Investigating the Neuroendocrine Control of Metabolism and Energy Homeostasis

INVESTIGATING THE NEUROENDOCRINE CONTROL OF METABOLISM AND ENERGY HOMEOSTASIS

Thesis submitted for the degree of Doctor of

Philosophy at Imperial College London

CHIOMA NMEREOBASI IZZI-ENGBEAYA

2018

Section of Investigative Medicine

Division of Diabetes, Endocrinology & Metabolism

Department of Medicine

Imperial College London Abstract

Obesity and associated metabolic disorders are major causes of morbidity and mortality in both developed and developing countries, but the options for treating these conditions are limited. Energy homeostasis and metabolism are regulated by a complex network of neuroendocrine systems, neural pathways, peripheral signals and circuits. Consequently, our knowledge of the regulation of energy homeostasis and metabolism remains incomplete. Two neuroendocrine systems were examined in this thesis, one involving glucocorticoids (a peripherally produced hormone acting centrally) and one involving kisspeptin (a centrally produced hormone acting peripherally).

Reduction of active glucocorticoids in the arcuate nucleus of post-pubertal male rats (via stereotactic injection of recombinant adeno-associated virus to reduce expression of

11βHSD1) resulted in less weight gain despite matched food intake to controls on normal chow diet, with higher brown adipose tissue weight. However, on a high fat diet, reduction in arcuate active glucocorticoids resulted in higher average daily food intake and a trend towards higher body weight than controls. No differences in body composition, plasma corticosterone, plasma insulin and plasma glucose were produced by reduction of arcuate glucocorticoids.

Administration of kisspeptin to healthy men resulted in increased glucose-stimulated insulin secretion (GSIS) during hyperglycaemia but not during euglycaemia. Kisspeptin enhanced

GSIS without affecting the levels of metabolically active gut hormones. Additionally, acute kisspeptin administration did not affect appetite and food intake in healthy men.

These results provide further insights into the neuroendocrine control of energy homeostasis and metabolism and may help guide the development of hormone-modulating therapies for the treatment of obesity and associated conditions.

Acknowledgements

I would like to thank God, who made all this possible.

I would like to thank my husband for his unwavering support and belief in me, and my family for their support and encouragement.

I would like to thank Prof Waljit Dhillo for his invaluable mentorship, and for providing the springboard for my academic career.

I would like to thank Dr James Gardiner for his supervision.

I would like to thank the members of the Kisspeptin Research Team, the Section of

Investigative Medicine and the Clinical Chemistry staff at Imperial College NHS Trust for their assistance with various aspects of my project.

I would like to thank the MRC for awarding a Fellowship to me, which provided the funding for my PhD.

Declaration of Originality

The work in this thesis was designed and performed by the author, with contributions from collaborators (detailed in Declaration of Contributors). All other work is appropriately referenced and a comprehensive list of references is located at the end of this thesis.

The copyright of this thesis rests with the author and is made available under a Creative

Commons Attribution Non-Commercial No Derivatives licence. Researchers are free to copy, distribute or transmit the thesis on the condition that they attribute it, that they do not use it for commercial purposes and that they do not alter, transform or build upon it. For any reuse or redistribution, researchers must make clear to others the licence terms of this work.

Declaration of Contributors

The work in this thesis was designed and performed by the author and assistance from collaborators is detailed below.

Chapter 2

- Intra-arcuate injection of rAAV was performed with the assistance of Dr Yue (David) Ma

and Dr Risheka R Ratnasabapathy.

- Decapitation of animals was performed with the assistance of Prof Kevin Murphy.

- Dissection of animals was carried out with the assistance of members of the Section of

Investigative Medicine (Miss Isabel Fernandes Freitas, Dr Yue (David) Ma, Miss Mariana

Norton, Dr Risheka Ratnasabapathy, Dr Rebecca Scott and Miss Gala Farooq).

- Brain punch biopsies were performed with the assistance of Dr Yue (David) Ma.

- Commands for generalised estimating equation analyses were developed with assistance

from Dr Ali Abbara and Miss Anne Jomard.

Chapter 3

- IVGTT and MMTT studies were performed with the assistance of the Kisspeptin Research

Team.

- Analysis of serum LH, FSH, testosterone, insulin, C-peptide and plasma glucose (using the

Abbott Architect machines in the Imperial College Healthcare NHS Trust Clinical Chemistry

Department) was performed with the assistance of Dr Sophie Clarke, Dr Sophie Jones,

Miss Anne Jomard and Dr Lisa Yang.

- RIA for the measurement of plasma kisspeptin was performed with the assistance of Dr

Ali Abbara, Dr Sophie Clarke, Dr Lisa Yang and Dr Sophie Jones.

- RIAs for measurement of plasma glucagon and GLP-1 were performed with the assistance

of Dr Paul Bech and Dr Sophie Jones.

- IVGTT insulin sensitivity index was calculated by Dr Ian Godsland (Wynn Reader in Human

Metabolism, Imperial College London) using MLAB software.

- Multilevel linear regression was performed by Paul Bassett (Statistician) on IVGTT and

MMTT glucose, insulin and C-peptide curves.

