating reduced gluconeogenesis,
which is in agreement with decreases in the expression and activity of G6Pase in the
liver.
One might postulate the dysregulation of gluconeogenesis is attributed to the
impairment in insulin secretion which is commonly found in patients with impaired
glucose tolerance or complete glucose intolerance and CGA may have stimulatory
effect on insulin secretion. However, in this diabetic animal model, the present author
observed fasting hyperinsulinemia, which is commonly associated with obesity and
insulin resistance [42]. Treatment with CGA, on the other hand, ameliorated
hyperinsulinemia. Significantly lower AUCglucose as well as AUCinsulin, improved
insulin tolerance and HOMAIR index by CGA further explain and suggest its insulin
sensitizing role, which may explain the paradoxical effect of coffee on glucose
intolerance and insulin resistance in T2DM [103, 194, 195].
There is no previous study to implicate CGA to peripheral glucose disposal until
Prabhakar and Doble (2009) showed for the first time that CGA stimulated glucose
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transport in myotubes via increased expression of GLUT 4 and PPAR-γ transcript
[128]. However, mere increase in GLUT 4 gene expression cannot fully explain the
increase in glucose transport and the study lacks clarification on the mechanisms
involved to enhance glucose transport in skeletal muscle. Decrease in fasting blood
glucose can be due to suppression of gluconeogenesis or/and increased glucose
disposal in peripheral tissues such as skeletal muscle. The present author has shown
that CGA inhibited the expression and activity of gluconeogenic G6Pase and hence,
soleus muscle was next isolated from Leprdb/db
mice and treated with CGA followed
by 2DG transport assay. CGA was shown to stimulate and enhance both basal and
insulin-mediated glucose transports and thus augmenting glucose utilization in the
muscle. Additive effect of CGA in insulin-mediated glucose transport suggests that
CGA may act through a significant pathway which is different from insulin signaling.
To investigate long-term effect of CGA on glucose uptake in skeletal muscles, we
isolated the skeletal muscles after 2-week treatment and 2DG transport was performed
on the muscles. Similarly, glucose uptake was increased in the skeletal muscles after
long-term treatment with CGA. Consistent with this, immunohistochemistry studies
revealed that 2-week treatment with CGA increased both expression and translocation
of GLUT 4, the major insulin-responsive glucose transporter found in peripheral
tissues. Also, quantitation of GLUT 4 using immunoblotting further supports these
findings.
To further support the data, the present author investigated the effect of CGA on
glucose transport in L6 myotubes and the possible mechanisms to execute its function
in glucose transport. The results of the present study showed that CGA stimulated
glucose transport in L6 myotubes in a dose- and time-dependent manner. Optimum
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increase (~1.5 fold) was observed at the dose of 2 mmol/l after 24-hour incubation
period, compared to vehicle-treated control. Using non-radioactive glucose transport
assay, Prakhabar and Doble (2009) demonstrated that CGA caused significant
increase of glucose transport at micromolar concentration [128]. However, he found
that micromolar amounts of CGA only mildly stimulated glucose transport (data not
shown).
4.3 Studies of antilipidemic effects of CGA
Another striking finding from the present study is the antilipidemic effect of CGA.
Consumption of coffee induces thermogenesis [196] and most of the researchers
believe that it was due to the presence of caffeine in coffee as studies have shown that
caffeine increased energy expenditure and lipid oxidation [197] and lipolysis [198].
However, in this study, we showed that CGA inhibits fatty acid synthesis both in vivo
and in vitro. Free fatty acids are essential blocks for the synthesis of more complex
lipid molecules such as triacylglycerol or triglyceride [44]. In obesity and T2DM, free
fatty acids flux is chronically increased and this increase is responsible for the non-
alcoholic hepatic steatosis [44], insulin resistance [45], decrease in skeletal muscle
glucose disposal [46] and increased hepatic glucose production [47]. CGA inhibited
fatty acid synthesis in HepG2 hepatocytes and decreased circulating free fatty acids
level in Leprdb/db
mice, which in turn result in decreased triglyceride and cholesterol
levels. Fat accumulation in liver and hepatoma cells was also reduced by CGA, in line
with the hypothesis that CGA inhibited lipogenesis in the liver through the inhibition
of fatty acid synthesis. Owing to these lipid-lowering effects of CGA, a significant
drop in body weight was observed in Leprdb/db
mice treated with CGA, consistent with
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the previous finding in a human study that consumption of coffee enriched with CGA
resulted in significant reduction in body weight and fat mass [199]. Possibly,
restoration of dysregulation of gluconeogenesis, peripheral glucose uptake and insulin
sensitivity can be partially attributable to the reduction of free fatty acids level in the
CGA-treated animals.
