Evaluation of Pharmacological Modification of Growth Hormone

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Evaluation of Pharmacological Modification of Growth Hormone Secretagogue Receptor in a Mouse Model of Non-Obese Type 2 Diabetes Mellitus Rasha Mosa Master of Medicine

A thesis submitted for the degree of Doctor of Philosophy at The University of Queensland in 2017 School of Medicine Abstract

While it appears that obesity is associated with the higher incidence of type 2 diabetes mellitus (T2DM), recent findings that T2DM affects non-obese people are becoming more noticeable. Many of those who are not obese by traditional weight measurements have an increased percentage of body fat distributed predominately in the abdominal and visceral regions. Disorders of lipid metabolism play a major role in the pathogenesis of this non- obese diabetic phenotype. For examples, lipodystrophic patients develop insulin resistance due to insufficient adipose tissue and aged lean people with diabetes have defects in mitochondrial lipid oxidative mechanisms. Such non-obese diabetic patients are at increased risk of developing cardiovascular (CVD) complications. The management of non-obese T2DM should focus not only on life style modifications or blood glucose control but also strategies for correction of lipid metabolic aberrations. Growth hormone (GH) exerts powerful influences on growth and body composition. In obese animal models and human, GH is known to correlate inversely with insulin concentrations. Some studies suggest that insulin may have a direct inhibitory effect on GH secretion, whereas others showed that insulin inhibits GH secretion via suppression of growth hormone releasing hormone (GHRH) or stimulation of somatostatin (SST). Although GH-insulin relationship is well documented in obesity, studies of this GH-insulin relationship in non-obese diabetes are sparse and contradictory. Thus, this study aimed to investigate the pulsatile GH profile and its regulatory factors in MKR mice which are non-obese diabetic mice generated by igf-1 receptor mutation in skeletal muscle. Our observations demonstrated that unlike obese mice, MKR mice had normal to higher pulsatile GH secretion at different age groups. Nevertheless, there were no detectable changes in gene expressions of hypothalamic GHRH and SST. Interestingly, hypothalamic orexigenic NPY gene expression was increased that might affect GH secretion. These findings suggest that the relationship between GH and insulin was altered in MKR mice, leading to higher GH concentrations despite hyperinsulinaemia. Given that MKR mice exhibited postnatal growth retardation, we anticipated that endogenous GH levels would increase to facilitate rapid linear growth and promote muscle development that assist nutrient uptake and utilization. Therapy using growth hormone secretagogues (GHS) that increase endogenous GH secretion through binding to the ghrelin receptor (GHS-R1a) might have beneficial effects on lipid metabolism with less adverse effects on glucose metabolism than exogenous GH administration. Recent studies have provided some interesting avenues for further exploration on how Hexarelin, one of synthetic peptide GHS, enhanced fat metabolism of white adipose tissue (WAT) via CD36 activation independently of GHS-R1a. To address the effects of Hexarelin in non-obese diabetes, MKR were received daily intraperitoneal (I.P.) injection of Hexarelin (200ug/kg body weight) for 12 days. Remarkably, MKR mice displayed improved glucose and insulin tolerance with Hexarelin treatment due to enhanced fat metabolism associated with increased adipocyte differentiation and up- regulation of fatty acid uptake and oxidation related genes of WAT. Moreover, our results showed that Hexarelin increased pulsatile GH secretion, reduced fat mass and improved the lipid profile without compromising glucose metabolism. Ghrelin, the natural ligand of GHS-R1a is an appetite-stimulating hormone inducing food intake and adiposity. Due to the observed association between plasma ghrelin and insulin levels, it was suggested that inhibition of ghrelin secretion and/or blockade of GHS-R1a could be useful targets for treatment of T2DM. In this respect, the current study also examined the possible effects of repeated injections of ghrelin antagonist [D-Lys3]-GHRP- 6 (200 nmol/mouse) for 12 days in MKR mice. [D-Lys3]-GHRP-6 reduced pulsatile GH secretion and body fat mass as expected, but unexpectedly worsened glucose and insulin intolerance and increased cumulative food intake. In addition, current observations demonstrated a significant decrease in glucose stimulated insulin secretion, an increase in blood glucose and a decrease in plasma insulin in MKR mice following long-term [D-Lys3]- GHRP-6 treatment, indicating a direct inhibition of insulin secretion. Immunofluorescence staining of pancreatic islets of long-term [D-Lys3]-GHRP-6 treated mice showed a proportional increase in SST areas with a decrease in insulin areas. Furthermore, observations showed that [D-Lys3]-GHRP-6 stimulated food intake via reduction of proopiomelanocortin (POMC) gene expression and antagonized GH secretion via reduced GHRH gene expression in hypothalamus. In conclusion, the inverse relationship between GH and insulin is not apparent in this non- obese diabetic model. Balance of these two important hormones under different metabolic conditions is critically important in the pathophysiology of metabolic disorders and could provide therapeutic approaches to control diabetes with or without obesity. Our results also raise the possibility that Hexarelin may be a novel drug candidate for correcting the lipid disorders associated with non-obese T2DM and open many avenues for future use in clinical trials. However, the effects of long-term [D-Lys3]-GHRP-6 treatment are not completely opposite to ghrelin and may not be a target as an anti-diabetic drug.

Declaration by author

This thesis is composed of my original work, and contains no material previously published or written by another person except where due reference has been made in the text. I have clearly stated the contribution by others to jointly-authored works that I have included in my thesis.

I have clearly stated the contribution of others to my thesis as a whole, including statistical assistance, survey design, data analysis, significant technical procedures, professional editorial advice, and any other original research work used or reported in my thesis. The content of my thesis is the result of work I have carried out since the commencement of my research higher degree candidature and does not include a substantial part of work that has been submitted to qualify for the award of any other degree or diploma in any university or other tertiary institution. I have clearly stated which parts of my thesis, if any, have been submitted to qualify for another award.

I acknowledge that an electronic copy of my thesis must be lodged with the University Library and, subject to the policy and procedures of The University of Queensland, the thesis be made available for research and study in accordance with the Copyright Act 1968 unless a period of embargo has been approved by the Dean of the Graduate School.

I acknowledge that copyright of all material contained in my thesis resides with the copyright holder(s) of that material. Where appropriate I have obtained copyright permission from the copyright holder to reproduce material in this thesis.

Publications during candidature

Peer-reviewed papers: First- authored papers Mosa RM, Zhang Z, Shao R, Deng C, Chen J, Chen C. Implications of ghrelin and hexarelin in diabetes and diabetes-associated heart diseases. Endocrine 2015; 49:307- 323. Mosa R, Huang L, Wu Y, Fung C, Mallawakankanamalage O, LeRoith D, Chen C. Hexarelin, a Growth Hormone Secretagogue, Improves Lipid Metabolic Aberrations in Nonobese Insulin-Resistant Male MKR Mice. Endocrinology 2017; 158:3174-3187. Mosa R, Huang L, Li H, Grist M, LeRoith D, Chen C. Long-term treatment with the ghrelin receptor antagonist, [D-Lys3]-GHRP-6 does not improve glucose homeostasis in non- obese diabetic MKR mice. American Journal of Physiology 2017. R-00157-2017R1.

Co-authored papers Webster JA, Yang Z, Kim YH, Loo D, Mosa RM, Li H, Chen C. Collagen beta (1-O) galactosyltransferase 1 (GLT25D1) is required for the secretion of high molecular weight adiponectin and affects lipid accumulation. Biosci Rep. 2017 May 17; 37(3).

Conference abstracts Rasha Mosa, Jiezhong Chen, Chao Lin, Chen Chen. Protective roles of Hexarelin, a synthetic growth hormone secretagogue on pancreatic beta cells in vitro and in type 2 diabetes MKR mice. SBMS Postgraduate Symposium, The University of Queensland, 2014.

Rasha Mosa, Lili Huang, Chung Fung, Michael A. Grist, Derek LeRoith and Chen Chen. Investigating GH-IGF-1 axis in a non-obese mouse T2DM model and the therapeutic applications. SBMS Postgraduate Symposium, The University of Queensland, 2015.

Rasha Mosa, Lili Huang, and Chen Chen. Effects of growth hormone secretagogue receptor agonist and antagonist in non-obese type 2 diabetic MKR mice. Asia Pacific 11th Diabetic conference, Brisbane, 2016.

Rasha Mosa, Lili Huang, and Chen Chen. Effects of growth hormone secretagogue receptor agonist and antagonist in non-obese type 2 diabetic MKR mice. The Annual Scientific Meeting of the Endocrine Society of Australia, Gold Coast, 2016 (Received travel grant).

Rasha Mosa, Lili Huang, and Chen Chen. Effects of growth hormone secretagogue receptor agonist and antagonist in non-obese type 2 diabetic MKR mice. SBMS Postgraduate Symposium, The University of Queensland, 2016. Publications included in this thesis

(R Mosa et al., Endocrinology, 2017-00168R1) - incorporated as Chapter 4. Contributor Statement of contribution Rasha Mosa Experimental design (70%), data collection (80%), interpretation of observations (80%) and preparation of the manuscript (80%) Lili Huang Experimental design (10%), interpretation of observations (10%) and preparation of manuscript (10%) Yeda Wu Data collection (5%) Chung Fung Data collection (10%) Oshini Mallawa Data collection (5%) Derek LeRoith Experimental design (5%) Chen Chen Experimental design (15%), interpretation of observations (10%) and preparation of manuscript (10%)

(R Mosa et al., American journal of Physiology, R-00157-2017R1) - incorporated as Chapter 5. Contributor Statement of contribution Rasha Mosa Experimental design (70%), data collection (90%), interpretation of observations (80%) and preparation of the manuscript (80%) Lili Huang Experimental design (10%), interpretation of observations (10%) and preparation of manuscript (10%) Hongzhuo Li Data collection (5%) Michael Grist Data collection (5%) Derek LeRoith Experimental design (5%) Chen Chen Experimental design (15%), interpretation of observations (10%) and preparation of manuscript (10%)

Contributions by others to the thesis

Professor Chen Chen (Principal Supervisor) and Dr Lili Huang (Associate Supervisor) contributed significantly to the design of the project and revising of manuscripts.

Statement of parts of the thesis submitted to qualify for the award of another degree

None.

Acknowledgements

I would like to pay special thankfulness and appreciation to all those who made my research successful and helped me at every point to accomplish my goal.

Firstly, I would like to express my sincere gratitude to My Principal Supervisor Prof. Chen Chen for his continuous support of My PhD study, for his patience, motivation, and immense knowledge. His guidance helped me in all the time of research and writing of this thesis. Thanks for giving me the opportunity to join your lab, supporting me with all the materials and animals for this study. I feel incredibly privileged to have him as My supervisor.

I would like to present my special thanks to My Associate Supervisor Dr Lili Huang whose help and sympathetic attitude at every point during my research helped me to achieve the goal. She inspired me with new ideas and taught me new techniques. I have learned a lot about how to do interesting and rigorous research through her supervision. Thanks very much for giving me time to revise all my milestone reports and conference presentations.

I am grateful to the entire lab group for the constructive feedback that they have provided to this project over the years, for being open and friendly to me. Special thanks go to Ms Chung Yan Fung who helped me in blood sample collection for 6 hour-pulse bleed experiments. I would like to thank Mr Yeda Wu who helped me in daily mice injections in the phenomaster. I want also to thank Mr Michael Alexander Grist who helped me with the analysis of immunofluorescence images. Also thanks to Hongzhuo Li for interesting discussions and solving any unsolvable problems.

I would like to thank the international scholarships of University of Queensland and international educational scholarships of My country Egypt for providing me with generous financial support throughout my doctorate.

My sincere thanks also go to Dr Derek LeRoith in National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, USA for providing us with transgenic mice (MKR mice). I am also grateful to Dr Johannes Veldhuis for deconvolution analysis of pulsatile GH data.

I have been blessed with a very loving and supportive family, My Mum, My sisters and My brother. Although I was away from them all My PhD, yet they have cherished with me every great moment and supported me whenever I needed it. I also owe a deep thank to all My friends and My church who have encouraged me along the way and supported me spiritually throughout My PhD and writing this thesis.

I owe huge thanks to the spirit of My father, without whom I was nothing. He always stressed the importance of education and I know that this respect for education has, formed my values and made me the person that I am today. He was always believing in me. I will never forget that he was calling me “Doctor” since My childhood.

I am grateful to My husband Dr Magdy Sedrak not just because he has given up so much to make my success a priority in our lives, but because he has seen me through the ups and downs of the entire PhD process. He has shared this entire amazing journey with me, so it only seems right that I dedicate this dissertation to him.

Beyond this, I need to thank My little boys for being such a bundle of joy and love. Without their sunny laugh, I would be a much grumpier person.

Last but not least, I would like to show My gratitude to all the faculty, staff members and lab technicians of School of Biomedical sciences and Australian Institute for Bioengineering and Nanotechnology Animal House whose services turned my research a success. I want to acknowledge Dr Darryl Whitehead, Mrs Melanie Flint and Dr Shaun Walters for their rich and supportive technical experiences that gave to me. I am immensely grateful to My thesis committee, Dr Mary-Louise Manchadi and Professor Karen Moritz who guided me through all these years.

Keywords non-obese, type 2 diabetes, hexarelin, growth hormone, lipodystrophy, ghrelin receptor antagonists, insulin, pancreatic islets, hypothalamus, insulin resistance

Australian and New Zealand Standard Research Classifications (ANZSRC) ANZSRC code: 111502 Clinical Pharmacology and Therapeutics, 60% ANZSRC code: 111603 Systems Physiology, 20% ANZSRC code: 110306 Endocrinology, 20%

Fields of Research (FoR) Classification FoR code: 1115 Pharmacology and pharmaceutical sciences, 60% FoR code: 1116 Medical physiology, 20% FoR code: 1103 Clinical sciences, 20%

Table of Contents 1) CHAPTER ONE: LITERATURE REVIEW, AIMS AND HYPOTHESIS ...... 1 1.1 Introduction ...... 1 1.2 Non-obese type 2 diabetes and lipid metabolic disorders ...... 3 1.3 The role of Growth hormone/IGF-1 in diabetes ...... 7 1.3.1 Growth hormone-insulin interrelationships in different metabolic conditions ...... 7 1.3.2 The regulatory role of GH in adipose tissue ...... 10 1.3.3 GH treatment in obesity ...... 11 1.3.4 Role of IGF-1 in modulating insulin sensitivity ...... 12 1.3.5 MKR mouse model ...... 14 1.4 Ghrelin ...... 15 1.4.1 Ghrelin receptors ...... 17 1.4.2 Effects of ghrelin on the hypothalamus-pituitary-GH Axis ...... 19 1.4.3 Effects of ghrelin on glucose metabolism ...... 20 1.4.4 Effects of ghrelin on food intake and energy metabolism ...... 22 1.5 Pharmacological modification of ghrelin system ...... 25 1.5.1 Hexarelin as a growth hormone releasing peptide ...... 27 1.5.2 Hexarelin as a CD36 agonist ...... 29 1.5.3 Ghrelin receptor antagonists ...... 30 1.6 Hypothesis and Aims ...... 32 2) CHAPTER TWO: MATERIALS AND METHODS ...... 34 2.1 Animal Care ...... 34 2.2 Serial blood sampling for assessment of pulsatile GH secretion ...... 34 2.3 Drugs and experimental design ...... 35 2.4 Glucose tolerance (GTT) and insulin tolerance (ITT) tests ...... 35 2.5 Indirect calorimetric assays ...... 36 2.6 Analysis of hormones and metabolites ...... 36 2.6.1 Analysis of GH and IGF-1 hormones using in-house Elisa ...... 36 2.6.2 Analysis of circulating hormones and metabolites using commercial Elisa and Multiplex kits...... 36 2.6.3 Measurements of muscle and liver triglycerides ...... 37 2.7 Paraffin embedding of tissues for histological or immunofluorescence staining ...... 37 2.8 Real-time quantitative polymerase chain reaction (qPCR) ...... 38 2.8.1 Real-time qPCR of hypothalamic gene expression ...... 38

2.8.2 Real-time qPCR of adipose tissue gene expression ...... 39 2.9 Data and statistical analysis ...... 39 3) CHAPTER THREE: INVESTIGATING THE PHENOTYPIC AND METABOLIC CHARACTERISTICS OF NON-OBESE TYPE 2 DIABETIC MKR MICE ...... 41 3.1 Brief introduction and rationale ...... 41 3.2 Material and methods ...... 41 3.2.1 Mice ...... 41 3.2.2 Characterizing pulsatile GH secretion in MKR mice ...... 42 3.2.3 Characterizing the phenotypic and metabolic profiles of MKR mice ...... 42 3.2.4 In-house GH sandwich Elisa ...... 42 3.2.5 In-house IGF-1 Elisa ...... 43 3.2.6 Hypothalamic GHRH, SST, NPY and POMC gene expression in MKR and FVB mice ...... 44 3.2.7 Statistical analysis ...... 44 3.3 Results...... 44 3.3.1 MKR mice though lean tended to eat more and had higher fat and lower lean mass ...... 44 3.3.2 MKR had significantly impaired glucose and insulin tolerance except at aged mice ...... 45 3.3.3 MKR mice have higher GH secretion at 5 but comparable to slightly higher GH secretion at 10 and 33 weeks old respectively ...... 48 3.3.4 Hormonal profiles of MKR mice ...... 49 3.3.5 Increased GH secretion in MKR mice occurs without changes in hypothalamic GHRH and SST but elevated NPY gene expressions ...... 51 3.4 Discussion ...... 52 4) CHAPTER FOUR: HEXARELIN, A GROWTH HORMONE SECRETAGOGUE, IMPROVES THE LIPID METABOLIC ABERRATIONS IN A NON-OBESE MALE MOUSE MODEL OF TYPE 2 DIABETES MELLITUS ...... 56 4.1 Introduction ...... 56 4.2 Materials and Methods ...... 58 4.2.1 Mice ...... 58 4.2.2 Peptide ...... 58 4.2.3 Glucose tolerance (GTT) and insulin tolerance (ITT) tests ...... 58 4.2.4 Effects of Hexarelin treatment on pulsatile growth hormone secretion ...... 58 4.2.5 Indirect calorimetric assays ...... 59 4.2.6 Blood glucose and Plasma hormonal analysis ...... 59 iii

4.2.7 Plasma lipid and tissues triglyceride analysis ...... 59 4.2.8 Quantitative real-time PCR ...... 59 4.2.9 Adipose tissue histology ...... 60 4.2.10 Statistical analysis ...... 60 4.3 Results...... 60 4.3.1 Effects of Hexarelin treatment on glucose and insulin tolerance in MKR Mice ... 60 4.3.2 Effects of Hexarelin treatment on GH secretion ...... 62 4.3.3 Effects of Hexarelin treatment on cumulative food intake, body weight and body composition ...... 62 4.3.4 Effects of Hexarelin treatment on respiratory exchange ratio (RER) and locomotor activity...... 64 4.3.5 Effects of Hexarelin on blood glucose and plasma hormones ...... 66 4.3.6 Effects of Hexarelin treatment on plasma lipids and tissues triglycerides ...... 67 4.3.7 Effects of Hexarelin treatment on the genes involved in fatty acid uptake and oxidative phosphorylation of white adipose tissue ...... 68 4.3.8 Effects of Hexarelin treatment on adipocytes morphology and size ...... 68 4.4 Discussion ...... 70 5) CHAPTER FIVE: LONG-TERM TREATMENT WITH THE GHRELIN RECEPTOR ANTAGONIST, [D-Lys3]-GHRP-6 DOES NOT IMPROVE GLUCOSE HOMEOSTASIS IN NON-OBESE DIABETIC MKR MICE ...... 74 5.1 Introduction ...... 74 5.2 Materials and Methods ...... 75 5.2.1 Mice ...... 75 5.2.2 Drug and experimental design...... 75 5.2.3 Effect of [D-Lys3]-GHRP-6 treatment on pulsatile GH secretion ...... 76 5.2.4 Glucose tolerance (GTT) and insulin tolerance (ITT) tests ...... 76 5.2.5 Metabolic parameters and indirect calorimetry ...... 77 5.2.6 Determination of hypothalamic mRNA expression ...... 77 5.2.7 Immunohistochemistry ...... 77 5.2.8 Data Analysis ...... 78 5.3 Results...... 78 5.3.1 Effect of [D-Lys3]-GHRP-6 treatment of on pulsatile GH secretions ...... 78 5.3.2 Impaired glucose and insulin tolerance in MKR Mice chronically treated with [D- Lys3]-GHRP-6 ...... 80 5.3.3 Effects on metabolic activities ...... 82

5.3.4 Long-term effects on hormonal and metabolic parameters ...... 85 5.3.5 Hypothalamus expression of neuropeptides and ghrelin receptor involved in regulating food intake and GH secretion in response to the [D-Lys3]-GHRP-6 treatment ...... 87 5.3.6 Morphology and analysis of insulin and somatostatin positive areas within pancreatic islets after long-term [D-Lys3]-GHRP-6 treatment ...... 89 5.4 Discussion ...... 91 6) CHAPTER 6. GENERAL DISCUSSION ...... 95 6.1 Overview...... 95 6.2 Major Findings ...... 96 6.2.1 MKR mice have an atypical form of type 2 diabetes ...... 96 6.2.2 MKR mice have normal to higher pulsatile GH secretion despite hyperinsulinaemia ...... 96 6.2.3 Hexarelin improves insulin intolerance through ameliorating lipid abnormalities in MKR mice ...... 98 6.2.4 [D-Lys3]-GHRP-6 is not a complete ghrelin receptor antagonist ...... 99 6.2.5 [D-Lys3]-GHRP-6 does not improve glucose or insulin intolerance in MKR mice ...... 100 6.2.6 Peripheral [D-Lys3]-GHRP-6 injections affect central hypothalamic regulatory neurons ...... 101 6.3 Conclusions and Implications of our findings ...... 101 6.4 Limitations of the study ...... 102 6.5 Future directions ...... 103 REFERENCES ...... 105

List of Figures

Figure ‎1.1 Mechanisms of increased ectopic lipid deposition in the liver and skeletal Muscle associated with metabolic syndrome...... 4 Figure ‎1.2 Interaction between growth hormone, insulin and the insulin-like growth factor system in obesity...... 9 Figure ‎1.3 The insulin receptors (IR-A and IR-B), the insulin like growth factor 1 receptor (IGF-1R), and the hybrid receptors (IGF-1R/IR-A and IGF-1R/IR-B)...... 13 Figure ‎1.4 Graphic representative of ghrelin's physiological effects...... 17 Figure ‎1.5 Mechanisms of ghrelin action on GHS-R1a receptors in the hypothalamus and pituitary...... 19 Figure ‎1.6 Regulation of food intake and adiposity by ghrelin...... 23 Figure ‎2.1 Coronal section of the brain...... 39 Figure ‎3.1 Assessment of cumulative food intake, and body composition in MKR and FVB mice at 10 weeks old...... 45 Figure ‎3.2 Glucose and insulin tolerance tests of MKR and FVB at different age groups...... 48 Figure ‎3.3 Growth parameters and pulsatile growth hormone profiles of MKR and FVB at different age groups...... 49 Figure ‎3.4 Measurements of metabolic hormones and inflammatory markers in MKR and FVB mice...... 51 Figure ‎3.5 Hypothalamic GHRH, SST, NPY and POMC mRNA expression in MKR and FVB mice...... 52 Figure ‎4.1 Effects of twice daily I.P. injection of Hexarelin (200 ug/Kg BW) or saline for 12 days on GTT, ITT and pulsatile GH secretion...... 61 Figure ‎4.2 Effects of Hexarelin treatment on cumulative food intake, body weight and body composition...... 63 Figure ‎4.3 Effects of Hexarelin treatment on indirect calorimetric parameters of MKR and FVB mice...... 65 Figure ‎4.4 Effects of Hexarelin treatment on blood glucose and plasma hormones...... 66 Figure ‎4.5 Effects of Hexarelin treatment on plasma and tissue lipids...... 68 Figure ‎4.6 Fold changes in gene expression levels of white adipose tissue gene expression after Hexarelin treatment in MKR and FVB mice...... 69 Figure ‎5.1 Effect of chronic [D-Lys3]-GHRP-6 treatment on pulsatile GH secretion in FVB and MKR mice...... 79 Figure ‎5.2 Effects of chronic [D-Lys3]-GHRP-6 treatment (200 nmol/mouse) or saline daily for 12 days on GTT, GSIS, and ITT in MKR and FVB mice...... 81 Figure ‎5.3 Acute effects of [D-Lys3]-GHRP-6 on food intake and indirect calorimetric parameters in MKR and FVB mice...... 83 Figure ‎5.4 Effects of chronic [D-Lys3]-GHRP-6 treatment on cumulative food intake, body weight and composition in FVB and MKR mice...... 84 Figure ‎5.5 Effect of chronic [D-Lys3]-GHRP-6 treatment in the metabolic profile of FVB and MKR mice...... 87 Figure ‎5.6 Fold change expression of hypothalamic genes after [D-Lys3]-GHRP-6 treatment...... 89 vi

Figure ‎5.7 Double immunofluorescence for insulin and somatostatin of pancreatic islets. 91 Figure ‎6.1 Schematic graph of the proposed Hexarelin action in regulating energy metabolism...... 104

List of Tables

Table ‎2.1 Details of commercial assay kits used to determine levels of circulating hormones and metabolites ...... 36 Table ‎4.1 Primer sequences used for gene expression analysis by qPCR ...... 60 Table ‎5.1 Primer sequences used for gene expression analysis by qPCR ...... 77 Table ‎5.2 Chronic effects on metabolic and hormonal parameters ...... 85

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List of Abbreviations

5-HT2b 5-Hydroxytryptamine receptor 2B

11β HSD1 11β-hydroxysteroid dehydrogenase type

ABCA1 ATP-binding cassette sub-family A member 1

ABCG1 ATP-binding cassette sub-family G member 1

AC–PKA Adenylate cyclase–protein kinase A

ACTH Adrenocorticotropic hormone

AG Acylated ghrelin

AgRP Agouti related peptide

AIBN Australian Institute for Bioengineering and Nanotechnology

Akt Phosphatidylinositol-3 kinase

AKT Protein kinase B

ANOVA Analysis of variance apoE Apolipoprotein E

ARC Arcuate nucleus

AUC Area under curve

BAT Brown adipose tissue

BBB Blood–brain barrier

BMI Body mass index

BSA Bovine Serum Albumin

BW Body weight

C/EBP CCAAT/enhancer binding protein cAMP Cyclic AMP

CART Cocaine and amphetamine related transcript cDNA Complementary DNA cFos The human homolog of the retroviral oncogene v-fos

CNS Central nervous system viii

CPT1b Carnitine Palmitoyltransferase 1B

DAG Diacylglycerol

DIO Diet induced obesity

DM Diabetes mellitus

EDTA Ethylenediaminetetraacetic acid

ER Endoplasmic reticulum

ERK1/2 Extracellular signal-regulated kinase 2

F1-ATP F1 portion of ATP synthase

FAT Fatty acid translocase

FATP1 Fatty acid transport protein 1

FATP4 Fatty acid transport protein 4

FFA Free fatty acids

FGF-21 Fibroblast growth factor 21 fIGF-I Free IGF-I

FTO Fat Mass and Obesity-Associated

GH Growth hormone

GHR Growth hormone receptor

GHRH Growth hormone releasing hormone

GHRPs Growth hormone releasing peptides

GHS Growth hormone secretagogues

GHS-R Growth hormone secretagogue receptor

GHS-R1 Growth hormone secretagogue receptor-1

GHS-R1a Growth hormone secretagogue receptor-1 alpha isoform

GHS-R1b Growth hormone secretagogue receptor-1 beta isoform

GLP-1 Glucagon like peptide-1

GLUT1 Glucose transporter 1

GLUT4 Glucose transporter 4

GOAT Ghrelin O-acyl-transferase

GPIV Glycoprotein IV

HCL Hydrochloric Acid

HDL High density lipoprotein cholesterol

HFD High Fat diet

HIV Human immunodeficiency virus

HMGR 3-hydroxy-3-methylglutaryl-CoA reductase

HOMA-IR Homeostasis model assessment for insulin resistance

HRP Horseradish peroxidase

HSL Hormone-sensitive lipase

I.P. Intraperitoneal

ICV Intracerebroventricular

IGFBP-1 IGF-binding proteins-1

IGFBPs IGF binding proteins

IGF-I Insulin-like growth factor 1

IGF-II Insulin-like growth factor II

IGF-IR Insulin-like growth receptor

IL-6 Interleukin 6

IL-β Interleukin 1 beta

LXRα Liver X receptor alpha

IP3 Inositol triphosphate

IR-A or IR-B Insulin receptor isoforms

IRS Insulin receptor substrates

ITT Insulin tolerance test

KCNJ11 Potassium Voltage-Gated Channel Subfamily J Member 11

LDL Low density lipoprotein cholesterol

LH Luteinizing hormone

LPL Lipoprotein lipase

LV Left-ventricle

MAPK Mitogen activated protein kinase

MBOAT Membrane-bound O-acyltransferase

MCFA Medium chain fatty acids

NAC Nucleus accumbens

NMR Nuclear magnetic resonance

NPY Neuropeptide Y

NTC Nucleus tractus solitaries ob/ob Leptin-deficient mice

OPD O-phenylenediamine oxLDL Oxidized low-density lipoprotein

PBS Phosphate Buffered Saline

PEPCK Phosphoenolpyruvate carboxykinase

PFT Personal fat threshold

PGC-1α PPAR-γ coactivator alpha

PIP2 Phosphatidylinositol 4, 5-diphosphate

PKCε Epsilon form of protein kinase C

PKCθ Theta form of protein kinase C

PLC–PKC Phospholipase C–protein kinase C

POMC Proopiomelanocortin

PPAR-γ Peroxisome proliferator activated receptor gamma

PVN Paraventricular nucleus qPCR Quantitative Real-time Polymerase Chain Reaction

RER Respiratory exchange ratio

RNA Ribonucleic acid

ROI Region of interest

RQ Respiratory quotient

SEM Standard error of the mean

SGA Small for gestational age

SNS Sympathetic nervous system

SST Somatostatin

SSTR Somatostatin receptor

T1DM Type 1 diabetes

T2DM Type 2 diabetes

TCF7L2 Transcription factor 7-like2

TG Triglyceride tIGF-I Total insulin-like growth factor-I

TNF-α Tumor necrosis factor-alpha

TSP Thrombospondin

UAG Unacylated ghrelin

UCP-1 Uncoupling protein 1

VO2 Oxygen consumption

VTA Ventral tegmental area

WAT White adipose tissue

WT Wildtype

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1) CHAPTER ONE: LITERATURE REVIEW, AIMS AND HYPOTHESIS

1.1 Introduction Diabetes mellitus (DM) is perceived as a heterogeneous metabolic disorder characterized by chronic hyperglycaemia with disturbances of carbohydrate, fat and protein metabolism resulting from defects in insulin secretion and/or insulin action (1). DM is a global and pandemic disease, which affects nearly 415 million patients according to international diabetes federation (IDF) (2). This disease is expected to impact approximately 642 million people worldwide by 2040 (2). This is in addition to 318 million people who have impaired glucose tolerance and are at high risk of developing diabetes as well. DM is classified into type 1 diabetes (T1DM), type 2 diabetes (T2DM) and gestational diabetes. T1DM is an autoimmune process of idiopathic aetiology that leads to pancreatic β cell destruction, and generally appears in adolescence (3). T2DM is the most prevalent form of diabetes that affects more than 90% of cases. Insulin resistance is the common pathological change in T2DM which eventually leads to pancreatic β cell dysfunction (4). Gestational diabetes is the occurrence of hyperglycaemia which recognized only during pregnancy with higher risk to develop diabetes later in life (5).