Table of Contents

Abstract ...... 2

Acknowledgements ...... 4

Declaration of Originality ...... 5

Declaration of Contributors ...... 5

Index of Figures ...... 12

Chapter 1 ...... 12

Chapter 2 ...... 12

Chapter 3 ...... 15

Index of Tables ...... 18

Chapter 2 ...... 18

Chapter 3 ...... 18

Abbreviations ...... 19

1 Chapter 1 General Introduction ...... 21

1.1 Physiology and Pathology of Metabolism and Energy Homeostasis ...... 22

1.2 Central Regulation of Metabolism and Energy Homeostasis ...... 24

1.2.1 Key Hypothalamic Areas ...... 25

1.2.2 Hormones of Interest and the Hypothalamus ...... 35

1.3 Summary ...... 38

2 Chapter 2 – The effects of glucocorticoids on the regulation of metabolism and energy homeostasis ...... 39

2.1 Introduction...... 39

2.1.1 11betaHSD enzyme system ...... 39

2.1.2 11betaHSD1 ...... 41

2.1.3 Summary ...... 50

2.2 Hypothesis and aims ...... 51

2.2.1 Hypothesis...... 51

2.2.2 Aims and objectives ...... 51

2.3 Materials and Methods ...... 52

2.3.1 Recombinant adeno-associated virus ...... 52

2.3.2 In vivo Methods ...... 52

2.3.3 Saponification of carcasses for body composition analysis...... 57

2.3.4 Assays ...... 57

2.3.5 Statistical analysis ...... 61

2.4 Results ...... 62

2.4.1 Confirmation of ARC-Specific effects on corticosterone levels following bilateral

AAV injection ...... 62

2.4.2 Normal chow ...... 63

2.4.3 High fat diet ...... 71

2.4.4 Comparison of effects of arcuate overexpression and under expression of

11BETAHSD1 on food intake and body weight on standard chow diet ...... 78

2.5 Discussion and Conclusions ...... 80

2.5.1 Summary of main findings and comparison with existing literature ...... 80

2.5.2 Strengths ...... 82

2.5.3 Limitations...... 83

2.5.4 Therapeutic implications ...... 85

2.5.5 Conclusions ...... 86

3 Chapter 3 – The effects of kisspeptin on the regulation of metabolism and energy homeostasis ...... 87

3.1 Introduction...... 87

3.1.1 Discovery of Kisspeptin, Kisspeptin Isoforms and Distribution of Kisspeptin and

its Receptor ...... 88

3.1.2 Brief overview of Kisspeptin and Reproduction ...... 89

3.1.3 Kisspeptin and Glucose Metabolism ...... 90

3.1.4 Kisspeptin and Energy Homeostasis ...... 93

3.1.5 Summary ...... 95

3.2 Hypothesis and aims ...... 96

3.2.1 Hypothesis...... 96

3.2.2 Aims...... 96

3.3 Materials and Methods ...... 96

3.3.1 Participants ...... 96

3.3.2 Kisspeptin-54 ...... 97

3.3.3 Infusions ...... 97

3.3.4 Effects of kisspeptin infusion during Intravenous Glucose Tolerance Tests

(IVGTT) 98

3.3.5 Effects of kisspeptin infusion during Mixed Meal Tolerance Tests (MMTT) ..... 99

3.3.6 Analysis of metabolites and hormones ...... 100

3.3.7 Statistical analysis ...... 105

3.4 Results ...... 108

3.4.1 Effects of kisspeptin during IVGTTs in healthy men ...... 108

3.4.2 Effect of kisspeptin during MMTTs in healthy men ...... 117

3.4.3 Discussion and Conclusions ...... 126

4 Chapter 4 – General Discussion and Conclusions...... 131

4.1 Discussion ...... 131

4.2 Summary of Future Work ...... 134

4.3 Conclusions...... 137

5 References ...... 138

6 Appendix ...... 159

6.1 Screening Proforma ...... 159

6.2 Taste Test ...... 167

6.3 Visual Analogue Scale ...... 168

7 Publications and Communications ...... 169

7.1 Izzi-Engbeaya and Comninos et al. - Manuscript under review at Diabetes ...... 169

7.2 Association of Physicians of Great Britain and Ireland Annual Meeting 2018,

Manchester – Abstract accepted for oral presentation ...... 220

Index of Figures

Chapter 1

Figure 1: Cross-sectional diagram of the rat brain.

Figure 2: Neuronal and hormonal pathways influencing food intake and satiety in the brain.

Chapter 2

Figure 1: Corticosterone levels in the arcuate nucleus 10 weeks after rAAV-GFP (n=5) or rAAV- siβHSD1 (n=6) injection into the arcuate nucleus.

Figure 2: Corticosterone levels in the ventromedial hypothalamus and the paraventricular nucleus 10 weeks after rAAV-GFP (n=4-5) or rAAV-siβHSD1 (n=6-7) injection into the arcuate nucleus.

Figure 3: Cumulative food intake during the 10-week period following rAAV-GFP (n=11) or rAAV-siβHSD1 (n=10) injection into the arcuate nucleus.

Figure 4: Average daily food intake during the 10-week period following rAAV-GFP (n=11) or rAAV-siβHSD1 (n=10) injection into the arcuate nucleus.

Figure 5: Body weight pre- and post- rAAV-GFP (N=11) or rAAV-siβHSD1 (n=10) injection into the arcuate nucleus.

Figure 6: Weight of interscapular brown adipose tissue dissected from male rat carcasses 11 weeks after rAAV-GFP (n=11) or rAAV-siβHSD1 (n=10) injection into the arcuate nucleus.

Figure 7: Weight of interscapular brown adipose tissue dissected from male rat carcasses expressed as a percentage of carcass weight 11 weeks after rAAV-GFP (n=11) or rAAV-siβHSD1

(n=10) injection into the arcuate nucleus.

Figure 8: Percentage protein per carcass of male rats culled 11 weeks after rAAV-GFP (n=11) or rAAV-siβHSD1 (n=10) injection into the arcuate nucleus.

Figure 9: Percentage triglyceride per carcass of male rats culled 11 weeks after rAAV-GFP

(n=11) or rAAV-siβHSD1 (n=10) injection into the arcuate nucleus.

Figure 10: Plasma corticosterone in trunk blood collected from male rats culled 11 weeks after rAAV-GFP (n=11) or rAAV-siβHSD1 (n=10) injection into the arcuate nucleus.

Figure 11: Plasma glucose in trunk blood collected from male rats culled 11 weeks after rAAV-

GFP (n=11) or rAAV-siβHSD1 (n=10) injection into the arcuate nucleus.

Figure 12: Plasma insulin in trunk blood collected from male rats culled 11 weeks after rAAV-

GFP (n=11) or rAAV-siβHSD1 (n=10) injection into the arcuate nucleus.

Figure 13: Cumulative food intake during the 10-week period following rAAV-GFP (n=12) or rAAV-siβHSD1 (n=12) injection into the arcuate nucleus.

Figure 14: Average daily food intake during 8 weeks of high fat diet starting 2 weeks after rAAV-GFP (n=12) or rAAV-siβHSD1 (n=12) injection into the arcuate nucleus.

Figure 15: (A) Body weight pre- and post- rAAV-GFP (n=12) or rAAV-siβHSD1 (n=12) injection into the arcuate nucleus in male rats initially fed a chow diet and then fed a high fat diet from

2 weeks post-surgery until the end of the study. (B) Increase in body weight from pre-surgery baseline in male rats post- rAAV-GFP (n=12) or rAAV-siβHSD1 (n=12) injection into the arcuate nucleus on HFD.

Figure 16: Mean peak temperature of skin overlying interscapular brown adipose tissue in male rats 9 weeks post- rAAV-GFP (n=12) or rAAV-siβHSD1 (n=12) injection into the arcuate nucleus fed a high fat diet for 8 weeks.

Figure 17: Percentage protein per carcass of male rats fed a high fat diet and culled 10 weeks after rAAV-GFP (n=12) or rAAV-siβHSD1 (n=12) injection into the arcuate nucleus.

Figure 18: Percentage triglycerides per carcass of male rats fed a high fat diet and culled 10 weeks after rAAV-GFP (n=12) or rAAV-siβHSD1 (n=12) injection into the arcuate nucleus.