4.4 Studies of molecular targets that mediate beneficial metabolic
changes by CGA
AMPK is a central regulator of cellular metabolism and is sensitive to cellular energy
changes. Its activation is triggered during energy deprivation and exercise [200].
Carey et al (2009) reviewed it as a potential exercise-linked target for “exercise-
mimetics”, an interesting concept of the development of pharmacological agents that
improve energy expenditure while concomitantly reduce body fat and improve
metabolic homeostasis [201]. The present author showed that AMPK activation is
vital in CGA-mediated inhibition of hepatic glucose production and fatty acid
synthesis. AMPK activation has been shown to inhibit gluconeogenic gene expression
and hepatic glucose production [31]. Besides, AMPK phosphorylation inactivates
ACC, leading to inhibition of fatty acids de novo and also cholesterol synthesis while
increases fatty acid oxidation [202]. His data showed that CGA phosphorylates
AMPK and ACC, resulting in similar metabolic changes in the liver. Activation of
AMPK also leads to translocation of GLUT 4 from intracellular membranes to plasma
membranes, thus increasing glucose transport [203]. Consistent with this, our results
showed a time- and dose-dependent increase in phosphorylation of AMPK and
likewise its activity, leading to an increase in the number of GLUT 4 on the plasma
135
membrane of L6 myotubes. However, no changes were observed on the translocation
of GLUT 1, suggesting that CGA may only affect insulin-sensitive tissues such as
skeletal muscles, liver and adipocytes.
CaMKKβ has been reported as an alternative upstream kinase for AMPK besides
cellular stress (decreased ATP:AMP ratio) and LKB-1 [155]. The present author
examined whether CGA causes any changes in ATP: AMP ratio but no significant
changes were observed (data not shown). On the other hand, pretreatment of cells
with STO-609 also abrogated CGA-inhibited hepatic glucose production and fatty
acid synthesis, indicating that CAMKK could be the upstream kinase that mediates
the CGA-activated AMPK. This was further supported by the observation that CGA
raised intracellular level of free calcium at the same time. He found out later that
CGA increased expression of CaMKKβ in both L6 myotubes and HepG2 cells,
strengthening his findings that the upstream target of CGA that activates AMPK is
CAMKKβ.
Interestingly, in the in vivo study, pretreatment with compound c did not fully block
glucose-lowering effect by CGA, suggesting that there could be other mediators being
activated at the same time. More importantly, after 2-week treatment with CGA,
inhibitory effect of compound c was significantly decreased in which a doubled dose
of compound c was required to achieve the inhibitory effect observed initially. This
suggests that there could be an intermediary molecule which is stimulated by CGA
and thus indirectly and chronically enhances the activation of AMPK. It is highly
possible that this molecule could be the anti-diabetic adipokine, adiponectin, which
has been reported to be decreased in obesity, insulin resistance and T2DM [204].
Adiponectin has also been shown to reverse insulin resistance [204], enhance hepatic
136
insulin action [205] and increase fatty acid oxidation [206]. Consistent with this,
restoration of adiponectin levels in vivo by CGA showed improved glycemia, insulin
sensitivity and fatty acid metabolism. Moreover, very recently, adiponectin has been
demonstrated to activate AMPK via CAMKK [207]. Therefore, restoration of
adiponectin in vivo by CGA could have enhanced the activation of AMPK and
resulted in the above-mentioned beneficial metabolic alterations (Figure 4.4).
On the other hand, in accord with the earlier report that CGA did not show any
significant effect on the expression of PI3K [128], the present author further
demonstrated that CGA did not activate PI3K as no significant association was
observed between p85 subunit of PI3K and IRS-1, suggesting a negative role of CGA
on phosphorylation of IRS. However, to our surprise, he observed phosphorylation of
Akt in the absence of PI3K activation. In rat heart, phosphorylation of Akt has been
shown to phosphorylate AMPKα1 subunit at Ser 485/491 and hence interfering with
the LKB-1-mediated activation of AMPK at its Thr 172 residue [208]. This indicates
that Akt negatively influenced AMPK activation and activity. Other groups of
researchers have reported that several stimuli such as adiponectin [209] and VEGF
[210], elicited parallel activation of both AMPK and Akt. Likewise, they proposed the
upstream nature of AMPK, by which Akt is activated by the phosphorylation of
AMPK at Thr 172. This is highly notable as both insulin-dependent and AMPK-
dependent pathways converge to activate Akt substrate of 160 kDa (AS160) and Akt
may be the point of convergence instead of AS160 (Figure 4.4). The present results
seem to favour the latter but subsequent studies to determine the activities of AMPK
and Akt are required to support the occurrence of this phenomenon.