T2DM is a polygenic disease with a strong hereditary basis and environmental factors, especially unhealthy diets and physical inactivity (6). While, there is a general agreement that T2DM is closely associated with obesity, yet the disease can affect subjects with normal or even lower body mass index (BMI) (7). It has been projected that some obese patients have normal insulin sensitivity and are metabolically healthy (8), whereas, several normal weight individuals are metabolically obese and characterised by insulin resistance, impaired glucose tolerance and hyperlipidaemia (9,10). This non-obese diabetic phenotype is very prevalent especially among some ethnic populations. According to UK Biobank data, South Asians with BMI of 22 kg/m2 have equivalent prevalence of T2DM to white Europeans with BMI of 30 kg/m2 (11). Analysis of the Framingham study have shown that over 40% of men and women had increased visceral fat, despite an average BMI of 27 kg/m2 in women and 28 kg/m2 in men (12). The pathophysiology and causes of non-obese T2DM will be discussed in details in section 1.2.

In general, the complications of DM are disabling and life-threatening, resulting in increased risks for myocardial ischemia, stroke, neuropathy, nephropathy, retinopathy and microvascular complications (13). The non-obese T2DM patients are at equivalently high

1 risks of these complications as the obese diabetics. It has been recently reported that normal weight adults at the time of diagnosis of diabetes have higher cardiovascular and non-cardiovascular mortality than adults who are overweight or obese (14,15). Intensive changes in lifestyle and diabetic control are often unable to fully correct the metabolic aberrations in patients with non-obese T2DM. This is not very surprising, as these metabolic defects reflect the underlying insulin resistance rather than deranged hyperglycaemia. Thus, new drugs and novel methods as possible adjuncts to therapeutic lifestyle change to reduce visceral adiposity and/or treat insulin resistance should be considered.

Growth hormone (GH) is an anabolic hormone which has profound effects not only on body growth but also body composition. GH-deficient individuals are often obese and have increased body fat (16). GH is mainly regulated by GH releasing hormone (GHRH), somatostatin (SST), growth hormone secretagogues (ghrelin and its analogues) and insulin-like growth factor-1 (IGF-I) (17). Although alternation of GH secretion and its therapeutic implications have been well studied in obese diabetics but little is known about GH secretion in non-obese T2DM. Moreover, while established that ghrelin is a GH stimulator with a potent orexigenic effect, it is not yet known about the exact role of ghrelin and its receptor in the action of insulin secretion and glucose metabolism. Studies in humans and rodents have found a negative correlation between ghrelin and insulin Levels (18). Treatment of healthy young volunteers with ghrelin decreased insulin secretion, resulting in hyperglycaemia (19). On the contrary, ghrelin deletion in leptin deficient (ob/ob) mice improved glucose tolerance and insulin secretion but did not change body weight (BW) (20). On the other hand, the recent discovery of synthetic growth hormone secretagogues (GHS) demonstrated potent similar effects as ghrelin in promoting GH release, however, GHS have many metabolic effects independent of GH make them interest targets therapy for metabolic disorders.

The recent development of tissue-selective knockout animal models has brought new insights to our understanding of the relative roles of the metabolic hormones in glucose homeostasis and fat distribution. The diabetic model used in this study is MKR mice overexpressing a dominant negative form of IGF-1 receptor in muscle (21). This resulted in attenuation of both IGF-1 and hybrid insulin/IGF-1 receptor function and thus development of severe insulin resistance and diabetes (21). Besides insulin resistance, dysfunction of IGF-1 signalling results in less weight gain and marked lipid abnormalities in MKR mice

(22). These characteristics make MKR mice a good model of studying non-obese T2DM with inherited lipodystrophy, dyslipidaemia, or genetic disorders.

This thesis assessed pulsatile GH secretion and GH regulatory factors in a mouse model of non-obese T2DM (MKR mice) as addressed in chapter 3. Second to this, this study investigated whether Hexarelin, a growth hormone secretagogue contributes to enhanced glucose and insulin tolerance in MKR mice via altered GH secretion and corrected lipid metabolic aberrations as addressed in chapter 4. Finally, this thesis investigated whether [D-Lys3]-GHRP-6 as a consequence of antagonising ghrelin’s effects on food intake, insulin secretion and adiposity contributes to improved glucose and insulin intolerance in MKR mice as shown in chapter 5.

1.2 Non-obese type 2 diabetes and lipid metabolic disorders T2DM is commonly considered as a disease of obesity which deemed to be a key driving force behind the global epidemic of T2DM (23). Obesity refers to BMI above 25 kg/m2. Individuals who have BMI between 18-25 kg/m2 are considered normal weight and less than 18 are underweight (1). There are some people who are not obese according to their BMI but have T2DM and they are referred as non-obese or lean type 2 diabetic patients. In fact, the prevalence of non-obese T2DM is not low. It represents approximately 13% of total diabetes in USA, 20% in the northern European countries and more than 50% in Asian countries (24,25). Zhao et al reported a U-shaped association between BMI and mortality rates among adults with diabetes, whereby the lowest mortality rates are detected among obese adults than those in the normal weight and the underweight groups (26). This obesity paradox in diabetes was also noticed in other chronic diseases (27).

Many non-obese individuals prone to T2DM appear to have higher abdominal and visceral fat due to longitudinal changes in regional fat distribution, adipocyte morphology and function (28-30). An example of this concept is lipodystrophy; individuals with deficit in subcutaneous adipose tissue, impaired ability to store fat, and consequently fat is accumulated in visceral and ectopic sites such as liver, muscle, pancreas and heart causing lipotoxicity and derangement in organ metabolism possibly increasing risk to insulin resistance (31,32). In lipoatrophic A-ZIP/F-1 mice, which lack white adipocytes, fat accumulates in the liver and skeletal muscle, and profound insulin resistance happens in these tissues (33). However, when fat obtained from wild-type (WT) littermates transplanted subcutaneously into these mice, reduction of ectopic fat content and improvement of insulin sensitivity were observed (33). The adipose tissue is now 3 recognised as an endocrine organ with an important role in the regulation of glucose metabolism. However, it is only when the adipose tissue becomes dysfunctional that is associated with the development of diabetes. Failure of adipocyte differentiation and enlarged adipocyte size are considered markers of adipocyte dysfunction (34). Hypertrophic adipocytes display reduced ability to store and release fatty acids (FFA) accompanied by a changed pattern of adipokines secretion. This induces a redistribution of lipids towards peripheral tissues and development of insulin resistance (35).

Figure ‎1.1 Mechanisms of increased ectopic lipid deposition in the liver and skeletal Muscle associated with metabolic syndrome.

Ectopic lipid deposition can occur due to congenital or acquired defects in adipocyte metabolism (defects in fat storage or increased lipolysis). Acquired reductions in mitochondrial fatty acids metabolism (as in aged people) or inherited reductions (as in persons with insulin resistance whose parents have T2DM) predispose to ectopic lipid accumulation and insulin resistance. Adapted from Shulman GI. 2014 (36)

Ectopic fat deposition in liver and muscles are strongly associated with dysfunctional adipose tissues among diabetic patients (37-41) (Figure ‎1.1). Specifically, excess hepatic fat appears to be related to hepatic insulin resistance. Individuals with hepatic steatosis are therefore unable to suppress hepatic gluconeogenesis in response to insulin, thus leading to hyperglycaemia and development of diabetes. The molecular mechanisms by which the dysfunctional adipose tissue contributes to the development of insulin resistance 4 with ectopic adiposity have some arguments. More than half a century ago, Randle and colleagues, suggested that an increase in FFA oxidation would result in an inhibition of mitochondrial glucose oxidation enzymes such as pyruvate dehydrogenase, phosphofructokinase and hexokinase activity, leading to an increase in intracellular glucose concentrations and decreased glucose uptake by muscle (42). On the contrary, another hypothesis proposed that the impairment of FFA oxidation would lead to accumulation of intracellular lipid metabolites such as triacylglycerol, diacylglycerol and ceramides. These lipid metabolites mediate insulin resistance by causing defects in insulin signalling and reduced insulin-stimulated glucose-transport activity (43,44). The latter hypothesis was supported by studies that showed transient increases in muscle diacylglycerol content and constant activation of the theta form of protein kinase C (PKCθ) after muscle lipid infusion or high fat diet (HFD) to mice, leading to activation of a serine– threonine kinase of insulin receptor substrate (IRS-1) and inhibition of insulin signalling (45). Moreover, such increase in serine phosphorylation of IRS-1, has been observed in muscle of persons with T2DM (46). Similarly, fatty liver has also been linked to the decrease in insulin-stimulated tyrosine phosphorylation of IRS-1 and IRS-2 caused by the epsilon form of protein kinase C (PKCε) activation, which ultimately inhibits insulin-induced activation of glycogen synthesis and suppression of glucose production in the liver (36).

Other risk factors have been interplayed into development of non-obese diabetes such as genetics (47), adverse intrauterine conditions (48), β-cell dysfunction (49), and distinct organ defects including muscle insulin resistance (50). The contribution of genetics in the development of lean T2DM is still under research. Only few genetic polymorphisms have been reported; namely, Transcription factor 7-like 2 (TCF7L2) and Potassium Voltage- Gated Channel Subfamily J Member 11 (KCNJ11) in impaired insulin secretion (51). Moreover, two other polymorphisms of peroxisome proliferator activated receptor gamma (PPAR-γ) and its co-transcriptional factor PPAR-γ coactivator alpha (PGC-1α) are associated with adipose tissue abnormalities and insulin resistance (52,53). Studies have reported that altered in-utero conditions play a significant role in predisposing to non- obese T2DM (54). Increased abdominal adiposity, decline of insulin secretion and reduced muscle glucose uptake and oxidation are all associated with low birth weight (55,56). Although it is often presumed that non-obese people with T2DM have greater insulin secretory deficits compared with overweight/obese patients (57), yet the inﬂuences of ethnicity, age and diabetes phenotype on the pathophysiological characteristics of non- obese diabetes, notably insulin secretion and insulin resistance, are still under debate. The 5 higher prevalence of diabetes in non-obese subjects is more noticeable among the Asian populations with higher body fat percentage for a given BMI (58-60). The pathophysiology of non-obese T2DM among different populations may be driven by the personal fat threshold (PFT) and its relation to the degree of β-cell function and insulin sensitivity (61). Gaining excess fat that exceeds their PFT will trigger the development of diabetes. Aging is another risk factor that leads to intrinsic mitochondrial metabolic derangements in tissues hindering their ability to handle excessive FFA ﬂuxes (Figure ‎1.1). Patients diagnosed with T2DM at old-age tend to have higher fat mass and sarcopenia due to accelerated loss of muscle and bone mass (62). Thus, assessing the body composition and waist circumference are likely to be more important than simple measurements of obesity such as BMI in assessing risks for metabolic diseases (63).

Giving the pathophysiological heterogeneity of non-obese T2DM, a particular challenge may be posed to treat those patients (64). In general, lifestyle modifications including caloric restriction and increased physical activity are considered the first line of intervention for treating diabetic individuals (65,66). However, questions arise about whether non- obese patients should be advised to lose weight, or whether those patients should be recommended to gain weight (67-69). Some concerns raised that additional weight loss could exacerbate both muscle and bone loss contributing to further sarcopenia. Moreover, subjects with high visceral fat and fatty liver have a lower chance of improving from lifestyle intervention and may require intensified lifestyle prevention strategies (70). Another important question is whether the abnormal pattern of fat distribution can be reversed and whether dysfunctional fat cells can be converted to healthy adipocytes. Although it has been reported that sulfonylureas, metformin, thiazolidediones and α- glucosidase inhibitors are effective at reducing serum glucose levels in lean diabetic patients, lean patients with T2DM were shifted out earlier to initiation of insulin therapy compared with obese diabetics (24). Furthermore, some of these pharmacological interventions may promote further weight loss, accelerate β cell failure or increase cardiovascular risks in lean patients (71,72). Therefore, the strategy for treating non-obese patients should be considered to cure the metabolic aberrations and potentially reduce the risk of developing diabetic complications.

1.3 The role of Growth hormone/IGF-1 in diabetes

1.3.1 Growth hormone-insulin interrelationships in different metabolic conditions GH is secreted in a pulsatile manner from somatotrophs in anterior pituitary gland and this process is mainly regulated by two types of hypothalamic neurons: GHRH neurons in the arcuate nucleus (ARC) and SST neurons in the periventricular nucleus (PVN) (73). The primary function of GH is to regulate somatic growth either directly or indirectly through IGF-I, which is synthesized mainly by the liver. Circulating IGF-1 binds to IGF binding proteins (IGFBPs), mainly IGFBP-3, IGFBP-5, and acid labile sub-unit (ALS) which increase IGF-1 bioavailability and stabilize IGF-1 in circulation. In addition to stimulating body growth, GH has important metabolic roles that are independent of IGF-I effects, including stimulation of lipolysis and inhibition of insulin ability in muscle to promote glucose uptake. It also increases gluconeogenesis in the liver (74,75).

GH mediates its intracellular effects via the GH receptor (GHR) which is a transmembrane receptor belonging to the class 1 cytokine receptor family. GH binding to GHR dimers results in a conformational change in the receptors and associated Jak2 molecules. Activated Jak2 then phosphorylates the cytoplasmic domains of the GHR which then recruits several downstream proteins (76). Of the various activated proteins, STAT5, particularly STAT5b, mediates the majority of the biological effects of GH, including the transcription of IGF-1 (77). Activation of GHR also stimulates the mitogen activated protein kinase (MAPK) pathway downstream of both Jak2 and Src (76). Of note, GHR signaling has been associated with activation of the phosphatidylinositol-3 kinase (PI3K)/Akt pathway in a Jak2/IRS-1 dependent manner, a pathway which is common with insulin and IGF-1-mediated signal transduction (78).

Besides the hypothalamic factors that control GH, there are some peripheral hormones regulate GH secretion. IGF-1 regulates GH secretion via negative feedback mechanisms through acting directly on somatotrophs and indirectly on hypothalamus to inhibit GH secretion (79,80). GH secretion is also regulated by insulin, leptin and ghrelin (81,82). Leptin and ghrelin positively regulate GH release. In fact, selective deletion of leptin receptors in somatotrophes results in GH deficiency and an increase in adiposity (83). Ghrelin as an endogenous GHS, exerts its effects via growth hormone secretagogue receptor-1 alpha (GHS-R1a) independent of GHRH and SST (84). On the other hand, insulin negatively regulates GH release (85). In cases of obesity with hyperinsulinaemia, both spontaneous and stimulated GH secretion are diminished (86,87). Cross-sectional 7 studies are limited in their ability to determine cause-effect relationship, as low GH secretion could further contribute to the accumulation of abdominal fat (75). Nonetheless, the reduction of GH levels in obesity, does not accompanied by a proportional change to total IGF-1 levels (88). Additionally, free IGF-1 was reported to be elevated in obesity and this could further suppress GH secretion by activating the negative feedback loop (89). Moreover, reduced IGFBP-1 expression has also been reported in obese subjects, which could result in increased IGF-1 bioactivity (88,90) (Figure ‎1.2). Consistent with this notion, overeating for a period of 2 weeks was associated with an increase in insulin levels, decreased IGFBP-1, increased free IGF-I and reduced GH (91). However, most of the decline in GH concentrations occurred before the increase in free IGF-I in the circulation suggesting that the process might be independent of IGFBP-1 (91). Another hypothesis suggests that insulin may have a direct inhibitory effect on GH secretion (92) (Figure ‎1.2). In vitro incubation of mouse pituitary cells with insulin inhibits GH secretion, independently of IGF-1 receptor (93). Similarly, administration of an oral glucose challenge to healthy subjects with a non-functional pituitary tumour resulted in a significant reduction in GH levels within the first hour (94). It is likely that central insulin sensitivity is preserved in obesity where insulin can directly inhibit pituitary GH, or indirectly through increasing hepatic GH responsiveness and suppressing hepatic IGFBP-1 secretion. In obese mice it is proposed that hyperinsulinaemia induced GHRH suppression which could inhibit GH secretion (95). Other studies hypothesized that a change in SST tone also plays a role in the decline of GH secretion in obesity (96,97) (Figure ‎1.2).

Figure ‎1.2 Interaction between growth hormone, insulin and the insulin-like growth factor system in obesity.

Insulin inhibits GH secretion directly from pituitary or indirectly through peripheral inhibition of IGFBP-1 secretion from liver or central hypothalamic effects. Inhibitory effects are shown in blue and stimulatory effects in red. GH, growth hormone; tIGF-I, total insulin-like growth factor-I; IGFBP-1, IGF-binding proteins-1; fIGF-I, free IGF-I.

Apart from insulin, increased circulating FFA levels in obesity may also have a suppressive effect on GH secretion. Incubation of GH3 rat pituitary tumour cells with cis-unsaturated fatty acids such as oleic acid reduced GH secretion (98). Moreover, lowering of systemic FFA concentrations by administration of the lipolysis inhibitor (acipimox) increased total and pulsatile GH secretion in obese women (99). Leptin and adiponectin might also affect GH expression and secretion in obesity. Hypoadiponectinemia and leptin resistance, associated with obesity, have been implicated in regulating of GH secretion (100). Incubation of primary rat pituitary cells with adiponectin increased GH secretion (101). While selective deletion of leptin receptors from somatotrophes in mice results in a reduction of GH expressing cells and consequently reduced GH secretion (102). Leptin induced GH secretion was associated with an increase in ghrelin levels suggesting that leptin augmented ghrelin action (103). Thus, obesity-induced changes in circulating levels of hormones and adipokines can contribute to decreased GH secretion.

The metabolic data regarding GH secretion in lean T2DM are scarce and not conclusive. The children who born small for gestational age and at higher risk to develop T2DM later in adulthood, showed higher GH secretion indicating that GH has an important role to stimulate intrauterine growth along with other growth promoting factors (104). GH treatment in those children induced catch-up growth and increased their adult heights (105). Moreover, it resulted in a decline in fat mass and an increase in lean mass, but showed a reversible decrease in insulin sensitivity (105). Another study has found that both obese and lean diabetic subjects had decreased GH responses to GHRH and pyridostigmine (an acetylcholinesterase inhibitor, stimulates GH release), compared with obese and normal-weight healthy subjects respectively (96). In this study, it was hypothesized that impaired GH secretion in both lean and obese T2DM could be related to the hyperglycaemia and hyperinsulinaemia that affect SST tone independent of BW. It is also noteworthy that reduced GH secretion in obese state has been associated with adverse lipoprotein profile and cardiovascular complications (106,107). Utz et al reported that adverse cardiovascular complications increase with the reduction in GH secretion (107). However, the cause-effect relationship is still to be clarified.

1.3.2 The regulatory role of GH in adipose tissue The influence of GH on adipose tissue is well documented. GH stimulates lipolysis in the adipose tissue, particularly the visceral and subcutaneous depots (108) by increasing hormone-sensitive lipase (HSL) activity of the adipose tissue in humans and rodents (109,110). Both GH and IGF-1 receptors are abundant on all cell types which are found in adipose tissue such as; adipocytes, preadipocytes, fibroblasts, various immune cells (macrophages), and endothelial cells suggesting a crucial role of GH and IGF-1 in preadipocyte proliferation, differentiation and senescence (111,112). Not only can GH affect adipocytes maturation but also regulate the secretion of adipokines in the circulation (113). GH stimulated adiponectin secretion from differentiated 3T3-L1 adipocytes under normal and high glucose conditions (114). However, human adipocytes incubated with GH demonstrated decreased adiponectin secretion (115). Thus, the exact effect of GH regulation of adiponectin secretion remains to be determined. White adipose tissue (WAT) has some adipocytes with a brown-adipocyte-like phenotype (beige adipocytes) that exhibit a specialized thermogenic function (116). Increasing amount of beige cells has the potential to protect against obesity and alter whole-body energy expenditure. Interestingly, some evidence suggests that GH can induce browning of adipocytes, by increasing the proportion of beige adipocytes in WAT (117). GH may also modulate glucocorticoid action 10 in the adipose tissue. Recent studies have shown that GH down-regulates 11β- hydroxysteroid dehydrogenase type (111β HSD1) expression in the adipose tissue (118). 11β HSD1 stimulates the conversion of inactive dehydrocorticosterone to active corticosterone. Mice with adipose tissue-specific over-expression of 11β HSD1 are insulin resistant; while knockout of 11β HSD1 protects mice from diet-induced obesity and insulin resistance (119).

1.3.3 GH treatment in obesity Elevated GH concentrations has been related to the development of insulin resistance (120). The specific mechanisms responsible for GH-induced insulin resistance remain unclear. However, this effect is thought to be secondary to stimulation of lipolysis and subsequent FFA release (120). Moller et al reported that, GH infusion induced a sustained increase in FFA and enhanced rate of lipid oxidation (121). Nevertheless, others have argued against this mechanism as GH treatment causes insulin resistance before FFA levels are elevated in the circulation, which suggests that other mechanisms could also be present (122). Despite worries that GH treatment can induce T2DM in individuals with obesity, a 12-week placebo-controlled trial of GH treatment combined with caloric restriction in obese and newly diagnosed T2DM patients, showed that GH treatment reduced visceral adipose tissue mass and levels of low density lipoprotein (LDL) cholesterol and also improved insulin sensitivity as measured by euglycaemic- hyperinsulinaemic clamp (123). Similarly, GH replacement in patients with obesity and poorly controlled diabetes resulted in a reduction in abdominal adipose tissue mass with improvement in insulin sensitivity (124). The improvement in insulin sensitivity noticed in these studies could be attributed to the increase in serum IGF-1 levels, the increase in skeletal muscle mass and the reduction in visceral adipose tissue mass.

GH replacement in adults with severe GH deficiency and obesity resulted in increased whole-body insulin sensitivity (125). Moreover, when patients with human immunodeficiency virus (HIV) infection who exhibit abdominal obesity as a result of antiretroviral therapy are treated with GH, they also show a preferential reduction in central adipose tissue mass and reduction of serum triglyceride (TG) concentration and diastolic blood pressure (126). Furthermore, resting energy expenditure was increased in response to GH treatment in individuals with obesity. Studies in adults with GH deficiency indicate that exercise capacity is increased during GH replacement (127,128). GH treatment in children of short stature resulted in catch up growth and correction of body composition but

11 lowered insulin sensitivity (129,130). Of note, the duration of GH treatment could differentially affect insulin sensitivity. Studies reported an adverse effect on glucose homeostasis and insulin sensitivity with GH therapy of short duration, whereas studies with long-term GH treatment show minor effects, as indicated in a meta-analysis (131).

1.3.4 Role of IGF-1 in modulating insulin sensitivity IGF-I is a peptide hormone that shares nearly 50% amino acid sequence identity with proinsulin. Similar to insulin, IGF-1 is composed of an alpha and a beta chain connected by disulfide bonds. Plasma concentration of IGF-1 is 100 times higher than that of insulin nonetheless the effects of IGF-1 in the glucose uptake is only 4-7% of that of insulin (132). The effects of IGF-I are mainly mediated by its binding to the IGF-IR. Similar to the insulin receptor, IGF-IR is comprised of two transmembrane alpha subunits and two intracellular beta subunits (133) (Figure ‎1.3). In addition, insulin and IGF-I can bind with low affinity to the receptors of each other but only at high concentrations. IGF-II can bind IGF-IR and IGF-IIR, however, the function of IGF-II is not well known (134). The binding of both IGF-I and insulin to their respective receptors results in the activation of the tyrosine kinase domain present in these receptors, and post-receptor phosphorylation of insulin receptor substrates (IRS) (135). The insulin receptor favourably phosphorylates IRS-1 and has a strong metabolic activity whereas IGF-I is a more potent mitogen, and plays a major role in regulating cell replication, differentiation, and survival (136). Interestingly, there are hybrid receptors composed of one alpha or one beta subunit of the IGF-IR, and one alpha or one beta subunit of the insulin receptor (Figure ‎1.3). These hybrid receptors vary both in their relative affinity for IGF-I, IGF-II, and insulin, as well as in their activity depending on the insulin receptor isoform present (either IR-A or IR-B) (137). It is possible, therefore, that tissue-specific differences in the activity of IGF-I could be partly due to the differences in receptor distribution.

Figure ‎1.3 The insulin receptors (IR-A and IR-B), the insulin like growth factor 1 receptor (IGF-1R), and the hybrid receptors (IGF-1R/IR-A and IGF-1R/IR-B).

Insulin binds mainly to IR-A and IR-B with lower affinity for IGF-1R. IGF-1 binds to the IGF- 1R and IGF-1R/IR-A and IGF-1R/IR-B hybrids. IGF-II binds to the IR-A, IGF-1, and IGF- 1R/IR-A hybrid receptor. Activation of IR-A by insulin or IGF-II mostly activates mitogenic signaling pathway. Activation of the IR-B or IGF-1R/IR-B hybrid receptor stimulates mainly the metabolic effects.