Figure 19: Plasma corticosterone levels in trunk blood collected from male rats fed a high fat diet and culled 10 weeks after rAAV-GFP (n=12) or rAAV-siβHSD1 (n=12) injection into the arcuate nucleus.

Figure 20: Plasma glucose levels in trunk blood collected from male rats fed a high fat diet and culled 10 weeks after rAAV-GFP (n=12) or rAAV-siβHSD1 (n=12) injection into the arcuate nucleus.

Figure 21: Plasma insulin levels in trunk blood collected from male rats fed a high fat diet and culled 10 weeks after rAAV-GFP (n=12) or rAAV-siβHSD1 (n=12) injection into the arcuate nucleus.

Figure 22: Food intake (A) and body weight (B) during 4 weeks of chow diet following rAAV-

GFP (n=12) and rAAV-siβHSD1 (underexpression group, n=10) injection into the arcuate nucleus. Data presented as mean±SEM.

Figure 23: Food intake (A) and body weight (B) during 4 weeks of chow diet following rAAV-

GFP (n=12) and rAAV-βHSD1 (overerexpression group, n=8) injection into the arcuate nucleus.

Data presented as mean±SEM.

Chapter 3

Figure 1: Intravenous glucose tolerance test (IVGTT) study visit protocol.

Figure 2: Mixed meal tolerance test (MMTT) study visit protocol.

Figure 3: Plasma glucose levels during intravenous glucose tolerance tests performed in 15 male volunteers with kisspeptin infusion and vehicle infusion.

Figure 4: Serum insulin levels during intravenous glucose tolerance tests with kisspeptin infusion and vehicle infusion.

Figure 5: Serum C-peptide levels during intravenous tolerance tests performed in 15 male volunteers with kisspeptin infusion and vehicle infusion.

Figure 6: Acute insulin response to glucose during intravenous glucose tolerance tests in 15 male volunteers with kisspeptin infusion and vehicle infusion.

Figure 7: Insulin sensitivity index during intravenous glucose tolerance tests in 15 male volunteers with kisspeptin infusion and vehicle infusion.

Figure 8: IVGTT disposition index with kisspeptin infusion and vehicle infusion in 15 male volunteers.

Figure 9: Circulating plasma glucagon during intravenous glucose tolerance tests in 15 male volunteers with kisspeptin infusion and vehicle infusion.

Figure 10: Mean circulating plasma GLP17-36 concentrations during intravenous glucose tolerance tests in 15 male volunteers with kisspeptin infusion and vehicle infusion.

Figure 11: Plasma kisspeptin levels during intravenous glucose tolerance tests in 15 male volunteers with kisspeptin infusion and vehicle infusion.

Figure 12 – Serum luteinising hormone (LH) levels during intravenous glucose tolerance tests with kisspeptin infusion and vehicle infusion.

Figure 13: Serum testosterone levels during intravenous glucose tolerance tests in 15 male volunteers with kisspeptin infusion and vehicle infusion.

Figure 14: Plasma glucose concentrations during mixed meal tolerance tests in 15 male volunteers with kisspeptin infusion and vehicle infusion.

Figure 15: Serum insulin concentrations during mixed meal tolerance tests in 15 male volunteers with kisspeptin infusion and vehicle infusion.

Figure 16: Serum C-peptide concentrations during mixed meal tolerance tests in 15 male volunteers with kisspeptin infusion and vehicle infusion.

Figure 17: Insulin secretion index during mixed meal tolerance tests in 15 male volunteers with kisspeptin infusion and vehicle infusion.

Figure 18: Insulin sensitivity index during mixed meal tolerance tests in 15 male volunteers with kisspeptin infusion and vehicle infusion.

Figure 19: Mixed meal tolerance tests disposition index in 15 male volunteers during kisspeptin infusion and vehicle infusion.

Figure 20: Change in hunger reported by 15 male volunteers from pre-infusion (T=-30mins) to pre-meal (T=30mins) during kisspeptin and vehicle infusions.

Figure 21: Food intake (in kilocalories) of 15 male volunteers during an ad libitum meal during kisspeptin and vehicle infusions.

Figure 22: Plasma kisspeptin levels during mixed meal tolerance tests in 15 male volunteers with kisspeptin infusion and vehicle infusion.

Figure 23: Serum lutenising hormone (LH) levels during mixed meal tolerance tests in 15 male volunteers with kisspeptin infusion and vehicle infusion.

Figure 24: Serum testosterone levels during mixed meal tolerance tests in 15 male volunteers with kisspeptin infusion and vehicle infusion.

Index of Tables

Chapter 2

Table 1 – Summary of effects of reduced 11βHSD1 expression and/or activity

Chapter 3

Table 1 – Details of intravenous glucose tolerance test participants

Table 2 – Details of mixed meal tolerance test participants

Abbreviations

11βHSD1 11-betahydroxysteroid dehydrogenase type 1 gene

11βHSD1 11-betahydroxysteroid dehydrogenase type 1 protein

11βHSD2 11-betahydroxysteroid dehydrogenase type 2 gene

11βHSD2 11-betahydroxysteroid dehydrogenase type 2 protein

ACTH Adenocorticotrophic hormone

AgRP Agouti-related peptide

ARC Arcuate nucleus

BAT Brown adipose tissue

BCA Bicinchoninic acid

BDNF Brain-derived neurotrophic factor

CART Cocaine and amphetamine regulated transcript

CRH Corticotrophin releasing hormone

DI Disposition Index

ELISA Enzyme-linked immunosorbent assay

FSH Follicle stimulating hormone

GDW Glass distilled water

GEE Generalized estimating equation

GnRH Gonadotrophin releasing hormone

GSIS Glucose-stimulated insulin secretion

HDL High density lipoprotein

HFD High fat diet

IVGTT Intravenous glucose tolerance test

19 kcal kilocalories

KISS1/Kiss1 kisspeptin gene

KISS1/Kiss1 kisspeptin gene product

KISS1R/Kiss1r kisspeptin receptor gene

KISS1R/Kiss1r kisspeptin receptor gene product

LH Luteinising hormone

LH Lateral hypothalamus

MCH Melanin concentrating hormone

MCR Melanocortin receptor

MMTT Mixed meal tolerance test

MMTT-IS Meal tolerance test insulin sensitivity index

MMTT-ISI Meal tolerance test insulin secretion index

NC Normal chow

NPY Neuropeptide Y

PBS Phosphate buffered saline

POMC Pro-opiomelanocortin

PVN Paraventricular nucleus

PYY Peptide YY

RIA Radioimmunoassay

RT-qPCR Quantitative reverse transcriptase polymerase chain reaction

Si Intravenous glucose tolerance test insulin sensitivity index

TRH Thyrotropin releasing hormone

UCP-1 Uncoupling protein-1

VMH Ventromedial hypothalamus

1 Chapter 1 General Introduction

The prevalence of obesity has trebled over the last 40 years and current estimates suggest

38% of adults and 18% of children (aged 5-19years) worldwide are obese or overweight

(http://www.who.int/mediacentre/factsheets/fs311/en/). Obesity rates are rising in both developed and developing countries

(http://www.who.int/mediacentre/factsheets/fs311/en/). Obesity is associated with a number of adverse metabolic disorders including insulin resistance, glucose intolerance, diabetes mellitus, hypertension, hypercholesterolaemia and atherosclerosis, which in turn significantly increase the risk of cerebrovascular and cardiovascular disease (Hubert et al.,

1983). Furthermore, obesity itself increases the risk of certain types of malignancies

(Kopelman, 2007) and both overweight and obesity are associated with a higher risk of all- cause mortality (Global et al., 2016).