137
Several studies have reported that AMPK activation by the well-known AMPK
activator, AICAR, led to cell cycle arrest which involved accumulation of tumor
suppressor protein p53 [211]. However, he did not detect any significant changes on
cell viability and cell cycle of L6 myotubes even after 24 hours of incubation in the
presence of 2 mmol/l CGA. This might be due to the parallel activation of pro-
survival Akt, as Akt regulates apoptosis through direct targets such as Bad, caspase 9,
the Forkhead family of transcription factors and the NF-κB regulator, IKK [212].
More importantly, the data eliminates the possibility that the increase in glucose
transport might be due to the effect of CGA on cell proliferation.
4.5 Possible cytotoxic effect of CGA
From Figure 3.3.9, CGA started to show cytotoxic effect on L6 myotubes at a dose of
≥4mM. This may certainly affect its clinical or practical significance in managing
T2DM. However, if the figure is studied more thoroughly, it is evident that CGA
(≥4mM) did not result in more than 20% cell death. In other words, CGA may not be
statistically or clinically cytotoxic at doses up to 10mM. Besides, even for the widely
used antidiabetic metformin, at a dose of 10mM, it caused ≈10% cell death in the
same cellular model (data not shown). In addition, the present author's in vivo toxicity
study showed that the LD50 of CGA in BALB/c mice is more than 3.5kg/g (data not
shown), which is ten times higher than our studied dose. Therefore, it is very unlikely
that CGA would cause toxicity in vivo and compromising its beneficial effects on
glucose and lipid metabolisms discussed above.
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4.6 VA vs CGA vs Met
At this point of studies, although both VA and CGA showed hypoglycemic effects in
different manners, the present author was unable to conclude whether VA or CGA can
be as effective as metformin for the treatment of T2DM. First, as discussed in Section
4.1, VA contains cytotoxic active constituents which theirs effects are unknown in
diabetic model. Second, the oral bioavailability of CGA remains unclear to date
(please refer to Chapter 5).
Besides CGA, the most abundant components in VA, di-CQAs, may contribute to its
glucose-lowering effect as well. To investigate about this, the present author
examined the effects of one of the di-CQA isoforms, 1.5-dicaffeoylquinic acid, on
glucose transport in L6 myotubes (details please see Chapter 5 and Appendix 4). We
observed a mild stimulation using up to 100μM 1,5-dicaffeoylquinic acid.
At a dose of 100 mg/kg (approximate equivalent dose of CGA in 400 mg/kg VA),
CGA still resulted in similar glucose-lowering effects pre- and post-glucose loading if
it was administrated intraperitoneally (higher AUC if compared to 250 mg/kg) [data
not shown]. However, if it was fed orally, the present author did not observed
glucose-lowering effects which are as strong as those in VA, if compared to DC. He
believe that it could be due to I) the low oral bioavailability of CGA, II) di-CQAs in
VA which could have better oral bioavailability, III) other active constituents in VA
that may be able to protect CGA from undergoing degradation or modification or IV)
active constituents in VA that may be able to enhance CGA absorption. Further works
are needed to explain these observations.
139
Figure 4.4. Cross-talk between insulin signalling & insulin-independent pathways and
schematic illustration of possible mechanism(s) of action of CGA to cause beneficial
metabolic outcomes
IR=insulin recptor, IRS=insulin receptor substrate, PI3K=phosphatidylinositide 3-
kinases.
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5 Conclusions and Future Perspectives
VA shows anti-hyperglycemic effect in the single-dose STZ-induced diabetic rat
model. This effect was most possibly mediated through inhibition of key hepatic
G6Pase and increase in expression and translocation of GLUT 4 in skeletal muscle.
Previous studies reported that VA increased insulin secretion. However, due to the
nature of our diabetic model, the massive destruction of pancreatic β-cells may mask
the stimulatory effect. Further studies are required to confirm whether VA increases
insulin secretion. VA also showed positive modulation of the lipid profile and
antioxidant defense. All these biological effects may be attributed to the polyphenols
in the extract, especially caffeoyl-quinic acids (CQA) and its isomers.
Owing to the abundance of caffeoyl-quinic acids and its isomers in VA extract, the
present author decided to choose one of the most common caffeoyl-quinics, CGA, to
study its potential antidiabetic effects. The present study demonstrated for the first
time that CGA regulates glucose and lipid metabolism via the activation of AMPK.