IGF-1 has an insulin-like action on glucose and lipid metabolism. IGF-1 may stimulate glucose uptake in the skeletal muscle by binding to hybrid receptors (138). IGF-1 has been shown to inhibit hepatic glucose production (139). Administration of recombinant human IGF-I has been reported to improve insulin sensitivity in healthy individuals as well as in patients with insulin resistance and T2DM (140). IGF-1 promoted fatty acids uptake into adipocytes and hepatocytes (141). Consistent with that, several studies reported that exogenous IGF-1 administration reduced FFAs levels in patients with and without T2DM (142). Pre-adipocytes have abundant IGF-1R expression and having an affinity for IGF-1 that is two times of magnitude greater than insulin, so it is suggested that IGF-1 plays a critical role in preadipocyte proliferation, differentiation and survival (143).

There are some genetic evidences of the role of IGF-1 system in obesity and glucose metabolism. In one human case with IGF-I deficiency related to a homozygous partial

13 deletion in the IGF-I gene has severe insulin resistance which is normalized by IGF-I therapy (144). Interestingly, IGF-1 knockout mice models develop T2DM with significant elevations in serum FFA and TG levels and increased TG deposits in liver and muscle, suggesting that hyperlipidaemia and accumulated lipids in tissues may be causative factors for the progression of diabetes in these mouse models (22). Overall, tissue specific IGF-1 knockout mouse models provide an evidence of the roles of IGF-I in lipid metabolism and insulin sensitivity

1.3.5 MKR mouse model Many different mouse models are available to understand the relationships of GH/IGF-1 and insulin resistance. For examples, liver IGF-I-deficient transgenic mouse model which has marked reduction in circulating IGF-I and elevated GH levels had been shown to develop muscle insulin resistance (145). MKR mice are transgenic mice generated by overexpression of a dominant negative IGF-1 receptor in the skeletal muscle containing the K1003R mutation (21). Expression of the mutant receptor results in the formation of hybrid receptors with endogenous IGF-1R and IR, leading to the impairment of the function of both the IGF-IR and the IR, as reflected by attenuation of their signalling pathways and development of insulin resistance and T2DM earlier in their life (21). The expression of the mutant human IGF-IR was restricted to skeletal muscle and no expression of the mutant receptor was detected in a variety of other tissues. Although MKR mice had the IGF-1 mutation merely in skeletal muscle, liver and adipose tissue also develop insulin resistance as indicated by increased hepatic glucose production and FFAs in the circulation (146). This was confirmed by hyperinsulinaemic-euglycaemic clamp that showed that total body glucose disposal, glycolysis, and glycogen synthesis were significantly reduced in the MKR mice, as compared to WT mice (21,147). In association with hyperglycaemia, MKR mice exhibited β cell dysfunction occurred in despite of compensatory increases in insulin content and β cell mass (148).

Apart from the insulin resistance, MKR mice showed growth retardation soon after birth up to 5 weeks of age, but after 5 weeks they catch up their siblings in body length, but not in body weight which remained lower by 10% in MKR compared with WT animals (149). MKR mice exhibited impaired skeletal muscle development and functional performance as characterized by reductions in myofibrils number and area as well as deficiencies in functional performance (150). Interestingly, recent studies have shown that MKR mice have some sort of metabolic inflexibility that enables them to utilise glucose during fasting

14 in spite of insulin resistance. This increased glucose uptake is most likely independent of the insulin-stimulated pathway and could be attributed to the compensatory increase in type II muscle fibres and GLUT1 and GLUT 4 glucose transporters (146). Therefore, MKR mice may be unusual by having normal fasting blood glucose despite marked insulin resistance.

In addition, MKR mice exhibit defects in FFAs oxidations pathways which leads to significant elevations in serum FFA and triglyceride levels and increased triglyceride deposits in liver and muscle, suggesting that hyperlipidaemia and accumulated lipids in tissue may be causative factors for the progression of T2DM in MKR mice (22). MKR mice had a significant down-regulation of many of the genes involved in fatty acid β oxidation genes involved in fatty acid oxidation in adipose tissue and skeletal muscles (151). Treating MKR mice with hypolipidaemic drug, WY-14643 (PPAR-α agonist), resulted in not only reduction of significantly elevated serum lipids and liver lipid content but also improvement of the whole-body insulin sensitivity (152). Treatment of adult MKR mice with the beta 3-adrenergic receptor agonist CL-316,243 led to improvement in metabolic parameters and altered expression of fatty acid oxidation genes (151). On the contrary, treating MKR mice with hypoglycaemic agents such as Phloridzin without correcting the hyperlipidaemia failed to improve glucose intolerance in these mice (153). These findings make MKR mice a useful model of non-obese T2DM with major lipid metabolic abnormalities.

1.4 Ghrelin Over the last 20 years, growing attention has been given to some peptides involved in the regulation of energy balance. Regulation of energy homeostasis requires precise coordination between peripheral food-sensing hormones and central regulatory neurons (154). Among many recently discovered peptides, ghrelin is the only peripheral orexigenic hormone that activates appetite centres in the hypothalamus (155). Consequently, ghrelin is predominantly considered as a central modulator of energy homeostasis. Ghrelin is 28 amino acids, synthesized and secreted mainly in the stomach from oxyntic mucosa X/A- like cells (156). However, low levels of ghrelin expression can also be found in other tissues such as the intestine, pancreas, kidney, ovary and brain (157).

Ghrelin was discovered In 1999 by Masayasu Kojima, Kenji Kangawa and their colleagues four years after the identification of its receptor, GHS-R1a (158) and nearly twenty years after the generation of a group of synthetic peptide and non-peptide agonists of the ghrelin 15 receptor that promote the release of GH from the anterior pituitary (159). Ghrelin is secreted in a pulsatile manner and also in relation to feeding. The circulating levels of ghrelin are elevated during fasting and before meals (160) and decline postprandial (161) which implies that ghrelin plays a significant role in initiation of food intake (162). Initially after its discovery, research focused on its roles as a hunger hormone and a GH stimulator from the anterior pituitary gland (163). Subsequently, numerous central and peripheral actions of ghrelin were discovered, including stimulation of gut motility and gastric acid secretion (164), taste sensation (165), regulation of glucose metabolism (166,167), modulation of sleep (168), enhancing learning and memory (169), reward seeking behaviour (170), increasing adiposity by reducing fat utilisation (171,172), modulation of depression and anxiety (173), protection against muscle degeneration (174), and improvement of cardiovascular functions such as vasodilatation and cardiac contractility (175,176) (Figure ‎1.4). Given this wide spectrum of biological activities, opened up many avenues for use of ghrelin in neuroendocrine and metabolic research.

Figure ‎1.4 Graphic representative of ghrelin's physiological effects.

Ghrelin receptor expressed in many organs and tissues of human body such as nervous system, heart, gastrointestinal tract and pancreas indicating a wide variety of ghrelin’s biological central and peripheral effects. Adapted from Ghrelin Mol Metab 2015; 4:437-460 (177) .

1.4.1 Ghrelin receptors In humans, the GHS-R1 gene codes for the full-length G-protein coupled seven transmembrane protein. GHS-R1a is the main ghrelin receptor, but a truncated isoform (GHS-R1b), which is not activated by ghrelin is also transcribed (178). Whether GHS-R1b is a functional receptor or not is not clear. However, a recent study has reported that the co-expression of GHS-R1b with GHS-R1a inhibits GHS-R1a activation, providing a possible explanation for the physiological function of the GHS-R1b (179). GHS-R1a is highly expressed in the hypothalamus and pituitary gland, consistent with the actions of ghrelin on the GH secretion. In addition to being expressed in GHRH neurons in ARC, GHS-R1a is co-localized in neurons that express neuropeptide Y (NPY) and Agouti related peptide (AgRP), which regulate food intake and satiety (180). Moreover, multiple peripheral organs, such as the stomach, intestine, pancreas, thyroid, gonads, adrenal, kidney, heart and vasculature as well as several endocrine and endocrine tumours and cell lines, have been found to express GHS-R1a (178). All these findings indicate that ghrelin has extensive functions beyond the control of GH release and food intake. It is noteworthy to mention that GHS-R1a is not expressed in tissues involved in lipid metabolism such as adipose tissue, liver and skeletal muscle (181-183).

To activate GHS-R1a receptor, ghrelin is required to be acylated by the attachment of a fatty acid side-chain (n-octanoic acid) to its serine 3 residue. Acylation is attained by the ghrelin O-acyl-transferase (GOAT), a member of the membrane-bound O-acyltransferase (MBOAT) family (184). Acylated ghrelin (AG) is considered to be the biologically active form of ghrelin. More specifically, the effects of ghrelin on energy balance and GH release were shown to be dependent on the acylation (185). Unacylated ghrelin (UAG), which is present in human serum in far greater quantities than AG, was initially considered to have no endocrine activity. However, some non-endocrine actions including cardiovascular and anti-proliferative effects have been assigned to UAG, probably by binding different GHS-R subtypes or receptor families (186,187). GOAT-ghrelin system acts as a nutrient sensor informing the body of the presence of nutrients specifically dietary lipids and links ingested lipids to energy expenditure. Studies showed that mice lacking GOAT when fed on

17 medium chain fatty acids (MCFA) supplemented diet, have lower BW and fat mass compared to WT mice. Whereas transgenic mice overexpressing ghrelin and GOAT show higher BW and fat mass and decreased energy expenditure, indicating a role for the endogenous AG in the regulation of energy balance and adiposity (188).

Many peptidyl and non-peptidyl molecules have been generated to manipulate the function of ghrelin receptor either by activation or inhibition. GHS are group of synthetic peptides that can bind to GHS-R1a and stimulate release of GH from the pituitary. In contrast, the ghrelin receptor antagonist (D-Arg1, D-Phe5, DTrp7, 9, Leu11)-substance P was found to be a high-potency full inverse agonist via decreasing the constitutive signalling of the ghrelin receptor (189). The GHS-R1a is unlikely to be the only ghrelin receptor. Other GHS-R subtypes or an unidentified receptor probably mediates GH-independent biologic activities of ghrelin and synthetic peptidyl GHS (190,191). GSK1614343, a fully ghrelin antagonist that could antagonize ghrelin action on the GHS-1a receptor inhibits ghrelin- induced GH secretion. However, GSK1614343 acts as a ghrelin agonist with regard to stimulating food intake and weight gain (192). The dissociation between the effects of GSK1614343 on GH secretion and food intake suggests unknown underlying regulations of the ghrelin system. Moreover unlike ghrelin which induced a prompt increase in glucose levels and decrease in serum insulin, Hexarelin, one of GHS, did not modify glucose and insulin levels despite its marked GH-releasing effect indicating that ghrelin may act through a different receptor to mediate insulin secretion and glucose uptake (193). Hexarelin has also been demonstrated to have cardioprotective effects through CD36 activation, a scavenger receptor that has a much lower affinity for ghrelin than for GHS (194).

Furthermore, Ghrelin seems to activate at least two signal transduction pathways on the same GHS-R1a receptor through binding to different ligand sites (195) (Figure ‎1.5). First, ghrelin increases cytoplasmic calcium through the adenylate cyclase-protein kinase A (AC- PKA) pathway in NPY expressing cells that in turn stimulates food intake (196). Second, ghrelin triggers GH release through the phospholipase C-protein kinase C (PLC-PKC) pathway that catalyses the hydrolysis of cell membrane phospholipids especially phosphatidylinositol 4, 5-diphosphate (PIP2) into inositol triphosphate (IP3) and diacylglycerol (DAG) which act as two different second messengers (197). The IP3 binds to 2+ IP3 receptor, which is a ligand-gated Ca channel of the endoplasmic reticulum (ER) and triggers the release of Ca2+ into the cytoplasm. Additional calcium also enters from the extracellular medium via voltage-operated L-type channels. The calcium ions causes

18 contraction of smooth muscles that leads to secretory changes in the somatotrophs followed by GH exocytosis (185). This indicates that different ghrelin agonists and antagonists may work differently according to their binging sites to the receptor.

Figure ‎1.5 Mechanisms of ghrelin action on GHS-R1a receptors in the hypothalamus and pituitary.

Ghrelin increases calcium fluxes through two signal transduction pathways; adenylate cyclase-protein kinase A (AC-PKA) pathway in NPY-expressing cells of the arcuate nucleus to stimulate food intake or phospholipase C-protein kinase C (PLC-PKC) pathway in the pituitary to stimulate GH release. Adapted from Front Neuroendocrinol. 2010; 31(1):44-60 (198).

1.4.2 Effects of ghrelin on the hypothalamus-pituitary-GH Axis GH is produced in the anterior pituitary, and its secretion is primarily regulated by two hypothalamic peptides: GHRH (stimulatory) and SST (inhibitory). Ghrelin, as well as synthetic analogues, also triggers GH release via activation of GHS-R1a at both hypothalamic and pituitary levels. In humans, acute intravenous injection or continuous 24 hour ghrelin infusion induces acute GH release and increases 24 hour pulsatile GH secretion respectively (199,200). This GH stimulatory effect of ghrelin is possibly mediated through functional inhibition of hypothalamic SST (201), stimulation of GHRH producing neurons in the ARC or augmentation of the effect of GHRH at the pituitary (202). One difference between ghrelin and GHRH is that the former mainly releases stored GH by exocytosis, but the latter can stimulate the synthesis of GH. Ghrelin and GHRH have been 19 shown to act synergistically on GH secretion (203). Other studies showed that an intact GHRH system is necessary for ghrelin induced GH secretion. The administration of antibodies against GHRH in rats decreases both pulsatile GH secretion and GH responsiveness to ghrelin and GHS in rats (204). Ghrelin has been shown not only to have stimulatory effects on GH secretion but also on other pituitary hormones such as adrenocorticotropic hormone (ACTH) and prolactin (205), while it inhibits luteinizing hormone (LH) (206).

On the contrary, other studies reported that ghrelin has no role in pituitary GH secretion. Circulating ghrelin levels was not changed during the GH peak in the rat (29). Immuno- neutralization against ghrelin did not alter pulsatile GH profile (207), while GHRH antibodies completely abolished endogenous pulsatile GH release (208). Moreover, recent studies with ghrelin knockout animals failed to show a significant effect on GH regulation (209). However, decreased GHS-R gene expression in the ARC reduced GH and IGF-I levels in transgenic models (210). Mutation of GHS-R1a may be associated with familial short stature (211). Thus, endogenous ghrelin might only amplify the basic pattern of GH secretion or adjust pituitary responsiveness to GHRH. It has also been suggested that the role of ghrelin in GH secretion becomes manifested during negative energy states (208). Therefore, further studies are necessary to reveal the exact physiological role of ghrelin in GH secretion.

1.4.3 Effects of ghrelin on glucose metabolism Ghrelin is a gastrointestinal peptide with a major role in the regulation of energy metabolism. Both ghrelin and GHS-R1a are also present in pancreatic islet cells (212) implying that ghrelin exerts paracrine and autocrine actions on the pancreas. Consequently, more interest has been directed towards investigating the role of ghrelin in the regulation of glucose homeostasis. From the literature, there are many contradictions regarding the action of ghrelin on plasma insulin levels and insulin sensitivity. These conflicting data have made it difficult to determine a key role of ghrelin in the control of glucose metabolism. In most animal studies, ghrelin induced hyperglycaemia and inhibits insulin secretion (166,193), and blockade of pancreatic-derived ghrelin enhances insulin secretion and ameliorates the development of diet-induced glucose intolerance (213). The inhibitory effect of ghrelin on insulin secretion was suggested to be due to a tonic inhibition of both pancreatic insulin and SST secretion (214). However, recently, it has been shown that ghrelin is produced in a new type of islet cell, the epsilon ε-cell, which would affect β

20 cells via a paracrine mechanism (215). Data in the literature have also indicated the existence of a feedback loop between ghrelin and glucagon. By this mechanism, ghrelin directly stimulates glucagon secretion from pancreatic α cells (216), while glucagon stimulates ghrelin secretion under nutrient-deficient conditions (217).

It is unclear whether such effects occur at physiologic or only at pharmacologic doses of ghrelin. Moreover, the reported effects of ghrelin on insulin secretion seem to correlate with the individual level of glycaemic control and nutritional status. Some studies reported that ghrelin stimulates insulin secretion in the presence of hyperglycaemia (218) and enhances glucose-induced insulin secretion in meal-fed sheep (219). However, others found no change in insulinaemia under normoglycaemic conditions (220). In addition, it has been reported that ghrelin has both inhibitory and stimulatory actions on glucose- induced insulin secretion after feeding and during starvation respectively (221). Other findings indicate a differential metabolic role of the ghrelin system in short-term and long- term treatments that request a further investigation of ghrelin’s long term role on glucose homeostasis (222).

Regarding the effects of ghrelin on insulin sensitivity, studies showed that ghrelin administration led to impaired glucose tolerance (166) and increase hepatic glucose production by activating the gluconeogenesis (223,224). In this context, studies using ghrelin knockout mice resulted in increased hepatic and peripheral insulin sensitivity (20). On the other hand, no effect on insulin sensitivity was observed in other study applying insulin tolerance test (ITT) (225). Specifically, ghrelin may directly influence adipose tissue insulin sensitivity. Ghrelin has been demonstrated to down-regulate the expression of adiponectin in differentiating adipocytes (226). Ghrelin increased insulin-stimulated deoxyglucose uptake in isolated white adipocytes. However, ghrelin administration in the absence of insulin had no effect on adipocyte deoxyglucose uptake, suggesting that ghrelin acts synergistically with insulin (227). The effects of ghrelin on adipocytes insulin sensitivity could be also at the central not only at the local level. When the animals received ghrelin treatment via intracerebroventricular (ICV) infusion, it triggered an increase in insulin-stimulated glucose utilization during euglycaemic-hyperinsulinaemic clamps in WAT as well as brown adipose tissue (BAT), but not in soleus muscle via activation of AMPK in the ARC (228).

Although UAG does not possess any biological activity on GH stimulation, several studies demonstrated a clear metabolic role for UAG on glucose homeostasis. Administration of 21

UAG alone did not induce any change in glucose and insulin levels compared to placebo (229). However, combined with ghrelin, UAG was able to inhibit the effects of ghrelin on glucose and insulin levels, but not on the stimulatory action on GH, prolactin, ACTH, and cortisol levels (230). Interestingly, these effects of UAG could not be antagonized by administration of synthetic GHS-R1a antagonists (231). These observations indicate that UAG binds to another receptor or GHS-R subtypes to increase insulin secretion and improve insulin sensitivity (195).

A recent study published by Gagnon et al. demonstrated a new aspect of ghrelin involvement in glucose regulation (232). Intraperitoneal injection of ghrelin into mice 15 minutes prior to oral glucose administration enhanced glucose-stimulated glucagon like peptide-1 (GLP-1) release and improved glucose tolerance, whereas the ghrelin receptor antagonist [D-Lys3]-GHRP-6 reduced plasma levels of GLP-1 and insulin and diminished oral glucose tolerance (232). Thus, improving glucose tolerance showed in this study was due to stimulated GLP-1 secretion by ghrelin. All these results emphasize the complexity of the relationship between ghrelin and insulin signalling; and new therapeutic approaches in diabetes that are based on manipulation of the ghrelin system must be addressed with utmost care.

1.4.4 Effects of ghrelin on food intake and energy metabolism The energy homeostasis is reached when there is a balance between energy intake and expenditure. As a result, weight loss will occur when the caloric intake is decreased and/or the energy expenditure is increased while weight gain will happen vice versa. The hypothalamus is considered the key region in the central nervous system (CNS) involved in feedback control of appetite and food intake. Although other brain regions have also been implicated such as Nucleus tractus solitaries (NTC) in the brain stem which serves as a gateway for neural signals from the gastrointestinal tract to the hypothalamic centres. The hypothalamic ARC is of special importance, as this region contains two distinct neuronal populations, i.e. orexigenic neuropeptide Y (NPY) and agouti-related peptide (AgRP) expressing neurons as well as anorexigenic cocaine and amphetamine related transcript (CART) and proopiomelanocortin (POMC) expressing neurons (233) (Figure ‎1.6). The interaction of these two systems seems to be the primary driving force in the regulation of energy homeostasis (234). These neurons are also components of the central melanocortin system that plays a critical role in the short-term regulation of energy intake (235).

Figure ‎1.6 Regulation of food intake and adiposity by ghrelin.

Circulating ghrelin may access the brain directly through the blood brain barrier (BBB). Neuropeptide Y (NPY)/agouti-related protein (AgRP)-expressing neurons, located mainly in the arcuate nucleus of the hypothalamus, are stimulated by ghrelin, which in turn stimulate effector pro-opiomelonocortin (POMC) neurons. These first order neurons in the Arc project to the paraventricular nucleus (PVN) and other hypothalamic neurons, which in combination, determine the behavioural and autonomic outputs involved in eating behaviour and metabolism. The mesolimbic dopaminergic pathway is closely associated with the regulation of food intake where elevated levels of dopamine in the ventral tegmental area (VTA) and nucleus accumbens (NAC) cause increased motivation to consume particularly energy-dense food. The nucleus tractus solitarius (NTS) also receives neuroendocrine signals from vagal afferents which are activated by a number of factors including mechanical distension of the gastrointestinal tract, ghrelin and nutrients which in turn activates PVN neurons. Ghrelin promotes adiposity through direct stimulation of PPAR-γ pathways of adipose tissue or indirectly through the melanocortin-sympathetic nervous system (SNS) pathway.

The role of ghrelin as an appetite stimulator and a meal initiator are well documented and supported by the following observations. Firstly, plasma ghrelin levels are dependent on recent food intake; they are increased by fasting and declined after eating (236). Secondly, ghrelin stimulates gastrointestinal motility, gastric and exocrine pancreatic secretions (237). Thirdly, ghrelin stimulates the secretion of NPY/AgRP in the ARC of the 23 hypothalamus (238) and ablation of the ARC inhibits the actions of ghrelin administration on feeding but not on GH secretion (239). Moreover, some ghrelin gene polymorphisms are associated with alterations in eating patterns (240). Ghrelin reaches ARC in three different ways; systemically by crossing the blood-brain barrier, via the vagal afferents and through local hypothalamic synthesis and secretion, thereby exerting paracrine actions (241,242) (Figure ‎1.6). Intact vagus nerve may be required for exogenous ghrelin to increase appetite and food intake in man (243).

In addition to meal initiation, ghrelin seems to drive the type of food. ICV administration of ghrelin forcefully enhanced fat intake over carbohydrate intake in both high carbohydrate and high fat preferring rats (244). Recent studies have demonstrated that ghrelin also plays a role in enhancing the food reward signal via activation of AMPK that increases the dopaminergic transmission in the mesolimbic reward neurons; ventral tegmental area (VTA) and the nucleus accumbens (NAC) (177,245) (Figure ‎1.6). On the other hand, the composition of ingested foods appears to influence ghrelin secretion, however, two contradictory conditions have been noticed. More obviously decreased levels of ghrelin after the ingestion of proteins and carbohydrates than those observed after the ingestion of lipids (246) and lower ghrelin levels after lipids than that after carbohydrate or protein intake (247).

For Long term coordination of energy metabolism, integration of complex central (orexigenic and anorexigenic) and peripheral (hormonal) pathways needs to be achieved, which result in energy homeostasis (248-250). Ghrelin also seems to be involved in the regulation of long term energy homeostasis (251). Ghrelin levels increase in cases of chronic energy deficits such as; low-energy diets, cancer anorexia, anorexia and bulimia nervosa, and cachexia of chronic diseases, suggesting that ghrelin directs the energy storage (252,253). In contrast to healthy individuals, obese subjects display reduced plasma ghrelin levels, together with low plasma GH and high plasma leptin levels (254). The lower levels of ghrelin may reflect a compensatory adaptation aiming at reducing a hunger stimulus and suggest that ghrelin itself is not the main cause of obesity. It remains unclear however, whether further inhibition of ghrelin would be of therapeutic value in obese patients. Although circulating plasma ghrelin levels are low in obese people, a lack of postprandial ghrelin suppression was observed, which could contribute to increased food intake in these people (255). Plasma ghrelin concentrations rise rapidly after fasting

24 in normal-weight animals, but this increase is delayed in obese ob/ob, db/db mice and fatty Zucker rats, suggesting that short-term regulation is modified by excess adiposity (251).

Likewise, peripheral or central chronic administration of ghrelin increases BW and adiposity (171,256) by decreasing energy expenditure (257,258), lipolysis and adipocyte apoptosis (259). The chronic ICV injection of ghrelin increased cumulative food intake and decreased energy expenditure, resulting in body-weight gain (260). This ghrelin’s adipogenic effects help to store more energy for later use in case of energy deficits. It is worth mentioning that these adipogenic effects appear to be independent from the orexigenic effect of ghrelin (224) indicating that the pathways that serve the orexigenic and adipogenic effects of ghrelin are different (Figure ‎1.6). Acute ghrelin injections induced an increase of the respiratory quotient (RQ) in mice suggesting increased utilisation of carbohydrate and reduced utilisation of fat was consistent with the observed increase in fat storage (171). Moreover, an inverse relationship has been reported between serum ghrelin and thermogenesis. Higher levels of ghrelin are associated with low levels of resting metabolic rate (RMR) and postprandial thermogenesis (261). The ablation of GHS-R increases energy expenditure by increasing thermogenesis in BAT through increasing uncoupling oxidative phosphorylation, which is independent of food intake or physical activity (262). Ghrelin suppresses energy expenditure and thermogenesis in BAT via its inhibitory effect on BAT sympathetic nerve activity (263) (Figure ‎1.6). Besides its actions in adipose tissue, chronic central infusion of ghrelin also increases plasma cholesterol levels, and more specifically HDL, an effect that is consistent with the observation that mice lacking both ghrelin and GHSR1a show lower plasma cholesterol levels than WT mice (264).

In conclusion, Ghrelin appears to target different CNS regions to modify energy balance and food intake. Hypothalamic circuits are the most known pathways so far, nonetheless, the relative contribution of these regions to energy regulation and their responses to changes in metabolism are still not clear warranting more studies to clarify the exact roles of these pathways.

1.5 Pharmacological modification of ghrelin system Manipulating ghrelin receptor could be clinically useful for different conditions related to energy homeostasis (265). Recent studies show that the lack of both ghrelin and its receptor GHS-R1a protects mice against diet induced obesity (DIO) (266). In addition, ghrelin immunization in rats has been reported to reduce BW gain (267). Thus, it was 25 suggested that pharmacological antagonism of ghrelin will be a useful way to treat obesity. [D-Lys3]-GHRP-6, a relatively selective antagonist reduced food intake and BW in mice and rats as would be desired as an anti-obesity drug (268). Nevertheless, the immunization against ghrelin failed to cause long-term BW reduction (267). Some studies with mice knockout for ghrelin or ghrelin receptor showed minor differences in food intake and BW under caloric restriction or HFD compared with the control mice (269). Furthermore, absence of ghrelin in ob/ob mice does not appear to decrease food intake or BW, although significantly lowering blood glucose (20). Given that ghrelin affects food intake and GH through separate pathways, newly GHS-R1a ligands have been developed lately to dissect the effects on food intake from GH mediated through GHS-R1a receptor (270). Ghrelin analogues have been examined as diagnostic agents for GH deficiency (271,272).

The utility of ghrelin in cancer cachexia has been demonstrated in rodent models. In a cross-over, randomized, placebo-controlled study in cancer patients with impaired appetite, acute administration of ghrelin led to a significant increase in food intake as compared to saline infusion (273). Moreover, some preclinical data have demonstrated that ghrelin prevents cytotoxic drugs-induced weight loss by restoring adipose tissue functionality (274) and ameliorating inflammation in the muscle (275). Based on these aspects, some researchers have anticipated the clinical use of ghrelin in cases in which increased levels of food intake may be favourable, such as elderly patients with nutritional deficiencies (276) or with anorexia associated with different catabolic diseases (277).

Ghrelin, in addition of inducing positive energy balance, has been demonstrated to induce beneficial cardiovascular effects. Chronic administration of ghrelin improved left-ventricular (LV) dysfunction and attenuated the development of LV remodelling and cachexia in a rat model of chronic heart failure (278). In a pilot study of 10 chronic heart failure patients, ghrelin administration was reported to decrease levels of norepinephrine, improve cardiac function and exercise capacity (279). Administration of ghrelin in persons with metabolic syndrome is shown to improve endothelial function by preventing atherosclerotic changes (280) and improving vasodilatation (281), via decreasing blood pressure without an increase in heart rate (176), besides, other beneficial haemodynamic effects such as increasing cardiac output (282).

Experimental studies have demonstrated the anti-inflammatory and cytoprotective effects of ghrelin. Application of ghrelin prior to the induction of acute pancreatitis significantly 26 attenuated the severity of the inflammation and reduced pancreatic tissue damage (283). Similarly, ghrelin protects against acute gastric ulceration and accelerates the healing of chronic gastric and duodenal ulcers (284). Ghrelin suppresses inflammation in sepsis and inflammatory bowel disease and attenuated chronic liver injury (285,286). In line with this, some studies suggest that ghrelin has positive trophic activity, protecting from β cell damage in experimental models of T1DM (287).