In the simplest terms, obesity results when energy intake exceeds energy expenditure over a period of time. However, the factors, which influence energy intake and energy expenditure

(such as hormones, accessibility and affordability of affordable energy dense food and drink, psychological factors, genetic polymorphisms and physical activity) are complex and interact with each other. Therefore, it is not surprising that our understanding of the aetiology and pathophysiology of obesity remains incomplete and consequently there are very few licensed, safe and effective medications available to treat obesity.

In this chapter, current concepts of the physiology and pathology of metabolism and energy homeostasis will be outlined, with a focus on the hypothalamus as well as the two hormones

(glucocorticoids and kisspeptin), which are the subject of subsequent chapters.

1.1 Physiology and Pathology of Metabolism and Energy Homeostasis

Energy homeostasis (to maintain a stable body weight) requires a balance between energy intake and energy expenditure. In humans, energy intake is influenced by the availability of food and drink, the composition of available food and drink, the palatability of available nutrition, the non-nutritive effects of calorie ingestion, hormones which suppress appetite

(i.e. anorexic hormones), hormones which stimulate appetite (i.e. orexigenic hormones), hormones which signal the energy status of the individual and a host of psycho-social factors

(including perceived effect of calorie ingestion on body image, dieting, religious observance, food trends promoted by the media, ‘sugar taxes’ levied by governments on high-sugar food and/or drink, cost of food, proportion of income available to spend on food and advertisements).

A handful of obesity syndromes are caused by gene mutations, which result in the absence of functional proteins or their receptors, such as leptin deficiency, leptin receptor deficiency, melanocortin-4 receptor (MC4R) deficiency and pro-opiomelanocortin (POMC) deficiency

(Farooqi et al., 2000, Farooqi et al., 2014). Therefore, these hormones are likely to be major determinants of appetite and food intake. However, in the vast majority of cases, obesity is not caused by single gene defects. Instead, some polymorphisms in genes encoding known anorexic hormone receptors (e.g. MC4R) have been found to protect against obesity (Young et al., 2007) and some polymorphisms in non-hormone encoding genes (e.g. FTO) have been found to be associated with simple obesity (Frayling et al., 2007).

Energy expenditure consists of resting energy expenditure (the basal metabolic rate), physical activity-related energy expenditure, diet-induced thermogenesis and brown adipose tissue

(BAT) thermogenesis. Resting energy expenditure is comprised of processes which enable the body to function, such as cardiac activity and metabolism of nutrient molecules. Glucose metabolism is an important component of resting energy expenditure and obesity is a common cause of abnormal glucose metabolism due to insulin resistance leading to diabetes mellitus. As a result, weight loss is often accompanied by an increase in insulin sensitivity and consequent improvement in glucose metabolism (Uusitupa et al., 2003).

Physical activity not only increases activity-related energy expenditure, but it also raises the basal metabolic rate (Melby et al., 1993). Interestingly, even when significant physical activity levels are maintained at the same level over 20 years, body weight and body mass index (BMI) increase (Williams and Wood, 2006), which suggests that basal metabolic rate declines with age. BAT thermogenesis is a significant component of total energy expenditure in rodents and human infants (Cypess and Kahn, 2010), but by the time humans reach adulthood, a significant proportion of BAT is no longer present. However, recent reports have demonstrated that metabolically active BAT depots are present in adult humans, which can be activated by cold exposure, adrenergic stimulation and circulating hormones like irisin and fibroblast growth factor-21 (Cypess et al., 2009, van Marken Lichtenbelt et al., 2009, Lee et al., 2014, Cypess et al., 2015).

In mice, when energy intake reduces (such as during fasting), stress-related glucocorticoid pathways are activated, which increases orexigenic stimuli resulting in greater food intake

(Pankevich et al., 2010). Additionally, in many individuals, when energy intake exceeds energy expenditure, anorexic stimuli do not appear to be powerful enough to reduce energy intake sufficiently to prevent weight gain as evidenced by the 0.5-0.96kg/year increase in body

23 weight in human adults reported by several longitudinal studies (Lewis et al., 2000, Brown et al., 2005).

In summary, the regulation of metabolism and energy homeostasis is very complex and there appears to be a predisposition towards increased body weight over time. Apart from lifestyle modification and bariatric surgery, there are very few medical options available to treat obesity. Currently only 6 medications are licensed for the treatment of obesity in America

(Srivastava and Apovian, 2018), and only 3 of these medications (i.e. orlistat, liraglutide and a naltrexone-bupropion combination) are licensed for obesity treatment in the UK. A better understanding of the neuroendocrine control of appetite, food intake and energy expenditure may lead to the development of additional therapeutic agents.

1.2 Central Regulation of Metabolism and Energy Homeostasis

Several areas of the brain and brainstem have been found to play key roles in the regulation of metabolism and energy homeostasis. For instance, the prefrontal cortex regulates decision-making and regulates behaviour (Miller and Cohen, 2001). Optogenetic stimulation of prefrontal D1-type dopamine receptor neurons increases food intake in mice and inhibition of these neurons reduces food intake (Land et al., 2014). Destruction of the posterior amygdala results in dramatic increases in food intake and body weight in rats compared to control rats who had undergone sham surgery (King et al., 1993).

Peripheral signals, which are increased by food ingestion (including glucose, insulin, peptide-

YY (PYY) and glucagon-like peptide-1 (GLP-1)) or released in proportion to fat mass (i.e. leptin),

24 are associated with reduced activation in the pre-frontal cortex, amygdala and insula, and PYY and GLP-1 reduce appetite (Zanchi et al., 2017). In contrast, ghrelin (a hormone released by the stomach during fasting, which stimulates appetite and increases food intake in rodents and humans) (Kojima et al., 1999, Wren et al., 2001a, Wren et al., 2001b) increases activation in these parts of the brain (Zanchi et al., 2017). Changes in baseline and food task-related activity in the prefrontal cortex and amygdala are detectable following bariatric surgery and some of these changes predict post-bariatric surgery weight loss (Holsen et al., 2017).