CGA inhibited hepatic G6Pase expression and activity, attenuated hepatic steatosis,
improved lipid profiles and skeletal muscle glucose uptake, which in turn improve
fasting glucose levels, glucose tolerance, insulin sensitivity and dyslipidemia. All
these suggest that CGA could be the main component that contributes to the beneficial
effect of coffee and also the paradoxical effect of coffee in T2DM subjects.
To date, the bioavailability of intact CGA remains unresolved. One group of
researchers reported that large amount of CQAs, di-CQAs and their isomers were
found in human plasma and urine after coffee consumption, indicating its high
absorption and bioavailability [213, 214]. However, CQAs have not been detected
after coffee consumption in other studies [215, 216], consistent with a very recent
141
study which demonstrated that most of the CQAs found in plasma were mainly
reduced and/or sulfated and/or methylated [217]. In another study that measured intact
CGA in plasma after 1 to 3 hours after coffee ingestion, a very low amount of CQAs
(10-40nm) was detected [218]. On the other hand, a study in rats has shown that CGA
was absorbed intact in stomach [219] while another group observed that CGA was
mostly absorbed as phenolic acids after hydrolysis by microbial enzymes [220]. Our
preliminary study (in which CGA was fed orally to Leprdb/db
mice) seems to favour
the fact of low bioavailability of CGA. Compared to ip administration, oral gavage of
CGA during an OGTT resulted in higher AUCglucose and diminished its hypoglycemic
effect before the glucose challenge as shown in Appendix 3. Further investigations are
needed to clarify this phenomenon. Also, a study on the bioavailability of CGA
should be performed following ingestion of CGA instead of coffee.
The present author has also tested the effect of one of the di-CQAs, known as 1,5-
dicaffeoylquinic acid (1,5DCQA), on glucose transport in L6 myotubes. Surprisingly,
he observed a mild stimulation using concentrations of up to 100μM (Appendix 4).
Further works are needed to investigate its effect(s) on glucose metabolism.
Although it is premature at this stage to conclude that CGA can be used as a
therapeutic tool for T2DM, from the studies discussed above, it is evident that the
impact of CGA on carbohydrate or glucose metabolism is extensive. This is highly
significant considering that obesity is positively correlated to metabolic diseases like
diabetes mellitus and increased intakes of CGA-rich foods not only are beneficial to
glucose homeostasis but have also been shown to be anti-lipidemic.
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6 References
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7 List of Appendices
Appendix 1. Criteria for the diagnosis of diabetes mellitus
Normal Impaired fasting glucose
(IFG)
Impaired glucose
tolerance (IGT)
Diabetes
Mellitus
Fasting blood glucose
mg/dl <100 100-125 - ≥126
mmol/l <5.6 5.6-6.9 - ≥7.0
2-hr post OGTT
mg/dl <140 - 140-199 ≥200
mmol/l <7.8 - 7.8-11.0 ≥11.1
Symptoms of diabetes with a random plasma glucose of ≥200 mg/dl or 11.1 mmol/l.
169
Appendix 2. Number of individuals with diabetes mellitus [by World Health
Organization (WHO) Regions]
Region 2000 2030
African region 7,020,000 18,234,000
Eastern Mediterranean 15,188,000 42,600,000
Region of the Americas 33,016,000 66,812,000
European 33,332,000 47,973,000
Southeast Asia 46,903,000 119,541,000
Western Pacific 35,771,000 71,050,100
Total 171,230,000 366,210,100
(Source: World Health Organization:
http://www.who.int/diabetes/facts/world_figures/en/)
170
Appendix 3. Acute effects of oral gavage of CGA on fasting blood glucose,
compared to i.p. CGA
Oral glucose tolerance test was performed in 6-hour fasted Leprdb/db
mice. Blood
samples were collected from the tail vein for fasting glucose measurement before
treatments (vehicle, ip 250 mg/kg CGA, oral gavage 250 mg/kg CGA). Ten minutes
after the treatments, blood samples were collected again followed by oral gavaging of
2 g/kg glucose. Blood samples were collected 15, 30, 60 and 120 minutes after the
glucose challenge.
Data were expressed as the means ± SE of three independent experiments.
DC=Diabetic Control.
171
Appendix 4. Effect of 1,5 DCQA on glucose transport in L6 myotubes
L6 myotubes were incubated with incremental concentrations of 1,5DCQA for 24
hours. 2-deoxyglucose uptake was measure over a 30-minute period, using liquid
scintillation counter. Readings are expressed as percentage increase over basal uptake
of cells incubated with vehicle.
Data were expressed as the means ± SE of three independent experiments. *P<0.05,
**P<0.01 compared with vehicle-treated control.