Chronic therapy with ghrelin or ghrelin agonists, however, is associated with weight gain, adiposity, and may be development of insulin resistance. Such observations have led to drug discovery efforts designed to inhibit ghrelin action with a goal of treating obesity and insulin resistance. Nonetheless, studies could not prove the therapeutic effects of ghrelin antagonists for treatments of diabetes and insulin resistance. In normal mice, GHS-R1a antagonist YIL-781 improves glucose tolerance by promoting insulin release, rather it did not affect insulin sensitivity (288). Similarly, in DIO rat, YIL-781 had no apparent effect on insulin sensitivity but improved glucose tolerance by stimulating insulin secretion (288). Other studies showed the greatest utility of UAG for the treatment of insulin resistance when injected combined with ghrelin. UAG and its analogue (AZP531) treatment prevented HFD-induced proinflammatory effects, and prevented development of a prediabetic metabolic state (289).

In fact, the clinical use of ghrelin in clinical has some limitation as ghrelin has a very short half-life. Thus, many attempts to how the peptide can be engineered for a more sustained delivery and better pharmacokinetic properties. Treatment with ghrelin by infusion may be indicated in very acute conditions such as prior to an elective surgery when a short-term anabolic state is required. The peptide and non-peptide GHS-R1a agonists, which developed prior to the discovery of ghrelin are orally bioavailable which results in sustained and more tolerable effects without increasing cortisol levels (290).

1.5.1 Hexarelin as a growth hormone releasing peptide Growth hormone secretagogues (GHS) are family of synthetic peptides (also called growth hormone releasing peptides (GHRPs) which stimulate the release of GH from the pituitary. The first in vivo bioactive GHRP called GHRP-6 (His-D-Trp-Ala-Trp-D-Phe-Lys- NH2) was synthesized in 1984 (291). Unfortunately, GHRP-6 and other peptidyl GHS that generated next had a very poor oral absorption and a short half-life (292). Tremendous effects had been then directed to generate peptidyl and non-peptidyl compounds that are more stable and have higher oral bioavailability. GHS stimulate GH secretion by a different pathway 27 than that of GHRH. The secretion of GH by GHS involved IP3/DAG pathway via binding to GHS-R1a (293) but the binding of GHRH to its receptor leads to the increase in intracellular cyclic AMP (cAMP) via stimulation of AC-PKA (294,295). Studies observed that combining GHRP-6 with GHRH had a synergistic effect on GH secretion in rats (296).

Hexarelin, a new synthetic hexapeptide has been first synthesized in 1994 by Vittorio Locatelli and colleagues in Italy (297). Hexarelin (His-d-2MeTrp-Ala-Trp-d-Phe-Lys-NH2) differs from GHRP-6 by having a methyl group in position 2 of the DTrp (298). This minor modification was beneficial, making Hexarelin more stable and longer acting in vivo (299). Hexarelin was well tolerated in humans through intravenous, subcutaneous, intranasal, and oral administration without reported side-effects and elicited a substantial elevation in plasma GH concentrations in a dose-dependent manner (297). Due to its anabolic effects on skeletal muscles, may partially via GH, Hexarelin draws attentions from the athletics as a performance enhancement drug (300). Hexarelin is a potent GH stimulator than GHRH and their co-administration has a synergistic effect (301). The underlying mechanism of Hexarelin-induced GH secretion is not clear yet. Some data supports the hypothesis that it could be through inhibiting SST activity and to a lesser extend through stimulating GHRH. However, the possibility that Hexarelin works through a different pathway could be present (302). This is confirmed by the studies that showed that GH-releasing activity of Hexarelin is partially refractory to inhibition by SST (303). Studies on long term daily administration of Hexarelin showed that GH decreased over 7 weeks of treatment in dogs and might be earlier than that in humans indicating desensitizing effects to Hexarelin upon prolonged treatment (304).

Besides GH stimulation, Hexarelin have shown food stimulating effects similar to ghrelin. Daily subcutaneous administration of Hexarelin for 8 weeks have shown to have strong orexigenic properties both in young and old rats (305). Similarly, dogs treated with Hexarelin for 6 weeks exhibited the same feeding patterns (306). This indicates that there is no tolerance to the orexigenic effects of Hexarelin unlike GH response, suggesting that these two effects could be mediated by different receptor moieties. Interestingly, in contrast to studies demonstrating that ghrelin and some GHS-R1a agonists increased adiposity and total BW. Hexarelin did not significantly alter total BW upon long term treatment in rats and elderly subjects (304,305). The failure of increase in BW could be related to the fat burning effects of Hexarelin on the adipose tissue (181) with corresponding increase in muscle mass (307).

1.5.2 Hexarelin as a CD36 agonist Previous studies showed that Hexarelin had a distinct binding pattern other than GHS-R1a (308). In 2002, CD36, the scavenger receptor was identified as the second receptor for Hexarelin (194). Due to its multi-ligand pattern recognition function, CD36 is also known as Glycoprotein IV (GPIV), thrombospondin (TSP) receptor, collagen receptor, and fatty acid translocase (FAT) (309-311). CD36 is highly expressed in cells involved in lipid metabolism such as adipocytes (312), hepatocytes (313) and enterocytes of the small intestine (314). Therefore, it is highly suggested that Hexarelin works on tissues lacking GHS-R1a expression such as adipocytes and hepatocytes through binding to CD36. The function of CD36 is largely defined by its ligand and by the cell type in which it is expressed. In recent years a greater concern on the impact of CD36 in the pathogenesis of metabolic disorders has been risen (315,316). As a fatty acid translocase (FAT), CD36 has the ability to bind and internalize long chain fatty acids (LCFA) (312). In addition, CD36 plays an important role in the coupling between FFA uptake and their efficient use for energy production (317). Interestingly, CD36 has been shown to control the expression of genes not only involved in fatty acid oxidation but also with fatty acid re-esterification such as Phosphoenolpyruvate Carboxykinase (PEPCK) in the adipose tissue (318). Peroxisome proliferator-activated receptor-γ (PPAR-γ) and CCAAT/enhancer binding protein (C/EBP) play a key role in the complex transcriptional cascade during adipocyte differentiation (319). Recently, FAT/CD36 expression was activated during 3T3-L1 adipocyte differentiation, and FAT/CD36 protein levels were positively correlated with C/EBP alpha and PPAR-γ (320). PPAR-γ and C/EBP play a key role in the complex transcriptional cascade during adipocyte differentiation (319).

Hexeralin as a CD36 agonist have been reported to have beneficial effects on fat metabolism (321-323). Studies by Rodrigue-Way et al showed that Hexarelin might bind to CD36 receptor independent from the GH secretion to enhance the activation of PPAP-γ in macrophages and adipocytes (181). In vitro incubation of differentiated THP-1 macrophages or mouse peritoneal macrophages with Hexarelin resulted in an increase in cholesterol efflux, which correlates with an enhanced expression of LXRα, apoE, and sterol transporters ABCA1 and ABCG1, all involved in promoting the cholesterol reverse pathway (324). This was also confirmed by in vivo studies when apolipoprotein E (apoE)- null mice maintained on a long-term high-fat and high cholesterol diet, a condition known to induce atherosclerosis, showed a significant regression in atherosclerotic plaque formation when treated with Hexarelin (324). In addition to enhancing PPAR-γ in 29 macrophages, the ability of Hexarelin to upregulate PPAR-γ-dependent downstream events in cultured adipocytes and in fat tissues from treated mice has been reported (181). In these studies, treatment of differentiated 3T3-L1 adipocytes with Hexarelin resulted in a depletion in TG cellular content, accompanied by profound changes in the gene expression profile of key markers of fatty acid metabolism such as LXR, FATP1, FATP4, CPT1b, and F1-ATP synthase (181). Interestingly, many of these genes were shared with a PPAR-γ agonist, troglitazone, an insulin sensitizing drug, indicating that PPAR-γ may be considered as a common regulator in both responses. Furthermore, Hexarelin not only promotes cholesterol efflux but also inhibited cholesterol synthesis in liver cells through the activation of AMPK pathway, reduction of 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR), and increase recruitment of the anchor proteins insulin-induced genes (Insig-1 and Insig-2) in the liver cells (325). HMGR is the rate-limiting enzyme in the cholesterol biosynthesis pathway. These findings support that Hexarelin plays a role in regulating fatty acid metabolism and reverse cholesterol transport via CD36 and PPAR-γ activation.

The cardioprotective effects of Hexarelin on heart are well documented (326). The cardiac action of Hexarelin was reported to be mediated in part by 1=1=11GHS-R1a and largely by activation of the CD36 receptor (327). Hexarelin-mediated activation of CD36 in perfused hearts increased coronary perfusion pressure in a dose-dependent manner. This effect was not observed in hearts from CD36-null mice and from spontaneously hypertensive rats genetically deficient in CD36 (194,328). Acute intravenous administration of Hexarelin induced a rapid increase in left ventricle ejection fraction (LVEF), cardiac output, and cardiac index, while reducing wedge pressure (322). Chronic administration of Hexarelin alleviates LV dysfunction, pathological remodelling, and cardiac cachexia in rats with congestive heart failure by suppressing stress-induced cardiomyocyte apoptosis (329). Moreover, chronic administration of Hexarelin to GH-deficient rats had a pronounced protective effect against ischemic and post-ischemic ventricular dysfunction (330). Hexarelin treatment of spontaneously hypertensive rats significantly reduced cardiac fibrosis by decreasing interstitial and perivascular myocardial collagen deposition (331). Ghrelin was far less effective at preventing ischemia-reperfusion damage. Therefore, the cardioprotective effects of Hexarelin are mainly due to binding to CD36 (327).

1.5.3 Ghrelin receptor antagonists GHS-R1a is a G protein-coupled receptor, which binds ghrelin and plays a considerable role in energy homeostasis, metabolism and regulation of BW (332). If ghrelin is assumed

30 to be an endocrine peptide, which has orexigenic, adipogenic and diabetogenic effects, blocking its receptors may have the opposite endocrine and metabolic effects (268). Several GHS-R1a antagonists have been developed to clarify the function of the ghrelin/GHS-R pathway in the regulation of feeding behaviour and GH secretion such as [d-Arg-1, d-Phe-5, d-Trp-7, 9, Leu-11]-substance P, BIM-28163, [D-Lys3]-GHRP-6, JMV2810, GSK1614343 and BIM-28163 (333,334). Although these ghrelin antagonists have all been clearly shown to antagonize exogenous ghrelin actions on GH secretion both in in vitro and in vivo, their biological effects on several biological parameters are opposing and their mechanisms of action remain poorly understood.

[D-Lys3]-GHRP-6, (His-DTrp-D-Lys-TrpD-Phe-Lys-NH2), made by a minor alteration in GHRP-6a via replacing D-lysine with alanine (335). [D-Lys3]-GHRP-6 is extensively used as a selective ghrelin receptor antagonist (336). It has been reported that [D-Lys3]-GHRP- 6 decreased food intake in postmenopausal mice fed both HFD and standard diet (337). Furthermore, it reduced BW and blood glucose, insulin and leptin (337). In fact, acute and ICV infusion of ghrelin receptor antagonists, [D-Lys3]-GHRP-6, or [d-Arg-1, d-Phe-5, d- Trp-7, 9, Leu-11]-substance P reduced food intake and BW gain in laboratory mice (268,333). In contrast, treatment with BIM-28163 in rats increased food intake and weight gain by inducing cFos activation in the dorso-medial nucleus of the hypothalamus (334). Similarly, another GHS-R1a antagonist, GSK1614343, showed unexpected stimulatory effects on feeding in rats and dogs (192). The appetite-mediated action of GSK1614343 was abolished in GHS-R null mice suggesting that the orexigenic effect of this compound is dependent on GHS-R1a (192).

Altogether these data are interesting and suggest the existence of alternative pathways mediating the effects of these ghrelin antagonists distinct from ones activated by ghrelin through either the GHS-R1a or another unknown receptor. In consistent with that, BIM- 28163 appears to have greater activity in the NTS, however, is not as effective as ghrelin in stimulating cFos in the arcuate neurons (338). Some studies postulated that blocking the GHS-R1a with the antagonist may allow UAG to stimulate feeding through a GHS-R1a independent pathway (339). Moreover, it is possible that GHS-R1a forms a dimer with other receptor so the orexigenic effects of some ghrelin antagonists may be due to binding to GHS-R1a heterodimers rather than GHS-R1a receptor (340). As we mentioned before that GHS-R1a is associated with multiple signal transduction pathways so it is possible

31 that these ghrelin antagonists could activate a specific pathway on the GHS-R1a that is independent from the one mediating GH-releasing activities.

Recent studies have reported that [D-Lys3]-GHRP-6 is not completely adverse to ghrelin. Infusion of [D-Lys3]-GHRP-6 in rats may increase serum glucose and cortisol concentration (341); this effect was not completely different from the ghrelin effect. In consistent with that, more recent studies did not ﬁnd a signiﬁcant impact of the infusion of ghrelin antagonist [D-Lys3]-GHRP-6 on food intake and appetite in a rat model (342). Another study also showed that this ghrelin receptor antagonism did not decrease stressed induced caloric intake, but paradoxically increased the intake of the HFD (343). The possibility of quick tolerance to [D-Lys3]-GHRP-6 should not be excluded as [D-Lys3]- GHRP-6 decreased ethanol intake, preference, and water intake only on the first day of treatment (342). It is also worthy to note that most studies tested [D-Lys3]-GHRP-6 on food intake and BW were on an acute or subacute basis (268,337). No much studies about the chronic or long term effects of [D-Lys3]-GHRP-6.

Although many studies have shown the effects GHS-R antagonist on food intake, cardiovascular effects of ghrelin antagonist remain unclear. There is a study demonstrated that subcutaneous injections of [D-Lys3]-GHRP-6 evoked a significant increase in arterial pressure and heart rate of conscious rats (344). It is possible that these effects were mediated by the activation of sympathetic nervous system or/and inhibition of the parasympathetic nervous system and suppression of the baroreflex. Based on these findings using ghrelin antagonist seems to modulate sympathetic activity and might possibly cause side effects in patients with metabolic syndrome.

In conclusion, understanding the mechanisms of action of these compounds that differentially affect feeding and GH secretion can be of clinical implications and whether the effects of these antagonists are completely adverse to the effects of ghrelin should be clarified. Further molecular studies are necessary to identify the physiological effects of the peptidyl GHS-R antagonist.

1.6 Hypothesis and Aims Alternation of GH-IGF1 plays an important role in the pathogenesis of type 2 diabetes. GH secretion was found to be inversely correlated with insulin in obese T2DM. Therefore, it was proposed that suppression of GH in obese T2DM helps to ameliorate diabetes. However, whether this correlation is kept in non-obese T2DM has not been well studied.

GHS are class of synthetic peptides working centrally on GHS-R1a to stimulate GH secretion. Evidence is growing that some of GHS can act independently from GH and exerts specific metabolic actions. In this context, studies reported that Hexarelin, one of GHS enhanced fat metabolism of adipocytes and WAT via CD36 activation independent of GHS-R1a. Ghrelin is the endogenous ligand for GHS-R1a. Although ghrelin is best known for its appetite and adiposity inducing effects, however, the role of the ghrelin in the regulation of insulin secretion and insulin action remains a controversial topic. For example, deletion of ghrelin in ob/ob mice improved glucose tolerance and in mice fed on HFD (20,266). By contrast, other studies showed that ghrelin enhanced insulin stimulated glucose disposal (345,346). Moreover, central ghrelin infusion in mice exhibited better glucose tolerance and increased insulin secretion (347). Thus, whether to use ghrelin receptor as a target for the treatment of diabetes remains vague and call for more studies to clarify ghrelin’s role on glucose homeostasis. Collectively, the observations detailed previously have led to the hypothesis that pulsatile GH secretion is modified in non-obese T2DM and ligands of growth hormone secretagogue receptor could modulate the progress of diabetes by affecting GH secretion and energy metabolism.

Thus, the main aim of this project is to investigate the effects of a ghrelin receptor agonist (Hexarelin) and a ghrelin receptor antagonist [D-Lys3]-GHRP-6 in non-obese type 2 diabetic mouse model. To address this, the specific aims of this project are:

1. To characterize GH secretion profiles and its regulatory factors in non-obese type 2 diabetic MKR mice. 2. To investigate the effects of Hexarelin, a GHS-R1a agonist, in non-obese type 2 diabetic MKR mice. 3. To investigate the effects of [D-Lys3]-GHRP-6, a selective GHS-R1a antagonist, in non-obese type 2 diabetic MKR mice.

2) CHAPTER TWO: MATERIALS AND METHODS This chapter includes general experimental protocols used throughout this thesis. Detailed information on specific experimental designs will be addressed in relevant chapters.

2.1 Animal Care Homozygous adult male MKR mice (FVB/N background) and their corresponding age and gender-matched wild type control FVB littermates were obtained from Mount Sinai School of Medicine, USA (21). Mice at different age groups (5, 10 and 33 weeks old) were used for studying the metabolic profiles as addressed in chapter 3. Mice at 7-15 weeks old were used to examine the effects of ghrelin-related drugs on different metabolic parameters as addressed in chapters 4 and 5. All mice were bred in Australian Institute for Bioengineering and Nanotechnology (AIBN) animal house within the University of Queensland and housed at 22 ± 2⁰C with 35 ± 4% humidity on a 12-hour light-dark cycle (dark period start at 6:00pm). Animals were grouped into two, housed in micro-isolating cages with separation of genotypes. Mice have free access to water and standard rodent chow (Specialty Feeds, Glen Forrest, WA, Australia) unless otherwise indicated. All experiments and procedures were approved by the University of Queensland Animal Ethics Committee (SBMS/031/15/NHMRC/UQ) and performed in accordance to national guidelines. Genotyping was performed by tail biopsy and standard PCR using red extract N-AMP tissue PCR kit (XNATR; Sigma, USA).

2.2 Serial blood sampling for assessment of pulsatile GH secretion Serial blood collection for assessment of pulsatile GH secretion was done according to the established method in our lab (348). Briefly, mice were pair-housed in individual cages for at least one week before the procedure. All mice were habituated to the procedure by daily handling of them inside cardboard rolls while holding and wiping the tail to mimic the procedure and to avoid the stress. During the procedure, 36 sequential tail tip blood samples (2μl) were collected from each mouse at 10-minute intervals for a period of 6 hours. Mice were handled in a circular cardboard roll, and held by the tail. The distal 0.5 mm of the tail was excised using a sterile surgical blade (ProsciTech, Australia) following applying the local anaesthetic cream (Emla lignocaine and prilocaine 5% cream, Astrazeneca, Australia). For each sample, 2 μl of whole blood was collected, and placed directly into 58 μl of 0.1M PBS supplemented with 0.05% Tween 20. Following blood collection, gentle pressure was applied to the tail-tip to stop blood flow and mice were

34 returned to the cage. For repeated collection, the tail-tip was briefly immersed in physiological saline (0.9% sodium chloride, Baxter, Australia), and gently wiped-dry with a paper towel. Samples were mixed by vortex, immediately placed on dry-ice and stored at - 80°C for future batch analysis using in-house GH Elisa.

2.3 Drugs and experimental design Hexarelin or [D-Lys3]-GHRP-6 were purchased from China Peptides (Shanghai, China) and Bachem AG (Switzerland) respectively. The drugs were prepared daily by diluting in 0.9% sterile saline. The drugs were administered I.P., at a concentration of 200 ug/kg BW for Hexarelin or 200 nmol/mouse for [D-Lys3]-GHRP-6. The doses of Hexarelin and GHS- R antagonists were chosen according to previous published studies on food intake and affinities for GHS-R receptor (268,305). For all experiments, animals were allowed to acclimate to their environment for one week. All mice received at minimum three saline injections (0.1 cc) prior to drug administration to habituate them to the procedure. Chronic drug treatment involved groups of mice receiving twice daily doses of drugs or saline I.P. injection at 08.00am and 6.00pm for 12 days. BW and food consumption were recorded on a daily basis. Pulsatile GH secretion was assessed following drug administration. Glucose and insulin tolerance tests were carried during the experimental period. Indirect calorimetric parameters and locomotor activity were recorded before, in the middle and at the end of experiments. At the end of the treatment of the respective groups of FVB and MKR mice, mice were culled following a single I.P. injection of sodium pentobarbitone (32.5 mg/ml, Virbac Animal Health, NSW, AUS)) under fed state. Terminal cardiac blood samples were collected into EDTA-coated tubes. Plasma was separated, stored at -80⁰C and assessed for metabolic and hormonal analysis. Immediately after culling, brains, adipose tissues (gonadal WAT), liver and muscles were collected and snap frozen at -80⁰c for gene expression and metabolic analysis. Adipose gonadal tissues and pancreas were fixed overnight by immersion at 4°C in 4% paraformaldehyde and prepared for histological and immunofluorescence staining respectively.

2.4 Glucose tolerance (GTT) and insulin tolerance (ITT) tests The mice were fasted for 6 hours during the daytime from 8 am to 2 pm and then received an I.P. injection of glucose (2 g/kg) for GTT or human insulin (0.75 U/kg, Insulin solution human, Sigma) for ITT. Blood glucose levels for GTT measured prior to the glucose (0 time point) and subsequently at 15, 30, 45, 60, 75, 90, 105 and 120 minutes. For ITT, blood glucose was measured at 0, 30, 60, 90, 120 minutes after the injection of insulin.

Measurement of blood glucose was carried out using a glucometer (Nova Stat Strip Xpress Glucose Hospital Meter, Nova Biomedical, UK) and glucose-test strips (42214, Nova Biomedical UK).

2.5 Indirect calorimetric assays Prior to experiments, mice were acclimatized to indirect calorimetry cages (TSE PhenoMaster, Bad Homburg, Germany) for 3 days. Mice were injected with drugs or vehicle twice a day (at 8:00am and 6:00pm) for 12 days inside these close-cages. Food and water were available throughout the experiments unless otherwise specified. BW and food intake were recorded on a daily basis. Respiratory exchange ratio (RER) was calculated as ratio between CO2 production (litres) and O2 consumption (litres). Oxygen consumption (VO2) was expressed in ml O2/min and normalized by body mass. Locomotor activity was monitored by using a multi-dimensional infrared light beam system. Body composition (lean mass, and fat mass) was determined by nuclear magnetic resonance (NMR) (Brukerminispec, Bruker, LF50H, Billerica, Massachusetts) at the end of treatment.

2.6 Analysis of hormones and metabolites

2.6.1 Analysis of GH and IGF-1 hormones using in-house Elisa The details of these assays will be addressed in chapter 3.

2.6.2 Analysis of circulating hormones and metabolites using commercial Elisa and Multiplex kits. The details of hormones and metabolites measured by commercial assay kits and the assays information are summarised in Table ‎2.1.

Table ‎2.1 Details of commercial assay kits used to determine levels of circulating hormones and metabolites

Hormones/metabolites Kits name Catalogue number Company Insulin Ultrasensitive mouse 90080 or Millipore insulin ELISA kit or MMHMAG-44K Milliplex Map kit ghrelin Rat/mouse Ghrelin EZRGRA-90K or Millipore (Active) ELISA or MMHMAG-44K Milliplex Map kit Inflammatory markers Milliplex Map kit MCYTOMAG-70K Millipore

(TNF-α, IL-6, IL-β) C-peptide Milliplex Map kit MMHMAG-44K Millipore Glucagon-like peptide 1 Milliplex Map kit MMHMAG-44K Millipore (GLP-1) Leptin Milliplex Map kit MMHMAG-44K Millipore Free fatty acids (FFA) NEFA C kit 279-75401 Wako Triglycerides Triglyceride colorimetric 10010303 Cayman assay kit chemical Total and free Cholesterol/cholesterol Ab65359 Abcam cholesterol ester quantification assay kit

2.6.3 Measurements of muscle and liver triglycerides Homogenized liver or muscle tissue (100 mg) were incubated with 350 μl ethanolic KOH overnight at 55 °C. The volume of sample mixture was adjusted to 1000 μl with H2O: EtOH (1:1) and centrifuged for 5 minutes at room temperature. The resulting supernatant was transferred to a new tube and the volume was adjusted to 1200 ul with H2O: EtOH (1:1).

Then, 200 μl of the new diluted mixture was incubated with 215 μl 1 M MgCl2 for 10 minutes at room temperature. Following centrifugation, the supernatant was transferred to a new tube for assessment of glycerol content. Glycerol content was determined using a glycerol standard (G7793, Sigma, St. Louis, MO). All tissue TG were converted to glycerol with TG reagent (Sigma-Aldrich, F6428), and analysed spectrophotometrically at 540 nm. The concentration of TG was estimated from a standard glycerol curve and corrected for tissue weight using the following formula: Liver TG content (in mg TG / gram liver) = Triolein equivalent glycerol concentration (mg/dL) × (415 / 200) × 0.012 (dL) / weight of tissue (g) × standard dilution factor.

2.7 Paraffin embedding of tissues for histological or immunofluorescence staining The tissues (gonadal fat or pancreas) were dissected from mice following euthanasia at the end of treatment and fixed overnight in 4% paraformaldehyde in 0.01 M PBS buffer, pH 7.4 overnight at 4°C (349). After rinsing with running water, specimens were dehydrated in ethanol, cleared in xylene and embedded in paraffin using Medite TES Valida Embedding Station. Sectioning of tissue (7 uM thickness) was done using Leica RM 2245 Rotary Microtome. De-paraffinization of the sections was done by placing slides in three changes of clearing agent (xylene) for 2 minutes. The sections were rehydrated through sequential

37 incubations in graded concentrations of ethanol (100%, 90% and 70%). Then the slides were washed with running water for 2 minutes. The adipose tissue was stained with Haematoxylin for 5 minutes as previously described (349). Then the slides were washed with running water, incubated with 70% Alcohol for 2 minutes and stained with Eosin for 30 seconds. The sections were dehydrated with alcohol and Xylene. The slides were then mounted with coverslips using DePex. The stained sections were viewed with Aperio XT Slide Scanner using a 10X objective and analysed for the surface area of adipocytes from three gonadal WAT samples per group and 25 cells per mouse using Image J software (Java (TM) Platform, Oracle). The pancreatic sections were immunofluorescence-stained and the details of this staining will be addressed in chapter 5.

2.8 Real-time quantitative polymerase chain reaction (qPCR)

2.8.1 Real-time qPCR of hypothalamic gene expression The hypothalamus from frozen brain tissues were dissected by cutting alongside its boundaries. The hypothalamus is roughly diamond shaped; although its boundaries are not sharply demarcated, its perimeters can be correlated using neuro-anatomic landmarks. In the coronal plane, the boundaries of the hypothalamus are more distinct. Superiorly, the hypothalamus is divided from the thalamus by a groove in the lateral wall of the third ventricle, the hypothalamic sulcus. The lateral surface is contiguous with the thalamus and subthalamus and is bordered by the internal capsule and optic tracts. Medially, the hypothalamus is bound by the ependyma of the third ventricle. Finally, the inferior surface is continuous with the floor of the third ventricle (350) (Figure ‎2.1). Total cellular RNA was extracted from dissected hypothalamus suspended in Trizol. Total amount of RNA in each sample was quantified using a spectrophotometer (NanoDrop, ND-1000, Thermo Scientific, Wilmington, DE, USA). cDNA was synthesised form 1ug of RNA using I script Tm cDNA synthesis kit (Bio-Rad Laboratories Pty Ltd, Gladesville, NSW, Australia)). Real- time qPCR was performed using Power Sybr green PCR master mix kit (Applied Biosystem, Australia). The forward and reverse primer sequences used in this assay were illustrated in Table 5.1. All primers were supplied from sigma Aldrich. QuantStudio 7 Flex system was used to detect fluorescence during each cycle of 10 ul reactions using 384- well PCR (Applied Biosystems, Australia). Initial step for the standard run were pre-set at thermal cycling conditions of 2 minutes at 150°C and 10 minutes at 95°C. The conditions for annealing and extending were set to 15 seconds at 95 °C and 1 minute at 60°C for 40 cycles. Changes in cycle threshold of the genes of interest were corrected to the

38 housekeeping gene (β-actin). The fold-change value of gene expression was calculated by using the 2-ΔΔCT method as described previously (351).

Figure ‎2.1 Coronal section of the brain.