Although the parts of the brain outlined above (as well as other areas, e.g. the hippocampus

(Sweeney and Yang, 2015)) play significant roles in the control of metabolism and energy homeostasis; from a neuroendocrine point of view, the hypothalamus is the main centre for the production and integration of hormonal and non-hormonal signals, which contribute to the regulation of energy homeostasis and metabolism. Therefore, the hypothalamus will be discussed in more detail below.

1.2.1 KEY HYPOTHALAMIC AREAS

The arcuate nucleus (ARC), lateral hypothalamus (LH), ventromedial hypothalamus (VMH) and periventricular nucleus (PVN) are hypothalamic nuclei, which have been found to have critical roles in energy homeostasis and regulation of metabolism in mammals.

1.2.1.1 ARCUATE NUCLEUS (ARC)

The ARC is adjacent to the median eminence (Figure 1), where the blood brain barrier is permeable due to fenestrated capillaries (Shaver et al., 1992). Consequently, ARC neurons

(unlike neurons in other regions of the brain) come into contact with hormones (and nutrient molecules) in the circulation, and can respond to these signals. Additionally, ARC neurons are connected to many of the other key appetite regulating regions within the hypothalamus and in other parts of the brain.

P P

L * L V V A A

Figure 1: Cross-sectional diagram of the rat brain illustrating the position of the arcuate nucleus (A) relative to the median eminence (ME), paraventricular nucleus (P), lateral hypothalamus (L), ventromedial hypothalamus (V) and third ventricle (*).

Two sets of ARC neurons with opposing effects on food intake have been identified and extensively studied. One set is anorexic and these neurons produce proopiomelanocortin

(POMC) and cocaine-amphetamine regulated transcript (CART). When these neurons are stimulated they reduce appetite (Boston et al., 1997, Ellacott and Cone, 2004). Conversely, when POMC neurons are ablated in adult mice, the mice develop hyperphagia and obesity

(Gropp et al., 2005). POMC is secreted as a precursor peptide, which is cleaved to generate

α-, β- and γ- melanocyte-stimulating hormone (MSH), β-endorphin and adenocorticotrophic hormone (ACTH). α- and β-MSH (but not γ-MSH) are potent agonists of melanocortin receptor subtypes 3 and 4 (MC3R and MC4R) (Adan et al., 1994, Abbott et al., 2000), which are expressed predominantly in the brain (Gantz et al., 1993a, Gantz et al., 1993b). MC3R is expressed by arcuate POMC and Agouti-related peptide neurons (Bagnol et al., 1999). MC4R is expressed in central regions known to be involved in appetite regulation including the amygdala, ARC, LH, and PVN (Kishi et al., 2003). Central administration of α-MSH in rats reduces food intake by up to 75% (Brown et al., 1998). POMC deficiency or absent MC4R signalling in rodents and humans results in severe obesity due to hyperphagia and reduced energy expenditure (Huszar et al., 1997, Krude et al., 1998, Ste Marie et al., 2000, Farooqi et al., 2000). Additionally, MC4R agonists stimulate BAT thermogenesis via activation of the sympathetic nervous system (Brito et al., 2007) and thereby increase energy expenditure.

POMC/CART neurons in the ARC express receptors for peripheral signals. For instance,

POMC/CART neurons in the ARC express GLP-1 receptors and agonism of these receptors within the ARC results in reduced food intake and weight loss in mice (Secher et al., 2014).

Leptin, a hormone produced by adipocytes in proportion to fat mass in non-fasting conditions

(Frederich et al., 1995a, Frederich et al., 1995b), increases ARC POMC expression (Schwartz

27 et al., 1997), activates ARC POMC neurons via the leptin receptor (Elias et al., 1999, Cowley et al., 2001) and reduces food intake (Farooqi et al., 1999). Ghrelin, a potent orexigenic hormone produced by the stomach during fasting (Kojima et al., 1999, Tschop et al., 2000,

Wren et al., 2001a, Wren et al., 2001b), reduces POMC neuronal firing (Cowley et al., 2003).

The other set of neurons is orexigenic, and these neurons secrete Agouti-related peptide

(AgRP) and neuropeptide Y (NPY) (Ollmann et al., 1997, Baskin et al., 1999). AgRP is an MC3R and MC4R antagonist (Ollmann et al., 1997) and/or MC4R inverse agonist (when this receptor is constitutively active) (Haskell-Luevano and Monck, 2001, Srinivasan et al., 2004), which dramatically increases food intake (Ollmann et al., 1997). NPY increases food intake (Stanley and Leibowitz, 1984, Clark et al., 1985) by inhibiting POMC neurons (Cowley et al., 2001). NPY also increases body weight by reducing energy expenditure via a reduction in BAT thermogenesis (Billington et al., 1991).

NPY neuron ablation in adult mice results in marked hypophagia and reduced body weight

(Gropp et al., 2005, Bewick et al., 2005, Luquet et al., 2005), but NPY neuron ablation in the early postnatal period does not affect food intake or produce a metabolic phenotype (Luquet et al., 2005). Therefore, maturity may be required for NPY to exert orexigenic effects, or a compensatory mechanism may counteract the effects of NPY neuronal deficits if they occur early in development. Additionally, NPY is also produced by non-ARC neurons (Allen et al.,

1983) and this may also have influenced the results obtained following early post-natal NPY neuronal ablation.

Similar to POMC/CART neurons, AgRP/NPY neurons also respond to peripheral signals. The anorectic hormone leptin reduces NPY expression (Mercer et al., 1997) and inhibits NPY

28 neuronal firing frequency (van den top 2004). Conversely, ghrelin increases the firing rate of

NPY neurons (Cowley et al., 2003). Fasting increases both NPY and AgRP expression in mice

(Hahn et al., 1998). However, while both acute and chronic food deprivation increases ARC

NPY expression, ARC AgRP expression is only upregulated by acute food derivation in rats (Bi et al., 2003).

Both the anorexic and orexigenic populations of neurons in the ARC have extensive functional connections to other appetite- and metabolism-regulating areas of the brain (e.g. PVN, LH and amygdala) (Wang et al., 2015). AgRP neuron activation reduces peripheral insulin sensitivity and BAT activity (via reduced sympathetic stimulation of BAT) (Steculorum et al.,

2016), while ARC POMC stimulation increases BAT thermogenesis (Fenselau et al., 2017).

Furthermore, leptin infusion into the ARC increases BAT glucose uptake (Toda et al., 2009). In addition, oral glucose ingestion is modulated by ARC glucokinase (an enzyme involved in glucose metabolism) (Hussain et al., 2015). Therefore, by responding to and modulating central and peripheral systems and signals, the ARC controls food intake, energy expenditure and metabolism.

