IN VITRO AND IN VIVO STUDIES ON THE ANTIDIABETIC AND ANTILIPIDEMIC EFFECTS OF CHLOROGENIC ACID ONG KHANG WEI NATIONAL UNIVERSITY OF SINGAPORE 2013 I 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 II 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 III 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. IV 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. V Contents LIST OF PUBLICATIONS……………………………………………………………i LIST OF ABBREVIATIONS……………………………………………………… ii LIST OF FIGURES………………………………………………………………… .iv LIST OF TABLES……………………………………………………………………vi LIST OF APPENDICES……………………………………………………… vii SUMMARY…………………………………………………………………………viii Chapter 1: Introduction . 1.1 Diabetes Mellitus . 1.2 Classification of Diabetes Mellitus . 1.3 Normal Glucose Homeostasis . 1.4 Insulin signaling vs AMPK-dependent pathway . 1.5 Pathogenesis of T2DM 1.5.1 β-cell Dysfunction 1.5.2 Insulin Resistance 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 Chapter 2: Materials and Methods 26 I 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 and GLUT . 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 II 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 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 III 3.1.7 Long-term effects of VA extract on expression of GLUT 1/ GLUT and cellular distribution of GLUT 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 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 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 IV 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 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 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Criteria for the diagnosis of diabetes mellitus Normal Impaired fasting glucose Impaired glucose Diabetes (IFG) tolerance (IGT) Mellitus Fasting blood glucose mg/dl [...]... 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 11 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... 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 6 Two incretin... 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... 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... 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 2 resistance The etiology of this form of diabetes is wide and complicated,... 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. .. 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... Fate of Glucose 4 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. .. 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 . 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. 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. I IN VITRO AND IN VIVO STUDIES ON THE ANTIDIABETIC AND ANTILIPIDEMIC EFFECTS OF CHLOROGENIC ACID ONG KHANG WEI NATIONAL UNIVERSITY OF SINGAPORE 2013