Cerebrum around the thalamus and hypothalamus was cut and discard, the central part (marked by the red square) which indicated by the median eminence was the desire region that used for experimental approaches. Adapted from Visceral Brain, Language and Thought.

2.8.2 Real-time qPCR of adipose tissue gene expression Gonadal fat mRNA was extracted using Trizol reagent (Ambion, Lifetechnology) and purified using Pure Link Tm RNA mini Kit (Invitrogen, Australia), and total RNA (1 μg) was reverse-transcribed into cDNA according to the manufacturer’s instructions (I script Tm cDNA synthesis kit, Bio-Rad, Australia). The details of this quantitative PCR assay and the primers used will be addressed in detail in section 4.2.8.

2.9 Data and statistical analysis Data were analysed by using GraphPad Prism 7 (graphPad, Inc., San Diego, CA). Student’s t-test was used to compare the differences between two independent groups. One-way or two-way analysis of variance (ANOVA) was used to compare more than two groups followed by Tukey’s test for multiple comparisons to detect differences between groups. The kinetics and secretory patterns of pulsatile GH secretion were determined by deconvolution analysis following parameters established previously (348). All data were

39 expressed as mean ± standard error of the mean (SEM). Statistical significance was considered at a level of P<0.05.

3) CHAPTER THREE: INVESTIGATING THE PHENOTYPIC AND METABOLIC CHARACTERISTICS OF NON-OBESE TYPE 2 DIABETIC MKR MICE

3.1 Brief introduction and rationale GH plays an important role in metabolism besides stimulating body growth (352). Studies showed that GH secretion is altered as a consequence of obesity. In genetically obese Zucker rats, reduced basal and stimulated GH secretion is due to decreased hypothalamic GHRH synthesis and secretion and increased IGF-I levels (95). Moreover, most studies concerning the association between GH and insulin concentrations have shown negative correlation especially in obese diabetics (353). However, alternation of GH secretion in non-obese T2DM has not been well studied. Thus, we aimed to characterize pulsatile GH secretion profiles and GH regulatory factors in non-obese type 2 diabetic MKR mouse model. To achieve this aim, glucose and insulin tolerance tests were assessed at 5, 10 and 33 weeks old. Growth parameters and pulsatile GH concentrations were measured at the same age groups. Blood glucose, metabolic regulatory hormones and inflammatory markers were assessed. Insulin resistance was estimated using the homeostatic model assessment of insulin resistance (HOMA-IR). Food intake and body composition were measured using indirect calorimetric chambers and NMR respectively. Hypothalamic GH and food intake regulatory gene expressions were measured by quantitative real time PCR.

3.2 Material and methods

3.2.1 Mice Homozygous adult MKR male mice (FVB/N background) and their corresponding age and gender-matched littermate FVB mice at three different ages (5, 10 and 33 weeks old) were used for the current study. The details of the housing conditions, genotyping method and the ethics were detailed in section 2.1. MKR mice developed hyperinsulinemia at 3 weeks old without overt hyperglycaemia representing an early diabetic stage then developed overt hyperglycaemia at 6 weeks of age that remained high to 31 weeks old (148). Moreover, MKR mice exhibited progressive increase in circulating and pancreatic insulin through life which was substantially increased at 31 weeks (148). We therefore studied the phenotypic and metabolic characteristics of MKR mice including GH secretion at 5, 10 and 33 weeks old which represent different stages of the disease.

3.2.2 Characterizing pulsatile GH secretion in MKR mice For measurement of pulsatile GH secretion, blood samples were collected and processed as detailed in section 2.2. Pulsatile GH secretion was assessed in MKR and FVB mice at 5, 10 and 33 weeks of age. Analysis of GH was performed using an in-house Elisa as described in section 3.2.4. Following collection of blood samples, mice were returned to their home cage and given 2 days to recover before assessment of other metabolic parameters.

3.2.3 Characterizing the phenotypic and metabolic profiles of MKR mice Body weight and body length (nasal-anal distance) of MKR and FVB mice were measured at 5, 10 and 33 weeks of age. To re-confirm different stages of diabetes in MKR mice, glucose tolerance test (GTT) and insulin tolerance test (ITT) were done at 5, 10 and 33 weeks old as described in section 2.4. In addition, cumulative food intake was measured for 3 days using indirect calorimetric chambers (TSE PhenoMaster, Bad Homburg, Germany) and whole body composition was assayed using NMR (Brukerminispec, Bruker, LF50H, Billerica, Massachusetts). Given the direct association between IGF-1, ghrelin and GH secretion, observations were extended to assess circulating total IGF-1 and acylated ghrelin in MKR and FVB mice. Mice were assessed for fed and fasting blood glucose and plasma insulin. The homeostasis model assessment for insulin resistance (HOMA-IR) was used for the determination of IR. HOMA-IR was calculated by multiplying the fasting blood glucose values (mg/dl) with the fasting plasma insulin values (µU/ml) (354). The mice were culled as detailed in section 2.3 and plasma were stored at -80°C for further analysis. Measurement of total IGF-1 using an in-house Elisa assay as detailed in section 3.2.5. Plasma insulin and acylated ghrelin were measured using commercial ELISA kits (Ultrasensitive mouse insulin Elisa kit and Rat/mouse Ghrelin (Active) Elisa; Millipore, respectively). Inflammatory markers (TNF-α, IL-6, IL-β) were assessed using Milliplex Map kit.

3.2.4 In-house GH sandwich Elisa Analysis of GH was determined using an in-house sandwich ELISA according to the well- established and published method (348). A standard curve of 9 points was generated using a mouse GH (mGH) reference preparation (AFP-10783B, National Institute of Diabetes & Digestive & Kidney Diseases-NIDDK-NHP) by two-fold serial dilution (8 ng/ml to 0.03 ng/ml), diluted in PBST supplemented with bovine serum albumin (0.2% BSA- PBST). A 96-well plate (Corning Inc., Corning, NY) was coated with 50 μl of capture

42 antibody (monkey anti-rat GH, AFP411S, NIDDK-NHPP) at a dilution of 1:40000 and incubated at 4°C overnight. To decrease non-specific binding, each well was incubated with blocking buffer (5% skim milk powder in 0.05% PBST) for 2 hours at room temperature. Following blocking, standards and samples were loaded into the pre-coated plate and incubated for 2 hours at room temperature. Bound standards and samples were incubated with 50 μl of detection antibody (rabbit antiserum to rat GH, AFP5672099, NIDDK-NHPP) at a final dilution of 1:40000. Bound complex were then incubated with 50 μl of horseradish peroxidase (HRP)-conjugated antibody (anti-rabbit, IgG, GE Healthcare, UK) at a dilution of 1:2000. 100 μl of O-phenylenediamine (OPD, 00.2003, Invitrogen, CA) were added to each well and resulted in an enzymatic colorimetric reaction. This reaction was stopped by the addition of 50 μl of 3 M hydrochloric acid (HCL, Sigma). The absorbance was read at a wavelength of 490 nm (reference wavelength set at 650 nm) on a TECAN Sunrise 96-well monochromatic microplate reader (TECAN, Switzerland). The concentration of GH in each well was calculated against a non-linear regression of the standard curve.

3.2.5 In-house IGF-1 Elisa The 96-well plate was incubated overnight with 50 µl of purified hamster monoclonal IgG anti-mouse antibody; capturing antibody (5 µg/ml), diluted in sterile 1xPBS at 4°C. After 3 times washing with 0.05% PBST, the wells were blocked using 180 µl of 5% skim milk powder for 1 hour at room temperature on a shaker. For the standards, recombinant mouse IGF-1 was diluted in assay buffer (50 mM sodium phosphate, 150 mM NaCl, 0.1% Tween 20, 0.25% Bovine Serum Albumin (BSA), pH=7.4) ranging from 0 ng/ml to 25 ng/ml. Plasma samples were acid/ethanol extracted to isolate IGF-1 from its binding proteins. 25 µl of each sample was mixed with 100 µl of acid/ethanol reagent (12.5% 2 M HCl, 87.5% ethanol, v/v) and incubated for 30 minutes at room temperature. Samples were then micro-centrifuged at 10000 rcf for 10 minutes. 50 µl of supernatant was pipetted out and neutralised using 25 µl of 1 M Trizma base. The samples were further diluted in assay buffer to a final dilution of 1/75. Following the incubation with blocking buffer, plate was washed 3 times using 0.05% PBS Tween. Then, 50 µl of prepared standards and samples were added to respective wells along with 50 µl of detecting antibody biotinylated mouse IGF-1 affinity purified goat IgG, which was diluted in 5% skim milk powder to a concentration of 1µl/ml. Plate was incubated for 2 hours in room temperature on a shaker, followed by washing 3 times using 0.05% PBS Tween. Streptavidin-HRP diluted in sterile 1 x PBS (1:10000) was added to each well (100 µl/well) and further incubated for 20 43 minutes. After washing the plate 4 times using 0.05% PBS Tween, 100 µl of photo sensitive OPD solution was pipetted to each well. Reaction was terminated after 10 minutes with 4 M HCl. Finally, the absorbance was read by a plate spectrophotometer (TECAN, Switzerland) at a wavelength of 490 nm.

3.2.6 Hypothalamic GHRH, SST, NPY and POMC gene expression in MKR and FVB mice Hypothalamic tissues were obtained as described in section 2.8.1 and directly lysed in Trizol. RNA in Trizol was extracted using the PureLink RNA Mini Kit (Ambion). Procedures of RNA concentration quantification and cDNA synthesis were the same as described in section 2.8.1. Master Mix was composed of Taq polymerase (Taqman) and each of the primers of GHRH, SST, NPY, POMC and β-actin are as follows: GHRH Mm00439100_m1, SST Mm00436671_m1, NPY Mm03048253_m1, POMC Mm00435874_m1, β-actin, Mm00607939_s1. Then, 10 ul of each cDNA master mix was loaded into a 384-well plate (Bio-Rad Laboratories). QuantStudioTM 7 Flex Real-Time PCR System (Applied Biosystem) was used to run the PCR cycle. The cycle was initially hold at 50°C for 2 minutes and 95°C for 10 minutes, then enter 40 cycles of annealing at 95°C for 15 seconds and extension at 60°C for 1 minute. The final amplification plot was analysed by QuantStudioTM 7 Flex Software (Applied Biosystem) and each data was normalised to the housekeeping gene β-actin.

3.2.7 Statistical analysis Data were presented and analysed as described in section 2.9

3.3 Results

3.3.1 MKR mice though lean tended to eat more and had higher fat and lower lean mass We studied growth parameters in MKR and WT mice by measuring total body weight and body length at 3 different ages 5, 10 and 33 weeks old. Compared with WT mice, MKR mice were significantly leaner at all age groups (Figure ‎3.3D). However, MKR mice were only shorter at 5 weeks of age, as compared with FVB mice but there was no difference in body length at 10 and 33 weeks old (Figure ‎3.3G). These data suggest that MKR mice had growth retardation early in life whereas they caught up growth later on. MKR mice consumed 2.807 ± 0.1377 g of food/day, whereas FVB ate 2.292 ± 0.3193 g of food/day, however, the AUC of cumulative food intake within 3 days did not reach statistical

44 significance (Figure ‎3.1A and B). Interestingly, analysis of body composition at 10 weeks old by MNR indicated that MKR mice had more fat mass by 30% (P=0.0034, Figure ‎3.1C) and less lean mass by 5% (P=0.0034, Figure ‎3.1D).

Figure ‎3.1 Assessment of cumulative food intake, and body composition in MKR and FVB mice at 10 weeks old.

Cumulative food intake was measured up to 72 hours in MKR mice compared to FVB mice (A). Area under curve (AUC) for cumulative food intake in MKR mice compared to FVB mice (B). Fat mass (% body weight) in MKR mice compared to FVB mice (C), and Lean mass (% body weight) in MKR mice compared to FVB mice (D). Data is expressed as mean ± SEM (n=6-10/group), **P<0.01 vs. FVB mice.

3.3.2 MKR had significantly impaired glucose and insulin tolerance except at aged mice MKR mice and WT FVB mice were fasted for 6 hours and injected I.P. with a bolus of glucose (2 mg of glucose/g of body weight) for GTT. MKR mice showed significantly higher glucose values during GTT at 5 (Figure ‎3.2B) and slightly higher glucose values at 10

45 weeks old (Figure ‎3.2D). At the age of 33 weeks old, there was no significant difference in the blood glucose levels after glucose injection between FVB and MKR mice (Figure ‎3.2F). AUC of GTT data showed significant differences at 5 weeks old (P<0.05) and 10 weeks old (P<0.05) but no significant difference at 33 weeks old (Figure ‎3.2H). Although the fasting glucose levels in MKR mice are higher than FVB controls at 5 weeks old (Figure ‎3.2A and B), they are similar at 10 weeks old (Figure ‎3.2C and D) and below that of FVB mice at 33 weeks old (Figure ‎3.2E). For ITT, mice were fasted for 6 hours and then I.P. injected with a bolus of insulin (0.75 m-units/g of BW). Insulin did not decrease blood glucose levels in MKR mice as efficiently as in FVB controls (Figure ‎3.2A, C and E), suggesting that MKR mice are insulin resistant. The total AUC are significantly higher in MKR mice compared to FVB mice at 5 and 10 weeks old but no significant difference at 33 weeks old (Figure ‎3.2G).

Figure ‎3.2 Glucose and insulin tolerance tests of MKR and FVB at different age groups.

ITT of MKR and FVB mice at 5 (A), 10 (C) and 33 weeks of age (E). GTT of MKR and FVB mice at 5 (B), 10 (D) and 33 weeks of age (F). Area under curve for ITT at 5, 10 and 33 weeks of age (G). Area under curve for GTT at 5, 10 and 33 weeks of age (H). Data is expressed as mean ± SEM (n=4-6/group). *P<0.05, **P<0.01, ****P<0.0001 vs. FVB mice.

3.3.3 MKR mice have higher GH secretion at 5 but comparable to slightly higher GH secretion at 10 and 33 weeks old respectively GH secretion in MKR mice was significantly higher at 5 weeks old compared to FVB mice (Figure ‎3.3A). This was characterized by a significant increase in total (Figure ‎3.3H), pulsatile (Figure ‎3.3E) GH release, and the mean peak of GH release per pulse (Figure ‎3.3F) at 5 weeks old. GH secretion patterns and the amount of GH released at 10 weeks old in MKR mice were comparable to that observed in FVB mice (Figure ‎3.3B, E, F, H and I). Pulsatile GH secretion was decreased in both aged FVB and MKR mice at 33 weeks old, however, MKR mice still have higher GH secretory profiles which were near statistical significance compared to FVB mice (Figure ‎3.3C, E, F, H and I).

Figure ‎3.3 Growth parameters and pulsatile growth hormone profiles of MKR and FVB at different age groups.

Representative examples of pulsatile GH profiles in FVB and MKR mice at 5 (A), 10 (B) and 33 (C) weeks of age as measured by deconvolution analysis. Total body weight in MKR mice at 5, 10 and 33 weeks old compared to FVB mice (D). Total body length in MKR mice at 5, 10 and 33 weeks old compared to FVB mice (G). Pulsatile GH secretion (E), Total GH secretion (H), Mass of GH secretion per burst (F), Number of GH secretory events (I) in MKR mice compared to FVB mice. Data are presented as mean ± SEM (n=5- 7/ group). *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 vs. FVB mice.

3.3.4 Hormonal profiles of MKR mice Consistent with previous studies, MKR mice were hyperglycaemic 23.55 ± 0.964 vs. 8.075 ± 0.2671, P<0.0001, Figure ‎3.4A), hyperinsulinaemia during fed 41.17 ± 6.079 vs. 3.057 ± 0.5609 P<0.0001, Figure ‎3.4B; and during fasting 0.7654 ± 0.1077 vs. 0.2305 ± 0.02916 P=0.0006, Figure ‎3.4C). HOMA-IR as an indicator of insulin resistance was significantly higher in MKR mice (1.571 ± 0.2934 vs. 0.3689 ± 0.05179, P=0.0023, Figure ‎3.4D). Total

IGF-1 was also significantly higher in MKR mice (416.2 ± 35.32 vs. 237.8 ± 25.09, P=0.0010, Figure ‎3.4E). However, plasma ghrelin was significantly lower in MKR mice (201.1 ± 43.31 vs. 442 ± 73.28, P=0.0391, Figure ‎3.4F). In addition, MKR mice showed significant higher plasma inflammatory cytokines such as IL-6 and IL-1B (Figure ‎3.4H and I). TNF-α is also higher but did not reach statistical significance (Figure ‎3.4G).

Figure ‎3.4 Measurements of metabolic hormones and inflammatory markers in MKR and FVB mice.

Blood glucose concentrations in fed mice (A), plasma insulin concentrations in fed (B), and fasted (C) mice, homeostasis model assessment (HOMA-IR) (D), circulating total IGF-1 (E), circulating acylated ghrelin (F), TNF-α (G), IL-6 (H), and IL-1β (I), in MKR mice compared to FVB mice. Data are presented as mean ± SEM (n=4-8/group). *P<0.05, **P<0.01, ***P<0.001, ****P<0.001 vs. FVB mice.

3.3.5 Increased GH secretion in MKR mice occurs without changes in hypothalamic GHRH and SST but elevated NPY gene expressions To investigate whether the increase in GH secretion was due to altered hypothalamic control of GH release. Hypothalamic GH and food intake regulatory genes of MKR and FVB mice were measured using quantitative real-time PCR. Compared to FVB mice, no discernible change in GHRH (Figure ‎3.5A) or SST (Figure ‎3.5B) mRNA expression was observed in MKR mice, suggesting that increased GH secretion in MKR mice may occur independent of changes in hypothalamic neurons in control of GH secretion. The amount of POMC mRNA reduced without reaching statistical significance (Figure ‎3.5D). However, the number of NPY mRNA in hypothalamus of MKR mice was significantly increased by 40% compared to that of FVB mice (Figure ‎3.5C).

Figure ‎3.5 Hypothalamic GHRH, SST, NPY and POMC mRNA expression in MKR and FVB mice.

GHRH (A), SST (B), NPY (C), and POMC (D) mRNA expression in hypothalamus of MKR mice and FVB mice. Data are presented as mean ± SEM (n=7-8/group). *P<0.05 vs. FVB mice.

3.4 Discussion The GH/IGF-1 axis represents an important physiological mechanism for regulating growth and metabolism (355). The somatic effects of GH are exerted mainly via the stimulation of IGF-1 production from liver. IGF-1 has similar effects to insulin to reduce glycaemia, mainly by increasing insulin sensitivity in peripheral tissues (356). Moreover, IGF system plays an essential role in the formation and maintenance of skeletal muscle (357). The

52 recent development of igf-1 knockout animal models has brought new insights to our understanding of the relative roles of these hormones in growth and carbohydrate homeostasis. MKR mice are transgenic mice generated by overexpression of a dominant negative igf-1 receptor in the skeletal muscle containing the K1003R mutation (21). These mice have growth retardation soon after birth till up to 5 weeks of age (149). They are lean but have severe insulin resistance merely in skeletal muscle because of local development of hybrid receptor between the mutated igf-1 and insulin receptor, however, liver and adipose tissue also develop insulin resistance as indicated by increased hepatic glucose production and FFAs in the circulation (146).

While GH/IGF-1-insulin relationship has been well studied in obese T2DM, the metabolic data regarding GH secretion in non-obese T2DM are scarce and not conclusive. In one study, GH levels were higher both in the fasting state and in response to glucose load in lean women carrying the Fat Mass and Obesity-Associated (FTO) haplotype and this increase in GH was accompanied with increased glucose, insulin and C-peptide levels (358). On the contrary, another study has found that obese diabetic subjects had slightly decreased GH responses to GHRH and pyridostigmine (an anticholinesterase suppress SST) compared with obese non-diabetic subjects whereas Lean T2DM showed a blunted GH release after GHRH and pyridostigmine compared with normal-weight healthy subjects (96). Thus, it was hypothesized that impaired GH secretion in both lean and obese T2DM could be related to the hyperglycaemia and hyperinsulinaemia that affect SST tone independent of BW.

Our data confirmed that the linear growth in MKR mice at 5 weeks of age was significantly lower to that observed in FVB mice but they have normal body lengths at 10 and 33 weeks old (Figure ‎3.3G). Their body weights remained lower in MKR compared with WT animals at all age groups (Figure ‎3.3D). Our data showed that total and pulsatile GH secretion in MKR mice was higher at 5 weeks old but comparable to that of FVB mice at 10 weeks old (Figure ‎3.3A and B). Assessment of GH release in older mice at 33 weeks old demonstrated a decline in GH secretion in both MKR and FVB mice, rather pulsatile GH secretion was still higher in MKR mice (Figure ‎3.3C). Blood glucose and plasma insulin were elevated in MKR mice, and the development of insulin resistance in these mice was confirmed by ITT and HOMA-IR (Figure ‎3.2 and Figure ‎3.4D). The increase in GH secretion in MKR mice occurred despite hyperinsulinaemia and the insulin resistance. This is on contradictory with the clinical data that observed decreased GH levels in both human

53 with increased circulating insulin level and obese diabetic patients with hyperinsulinaemia (91). There was a negative relationship between GH and insulin in obese mouse models, ob/ob mouse and the diet-induced obese (DIO) mouse (359). Insulin is proposed to act directly at the pituitary gland via the insulin receptor to suppress GH release (93). However, this inverse relationship between GH and insulin was omitted in MKR mice. This could be probably to facilitate growth in these mice which exhibit growth retardation earlier in life.

To investigate whether the increase in GH secretion is coupled with a change in the GH regulatory hormones, circulatory levels of IGF-1 and ghrelin were assessed. Circulating levels of IGF-1 was increased in MKR mice that could be as a consequence of increased GH but increased IGF-1 failed to suppress GH secretion in MKR mice (Figure ‎3.4E). Furthermore, ghrelin was decreased in MKR mice (Figure ‎3.4F). Therefore, it is unlikely that the rise in GH is due to stimulatory effects of ghrelin in this model. In another animal model with deletion of liver-specific expression of the igf-1 gene they had low serum IGF-1 by 15-25% and increased GH levels by six-fold (360). These observations suggest that there is a dissociation of GH/IGF-1 axis and insulin in this mouse model.

To examine the molecular mechanisms responsible for the increase in GH secretion in MKR mice, we assessed gene expression levels of a number of GH target genes, including those involved in food intake regulation. Our results showed that there were no observable changes in gene expression of GHRH and SST in the hypothalamus between MKR mice and FVB mice. However, the number of NPY mRNA in hypothalamus of MKR mice was significantly increased compared to that of FVB mice. Other Studies showed that NPY could affect GH secretion through regulation of hypothalamic GH regulatory neuropeptides, GHRH and SST (361). In addition, activated NPY neuron during fasting has a stimulatory effect on SST neuron activity thus suppressing GH secretion (362). Interestingly, we found that hypothalamic NPY gene expression was increased without altering SST mRNA in MKR mice (Figure ‎3.5). Thus, we proposed that neurohormonal activity between NPY neuron and SST neuron was disrupted, while NPY lost its ability to trigger SST. In genetically obese Zucker rats, with hyperinsulinaemia, impaired GH secretion is due, at least, decreased hypothalamic GHRH synthesis and secretion and increased IGF-I levels (95). However, hyperinsulinaemia in MKR mice has no effect on hypothalamic GHRH and SST gene expression (Figure ‎3.5A and B).

Although MKR mice had lower BW at all age groups compared to control mice, they have higher fat mass and lower lean mass (Figure ‎3.1C and D). Moreover, they have higher levels of inflammatory cytokines in plasma such as TNF-α, IL-1β and IL-6 compared to wild type control mice (Figure ‎3.4G-I). The hyperlipidaemia and elevated inflammatory markers could participate in the dissociation between insulin and GH at the level of pituitary or hypothalamus. Hypothalamus of DIO mice was insulin resistance with increased accumulation of JNK and other inflammatory cytokines induced by hyperlipidaemia (363). Further studies are warranted to clarify that effects of these metabolites on GH-insulin signalling in MKR mice.

GTT data showed that MKR mice were glucose-intolerant except at 33 weeks old where there was no significant difference of glucose tolerance. Fasting blood glucose was higher at 5 weeks old but normal to lower levels at 10 and 33 weeks old (Figure ‎3.2) despite the severe insulin resistance that MKR mice have been developed. Consistent with our results, another study reported increased glucose disposal in MKR mice by doing a stable isotope flux phototyping GTT using [6,6-2H2]glucose (146). They suggested that MKR mice had metabolic inflexibility that may result from the compensatory increase in type II muscle fibres and increased levels of GLUT1 and GLUT4 in skeletal muscle that enable them to utilise more glucose especially during fasting (146). Similarly, the chow-fed Pparα- null mouse that a diminished capacity to utilise fatty acids results in a compensatory increase in glucose disposal, as assessed by a stable isotope I.P. glucose tolerance test (364).

In conclusion, the cause of the dissociation of GH/IGF-1 axis and insulin in this non-obese diabetic model is unclear. Elevated GH secretion in MKR could be an essential physiological adaption that promote muscle development and facilitate nutrient uptake and utilization in the muscle to ensure adequate growth (365). Furthermore, it could arise from increased NPY expression in MKR mice that could increase GH secretion or lack of negative feedback between GH and IGF-1 at the hypothalamic level. The possibility that hyperlipidaemia and higher inflammatory cytokines could affect GH profile in these mice is also present. Nonetheless, the interpretation of our findings is somewhat limited by the use of transgenic mouse model. A more comprehensive assessment of GH in non-obese type 2 diabetic patients and other animal models is highly warranted.

4) CHAPTER FOUR: HEXARELIN, A GROWTH HORMONE SECRETAGOGUE, IMPROVES THE LIPID METABOLIC ABERRATIONS IN A NON-OBESE MALE MOUSE MODEL OF TYPE 2 DIABETES MELLITUS This chapter was reproduced from published manuscript (Mosa et al. 2017, Endocrinology)

4.1 Introduction Hyperlipidaemia and ectopic fat deposition are strongly associated with insulin resistance and are well documented risk factors for cardiovascular diseases (45,366). Although ectopic lipid accumulation is typically observed in most obese individuals (367), it is also encountered in lean subjects, as in the lipodystrophies (38,368). It has been suggested that adipose tissue dysfunction contributes to ectopic lipid accumulation associated with insulin resistance through different mechanisms (369). Firstly, insufficient adipose tissue mass in inherited or acquired forms of lipodystrophy leads to excessive storage of ingested fat in muscle and liver and the development of insulin resistance in these organs (370- 372). Secondly, lipolysis of visceral adipose tissues is closely associated with insulin resistance (373). Thirdly, impairment in differentiation capacity of adipocytes leads to formation of large insulin resistant adipocytes with diminished capacity to accumulate fat (374,375). Importantly, if fat oxidation fails to adaptively compensate for the increased influx of lipid within these tissues, intracellular accumulation of lipids will occur (376,377). The relative contribution of these factors to ectopic lipid accumulation may vary in different pathological conditions and in different tissues. In lipodystrophy, excess lipid influx would appear to be predominantly associated with ectopic lipid accumulation (31), whereas in lean insulin resistant offspring of type 2 diabetics, impaired mitochondrial fatty acid oxidation may play a major role in this process (378).

Intensive changes in lifestyle and glycaemic control are unable to fully correct the metabolic aberrations in patients with lipodystrophy or lean type 2 diabetes (67,379). Therefore, lipid-modifying therapy is warranted in patients with disorders of fat metabolism to restore adipose tissue functionality and correct dyslipidaemia. GH replacement showed some promising effects for lipodystrophy population by decreasing visceral adiposity and increasing lean mass with transient side effects including reversible glucose intolerance (380,381). However, the effects of GH releasing peptides have not been well studied with respect to dyslipidaemia and related metabolic disorders. Hexarelin (Hex), a synthetic peptide GH secretagogue, stimulates the release of GH through binding to growth hormone secretagogue receptor (GHS-R1a) in the pituitary and hypothalamic regions

56 (382). Due to its anabolic effects on skeletal muscles (may partially via GH), Hexarelin has received attentions from the athletics as a performance enhancement drug (300). Moreover, the cardioprotective effects of Hexarelin are well documented (323,326). Acute intravenous administration of Hexarelin induced a rapid increase in left ventricle ejection fraction (LVEF), cardiac output, and cardiac index (322). Chronic administration of Hexarelin to GH-deficient rats has a pronounced protective effect against ischemic and post-ischemic ventricular dysfunction (330).