1.2.1.2 LATERAL HYPOTHALAMUS (LH)

Following destruction of the lateral hypothalamus rats become hypophagic and consequently lose weight (Anand and Brobeck, 1951). Some neurons in the LH secrete orexin (A and/or B) while others secrete melanin concentrating hormone (MCH) (Mickelsen et al., 2017), both of which are potent stimulators of food intake. Orexin and MCH neurons have reciprocal connections to each other and with ARC POMC and NPY neurons (Muroya et al., 2004, Gao

29 and Horvath, 2007). Orexin-A reduces ARC POMC and α-MSH expression and there is significant negative correlation between orexin-A and serum levels of α-MSH in obese men

(Morello et al., 2016). Conversely, orexin-A expression is reduced by α-MSH (Lopez et al.,

2007). Both orexin-A and orexin-B activate ARC NPY neurons and inhibit ARC POMC neurons

(Muroya et al., 2004).

Central administration of orexin-A or orexin-B increases food intake in rats (Sakurai et al.,

1998) but the orexigenic effect of these peptides may be dependent on the circadian rhythm

(Haynes et al., 1999). Additionally, central orexin-A administration stimulates the ingestion of high fat food in preference to low fat food via a mechanism involving opioid receptors (Clegg et al., 2002). Central blockade of orexin-A dose-dependently suppresses hyperphagia

(Yamada et al., 2000). Fasting increases orexin expression (Sakurai et al., 1998) and increases orexin-neuron activation (Diano et al., 2003). Similarly, ghrelin activates orexin neurons and the ability of ghrelin to induce feeding is reduced when orexin signalling is defective (Toshinai et al., 2003).

Acute and chronic MCH receptor agonism results in increased food intake and body weight in rats (Qu et al., 1996, Shearman et al., 2003). Central administration of an MCH receptor agonist increases food intake, which is partially blocked by co-administration of an MCH receptor antagonist (Shearman et al., 2003). Mice which overexpress MCH are hyperphagic and obese (Ludwig et al., 2001). MCH knockout mice are hypophagic and have increased energy expenditure (Shimada et al., 1998, Alon and Friedman, 2006), which may in part be due to the absence of MCH-mediated inhibition of thyroid stimulating hormone release

(Kennedy et al., 2001). MCH antagonises α-MSH, and this may be the mechanism for some of its orexigenic effects. However, neither MCH receptor agonism nor antagonism alter arcuate

NPY and POMC mRNA expression (Shearman et al., 2003). MCH neurons also respond to peripheral signals as both leptin deficiency and increased ghrelin/fasting stimulate MCH neurons (Qu et al., 1996).

1.2.1.3 VENTROMEDIAL HYPOTHALAMUS (VMH)

There is a close relationship between the VMH and the ARC. VMH neurons project to ARC

POMC neurons and stimulate ARC POMC neurons, and this stimulation is decreased by fasting

(Sternson et al., 2005). Melanocortin and NPY receptors are present in the VMH (Bouali et al.,

1995, Toda et al., 2009). Fasting increases NPY in the VMH and infusion of NPY into the VMH stimulates food intake (Bouali et al., 1995). Additionally, both AgRP and fasting reduce the electrophysiological response of VMH neurons to α-MSH compared to the responses generated in animals with ad libitum food intake (Li and Davidowa, 2004).

Leptin receptors are present in ARC and VMH neurons (Elmquist et al., 1998). Selective deletion of the leptin receptor gene from ARC POMC neurons produces mildly obese animals with similar food intake to controls (Balthasar et al., 2004) while selective deletion of the leptin receptor gene from VMH neurons produces both hyperphagia and obesity (Majdic et al., 2002, Dhillon et al., 2006). Additionally, mice with non-functional leptin receptors in both the ARC POMC neurons and VMH neurons have a more severe obese phenotype than mice with defective leptin receptors in only one of these sets of neurons (Dhillon et al., 2006).

Therefore, the VMH may be as important as the ARC for leptin-mediated anti-obesity effects.

Other appetite-regulating hormones exert their effects within the VMH. Oxytocin, produced in the VMH (as well as by the posterior pituitary), reduces appetite, increases energy expenditure (Noble et al., 2014) and reduces reward-motivated food intake in humans (Ott et al., 2013). Brain-derived neurotophic factor (BDNF) is produced in large quantities in the

VMH and deficiency of functional BDNF or its receptor results in hyperphagia and obesity in rodents and humans (Xu et al., 2003, Rios et al., 2001, Gray et al., 2006). In leptin receptor- deficient mice (which are hyperphagic, obese and diabetic) BDNF reduces food intake, attenuates hyperglycaemia, improves insulin sensitivity and lowers circulating lipid levels

(independently of changes in weight), and increases energy expenditure via increased oxygen consumption and stimulation of BAT thermogenesis (Nakagawa et al., 2000, Tsuchida et al.,

2002). The anorexic effects of BDNF occur independently of the melanocortin system as BDNF lowers food intake and body weight in mice lacking the MC4R (Xu et al., 2003).

Apart from regulating food intake, VMH neurons increase energy expenditure as knockout of

VMH steriodogenic factor-1 neurons in mice result in reduced energy expenditure related to lower BAT UCP-1 (Kim 2011). Similar to other hypothalamic nuclei, the neurons in the VMH are sensitive to peripheral signals. For instance, leptin increases BDNF production (Komori et al., 2006) and fasting decreases BDNF in the VMH (Xu et al., 2003).

1.2.1.4 PARAVENTRICULAR NUCLEUS (PVN)

The melanocortin system is important for the energy homeostasis and metabolism-regulating roles of the PVN. Administration of MC4R agonists into the PVN reduces food intake (Giraudo et al., 1998) and increases energy expenditure via activation of BAT thermogenesis (Monge-

Roffarello et al., 2014). Expression of MC4R in the PVN in MC4R knockout mice reduces body weight by lowering food intake (Balthasar et al., 2005, Shah et al., 2014).

PVN neurons produce several anorexic peptides and their receptors including CART, cholecystokinin and nesfatin-1 (Koylu et al., 1998). Administration of CART into the PVN reduces food intake, stimulates hypothalamic NPY release and ACTH release from the pituitary (with subsequent elevation in plasma corticosterone) (Stanley et al., 2001). High fat feeding reduces central cholecystokinin production (Morris et al., 2008) while fasting increases hypothalamic cholecystokinin (Saito et al., 1981). Antagonism of central cholecystokinin receptors increases food intake (Dourish et al., 1989). PVN neurons also produce the orexigenic peptide NPY. Central insulin administration reduces NPY expression in the PVN (but not the ARC) (Schwartz et al., 1992) and reduces food intake (Woods et al.,

1979).