Interestingly, there are arising evidences that Hexarelin might have beneficial effects on fat metabolism. Studies by Rodrigue-Way et al showed that Hexarelin might bind to a scavenger receptor class B (CD36) independent from the GH secretion to enhance the activation of peroxisome proliferator-activated receptor gamma (PPAR-γ) in macrophages and adipocytes (181). CD36 plays an important role in the pathogenesis of metabolic disorders (315,316). As a fatty acid translocase (FAT), CD36 binds and internalizes long chain fatty acids to facilitate energy production (312,317). Moreover, CD36 expression is activated during adipocytes differentiation, and its protein levels are positively correlated with CCAAT/enhancer binding protein (C/EBP alpha) and PPAP-γ which are critical transcription factors in lipid metabolism and adipocytes differentiation (320,383,384). Hexarelin is proposed to act through CD36 to inhibit accumulation of oxidized low-density lipoprotein (oxLDL) cholesterol in macrophages resulting in a decreased the atherosclerotic lesions in apolipoprotein E-deficient mice fed with atherogenic diet (385).

In the present study, we investigated the effects of Hexarelin on glucose and lipid metabolism in non-obese insulin-resistant MKR mice. MKR mice with an over-expression of a dominant-negative IGF-1 receptor in skeletal muscle showed impaired insulin signalling pathways in skeletal muscle due to hybrid formation of the mutated IGF-1R with the endogenous IGF-1 and insulin receptors (21). This defect results in insulin resistance not only in skeletal muscle but also in adipose tissue and liver; causing beta cell dysfunction and hyperglycaemia. Importantly, MKR mice exhibit defects in free fatty acids (FFA) oxidation pathway, which leads to elevations in serum FFA and TG, and increased TG deposits in liver and muscle tissues; suggesting that hyperlipidaemia and accumulated lipids in tissues may be causative factors for the progression of type 2 diabetes in MKR mice, replicating human cases with inherited lipodystrophy or dyslipidaemia (146,151). Our results showed that Hexarelin reversed the abnormal lipid metabolic states of MKR mice through modulation of genes related to fatty acid uptake and oxidation, and enhancement of adipocytes differentiation. Administration of Hexarelin for 12 days alleviated the glucose

57 and insulin intolerance in MKR mice without affecting the levels of blood glucose, plasma insulin, or body weight.

4.2 Materials and Methods

4.2.1 Mice Homozygous adult MKR male mice (FVB/N background, 10-15 weeks old) and their corresponding age and gender-matched WT homozygous littermate FVB mice were used for the current study (21). The details of the housing conditions, genotyping method and the ethics were discussed in section 2.1.

4.2.2 Peptide Hexarelin, supplied by China Peptides (Shanghai, China), was used and prepared freshly by dissolving in physiological saline (0.9% NaCl). The drug was administered I.P., at a concentration of 200 μg/kg BW. Dose was chosen according to previous published results showing that Hexarelin maximally stimulated both GH secretion and food intake at a range of concentrations 80–320 μg/kg (270,305,386). Mice were injected twice daily at 8:00am and 18:00pm with a dose volume of 0.1 ml/20g BW of Hexarelin or saline. Mice were divided into 4 groups namely; FVB saline (FVB mice injected with saline), FVB Hex (FVB mice injected with Hexarelin), MKR saline (MKR mice injected with saline) and MKR Hex (MKR mice injected with Hexarelin).

4.2.3 Glucose tolerance (GTT) and insulin tolerance (ITT) tests GTT and ITT were conducted following 6 hours of fasting (food withdrawn at 8.00am) on days 8 and 11 of experimental period respectively. The details of these measurements were addressed in section 2.4.

4.2.4 Effects of Hexarelin treatment on pulsatile growth hormone secretion As Hexarelin is known to stimulate GH secretion, we further assessed plasma GH levels across 2 hours blood collection post injection on day 7 of treatment. Whole blood (2 μl) was withdrawn from the tail vein at -15, 0, 15, 30, 45, 60, 75, 90, 105, and 120 min relative to Hexarelin or saline injections (0 time) and homogenized in 58 μl of GH buffer (PBS, 0.05%Tween). GH concentrations were measured with an in-house mouse GH ELISA according to our well-established method (387). The details of GH Elisa were addressed in section 3.2.4.

58 4.2.5 Indirect calorimetric assays Animals were individually placed in calorimeter chambers (TSE PhenoMaster, Bad Homburg, Germany) containing food and water. Mice were acclimatized to indirect calorimetry cages for 3 days before start of injection. Mice received I.P. injection of either saline or Hexarelin at day 0 of calorimeter recording. BW and food intake were recorded daily. Respiratory exchange ratio (RER) and locomotor activity were measured during dark and light cycles for individual mice on days 0, 5 and 10 of treatment. Body composition was determined by nuclear magnetic resonance (NMR) (Brukerminispec, Bruker, Billerica, Massachusetts) at the end of experimental period (day 12).

4.2.6 Blood glucose and Plasma hormonal analysis At the end of treatment, all mice were culled following a single I.P. injection of sodium pentobarbitone (32.5 mg/ml) under fed state. Measurement of blood glucose from tail blood was carried out as described in section 2.4. Terminal blood samples were collected via cardiac puncture into EDTA-coated tubes. Plasma was separated and stored at -80⁰C for assessment of insulin, c-peptide, ghrelin, GLP-1 and leptin by commercially Milliplex Map kit (Millipore, MMHMAG-44K) using Luminex XMap analyser (Magipex).

4.2.7 Plasma lipid and tissues triglyceride analysis Plasma non-esterified fatty acids (NFFAs) were measured using a colorimetric determination kit (Wako NEFA C kit, USA). TG and cholesterol were measured in plasma by enzymatic colorimetric methods using Triglyceride colorimetric assay kit (Cayman chemical, USA) and Cholesterol / cholesterol ester quantification assay kit (Abcam, UK) respectively. Hepatic and muscle TG content were assayed as previously described in section 2.6.3. Briefly, tissues TG were extracted from frozen liver and muscle by saponification in ethanoic KOH overnight at 55°C and neutralized with MgCl2. All tissue TG were converted to glycerol with a TG reagent-F6428 (Sigma-Aldrich, Australia), and analysed spectrophotometrically at 540nm. The concentration of TG was estimated from a standard glycerol curve and corrected for tissue weight.

4.2.8 Quantitative real-time PCR White adipose tissue mRNA was extracted using Trizol reagent (Ambion, Life technology) and purified using Pure Link Tm RNA mini Kit (Invitrogen, Australia), and total RNA (1μg) was reverse-transcribed into cDNA according to the manufacturer’s instructions (I script Tm cDNA synthesis kit, Bio-Rad, Australia). For the quantitative PCR assays, cDNAs were amplified by real-time PCR in triplicates with a Power Sybr green PCR master mix kit (Applied Biosystem, Australia) using QuantStudio7 384-well PCR (Applied Biosystems,

59 Australia). The forward and reverse primer sequences used in this assay are illustrated in Table ‎4.1. All optimal cycling reactions were performed in the same manner: 95°C for 10 minutes, followed by 40 cycles of 95°C for 15 seconds and 60°C for 1 minute. Changes in cycle threshold of the genes of interest were corrected to the housekeeping gene (β-actin). The fold-change value of gene expression was calculated by using the 2-ΔΔCT method as described previously (351).

Table ‎4.1 Primer sequences used for gene expression analysis by qPCR

Gene Forward Reverse PPAR-γ 5’-CGGTTTCAGAAGTGCCTTG-3’ 5’-GGTTCAGCTGGTCGATATCAC-3’ PGC1α 5’-TATGGAGTGACATAG AGTGTGCT-3’ 5’-CCACTTCAATCCACCCAGAAAG-3’ HSL 5’-GGCTCACAGTTACCATCTCACC-3’ 5’-GAGTACCTTGCTGTCCTGTCC-3’ CD36 5’-GGCGTGGGTCTGAAGGACTGGAA-3’ 5’-GGAGGCACGGGGTCTCAACCA-3’ LPL 5’-CTGCTGGCGTAGCAGGAAGT-3’ 5’-GCTGGAAAGTGCCTCCATTG-3’ Fatp1 5’-CGCTTTCTGCGTATCGTCTG-3’ 5’-GATGCACGGGATCGTGTCT-3’ β-actin 5’-CTGAATGGCCCAGGTCTGA-3’ 5’-CCCTGGCTGCCTCAACAC-3’ UCP-1 5’-ACTGCCACACCTCCAGTCATT-3’ 5’-CTTTGCCTCACTCAGGATTGG-3’

4.2.9 Adipose tissue histology The left gonadal fat pads from the mice were removed following euthanasia at the end of treatment and fixed overnight in 4% paraformaldehyde in PBS buffer. The tissues were then transferred to 70% ethanol and embedded in paraffin. Sections (7 μm thick) were stained with haematoxylin-eosin and were mounted on glass slides as described in section 2.7. The stained sections were viewed with Aperio XT Slide Scanner and analysed for the surface area of adipocytes using Image J software (Java (TM) Platform, Oracle).

4.2.10 Statistical analysis Data were presented and analysed as described in section 2.9

4.3 Results

4.3.1 Effects of Hexarelin treatment on glucose and insulin tolerance in MKR Mice Following eight days of treatment, GTT results showed that Hexarelin significantly improved glucose excursions (Figure 4.1A) and reduced area under curve (AUC) of glucose in MKR mice (p=0.0423, Figure 4.1B). ITT performed on day 11 of treatment showed a significant reduction of glucose levels (Figure 4.1C) and AUC in response to insulin injection in Hexarelin-treated MKR mice (P=0.0105, Figure 4.1D). There were no

60 changes of glucose and insulin tolerances in FVB mice following Hexarelin treatment (Figure 4.1A-D).

Figure ‎4.1 Effects of twice daily I.P. injection of Hexarelin (200 ug/Kg BW) or saline for 12 days on GTT, ITT and pulsatile GH secretion.

Glucose tolerance test (A), insulin tolerance test (C), and pulsatile GH secretion (E) in FVB saline (black squares), FVB Hex (white squares), MKR saline (black circle) and MKR Hex (white circles). Area under the curve (AUC) of GTT, ITT, and pulsatile GH secretion (B, D and F respectively). Data is expressed as mean ± SEM (n=5-6/group). *P<0.05; **P<0.01; ****P<0.0001 vs. MKE saline group. #P<0.05, ##P<0.01, ###P<0.001, ####P<0.0001 vs. FVB saline group.

61 4.3.2 Effects of Hexarelin treatment on GH secretion Pulsatile GH levels were significantly increased after 15 minutes of Hexarelin injection in both FVB and MKR mice compared with vehicle-treated groups (P<0.001, Figure. 4.1E). This increase in pulsatile GH lasted for nearly 15-30 minute post-injections and then returned to baseline. There is no significant difference in total AUC of GH between Hexarelin and vehicle treatment groups (Figure 4.1F).

4.3.3 Effects of Hexarelin treatment on cumulative food intake, body weight and body composition Food intake of Hexarelin‐treated MKR mice on days 0 to 12 of treatment was significantly increased relative to vehicle-treated MKR mice (Figure 4.2B). There is no difference in the food intake between FVB saline and MKR saline groups. At the end of treatment, the total cumulative food intake of Hexarelin-treated MKR mice was significantly increased by 24% compared to vehicle-treated MKR mice. Interestingly, the orexigenic effect of Hexarelin was not observed in FVB mice (Figure 4.2A). MKR mice had lower body weights compared to FVB mice. However, body weights remained unchanged in both MKR and FVB mice after Hexarelin treatment (Figure 4.2C and D). Analysis of body composition by NMR on day 12 of treatment revealed that MKR mice tended to have higher fat mass and lower lean mass compared to FVB mice (Figure 4.2E and F). With Hexarelin treatment, there was a 13% decrease in body fat (P=0.0278, Figure 4.2E) and a 3.3% increase in lean mass in MKR mice (P=0.0278, Figure 4.2F). There was no significant change in body composition of Hexarelin-treated FVB mice compared to vehicle-treated FVB mice.

Figure ‎4.2 Effects of Hexarelin treatment on cumulative food intake, body weight and body composition.

Cumulative food intake of FVB mice (A) and MKR mice (B) measured from day -3 to day 12 of treatment in indirect calorimetric cages. Change in body weight of FVB mice (C) and MKR mice (D) measured from day 0 (start of treatment) to day 12 of treatment. Fat mass (% BW) (E) and lean mass (% BW) (F) measured at day 12 of treatment. Data is expressed as mean ± SEM (n=4-5/group), *P<0.05, **P<0.01, ***P<0.001 vs. MKR saline group.

63 4.3.4 Effects of Hexarelin treatment on respiratory exchange ratio (RER) and locomotor activity Indirect calorimetry revealed that Hexarelin decreased RER of MKR mice during the dark phase but not during the light phase at days 5 and 10 of treatment (P=0.0173 and 0.0420, respectively), demonstrating an increase in fat oxidation (Figure 4.3D-F). There was no change in RER in FVB mice after Hexarelin treatment (Figure 4.3A-C). Analysis of locomotor activity showed that there was a decrease in total activity among the vehicle- treated FVB mice which was significant on day 5 during the dark phase (40.5% decrease, P=0.0079, Figure 4.3I), and day 10 during the light phase compared to day 0 (41% decrease, P=0.0064, Figure 4.3H). This decrease was however unnoticed among Hexarelin-treated FVB mice; indicating that Hexarelin might have stimulatory effects on locomotor activity (Figure 4.3G-I). Hexarelin-treated MKR mice showed a progressive increase in the locomotor activity during the light phase although not reaching statistical significance (Figure 4.3K-M).

Figure ‎4.3 Effects of Hexarelin treatment on indirect calorimetric parameters of MKR and FVB mice.

Respiratory exchange ratio (RER) in FVB mice (A), RER in MKR mice (D), total locomotor activity in FVB mice (G) and total locomotor activity in MKR mice (J) at days 0, 5 and 10 of treatment during dark phase (18pm-6am) marked by shaded area in the graphs and light phase (6am-18pm). AUC of RER during the light and dark phase of day 0, 5 and 10 of treatment in FVB mice (B and C respectively). AUC of RER during the light and dark phase of day 0, 5 and 10 of treatment in MKR mice (E and F respectively). AUC of

65 locomotor activity during the light and dark phase of day 0, 5 and 10 of treatment in FVB mice (H and I respectively). AUC of locomotor activity during the light and dark phase of day 0, 5 and 10 of treatment in MKR mice (K and L respectively). Data are expressed as mean ± SEM (n=4/group). *P<0.05 vs. MKR saline group, ##P<0.01 vs. FVB saline group at day 0.

4.3.5 Effects of Hexarelin on blood glucose and plasma hormones Treating MKR mice with Hexarelin for 12 days did not change fed blood glucose, plasma levels of insulin, C-peptide or leptin (Figure 4.4A-C and F respectively). However, Plasma ghrelin and GLP-1 showed a slight increase after Hexarelin treatment in both FVB and MKR mice but did not reach statistical significance (Figure 4.4D and E).

Figure ‎4.4 Effects of Hexarelin treatment on blood glucose and plasma hormones.

Blood glucose (A), plasma concentrations of insulin (B), C-peptide (C), ghrelin (D), GLP-1 (E) and leptin (F) at the end of treatment from terminal blood samples during fed

66 conditions. Data are expressed as mean ± SEM (n=4-6/group). ###P<0.001, ####P<0.0001 vs. FVB saline group.

4.3.6 Effects of Hexarelin treatment on plasma lipids and tissues triglycerides Compared with FVB mice, MKR mice had nearly double plasma TG (Figure 4.5B), 39% increase in free cholesterol (Figure 4.5D) and 44% increase in liver TG (Figure 4.5E); indicating the presence of hyperlipidaemia and fatty liver in these mice. MKR mice exhibited however similar plasma NEFA, total cholesterol and muscle TG to that in FVB mice (Figure 4.5A, C and F, respectively). Hexarelin treatment significantly reduced plasma TG by 28% (P=0.0147, Figure 4.5B) and liver TG by 32% (P=0.0112, Figure 4.5E) in MKR mice than that in vehicle-treated MKR mice. Hexarelin treatment did not change plasma FFA, total or free cholesterol (Figure 4.5A, C and D, respectively) in FVB and MKR mice. Thus, improved insulin sensitivity in MKR mice in response to Hexarelin treatment may be due to the reduced TG levels observed in plasma and liver.

67 Figure ‎4.5 Effects of Hexarelin treatment on plasma and tissue lipids.

Plasma FFA (A), plasma TG (B), plasma total cholesterol (C) and plasma free cholesterol (D). Quantitative analyses of TG in liver (E) and muscles (F). Data are expressed as mean ± SEM (n=4-7/group). *P<0.05 vs. MKR saline group; #P <0.05, ##P<0.01 vs. FVB saline group.

4.3.7 Effects of Hexarelin treatment on the genes involved in fatty acid uptake and oxidative phosphorylation of white adipose tissue Several genes required for fatty acid transport and oxidation in WAT, such as PGC-1, LPL, HSL and UCP-1, were significantly up-regulated by Hexarelin treatment in MKR mice (Figure 4.6B). PPAR-γ and CD36 did not reached but very close to statistical significance (P=0.053 and 0.059 respectively). However, in FVB mice, only CD36 was slightly increased by Hexarelin treatment (P=0.066, Figure 4.6A). These data, combined with indirect calorimetry data (Figure 4.3), further supported the conclusion that Hexarelin treatment might result in the enhancements of lipid metabolism in MKR mice.

4.3.8 Effects of Hexarelin treatment on adipocytes morphology and size Histological examination revealed that the average adipocyte size was 8% larger in MKR mice compared with FVB mice (Figure 4.6C). Hexarelin treatment markedly reduced the size of adipocytes in MKR mice. The average size of adipocytes from Hexarelin-treated MKR mice was 11% smaller than that from vehicle-treated MKR mice (Figure 4.6D). There was no significant change in the adipocyte size in FVB mice after Hexarelin treatment. These results indicate possible pro-differentiation effects of Hexarelin treatment on adipocytes in MKR mice that could be linked to PPAR-γ activation.

Figure ‎4.6 Fold changes in gene expression levels of white adipose tissue gene expression after Hexarelin treatment in MKR and FVB mice.

Fold changes in gene expression levels of PPAR-γ, PCG-1, FATP-4, CD36, LPL, HSL and UCP-1 of white adipose tissue of FVB (A) and MKR mice (B) after treatment. The data was calculated using the 2-ΔΔCt method. The results are presented as mean ± SEM (n=5- 6/group). *P<0.05, **P<0.01 vs. MKR saline group. Haematoxylin and eosin staining of the gonadal fat pads from MKR and FVB mice treated with saline or Hexarelin 12 days (C). Magnification 10X, scale bars=100μm. Quantitative analysis of mean adipocytes area from

69 25 adipocytes per mouse (D). Data are expressed as mean ± SEM (n=3/group). **P<0.01 vs. MKR saline group. #P <0.05 vs. FVB saline group.

4.4 Discussion In this study, we demonstrated that Hexarelin ameliorated the lipid metabolic abnormalities, along with improvement in glucose and insulin intolerances in non-obese insulin-resistant MKR mice, in which lipotoxicity played a significant role in the exacerbation of insulin resistance (153). Following 12 days with Hexarelin treatment, there was a significant reduction of plasma TG in MKR mice but total cholesterol levels remained unchanged (Figure ‎4.5B-D). This may be due to the increase in ratio of HDL/LDL cholesterols. In fact, Hexarelin suppressed high lipid diet and vitamin D3-induced atherosclerosis in rats, through probably an increase in HDL/LDL ratio (388). In addition, Hexarelin treatment resulted in a decrease in hepatic TG in MKR mice (Figure ‎4.5E). This effect may be secondary to the enhancement of fat metabolism, which prevents excessive flux of fatty acids to the liver. In addition, direct stimulation of CD36 in the liver may lead to improved hepatic insulin sensitivity (389). Unlike the liver, TG content in muscle remained unchanged. This may be due to a lack of functional signalling pathways employed by the mutated IGF-I receptor in skeletal muscle of MKR mice. Hepatic TG content is directly correlated with fasting endogenous hepatic glucose production (390). Thus, lowering fasting blood glucose noticed during ITT after Hexarelin treatment in MKR mice could be related to improvement of liver insulin sensitivity. However, fed blood glucose depends mainly on muscle glucose uptake and insulin secretion from pancreas (391). In this study, there were no observable changes in muscle TG, plasma insulin or c-peptide levels after Hexarelin treatment.

It is well known that Hexarelin boosts GH secretion in humans and other species (297,306,392), we also showed that administration of Hexarelin induced a transient increase in GH secretion, in both MKR and FVB mice after injection (Figure ‎4.1E). Previous study showed that loss of muscle GH receptor signalling in MKR mice did not improve their glucose homeostasis or insulin sensitivity (393). Exogenous GH treatment was reported to improve lipid parameters in some lipodystrophy patients (394,395). Here, we also reported that Hexarelin treatment in MKR mice resulted in the correction of body composition abnormality in these mice as illustrated by decreasing fat mass and increasing lean mass (Figure ‎4.2E and F). However, it is not known whether Hexarelin- induced GH response contributes to the correction of abnormal body composition observed in MKR mice.

70 According to our results, the possible mechanism by which Hexarelin improved glucose and insulin tolerances is likely through enhancement of lipid metabolism via CD36 and PPAR-γ activation. In this study, activation of PPAR-γ was indicated by up-regulation of its transcription coactivator (PCG-1) and the increase in the expression of other genes that facilitate entry and acylation of FFA in adipocytes, including CD36 and LPL (Figure 4.6B). In line with our results, Hexarelin was reported to induce the gene expression profiles of fatty acid metabolism in cultured adipocytes and in mouse adipose tissue through CD36 (181). In addition, the in-vivo effects of Hexarelin were absent in CD36 null mice, a finding which confirms that Hexarelin action requires CD36 receptor (396). Interestingly, when MKR mice were crossed bred with mice overexpressing CD36 in skeletal muscle, the double-transgenic MKR/CD36 mice showed a normalization of the hyperglycaemia and hyperinsulinemia; as well as a marked improvement in muscle fatty acid metabolism (397). These findings support our hypothesis that Hexarelin through activation of CD36 and PPAR-γ receptors may correct the lipid metabolic derangements associated with insulin resistance.

In addition, Hexarelin treatment enhanced thermogenic gene expression of UCP-1 (Figure 4.6B). UCP-1 is the hallmark of thermogenesis and browning of WAT. Recent studies showed that browning of WAT had beneficial metabolic effects (398). Transgenic mice overexpressing UCP1 in their skeletal muscle or WAT develop a resistance to diet- induced obesity and T2DM, and have a marked stimulation of fatty acid oxidation in muscles (399). Tiraby et al reported that the adenovirus-mediated expression of human PGC-1α increased the expression of UCP1, respiratory chain proteins, and fatty acid oxidation enzymes in human subcutaneous white adipocytes (400). Thus, changes in the expression of genes in this study were consistent with browning adipocyte mRNA expression profile. This is in consistent with previous study showed that Hexarelin induced the expression of genes associated with fatty acid oxidation and browning of differentiated 3T3-L1 adipocytes (401).

Moreover, the adipose tissues from Hexarelin-treated MKR mice showed a reduction in the sizes of adipocytes (Figure ‎4.6C). Recent reports have implied the correlation between adipocyte size and insulin sensitivity (402,403). The hypertrophic adipocytes become oversaturated with lipids and exhibit changes in the secreted adipokines (404). Accordingly, lipids that cannot be stored in the engorged adipocytes may deposit ectopically in other organs such as liver and muscle. Activation of PPAR-γ reduces the size of adipocytes in WAT and increases adipocyte ability to actively take up and efficiently

71 retain lipid (405). Consistent with our results, mature adipocytes treated with Hexarelin showed a significant decrease in total lipid amount, compared with untreated cells. Such decrease was comparable with cells treated with troglitazone, a specific PPAR-γ ligand (181).

The improvements in glucose and insulin tolerances (Figure ‎4.1A-D) occurred without significant changes in circulating levels of fed blood glucose, plasma insulin, c-peptide or leptin but slight increase in GLP-1 and ghrelin (Figure ‎4.4). In agreement with our findings, it was reported that plasma insulin and glucose concentrations did not change after Hexarelin treatments in lean Zucker rats and humans (193,406). Other studies showed that treatment of MKR mice with PPAR-γ agonists, such as rosiglitazone and pioglitazone, markedly improved lipid metabolic profiles and adipose tissue insulin sensitivity, yet failed to reverse the hyperglycaemia and hyperinsulinaemia, similar to what observed in this study (407). Collectively, our results showed that Hexarelin increased pulsatile GH secretion, reduced fat mass and improved the lipid profile without compromising glucose metabolism in MKR mice.

Our results also demonstrated that Hexarelin had potent orexigenic effects in MKR diabetic mice over the experimental period with no change in BW (Figure ‎4.2B). In agreement with our data, similar feeding and BW patterns have also been reported in healthy 24-month-old rats treated chronically with Hexarelin for 8 weeks (305). However, it is worthy to mention that in FVB mice, Hexarelin did not show such an orexigenic effect (Figure ‎4.2A). The underlying causes of such difference of the feeding response to Hexarelin in FVB and MKR mice are not clear yet. In this study, we showed for the first time the effects of Hexarelin in non-obese T2DM model on energy balance and locomotor activity during chronic treatment. It was demonstrated that RER was decreased during dark cycles in Hexarelin-treated MKR mice, further indicating an increased lipid utilization (Figure ‎4.3F). These findings explain well that Hexarelin treatment caused an increase in food intake without a related increase in BW. In addition, Hexarelin treatment tended to increase locomotor activity in both FVB and MKR mice, contributing to the energy balance by enhancing activity thermogenesis. Recent studies suggest that enhancement of spontaneous physical activity is important to counteract the sedentary life style and lack of voluntary exercise; which are the major contributors of adiposity and metabolic syndrome (408).

In Summary, Hexarelin, a GH secretagogue as well as a CD36 ligand, could ameliorate dyslipidemia in non-obese insulin-resistant MKR mice. This was alongside with

72 improvement in glucose and insulin intolerances, and correction of body composition. Besides the efficacy in the prevention of heart diseases as indicated by previous studies, outcomes from the current study suggest that Hexarelin can be used as a potential therapeutic drug in the treatment of dyslipidaemia associated with metabolic syndrome. A limitation of this study is the use of a transgenic insulin-resistant mouse model, which may not completely replicate the human conditions. Thus, further investigation of longer duration regimens would be essential to confirm Hexarelin efficacy in clinical trials of relevant patient populations.

73 5) CHAPTER FIVE: LONG-TERM TREATMENT WITH THE GHRELIN RECEPTOR ANTAGONIST, [D-Lys3]-GHRP-6 DOES NOT IMPROVE GLUCOSE HOMEOSTASIS IN NON-OBESE DIABETIC MKR MICE This chapter was reproduced from accepted manuscript (Mosa et al. 2017, American journal of Physiology)

5.1 Introduction Over the last two decades, a growing attention has been directed towards the role of some endogenous peptides and related synthetic ligands in the regulation of energy balance (409). Ghrelin is a peptide hormone, which is mainly secreted from the oxyntic cells of stomach (158). Noteworthy, ghrelin has been identified as the only orexigenic hormone that stimulates appetite and food intake through activation of orexigenic NPY/AgRP neurons and inhibition of anorexigenic POMC/CART neurons in the hypothalamus (410,411). Moreover, ghrelin contributes to the regulation of BW and composition by stimulating growth GH secretion from the pituitary gland, increasing adiposity and reducing energy expenditure (171,412,413). The adipogenesis effects of ghrelin are thought to be independent of its appetite stimulating action (414). The expressions of ghrelin and its receptor, GHS-R1a have been demonstrated in many peripheral organs and tissues such as the stomach, intestine, pancreas, thyroid, gonads, adrenal, kidney, heart and blood vessels. It is suggested that ghrelin may have many biological activities other than the stimulation of appetite and GH secretion (178). In particular, the expressions of ghrelin and GHS-R1a in pancreatic islets have indicated the possible roles of ghrelin in regulating glucose homeostasis (415).

Although the effect of ghrelin on GH secretion is well established, the effects on insulin and glucose metabolism are far from clear with reported controversies. Many studies have shown that ghrelin induces hyperglycaemia and impairs glucose tolerance through direct inhibition of insulin secretion and enhancement of hepatic glucose production (416). Studies using ob/ob mice demonstrated that the ablation of ghrelin improved the diabetic phenotype with increased insulin secretion, reduced blood glucose and improved glucose tolerance (20). Based on these observation, GHS-R antagonists were proposed to be promising targets for the treatment of obesity and T2DM (257,417). On the other hand, a recent study has shown that GHS-R ablation in ob/ob mice aggravated the hyperglycaemia, decreased insulin levels, and impaired glucose tolerance (418). Moreover, GHS-R ablation in ob/ob mice reduced the expression of β-cell proliferative

74 regulators (such as hypoxia inducible factor (HIF-1α), Fibroblast growth factor 21 (FGF- 21), and Pancreatic duodenal homeobox 1 (PDX-1)) in pancreatic islets and consequently impaired pancreatic β-cell mass and function (418). These paradoxical effects of GHS-R ablation in ob/ob mice emphasize the complexity of the ghrelin-signalling pathway and reinforce the notion that the effects of ghrelin on insulin secretion may be independent of GHS-R. Another recent study showed that GHS-R1a forms heteromeric complex with other receptors such as somatostatin receptor (SSTR) in pancreatic islets to influence insulin secretion according to the ratio of ghrelin to SST (419).

Most of GHS-R1a antagonists are known to decrease appetite and BW (420). Only few exceptions have been reported to have an unexpected increase in food intake such as BIM-28163 and GSK1614343; suggesting a different receptor or alternative signalling pathways targeted by these ghrelin antagonists (192,334). Of various ghrelin antagonists, [D-Lys3]-GHRP-6 is extensively used in vivo and in vitro as a selective ghrelin receptor antagonist (336). [D-Lys3]-GHRP-6 has been reported to decrease food intake and improve hyperglycaemia in ob/ob mice and obese postmenopausal disorders (337,421). However, most of these studies were performed on a short term basis and in obese diabetic models (268,337). Therefore, it is necessary and clinically relevant to establish the efficacy of this GHS-R1a antagonist over long term treatment of non-obese diabetic models.

Present study was designed to (1) assess the long-term effects of [D-Lys3]-GHRP-6 on glycaemic control, (2) compare the differences between the acute and long-term effects of [D-Lys3]-GHRP-6 on food intake and energy expenditure, (3) demonstrate the involvement of hypothalamic neurons in the action of peripheral [D-Lys3]-GHRP-6 administration, and (4) investigate the effects of [D-Lys3]-GHRP-6 on pancreatic islets structure and composition in non-obese diabetic MKR mice.

5.2 Materials and Methods

5.2.1 Mice In this study, male MKR mice and their WT controls (FVB) at 7-12 weeks old. The details of the housing conditions, genotyping method and the ethics were detailed in section 2.1.

5.2.2 Drug and experimental design [D-Lys3]-GHRP-6 was purchased from Bachem AG (Bubendorf, Switzerland) and diluted in 0.9% sterile saline with a stock concentration of 25 mg/ml. The drug was prepared daily at a concentration of 200 nmol/mouse. The dose of the antagonist was chosen according

75 to previous published studies on food intake and affinities for GHSR receptor (268,422). For all experiments, animals were allowed to acclimate to the experimental environment for one week. All mice received at least three saline injections (0.1 cc) prior to drug administration to habituate them to the procedure. The acute effects of a single dose of [D- Lys3]-GHRP-6 (200 nmol/mouse) on food intake and RER were assessed in FVB and MKR mice. Chronic drug treatment involved twice daily I.P. injections of [D-Lys3]-GHRP-6 or equivalent volume of saline for 12 days. Accordingly, the mice were divided into four groups, namely FVB saline group (FVB mice with saline administration), FVB Lys group (FVB mice with [D-Lys3]-GHRP-6 administration), MKR saline group (MKR mice with saline administration) and MKR Lys group (MKR mice with [D-Lys3]-GHRP-6 administration). Body weight and food consumption were recorded on a daily basis. Measurements of pulsatile GH secretion, GTT and ITT were applied on days 7, 9 and 11 of experimental period, respectively. At the end of the treatment, mice were culled following a single I.P. injection of sodium pentobarbitone (32.5 mg/ml) under fed state. Terminal cardiac blood samples were collected into EDTA-coated tubes. Plasma was separated, stored at -80°C and assayed for metabolic and hormonal analysis as addressed in section 4.2.6 and 4.2.7. Immediately after culling liver and muscles were collected and snap frozen at -80°c for metabolic analysis as addressed in section 2.6.3. Pancreas were fixed overnight by immersion at 4°C in 4% paraformaldehyde and prepared for immunofluorescence staining.

5.2.3 Effect of [D-Lys3]-GHRP-6 treatment on pulsatile GH secretion Drug or saline was given 15 minutes after the initial blood sample, subsequent blood samples (2 μl) were withdrawn from the tail vein into 58 μl of GH assay buffer (PBS, 0.05%Tween) at 15, 30, 45, 60, 75, 90, 105 and 120 minutes after drug administration for pulsatile GH analysis. Whole blood GH concentrations were measured using an in-house mouse GH sandwich ELISA as described in section 3.2.4.

5.2.4 Glucose tolerance (GTT) and insulin tolerance (ITT) tests To test the chronic effects of the drug on glucose and insulin tolerances, GTT and ITT were carried out on day 9 and day 11 of experimental period respectively. The details of these measurements were addressed in section 2.4. Furthermore, 20 ul blood samples were drawn at time points 0, 15 and 30 minutes of GTT in heparin-coated capillary tubes, centrifuged and snap frozen for insulin measurements using Ultra-Sensitive Mouse Insulin Elisa Kit (Crystal Chem, Cat # 90080).

76 5.2.5 Metabolic parameters and indirect calorimetry MKR and FVB mice were placed into in calorimeter chambers (TSE PhenoMaster, Bad Homburg, Germany). Before experiments, the mice were initially accustomed to the experimental rooms for three days prior to the experiment. To test the acute effects of [D- Lys3]-GHRP-6, mice were fasted overnight (14 hours) with free access to water and then were injected I.P. with [D-Lys3]-GHRP-6 or saline. Ten minutes after the injection, mice were given food pellets. Food intake was followed, and cumulative food intake was registered for 7 hours post-injection. RER and Locomotor activity were recorded for the entire 7-hour session. For chronic treatment, mice were injected with [D-Lys3]-GHRP-6 at doses of 200 nmol/mouse or saline twice a day (at 8:00 am and 6:00 pm) for 12 days. Food and water were available throughout the experiment. Body weight and food intake were recorded on a daily basis. 24h-RER and locomotor activity were obtained before start of treatment and on day 6 and day 11 of treatment. Body composition (lean mass, and fat mass) was determined by NMR (Brukerminispec, Bruker, LF50H, Billerica, Massachusetts) before and at the end of treatment.

5.2.6 Determination of hypothalamic mRNA expression The details of hypothalamic dissection, mRNA extraction, cDNA synthesis and real-time PCR assays were mentioned in section 2.8.1. The primer sequences of target genes were given in Table ‎5.1. The fold-change value of gene expression was calculated by using the 2-ΔΔCT method.

Table ‎5.1 Primer sequences used for gene expression analysis by qPCR

Gene Forward Reverse NPY 5’-GTGTTTGGGCATTCTG-3’ 5’-TTCTGTGCTTTCCTTCAT-3’ POMC 5’-AGAACGCCATCATCAAGAAC-3’ 5’-AAGAGGCTAGAGGTCATCAG-3’ GHRH 5’-TGCCATCTTCACCACCAAC-3’ 5’-TCATCTGCTTGTCCTCTGTCC-3’ SST 5’-TCTGCATCGTCCTGGCTTT-3’ 5’-CTTGGCCAGTTCCTGTTTCC-3’ GHSR 5’-TCAGGGACCAGAACCACAAA-3’ 5’-CCAGCAGAGGATGAAAGCAA-3’

5.2.7 Immunohistochemistry Pancreatic tissues were fixed overnight with 4% paraformaldehyde, dissected and embedded in paraffin as mentioned in section 2.7. Pancreatic sections were cut at 7 μm. After dewaxing, slides were washed, then antigen retrieval was performed using sodium citrate buffer (10mM sodium citrate, 0.05% Tween 20, PH=6) at 80°C for 30 minutes. Mounted tissue sections were blocked with 3% normal goat serum (NGS, Sigma-Aldrich,

77 NSW, AUS) / 0.5% BSA in PBS for 1 hour before incubation with primary antibodies of anti-insulin, clone E11D7 (mouse monoclonal, 1:500, Lot 2586418, Merk, Temecula, California) and anti-somatostatin-14 (rabbit polyclonal, 1:1000, Abcam, Boston, MA, USA) diluted in blocking buffer overnight at 4°C. After three times of 10-minute washes in PBS, the sections were incubated with an Alexa Fluor® 555-conjugated secondary goat anti- rabbit antibody (1:1000, Life technology, USA) and Alexa Fluor® 488-conjugated secondary goat anti-mouse antibody IgG, IgM (H+L) (1:1000, Life technology, USA) diluted in blocking buffer at RT for 2 hours, followed by three 5 minute washes in 0.01M PBS. The sections were then air dried, mounted with Pro-Long® Gold anti-fade reagent with DAPI (Molecular Probes®, Invitrogen, USA), covered with a coverslip at RT for 2 hours before sealing with nail polish and stored at -20°C. Stained sections were viewed on confocal laser-scanning microscope (Olympus, FV 1000, USA), and morphometric measurement was performed using Imaris 8.1.2 software. At least 3 non-consecutive sections per pancreas, collected from three animals per group. Islets used for quantitation were defined as the region of interest (ROI) by marking the islet border with the computer interfaced freehand tool. The border of the islet was defined by the outer margin of insulin immunofluorescence. The area of the islet in arbitrary units was recorded. Measurements of insulin and SST positive areas were then expressed as a percentage of total islet area. All microscope parameters and settings were kept constant throughout each microscopy viewing to maintain consistency within the experiment.

5.2.8 Data Analysis Data were presented and analysed as described in section 2.9.

5.3 Results

5.3.1 Effect of [D-Lys3]-GHRP-6 treatment of on pulsatile GH secretions [D-Lys3]-GHRP-6 treatment inhibited GH secretion after 15 minutes of injection that sustained throughout the 2-hour measurement of pulsatile GH secretion in MKR mice as shown by a significant decrease of total AUC of GH levels in MKR Lys mice (P<0.05, Figure 5.1B and C). In FVB Lys mice, GH was also suppressed after the treatment but total AUC did not reach statistical significance between treated and control mice (P= 0.0786, Figure ‎5.1A and C).

Figure ‎5.1 Effect of chronic [D-Lys3]-GHRP-6 treatment on pulsatile GH secretion in FVB and MKR mice.

Time course response of plasma GH from tail blood collected at the indicated time points after drug injection on day 7 of treatment from FVB mice (A), and MKR mice (B). AUC of pulsatile GH secretion after drug injection (C). Data is expressed as mean ± SEM (n=5- 7/group). ﬃP<0.05 compared to MKR saline group.

79 5.3.2 Impaired glucose and insulin tolerance in MKR Mice chronically treated with [D-Lys3]-GHRP-6 To evaluate whether long-term injections of [D-Lys3]-GHRP-6 can alter glucose tolerance and insulin sensitivity, GTT and ITT were performed in MKR and FVB mice on day 9 and day 11 respectively of experimental period respectively. During GTT, blood glucose peaked at 30-60 minutes after the I.P. glucose challenge in MKR saline compared to FVB controls. [D-Lys3]-GHRP-6 treatment significantly increased the blood glucose response at all-time points relative to FVB controls (Figure 5.2A). However, there was no significant difference in total glucose AUC between MKR lys and MKR saline groups (Figure 5.2B). Glucose-stimulated insulin secretion (GSIS) in vivo was assessed from plasma before and at 15 and 30 minutes after glucose challenge. Insulin levels in response to glucose were significantly reduced at 15 minute in FVB mice treated with [D-Lys3]-GHRP-6 (P<0.05, Figure 5.2C) and at 15 and 30 minutes in MKR mice treated with [D-Lys3]-GHRP-6 (P<0.05 and P<0.0001 respectively, Figure 5.2D). These data were consistent with the impaired glucose tolerance observed in MKR mice after chronic drug administration. During ITT, administration of insulin produced significant hypoglycaemia in FVB mice at 30, 60, 90 minutes (P<0.01, P<0.0001 and P<0.0001 respectively) (Figure 5.2E). MKR saline mice showed higher blood glucose after insulin injection, indicating significant insulin intolerance (Figure 5.2E). [D-Lys3]-GHRP-6 treatment further increased the blood glucose levels in response to insulin injection; suggesting that the [D-Lys3]-GHRP-6 treatment exacerbated insulin intolerance in MKR mice (Figure ‎5.2E). However, the corresponding blood glucose level AUC of ITT did not reach statistical significance between MKR lys and MKR saline groups (Figure 5.2F).

Figure ‎5.2 Effects of chronic [D-Lys3]-GHRP-6 treatment (200 nmol/mouse) or saline daily for 12 days on GTT, GSIS, and ITT in MKR and FVB mice.

81 Effects on GTT (n=6-7/group) (A). AUC for glucose changes during GTT (B). Effects on GSIS (n=4-5/group) (C and D); plasma insulin at 0, 15 and 30 minutes after glucose injection in FVB mice (C) and in MKR mice (D). Effects on ITT (n=6-7/group) (E). AUC for glucose changes during ITT (F). Blood glucose levels were measured at indicated time- points for 120 minutes. Data is expressed as mean ± SEM. *P<0.05, **P<0.01, ***P<0.001 MKR saline vs. FVB saline groups. #P<0.05, ##P<0.01, ###P<0.001, ####P<0.0001 MKR Lys vs. FVB saline groups. $P<0.05 vs. FVB Lys group at time 0 point. ffiP<0.05, ffiffiffiffiP<0.05 vs. MKR Lys group at time 0 point.

5.3.3 Effects on metabolic activities Acute [D-Lys3]-GHRP-6 treatment tended to delay the onset of eating after over-night fasting in FVB mice, as demonstrated by a reduction of the total food intake during the first two hours after injections compared to FVB saline mice (Figure 5.3A). This was followed by a change in the meal patterns in both FVB and MKR mice as demonstrated by hyperphagia during the 3-5 hour post-injections, then hypophagia during the 6-7 hour post- injections compared to the control mice (Figure 5.3A& B). In FVB mice, acute [D-Lys3]- GHRP-6 injection reduced RER during the 6-hour post injection significantly at 5 hour post injection (P<0.05), indicating increased fat utilization (Figure 5.3C); whereas such acute injection had no effects on RER in MKR mice (Figure 5.3D). There was no significant change in oxygen consumption (VO2) after acute injection in both FVB and MKR mice (Figure 5.3E and F). Acute [D-Lys3]-GHRP-6 did not significantly alter the normal locomotion observed in saline injected FVB and MKR mice (Figure 5.3G and H). Long- term injection with [D-Lys3]-GHRP-6 significantly increased cumulative food intake in MKR mice by nearly 20% (P<0.0001) at the end of the study (day 12) (Figure 5.4B) and to a lesser extend in FVB by 8% without statistically significant (Figure 5.4A). Long-term [D- Lys3]-GHRP-6 injection did not affect body weights in both FVB and MKR mice (Figure 5.4C and D). However, lean body mass was increased by 4% in FVB mice (P=0.0576) and by 5% in MKR mice (P<0.05) (Figure 5.4F) whereas the fat mass was reduced by 17.8% in FVB mice (P=0.0539) and by 18% in MKR mice (P<0.05) (Figure 5.4E). Analysis of the 24h-indirect calorimetry before start of treatment (day 0) and at day 6 and day 11 of experimental period demonstrated that long-term [D-Lys3]-GHRP- 6 treatment did not alter RER during both the light and dark cycles in MKR mice suggesting that long-term exposure to [D-Lys3]-GHRP-6 did not alter nutrient partitioning (Figure 5.5D-F). However, RER was increased during dark cycles by [D-Lys3]-GHRP-6 treatment in FVB mice on day 6 and day 11 compared to pre-treatment RER (day 0) indicating an increase in glucose utilization (P<0.05 and P<0.01, respectively, Figure 5.5A-C). In addition, there was no difference in either dark or light cycle locomotor

82 activity between groups (Figure 5.5G-L). Taken together, these results indicate that [D- Lys3]-GHRP-6 treatment induced differential metabolic effects between acute and long- term treatments.

Figure ‎5.3 Acute effects of [D-Lys3]-GHRP-6 on food intake and indirect calorimetric parameters in MKR and FVB mice.

Food intake in FVB mice (A), food intake in MKR mice (B), respiratory exchange ratio in

FVB mice (C), respiratory exchange ratio in MKR mice (D), Oxygen consumption (Vo2) in

83 FVB mice (E), Oxygen consumption (Vo2) in MKR mice (F), locomotor activity in FVB mice (G) and Locomotor activity in MKR mice (H). Presented are hourly means ± SE (n=4/group). *P<0.05 vs. FVB saline group.

Figure ‎5.4 Effects of chronic [D-Lys3]-GHRP-6 treatment on cumulative food intake, body weight and composition in FVB and MKR mice.

Mice received daily injections of [D-Lys3]-GHRP-6 or saline beginning on the day indicated by the arrow. Cumulative food intake in FVB mice (A), and in MKR mice (B). Change in

84 body weight of mice expressed as percentage change from baseline in FVB (C), and in MKR mice measured from day 0 to day 12 of treatment (D). Fat mass (% BW) (E), and lean mass (% BW) measured before start and at day 12 of treatment (F). Data is expressed as mean ± SEM (n=4-5/group), *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 vs. MKR saline group. ﬃP<0.05 vs. MKR Lys group before start of treatment.

5.3.4 Long-term effects on hormonal and metabolic parameters There was a significant increase in blood glucose (P<0.01) with a decrease in plasma insulin and c-peptides levels (P<0.05) in MKR mice by [D-Lys3]-GHRP-6 treatment, whereas GLP-1, leptin, ghrelin, plasma and tissues lipid levels remained unchanged following the treatment (Table 5.2). For FVB mice treated with [D-Lys3]-GHRP-6, no significant changes of any of above mentioned parameters were observed, except for reduced total and free cholesterol levels (P<0.01 and P<0.05, respectively) (Table 5.2).

Table ‎5.2 Chronic effects on metabolic and hormonal parameters

Hormone/metabolite FVB saline FVB Lys MKR saline MKR Lys Fed glucose (mmol/L) 9.1 ± 0.6197 10.2 ± 0.8879 12.9 ± 1.184$ 23.78 ± 1.991**

Insulin (pg/ml) 660.6 ± 110.7 399.3 ± 52.55 11437± 1886§§§§ 8188 ± 1026ffiffiffi

C-peptide (pg/ml) 2172 ± 182.5 1935 ± 159.1 8920 ± 1267§§§§ 6076 ± 724.1ﬃﬃ/*

Ghrelin (pg/ml) 328.2 ± 117 409.1 ± 56.19 171.1 ± 47.43 300.2 ± 56.4

GLP-1 (pg/ml) 93.66 ± 13.39 103.2 ± 19.19 94.47 ± 21 118 ± 23.88

Leptin (pg/ml) 1569 ± 187.8 1627 ± 334.6 1428 ± 140.3 1317 ± 151.8

FFA (mEq/I) 0.3318 ± 0.04 0.2333 ± 0.03 0.3755 ± 0.04 0.3599 ± 0.04

Plasma TG (mg/dl) 173.2 ± 39.67 282.4 ± 59.13 337.3 ± 14.47§§ 302.1 ± 28.51

Plasma T.cholestrol 144.1 ± 2.478 124.3 ± 4.397 †† 146.5 ± 2.031 129.3 ± 12.47 (mg/dl) Plasma F.cholestrol 48.98 ± 1.59 41.25 ± 2.112 † 66.27 ± 3.282§§ 52.89 ± 7.904 (mg/dl)

85 Liver TG (mg/gm) 8.855 ± 0.6724 11.41 ± 0.4377 12.39 ± 0.9227§ 10.94 ± 0.7616

Muscle TG (mg/gm) 20.71 ± 4.851 26.74 ± 2.677 25.04 ± 5.664 22.35 ± 2.269 Values are means ± SEM (n=5-7/group). ∗Significant difference between MKR Lys vs. MKR control (P<0.05). ∗∗Significant difference between MKR Lys vs. MKR control (P<0.05). †Significant difference between FVB Lys vs. FVB control (P<0.05). ††Significant difference between FVB Lys vs. FVB control (P<0.01). §Significant difference between MKR saline vs. FVB saline (P<0.05). §§Significant difference between MKR saline vs. FVB saline (P<0.01). §§§§Significant difference between MKR saline vs. FVB saline (P<0.0001). ffiffiSignificant difference between MKR Lys vs. FVB saline (P<0.01). ffiffiffiSignificant difference between MKR Lys vs. FVB saline (P<0.001).

86 Figure ‎5.5 Effect of chronic [D-Lys3]-GHRP-6 treatment in the metabolic profile of FVB and MKR mice.

RER in FVB mice (A), RER in MKR mice (D), total locomotor activity in FVB mice (G), and total locomotor activity in MKR mice at days 0 (before start of treatment), 6 and 11 of treatment during dark phase (18pm-6am) marked by shaded area in the graphs and light phase (6am-18pm) (J). AUC of RER during the light and dark phase of day 0, 6 and 11of treatment in FVB mice (B and C respectively). AUC of RER during the light and dark phase of day 0, 6 and 11 of treatment in MKR mice (E and F respectively). AUC of locomotor activity during the light and dark phase of day 0, 6 and 11 of treatment in FVB mice (H and I respectively). AUC of locomotor activity during the light and dark phase of day 0, 6 and 11 of treatment in MKR mice (K and L respectively). Data are expressed as mean ± SEM (n=4-5/group). *P<0.05 vs. FVB saline.

5.3.5 Hypothalamus expression of neuropeptides and ghrelin receptor involved in regulating food intake and GH secretion in response to the [D-Lys3]-GHRP-6 treatment As our results have shown changes of cumulative food intake and GH secretion by the [D- Lys3]-GHRP-6 treatment, we further evaluated the effects of this treatment on the expression of some neuropeptides and growth hormone secretagogue receptor (GHSR1) involved in regulating food intake and GH secretion in hypothalamus. In MKR mice, POMC expression was significantly decreased by long-term [D-Lys3]-GHRP-6 injections (P<0.05) confirming the inhibition of anorexigenic pathways (Figure 5.6B). However, there was no change in the expression of mRNA encoding for the orexigenic NPY (Figure 5.6A). Furthermore, GHRH expression was significantly decreased in MKR mice (P<0.05) by the long-term treatment (Figure 5.6C). Although there was a tendency of an increase in the SST expression levels by 13% and 16% in the [D-Lys3]-GHRP-6 treated MKR and FVB mice respectively (Figure 5.6D), and a tendency of decrease in GHSR expression in [D- Lys3]-GHRP-6 treated MKR mice by 32% (Figure 5.6E), however, these changes did not reach a statistically significant value. Unlike MKR mice, the gene expression levels of, GHRH and GHSR tended to be increased in FVB mice by the treatment. Thus, the gene expressions of GHRH and ghrelin receptor were differentially regulated by [D-Lys3]- GHRP-6 treatment between diabetic MKR and WT control FVB mice.

88 Figure ‎5.6 Fold change expression of hypothalamic genes after [D-Lys3]-GHRP-6 treatment.

Hypothalamic expression of neuropeptide Y (NPY) (A), proopiomelanocortin (POMC) (B), growth hormone releasing hormone (GHRH) (C), somatostatin (SST) (D) and growth hormone secretagogue receptors (GHSR) (E) were determined using real-time PCR. Relative mRNA expression levels were calculated as the ratio of each gene expression level to the β-actin expression level in the same sample. Data are expressed as mean ± SEM (n=6-7/group). *P<0.05, **P<0.01, and ***P<0.001.

5.3.6 Morphology and analysis of insulin and somatostatin positive areas within pancreatic islets after long-term [D-Lys3]-GHRP-6 treatment The decreased GSIS, fed insulin and c-peptide levels from long-term [D-Lys3]-GHRP-6 treated mice suggest inhibitory effects of the treatment on insulin secretion from pancreatic β cells. Thus, immunofluorescence analyses were performed on sectioned pancreatic tissues from experimental mice. Immunofluorescence staining for insulin and SST showed that pancreatic islets from MKR mice had a significant decrease in SST producing cells compared with that from FVB mice (P<0.0001, Figure 5.7G). Moreover, the immunofluorescence staining demonstrated altered islet morphology in [D-Lys3]-GHRP-6 treated FVB and MKR mice (Figure 5.7F and L). Quantitative analysis of pancreatic islet insulin and SST positive areas revealed reduced insulin area (P<0.0001, Figure ‎5.7M) and increased SST areas (P<0.05, Figure 5.7N) in [D-Lys3]-GHRP-6-treated FVB and MKR mice. The increase in SST content within the pancreatic islets after long-term [D- Lys3]-GHRP-6 treatment may have a direct inhibitory effect on insulin secretion from pancreatic β cells.

90 Figure ‎5.7 Double immunofluorescence for insulin and somatostatin of pancreatic islets.

Representative confocal images of pancreatic sections. From left to right: SST immunofluorescence (red), insulin immunofluorescence (green) and merged immunofluorescence. Representative images of pancreatic sections from FVB mice administered with saline (A-C). Representative images of pancreatic section from FVB mice administered with [D-Lys3]-GHRP-6 (D-F). Representative images of pancreatic section from MKR mice administered with saline (G-I). Representative images of pancreatic section from MKR mice administered with [D-Lys3]-GHRP-6 (J-L). Scale bar, 50 μm. Quantitative analysis of thirty pancreatic islets from 3 stained pancreas sections from each mouse for insulin and somatostatin areas (M, N respectively), n = 3 mice/group. Data are expressed as mean ± SEM (n=3/group). *P<0.05, **P<0.01, ****P<0.0001.

5.4 Discussion In this study, we reported that chronic treatment with a ghrelin antagonist, [D-Lys3]-GHRP- 6 deteriorated glucose and insulin tolerances in non-obese diabetic MKR mice. This impairment of glucose tolerance was accompanied by an inhibition of glucose stimulated insulin response. Our immunofluorescence data demonstrated increased SST content and decreased insulin content in pancreatic islets with altered islet morphology (Figure ‎5.2). These structural and functional changes of pancreatic islets may be responsible for worsening of glucose and insulin intolerances in MKR mice after long-term treatment with [D-Lys3]-GHRP. On the contrary, some studies have highlighted the ability of GHS-R1a antagonists to stimulate insulin secretion and improve glucose tolerance. Nonetheless, neither GHS-R1a antagonists nor exogenous ghrelin significantly altered the insulin action by ITT experiments (167). Moreover, most of these studies were performed only on acute or short-term treatments (5-7 days) (167,288). There is no much GTT and ITT data upon long-term treatment with GHS-R1a antagonists to support the long-term glycaemic control of such receptor inhibition. Repeated injections of [D-Lys-3]-GHRP-6 for 5 days significantly lowered body weight gain and blood glucose concentrations, accompanied by a moderate decrease in serum insulin levels (268). The discrepancy between the short- term effects in previous studies and the long-term effects of [D-Lys-3]-GHRP-6 in this study could be explained by a duration dependent difference in ghrelin signalling pathways. In addition, potential adverse effects may arise from chronic dosing of [D-Lys- 3]-GHRP-6 that affect glucose homeostasis. Ghrelin is the only peripheral secreted hormone that stimulates food intake centrally. Recent reports have suggested that the effects of ghrelin receptor antagonists on insulin sensitivity might be mediated by the reduction of food intake. Our data demonstrated that whereas ghrelin receptor antagonist tended to inhibit food intake during the first two hours after injection (Figure 5.3A and B), it obviously increased cumulative food intake in MKR mice and tended to increase it in

91 FVB mice through long-term treatment (Figure 5.4A and B). In consistent with that, a recent study showed that [D-Lys3]-GHRP-6 did not decrease stress-induced caloric intake, but paradoxically increased the intake of HFD (343). Moreover, the infusion of [D-Lys3]- GHRP-6 in a rat model did not have a significant impact on food intake and appetite (342). Thus antagonizing the ghrelin system may reduce food intake and RER in the short term, however, compensatory mechanisms may override inhibitory effects in the long term (347). Moreover, the possibility of quick tolerance to [D-Lys3]-GHRP-6 should not be excluded as [D-Lys3]-GHRP-6 decreased ethanol intake, preference, and water intake in mice only on the first day of treatment (342). Similarly, there have even been a number of reported GHS-R1a antagonists which exhibit orexigenic effects. Although the antagonist BIM-28163 inhibits GH secretion through blocking ghrelin-induced GHS-R1a activation, this compound elicits increases in food intake and body weight (338). Furthermore, GSK1614343 increased food intake and body weight in vivo, but knockout of the GHS-R1a abolished this effect, indicating that the antagonist was working via this receptor (192). Despite increasing food intake in MKR mice after chronic treatment, ghrelin receptor antagonist nonetheless decreased adiposity (Figure 5.4E) and failed to increase body weight (Figure 5.4C and D) or change respiratory exchange ratio (Figure 5D-F). In this context, it was reported that the control of lipid metabolism by ghrelin might be independent of the orexigenic effects (414).