Oxytocin neurons in the PVN express the leptin receptor (Blevins et al., 2004) and are activated by leptin (Perello and Raingo, 2013). Additionally, GLP-1 (an insulin secretagogue and anorexic hormone produced by L-cells in the small intestine as well as by the solitary tract nucleus in the brain (Trapp and Richards, 2013)) receptor antagonism within the PVN increases food intake in fed but not fasted rodents (Turton et al., 1996).

Figure 2 illustrates the complex circuitry incorporating the hypothalamic nuclei discussed above.

Figure 2: Neuronal and hormonal pathways influencing food intake and satiety in the brain. Complex neuro–hormonal pathways, gut hormones and adiposity signals reciprocally interact between the hypothalamus, brainstem, higher cortical areas and limbic system to control appetite regulation. Neuropeptide Y–agouti-related protein (NPY–AgRP; orexigenic) and pro-opiomelanocortin–cocaine- and amphetamine-related transcript (POMC–CART; anorexic) neurons reside within the arcuate nucleus (AC) of the hypothalamus. The cumulative effect of either inhibition or activation of these orexigenic and anorexigenic neurons from various signals in the bloodstream through the incomplete blood–brain barriers (median eminence and area postrema) or neural pathways influences food intake and satiety. α-MSH, α-melanocyte-stimulating hormone; CRH, corticotropin-releasing hormone; GHSR, growth hormone secretagogue receptor; GI, gastrointestinal; GLP1, glucagon-like peptide 1; GLP1R, GLP1 receptor; IR, insulin receptor; LHA, lateral hypothalamic area; LR, leptin receptor; MCH, melanin-concentrating hormone; MC3R, melanocortin receptor 3; NST, nucleus of the solitary tract; PVN, paraventricular nucleus; PYY, peptide YY; TRH, thyrotropin-releasing hormone; Y1R, Y1 receptor; Y2R, Y2 receptor (Srivastava and Apovian, 2018).

1.2.2 HORMONES OF INTEREST AND THE HYPOTHALAMUS

Evidence is emerging that some of the appetite-regulating systems outlined above are also involved in the response to stress (Kyrou and Tsigos, 2009) and the control of reproduction

(Tilbrook et al., 2000). However, the hormones which link appetite to stress and reproduction have, to date, remained poorly understood. Recent evidence suggests that glucocorticoids may be involved in regulating stress (Sapolsky et al., 2000) as well as appetite (Tataranni et al., 1996). Similarly, kisspeptin is a recently identified hormone which is vital for normal reproductive function (Seminara et al., 2003, Topaloglu et al., 2012) but which may also have effects on metabolism (Tolson et al., 2014). This is discussed in further detail below.

1.2.2.1 GLUCOCORTICOIDS

Hypothalamic CRH (produced by parvocellular PVN neurons) stimulate the pituitary gland to secrete ACTH, which in turn stimulates the adrenal glands to produce glucocorticoids

(Stratakis and Chrousos, 1995). Glucocorticoids exert negative feedback on the hypothalamic- pituitary-adrenal axis by inhibiting CRH and ACTH release (Stratakis and Chrousos, 1995).

Glucocorticoids activate the glucocorticoid receptor, which is widely expressed, and alter gene expression. Glucocorticoids have been extensively studied and have a wide variety of effects on virtually every system of the body.

With respect to energy homeostasis and metabolism, in both rodents and humans, glucocorticoid administration increases food intake (Freedman et al., 1985, Tataranni et al.,

1996). Additionally, glucocorticoid administration decreases energy expenditure in rodents

(Strack et al., 1995) but increases energy expenditure in humans (Tataranni et al., 1996). 35

Interestingly, acute glucocorticoid administration increases BAT thermogenesis in humans

(Scotney et al., 2017) while chronic glucocorticoid ingestion decreases BAT thermogenesis

(Thuzar et al., 2016).

Glucocorticoids increase ARC NPY and AgRP gene expression (Shimizu et al., 2008), whilst

AgRP blocks α-MSH mediated glucocorticoid production (Dhillo et al., 2003). Deletion of the glucocorticoid receptor from AgRP neurons results in higher energy expenditure (via increased oxygen consumption and BAT UCP-1 expression) and lower body weight in mice fed a high fat diet (Shibata et al., 2016). Additionally, glucocorticoids are required for ghrelin- induced increase in NPY expression and increased food intake following fasting (Spinedi et al.,

2006). This data suggests that glucocorticoids in the ARC may have a physiological role in the control of energy homeostasis. However, the effects on energy homeostasis of a reduction in active glucocorticoids specifically in the ARC of adult animals is not known.

1.2.2.2 KISSPEPTIN

Within the central nervous system kisspeptin is produced by neurons in the ARC (in rodents), the infundibular nucleus (the human homologue of the ARC), the preoptic area of the hypothalamus, amygdala and pituitary (Muir et al., 2001, Rometo et al., 2007, Hrabovszky et al., 2010). Its receptor (KISS1R) is expressed in the hypothalamus, hippocampus, amygdala, cerebral cortex and pituitary (Lee et al., 1999, Kotani et al., 2001, Muir et al., 2001).

Evidence is beginning to emerge in support of a role for kisspeptin in the regulation of metabolism and energy homeostasis. Within the ARC, reciprocal and functional connections

36 have been described between kisspeptin and AgRP/NPY neurons as well as between kisspeptin and POMC/CART neurons (Backholer et al., 2010, Manfredi-Lozano et al., 2016)

(described in greater detail in Chapter 3 of this thesis). Furthermore, hypothalamic kisspeptin neurons express the glucocorticoid receptor (Takumi et al., 2012) and glucocorticoids inhibit hypothalamic kisspeptin expression (Luo et al., 2016a).

Fasting reduces ARC kisspeptin expression (with resultant suppression of LH secretion and pulsatility) (Luo et al., 2016b). Leptin deficiency or resistance results in reduced kisspeptin

(Smith et al., 2006a) and hypothalamic hypogonadism (Farooqi and O'Rahilly, 2014). ARC kisspeptin neurons express leptin receptors and leptin administration to leptin deficient mice increases arcuate kisspeptin expression (Smith et al., 2006a). Neurons, which produce GLP-1 are in close apposition to ARC kisspeptin neurons and activation of GLP-1 receptors on ARC kisspeptin neurons results in their stimulation (Heppner et al., 2017). Female kisspeptin receptor knockout mice are hypophagic but have higher body weights than ovariectomised controls (Tolson et al., 2014), which suggests kisspeptin may play a role in regulating both appetite and energy expenditure. Additionally, kisspeptin appears to have a role in modulating glucose-stimulated insulin secretion (GSIS) with both enhancement and inhibition of GSIS reported in vitro and in vivo in animal models (Song et al., 2014).