Ghrelin was initially discovered as the endogenous ligand of GHS-R1a. Ghrelin triggers GH release through activation of GHS-R1a. This action is mediated primarily through inhibition of hypothalamic SST neurons and to a lesser extent via a direct effect on the anterior pituitary gland (201). Our results showed that the injections of ghrelin receptor antagonist significantly suppressed pulsatile GH secretion (Figure 5.1A-C). Similarly, another ghrelin receptor antagonist BIM-28163 was reported to inhibit ghrelin-induced GH secretion but not ghrelin-induced food consumption (338). The reason for why GH secretion was inhibited while food intake was not inhibited upon chronic treatment in this study, is not clear. Previous reports have advised that the ability of ghrelin to regulate food intake or adiposity is independent of GH secretion (423). Moreover, individual antagonist- receptor interactions may cause distinct pharmacodynamics outcomes by stimulating some signalling pathways while having no effects or inhibiting other signalling pathways could be present (424).

The hypothalamus is a critical component of the CNS in the regulation of feeding and GH secretion. Ghrelin targets the arcuate nucleus, from where GHRH neurons trigger GH

92 secretion. This hypothalamic nucleus also contains NPY neurons, which play a master role in the effect of ghrelin on feeding (235,425). Interestingly, connections between NPY and GHRH neurons have been reported, leading to the hypothesis that the GH axis and the feeding circuits might be co-regulated by ghrelin. We examined the gene expression of neural peptides regulating feeding that are targeted by ghrelin such as NPY and POMC (Figure 5.6). Interestingly, the anorexigenic POMC gene was only down-regulated in MKR mice after long-term [D-Lys3]-GHRP-6 treatment which is consistent with increasing cumulative food intake in these mice. However, there was no significant change in NPY expression after treatment. This mechanism could raise the possibility that compensatory mechanisms may override the acute effects in the long term. GHRH was down-regulated and GHSR tended to be down-regulated in MKR treated mice while SST expression appeared to increase in both MKR and FVB mice by long-term [D-Lys3]-GHRP-6 treatment which explained the inhibitory effect of [D-Lys3]-GHRP-6 on GH secretion (Figure 5.6). The inhibitory effects of GHS-R1a antagonist on feeding and GH in MKR mice appear through central action on hypothalamus. However, in FVB mice, there was no significant change in the gene expression of these neuropeptides consistent with the differential effects of this ghrelin antagonist on food intake and respiratory exchange ratio in FVB and MKR mice. Such difference support the notion that the effects of ghrelin receptor ligands might depends on the metabolic states such as the glycaemic levels in rodents (426).

Fed blood glucose was higher with lower plasma levels of insulin and c-peptide levels in MKR mice at the end of [D-Lys3]-GHRP-6 treatment (Table ‎5.2). In agreement with this, a considerable increase in blood glucose levels was observed after infusion of different dosages of a GHS-R antagonist [D-Lys3]-GHRP-6 in Wistar rats (341). On the contrary, repeated injections of [D-Lys-3]-GHRP-6 for 5 days significantly lowered blood glucose and FFA levels in ob/ob obese mice (268). We did not observe changes of ghrelin, leptin or GLP-1 after [D-Lys-3]-GHRP-6 treatment (Table ‎5.2). Our findings showed that pharmacological blockade of ghrelin receptor improved the plasma lipid profile in FVB mice as demonstrated by a decrease in total and free cholesterol levels (Table ‎5.2). This raises the possibility that ghrelin antagonism might regulate cholesterol metabolism. Recently, it is reported that the gut-brain axis including ghrelin and the central melanocortin system directly regulates the hepatic synthesis and re-uptake of cholesterol (264).

93 This study showed that peripheral long term treatment with [D-Lys-3]-GHRP-6 had detrimental effects on pancreatic islets as showed by altered morphology, decreased insulin positive cells and increased SST positive cells (Figure 5.7). SST and ghrelin are two highly interconnected neuropeptides with a wide range of biological actions at central and peripheral actions (427). Interestingly, ghrelin and SST are expressed at the endocrine pancreas and are actively involved in the regulation of insulin secretion. Furthermore, under certain metabolic conditions such as T2DM, their pattern of distribution is severely disturbed (428). In this study, we showed that SST content was reduced in pancreatic islets of diabetic MKR mice (Figure 5.7G). By long-term ghrelin antagonist treatment, the SST content was increased which might inhibit insulin secretion and consequently worsened glucose tolerance in MKR mice. However, the molecular mechanisms underlying such action of ghrelin antagonist on SST secretion are unclear. Ghrelin exerts an anti-apoptotic effect in many cell types through activation of ERK1/2 and AKT, such as HIT-T15 pancreatic β-cell line, and macrophages (429-431). However, increased phosphorylation of ERK1/2 or AKT induced by ghrelin was suppressed after pre- incubated with [D-Lys3]-GHRP-6 (432). Consequently, it may be possible that GHS-R1a antagonists inhibit β cell survival that might responsible for alteration of islet morphology shown by [D-Lys3]-GHRP-6 treatment in this study.

In conclusion, this study provides an evidence that long-term antagonizing the ghrelin receptor GHS-R1a might not be an effective option for T2DM treatment. GHS-R1a belongs to the class a G-protein-coupled receptor family which display heterogeneous signalling cascades including phospholipase C, protein kinase C, protein kinase A (185) intracellular and extracellular Ca2+, and MAPK (433). All of which highlight the complexity of GHS-R1a activation and constitute important considerations for using ghrelin antagonists in diabetic therapy (424,434). Further research into the potential use of pharmacological ghrelin antagonists in an effort to regulate energy homeostasis is highly required.

94 6) CHAPTER 6. GENERAL DISCUSSION

6.1 Overview Obesity is associated with blunted GH secretion (359). GH treatment in obese cases has demonstrated favourable effects on most of the features of adiposity. However, it is not known about GH levels in non-obese type 2 diabetes and whether modulation of growth hormone secretagogue receptor (GHS-R1a receptor) can improve some of the metabolic aberrations observed in non-obese type 2 diabetes. While established that ghrelin is a GH stimulator with a potent orexigenic effect, it is not yet clear the exact role of ghrelin and its receptor in action of insulin secretion and glucose metabolism. Studies in humans and rodents have found a negative correlation between ghrelin and insulin Levels (18). Ghrelin deletion in ob/ob mice improved glucose tolerance and insulin secretion (20). Moreover, ghrelin was reported to increase pancreatic islet survival and induce protection against apoptosis in a pancreatic β-cell line and in human islets (435). Inspired by recent findings, Hexarelin, a GHS-R1a receptor agonist decreased lipid contents of cultured adipocytes (181) and reduced cholesterol efflux in macrophages through PPAR-γ-dependent pathway (324). Therefore, ghrelin receptor might, represent a valid drug target for diabetes. This thesis sought to investigate the phenotypic and metabolic characteristics of a mouse model of non-obese type 2 diabetes (MKR mice), more specifically, to identify and characterize patterns of pulsatile GH secretion and GH regulatory factors. Second to this, we sought to explore the effects of ghrelin receptor modulation in this mouse model with the goal of developing new drug targets for diabetes. Thus, we aimed to investigate the effects of Hexarelin as a ghrelin mimetic and [D-Lys3]-GHRP-6 as a ghrelin receptor antagonist in this mouse model and to understand the underlying molecular mechanisms involved. In addressing these notions, major findings in this thesis were observed as follows: 1) MKR mice have an atypical form of type 2 diabetes, 2) MKR mice have normal to higher pulsatile GH secretion despite hyperinsulinaemia, 3) Hexarelin improves insulin intolerance through ameliorating lipid abnormalities in MKR mice, 4) [D-Lys3]-GHRP-6 is not a complete ghrelin receptor antagonist, 5) [D-Lys3]-GHRP-6 does not improve glucose or insulin intolerance in MKR mice and 6) Peripheral [D-Lys3]-GHRP-6 injections affects central hypothalamic regulatory neurons.

95 6.2 Major Findings

6.2.1 MKR mice have an atypical form of type 2 diabetes By studying the phenotypic and metabolic profiles of MKR mice (Chapter 3.3), we showed that MKR mice had lower body weights at all age groups compared to control mice. Though being lean, they had higher fat mass and lower lean mass (Chapter 3.3). The development of insulin resistance in these mice was confirmed by ITT and HOMA-IR. Despite the marked insulin resistance that was exhibited by these mice, they had relatively normal fasting blood glucose at 10 weeks old as showed by GTT results in Chapter 3.3. This is likely a consequence of the metabolic inflexibility in MKR mice that enable them to utilise glucose on the expense of fat during fasting (146). This metabolic inflexibility indicates the severe impairment of fat metabolism in these mice. The association between the lack of IGF-1 and disturbed lipid metabolism of diabetic patients has been demonstrated in many studies (436). IGF-1 stimulates fatty acid transport in muscle and its inhibition causes redistribution of circulating FFA to the liver, which eventually leads to hepatic steatosis (437). Furthermore, adult mice with partial igf-1 deletion displayed impaired fat metabolism as indicated by decreased expression of genes involved in lipid metabolism (ATP-citrate lyase, acetyl-CoA acyltransferase 1B, acetyl-CoA acetyltransferase 1) and cholesterol synthesis and transport (HMG-CoA reductase and synthase, LDL-related protein 1) resulting in dyslipidaemia (438). Overall, all these findings in conjunction with our findings indicate that IGF-1 deficiency could be responsible for altering fat metabolism that participate in the development of this non-obese diabetic phenotype with lipid metabolic aberrations. Interestingly, MKR mice showed no significant difference in glucose and insulin tolerance at 33 weeks old which needs further study.

6.2.2 MKR mice have normal to higher pulsatile GH secretion despite hyperinsulinaemia Given the proposed interactions between GH and IGF-1 in maintaining normal growth and metabolism, and because MKR mice had been shown by previous studies to exhibit postnatal growth retardation (149), this study aimed to examine the growth parameters and characterize pulsatile GH secretion at different age groups in MKR mice. Our data confirmed that the linear growth in younger MKR mice at 5 weeks of age was significantly lower to that observed in FVB mice but they have normal body lengths at 10 and 33 weeks old. Moreover, our observations demonstrated increased total, mass and pulsatile GH secretion in MKR mice at 5 weeks of age but these parameters were comparable to FVB mice at 10 weeks old and slightly higher at 33 weeks old (Chapter 3.3). In spite of the

96 increased GH secretion, plasma total IGF-1 and insulin levels are significantly higher in MKR mice (Chapter 3.3). These findings were in contrast with the notion that hyperinsulinaemia is associated with hyposecretion of GH, and vice versa (439-441). In this issue, it was proposed that the suppression of GH secretion in obese diabetics may result from hyperinsulinaemia that stimulates hepatic IGF-I synthesis which in turn inhibits GH secretion by a negative feedback mechanism (442). On the other hand, patients with T1DM have higher pulsatile GH secretion due to decreased pancreatic β cells production of insulin, which leads to a decrease of the hepatic production of IGF-1. Thus, lower IGF-I levels may explain the increased GH secretion in T1DM. Moreover, the elevated levels of GH had been found in individuals born small for gestational age (SGA) with catch up growth and insulin resistance (443). Indeed, changes in glucose and insulin secretion result in counter-regulatory change of GH secretion. Thus, suppression of GH release in obese diabetics may have a beneficial effect to ameliorate insulin resistance associated obesity. Whether the increase in GH secretion in MKR mice is pathological or adaptive to facilitate rapid linear growth due to igf-1 receptor mutation and to promote muscle development is unclear. The possibility that diabetes alters hypothalamic function, which in turn, modulates the response characteristics of somatotrophs is also existent. The enhanced pulsatile GH concentrations in MKR mice could be due to hypothalamic dysfunction associated with deregulation of SST and/or GHRH secretion. However, by assessing the gene expression levels of GH regulatory factors, including those involved in food intake regulation, there were no observable changes in gene expressions of GHRH and SST in the hypothalamus between MKR mice and FVB mice. However, the number of orexigenic NPY mRNA in hypothalamus of MKR mice was significantly increased and anorexigenic POMC expression was slightly inhibited compared to that of FVB mice which was consistent with increased food intake noticed in these mice. It is possible that changes in GH secretion occurs due to disruption in the connection between GH regulatory neurons and food intake control centres in the hypothalamus. Recent studies have shown that increased NPY neuron inhibits SST expression (362) which was not observed in MKR mice. It is also likely that dissociation of GH/IGF-1 axis and insulin happened with loss of negative feedback control between IGF-1 and GH. Ultimately, it is not clear why GH increased in MKR mice in spite of hyperinsulinaemia. Thus, there will be renewed interest in defining the mechanisms that subserve the abnormalities of GH secretion in diabetes and understanding the relative roles of GH in modulating responsiveness to insulin.

97 6.2.3 Hexarelin improves insulin intolerance through ameliorating lipid abnormalities in MKR mice Growth hormone secretagogues (GHS) have the advantage over GH or GHRH therapy of a physiologically increased endogenous GH pulsatile secretion with decreased risk for side effects (444). GHS is generally used to refer to ghrelin and its analogues through acting on GHS-R1a receptor rather than to GHRH (291). The GHS family nowadays keeps growing and varies both in number and chemical heterogeneity. Given this diverse pharmacological development, most GHS are totally unrelated from the structural point of view and have many effects independent of GHS-R1a. In this study, we examined the effect of Hexarelin, one of peptidyl GHS. Hexarelin was identified as a ligand to CD36, in addition to its GHS- R1a agonistic activity (324,445). Hexarelin has been shown to have potent effects on adipose tissue regulation and fat metabolism (181). In our study, we demonstrated that Hexarelin improved glucose and insulin intolerance in MKR mice after 12 days of treatment whereas it was not able to alter the hyperglycaemia or insulinaemia (Chapter 4.3). Although Hexarelin increased total food consumption, this effect did not contribute to the weight gain in MKR mice (Chapter 4.3). This could be attributed to decreasing fat mass with corresponding increase in lean mass as noticed in this study. Moreover, our results showed that Hexarelin efficiently increased fatty acid oxidation as demonstrated by a reduction in fat mass and decreased RER in MKR mice. In this study, Hexarelin was demonstrated to potently reduce plasma concentrations of TG and normalize liver TC (Chapter 4.3). Thus, the improvement of insulin sensitivity by Hexarelin likely involves their ability to enhance fat metabolism. This was confirmed by up-regulating WAT expression of PPAR-γ and its downstream target genes that control fatty acid uptake, sequestration and oxidation in WAT. In specific, up-regulating UCP-1 allows the use of the energy derived from fatty acid oxidation for the generation of heat. Additionally, there was a slight increase of CD36 expression, a trans-membrane protein that thought to play an important role in mediating fatty acid transport into adipose tissue (446). These findings are consistent with previous work demonstrating controlling effects of gene expression by Hexarelin in cultured adipocytes (181). These lipid-lowering effects of Hexarelin could be derived from enhanced adipocytes differentiation as illustrated by our histological examination (Chapter 4.3). Whether enhancing lipid metabolism and changing the body composition in MKR mice related to Hexarelin-induced GH effects is not clear. Hexarelin increased GH secretion; however, this effect did not last long and GH returned to baseline levels within 30 minutes after injections (Chapter 4.3). In our study, Hexarelin did not change total cholesterol but had a tendency to decrease free cholesterol. Other studies showed that

98 Hexarelin had beneficial effects on cholesterol metabolism. Hexarelin stimulated cholesterol removal from macrophages through activation of the PPAR-γ-LXRα axis and up-regulation of ABCA1 and ABCG1 sterol transporters results in significant reduction in plaque formation of atherosclerotic mice (388). Our findings in conjunction with previous studies suggest that Hexarelin may regulate peripheral fatty acid metabolism and adipocyte differentiation through its effects on PPAR-γ-dependent genes; and propose a potential therapeutic utility of Hexarelin in the treatment of dyslipidemia associated with insulin resistance. The actions of Hexarelin in the regulation of energy metabolism as proposed by this study are summarized in Figure ‎6.1.

6.2.4 [D-Lys3]-GHRP-6 is not a complete ghrelin receptor antagonist Ghrelin stimulates GH secretion and has potent orexigenic effects (189). Both effects are mediated by the GHS-R1a, in addition, ghrelin promotes a positive energy status through the stimulation of adipogenesis via independent pathways (231). Ghrelin is also recognized as an important endogenous regulator of glucose and lipid homeostasis. Ghrelin induces hyperglycaemia and insulin resistance independent of GH stimulatory effects (447). There are in vivo and in vitro works suggest that ghrelin suppresses glucose stimulated insulin secretion (448), whereas the ghrelin antagonists do the opposite (213). Thus, efforts to utilize ghrelin antagonists in the treatment of T2DM and obesity have increased recently. However, activation of GHS-R1a may produce a variety of signalling mechanisms and subsequent different physiological responses dependant on the levels of transcription, receptor interaction and internalization (449). Studies on a short term basis showed that [D-Lys3]-GHRP-6 could have some of the anti-diabetic effects including decline of blood glucose, inhibition of food intake, slowing of gastric emptying and reduction of body weight in diabetic ob/ob mice (268). Nevertheless, there was no data to support the effects of [D-Lys3]-GHRP-6 on insulin sensitivity and insulin secretion on long term treatment and ob/ob mice do not completely replicate the human diabetes. In the present study, we investigated a variety of metabolic consequences following chronic treatment of [D-Lys3]-GHRP-6 in MKR mice to explore the potential therapeutic utility of this ghrelin receptor antagonist in diabetes. We employed MKR mice model to provide data with more relevance to human non-obese T2DM. Our results clearly demonstrate that chronic [D-Lys3]-GHRP-6 treatment suppressed pulsatile GH secretion as expected from antagonizing the ghrelin receptor (Chapter 5.3). However, such ghrelin antagonistic activity invoked by [D-Lys3]-GHRP-6 has not been observed in food intake and glucose metabolism in MKR mice (Chapter 5.3). For the effects of [D-Lys3]-GHRP-6 on food intake, we showed that [D-Lys3]-GHRP-6 decreased food intake only within the first 2

99 hours after acute injections, however, our chronic studies demonstrated that cumulative food intake has been increased significantly in MKR mice at the end of treatment. In consistent with this, a study showed that [D-Lys3]-GHRP-6 did not decrease stressed induced caloric intake, but paradoxically increased the intake of HFD (343). Moreover, decreased ethanol intake, preference, and water intake were noticed only during the first 4 hours of [D-Lys3]-GHRP-6 injection in C57BL6J mice (342). These results in conjunction with our results suggested a possibility of quick tolerance to [D-Lys3]-GHRP-6 or the presence of different signalling pathways mediating the acute and chronic effects of [D- Lys3]-GHRP-6.

6.2.5 [D-Lys3]-GHRP-6 does not improve glucose or insulin intolerance in MKR mice Our results showed that glucose and insulin intolerance in [D-Lys3]-GHRP-6-treated MKR mice were deteriorating while decreasing glucose-stimulated insulin release and pancreatic insulin content (Chapter 5.4). Pancreatic β-cells adapt to situations of chronic fuel over-supply and insulin resistance by increasing their mass and alterations in several parameters related to β-cell function. Other studies showed that ghrelin appears to have a paracrine/autocrine role in the regulation of pancreatic hormonal secretion and islet growth and differentiation (215). In this study, we showed that MKR mice had lower SST contents in pancreatic islets to allow more insulin secretion to overcome insulin resistance in MKR mice. After 12 days of [D-Lys3]-GHRP-6 treatment, alterations of these parameters have been observed as indicated by a reduction in insulin area with an associated increase in SST areas and islet morphological changes (Chapter 5.3). Collectively, these findings could be attributable to decreased insulin, diminished beta cell responsiveness to the glucose stimulus, or impaired conversion of proinsulin as indicated by decreasing c- peptide in MKR mice. In this study, we did not observe any significant changes in FFA, plasma or tissues TG, however, decreased total and free cholesterol were noticed after chronic [D-Lys3]-GHRP-6 treatment in FVB mice (Chapter 5.3). Another study observed a considerable increase in blood glucose levels in Wistar rats after I.P. infusions of [D-Lys3]- GHRP-6 (341). However, total cholesterol, triglycerides, and albumin showed no significant changes (341). Thus, the effects of [D-Lys3]-GHRP-6 on lipid metabolism await further investigations.

100 6.2.6 Peripheral [D-Lys3]-GHRP-6 injections affect central hypothalamic regulatory neurons It is well established that the gastric hormone, ghrelin acts on CNS neurons to modulate GH secretion, blood glucose homeostasis, and gastrointestinal tract motility (185). However, the mechanisms of action of several GHS-R1a agonists and antagonists in the CNS remain poorly understood. To bring insight into the understanding of the central activity of peripheral [D-Lys3]-GHRP-6 injections, we examined the gene expression levels of feeding and GH regulatory hormones in hypothalamus. In contrary to the expectations, chronic GHS-R antagonist treatment itself was not inhibitory to NPY gene expression and even inhibited the expression of anorexigenic neuron of POMC in MKR mice. These findings confirm the unexpected food stimulatory effects of [D-Lys3]-GHRP-6 noticed in this study after chronic treatment. In another study, D-Lys3-GHRP-6 was shown to reduce preference to alcohol in male C57BL/6J mice by decreasing the activation of c-Fos in some brain regions containing ghrelin receptor (450). Current observation also showed that GHRH was down-regulated while SST had a tendency to be up-regulated which explained the inhibitory effect of [D-Lys3]-GHRP-6 on GH secretion in MKR mice. Interestingly, the genes of GHRH and GHSR were expressed differently in MKR and FVB mice after chronic [D-Lys3]-GHRP-6 treatment. Based on these findings, it is likely that metabolic status has an impact on the central action of this peptide and future work could be directed at investigating the potential differential effects of ghrelin receptor blockage in different metabolic and feeding conditions. A better understanding of the molecular pathways activated by these compounds could be useful in developing future therapeutic applications.

6.3 Conclusions and Implications of our findings Diabetes mellitus clearly causes derangements in the GH secretion, which has been shown to increase or decrease according to the level of insulin and degree of obesity. It is not clear if these alternations play a role in the pathogenesis or adaptation to the disease. While our present data showed that MKR diabetic mice with impaired insulin sensitivity had normal to higher concentrations of GH levels suggested of dissociated GH regulation at the hypothalamic/pituitary level or altered feedback regulatory mechanisms, it is not clear yet whether the increase of pulsatile GH secretion in MKR mice is beneficial or harmful. However, we speculated that these mice need such increase in GH to partially compensate for the impaired muscle development caused by igf-1 mutation. Similarly, the increased of GH secretion noticed in children born SGA or low birth weight to help them

101 catch-up growth. Pharmacologic interventions aimed at adjusting GH secretion in these mice might reveal a sign towards benefit or harm from increased GH secretion.

Modulation of GHS-R1a with ghrelin agonists and antagonists has been identified as a pathway for new therapeutic options. The data from this thesis revealed a novel role of Hexarelin in the control of lipid aberrations-associated with non-obese type 2 diabetes such as in lipodystrophic cases. This was achieved with improving glucose and insulin tolerance, neutral effect on body weight and satisfactory body composition. Besides the previous evidences of Hexarelin’s efficacy in the prevention of heart diseases, give this novel compound the therapeutic advantages in the treatment of dyslipidaemia associated metabolic diseases. However, the need for further investigation of multiple dosing regimens of longer duration would be essential to demonstrate Hexarelin efficacy in clinical trials of relevant patient populations.

In spite of new hope that ghrelin-receptor antagonists might become a treatment for diabetes, this study showed that long term treatment with [D-Lys3]-GHRP-6 worsened the diabetic conditions and increased cumulative food intake in non-obese diabetic mice. Thus, our findings question [D-Lys3]-GHRP-6 as an effective GHS-R1a receptor antagonist in management of diabetes. The history of ghrelin receptor antagonist development includes many examples of promising candidates that failed to reach the clinic in the treatment of obesity. The complexity of the ghrelin system also carries the risk for such failures; however, the potential therapeutic application of other ghrelin antagonists remains broad and may imply clinically relevant perspectives.

6.4 Limitations of the study Possible limitations of this study are the use of MKR mice with muscle specific igf1 mutation. IGF-I co-ordinately links GH and insulin actions on tissues and organs. The actions of IGF-I are regulated with GH in order to utilize fat as an energy source for growth. It would be possible that such igf-1 mutation could interfere with insulin/GH relationship and more studies are needed to confirm insulin/GH relationship in non-obese type 2 diabetes. An additional limitation is that the ACTH/cortisol and prolactin releasing effects of Hexarelin have not been investigated in this study. Although up to date, no adverse consequences of long-term use of Hexarelin have been reported, it is advisable to rigorously follow up the long-term effects of Hexarelin treatment in clinical trials and other diabetic models. Based on compelling evidence provided here in this study, the impairment of insulin intolerance and glucose stimulated insulin secretion in response to chronic [D-Lys-3]-GHRP-6 injections may be due to stimulation of SST locally in pancreatic

102 islets. However, cellular mechanisms linking GHS-1Ra antagonist to insulin production were not examined. Furthermore, whether GHS-1Ra antagonist influences the response of pancreatic β cells to other secretagogues such as incretins was not studied. [D-Lys-3]- GHRP-6 has been reported to interact with the 5-HT2b receptors in the gut (451) although the brain expression of this receptor is very low compared to the gut. Given the multi- ligand, [D-Lys-3]-GHRP-6 could have potential off-target effects that might affect other signalling pathways in the body. Thus, testing more specific ghrelin receptor antagonists on long-term studies in diabetes is highly recommended.

6.5 Future directions Further research about the interaction of GH with glucose metabolism is clinically important for broadening our general understanding of the role of GH in such important condition as T2DM. This may in turn shed light on the development of intervention strategies to prevent adverse pathophysiological consequences associated with T2DM. Further investigations of the metabolic changes of some hormones or metabolites such as leptin, adiponectin, free IGF-1 or IGFBPs that affect GH signalling may deepen current knowledge regarding modification of GH-insulin relationship in T2DM.

In this study, presented findings provide valuable insights into the use of Hexarelin in management of lipid abnormalities. Hexarelin has therapeutic advantages over some lipid lowering medications in diabetic condition as illustrated by our findings, such as improving glucose intolerance, not provoking hyperglycaemia, and avoiding weight gain. The challenge is whether these results in mice can be translated to humans. The need of long term clinical trial studies data between possible adverse effects and the beneficial effects on lipid profile should be determined from treating patients.

Although our data showed that the peripheral injected [D-Lys-3]-GHRP-6 had central regulatory effects on hypothalamic neurons expressions, the transport of GHR-1Ra antagonist across the blood-brain barrier and their targets in the hypothalamus and other regions of the brain should be clearly identified in future studies. Moreover, the underlying mechanisms of the differential effects of GHS-1Ra antagonist on energy expenditure and fuel oxidation on acute and long-term effects should be more elucidated. Given the broad distribution of ghrelin receptor, studies are needed to determine the pleiotropic actions of GHS-R antagonists on cardiovascular functions, gastrointestinal motility, cognitive function and cell proliferation, etc. for a careful evaluation of potential side effects in clinic use.

103

Figure ‎6.1 Schematic graph of the proposed Hexarelin action in regulating energy metabolism.

Hexarelin binds to CD36 receptor in adipose tissue to enhance lipid uptake and metabolism through upregulating PPAR-γ and downstram signalling molecules. This results in decrease free fatty acids (FFA) release from adipose tissue, decrease circulating FFA and decrease the ectopic fat deposition in muscles and liver. Besides its CD36 agonistic activity, Hexarelin binds to GHS-R1a in hypothalamus and pituitary gland to stimulate GH release. GH in turn acts on muscle to increase protein synthesis, on adipose tissue to stimulate lipolysis and decrease fat mass and on the liver to stimulate IGF-1 production which has insulin like action on muscle and adipose tissues. The direct action of Hexarelin on liver gluconeogenesis is not clear yet.

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