Together this data in animals suggests that kisspeptin may play an important role in energy homeostasis in animals. However, the effects of kisspeptin administration on energy homeostasis in humans is not known.

1.3 Summary

The control of energy homeostasis and metabolism is multi-faceted and involves the integration of peripheral and central systems via hormonal and non-hormonal signals. In this thesis, I investigated:

(i) the effects of glucocorticoid reduction in the ARC on energy homeostasis in

rodents; and

(ii) the effects of peripheral administration of kisspeptin on energy homeostasis and

metabolism in healthy young men.

2 Chapter 2 – The effects of glucocorticoids on the regulation of

metabolism and energy homeostasis

2.1 Introduction

A rare cause of obesity is Cushing’s syndrome, and this disease may provide clues to the pathophysiology of simple obesity. In Cushing’s syndrome, excess glucocorticoid hormones

(cortisol in humans and corticosterone in rodents) are produced, which lead to elevated circulating levels of these hormones. This leads to increased food intake, alterations in energy expenditure, increased visceral fat accumulation, increased blood pressure, insulin resistance, depression, hypothalamic hypogonadism and decreased bone mineral density (Boscaro et al.,

2001). Interestingly, a similar phenotype is seen in simple obesity. However, whole body cortisol production is not elevated in obese men and women (Strain et al., 1980) and circulating cortisol levels are not elevated in non-Cushing’s obesity (Strain et al., 1982).

Therefore, tissue-specific levels of glucocorticoids may play a role in the metabolic phenotype of obesity. Intra-tissue concentrations of glucocorticoids are controlled by the 11β- hydroxysteroid dehydrogenase (11βHSD) enzyme system and investigation of this enzyme system in a key appetite controlling hypothalamic nucleus (i.e. the ARC) may increase our understanding of the development of obesity.

2.1.1 11BETAHSD ENZYME SYSTEM

Glucocorticoids exist in active forms (cortisol in humans and corticosterone in rodents), which can activate the glucocorticoid receptor (GR) (and the mineralocorticoid receptor), and 39 inactive forms (cortisone in humans and 11-dehydrocorticosterone in rodents), which do not activate the glucocorticoid receptor (or the mineralocorticoid receptor) (Chapman et al.,

2013). In both rodents and humans, the reactions required to convert one form to the other are catalysed by the two 11βHSD enzymes (11βHSD1 and 11βHSD2).

In vitro, 11βHSD1 can catalyse both the conversion of inactive glucocorticoids to active glucocorticoids and the conversion from active to inactive glucocorticoids (Agarwal et al.,

1989). However, in vivo, likely due to the availability of excess reduced nicotinamide adenine dinucleotide phosphate generated by hexose-6-phosphate dehydrogenase (which is associated with 11βHSD1 in the endoplasmic reticulum), 11βHSD1 almost exclusively catalyses the conversion of inactive glucocorticoids to active glucocorticoids (Chapman et al.,

2013). Therefore, 11βHSD1 serves to increase the local concentrations of active glucocorticoids in tissues. In rodents and humans, 11βHSD1 is present in the liver, kidney, lung, testes, ovary, adipose tissue, uterus, macrophages, brain (including in the ARC in rodents and the infundibular nucleus, the human homologue of the ARC), vascular smooth muscle, the heart and colon (Agarwal et al., 1989, Moisan et al., 1990, Tannin et al., 1991,

Benediktsson et al., 1992, Lakshmi et al., 1991, Whorwood et al., 1993, Bujalska et al., 1997,

Bisschop et al., 2013). Due to the association of 11βHSD1 with obesity and metabolic pathology (discussed in more detail in 2.1.2 below), 11βHSD1 will be the main focus of this chapter.

11βHSD2 catalyses the conversion of active glucocorticoids to inactive glucocorticoids. It is found predominately in the kidneys (but not in the liver, adipose tissue or brain) (Chapman et al., 2013), and it protects the mineralocorticoid receptor from activation by active glucocorticoids. It is also produced in sweat glands, salivary glands, colon, exocrine pancreas,

40 adrenal cortex, placenta, skin, lung and vascular epithelium (Krozowski et al., 1990, Albiston et al., 1994, Roland and Funder, 1996, Brem et al., 1998, Hirasawa et al., 1999). There is limited evidence for a role for 11βHSD2 in the regulation of energy homeostasis and metabolism. However, when 11βHSD2 is overexpressed in adipose tissue using the aP2 promoter in male mice, their food intake and fat mass decreases, energy expenditure increases, serum corticosterone levels are unaffected and they do not develop diet-induced obesity on a high fat diet (Kershaw et al., 2005).

2.1.2 11BETAHSD1

2.1.2.1 ASSOCIATION WITH METABOLIC DISORDERS

Elevated 11βHSD1 expression and activity are found in adipose tissue of obese rodents, obese humans and women with polycystic ovarian syndrome (which is characterised by oligomenorrhea, hyperandrogenism, polycystic ovaries, and insulin resistance) (Livingstone et al., 2000, Rask et al., 2001, Svendsen et al., 2009). 11βHSD1 expression in adipose tissue is positively correlated with obesity and insulin resistance (Purnell et al., 2009, Woods et al.,

2015). In contrast, lower 11βHSD1 activity is found in livers of obese humans and genetically obese rats (Woods et al., 2015).

Weight loss alters 11βHSD1 expression in adipose tissue in obese people (Tomlinson et al.,

2004, Purnell et al., 2009) and increases 11βHSD1 activity in the liver (Woods et al., 2015).

Furthermore, compared to wild type controls, 11βHSD1 global knockout mice have greater brown adipose tissue (BAT) mass, higher uncoupling protein-1 (UCP-1) mRNA and protein and do not develop the metabolic sequelae of chronically elevated levels of circulating

41 glucocorticoids (Kotelevtsev et al., 1997, Morton et al., 2001, Morton et al., 2004). These observations indicate that not only is 11βHSD1 implicated in the pathogenesis of obesity and associated adverse metabolic phenotypes, there are differences in tissue-specific levels of

11βHSD1 and consequently levels of active glucocorticoids within tissues.

2.1.2.2 11BETAHSD1 OVEREXPRESSION RODENT MODELS

2.1.2.2.1 Adipose tissue-specific overexpression

Two rodent models have been developed and studied to explore the effects of increased expression of 11βHSD1. Using the aP2 promoter (expressed in adipocytes and macrophages),

11βHSD1 expression was increased 2- to 3-fold in subcutaneous fat (similar to the increase reported in leptin deficient mice and obese humans), as well as in interscapular brown adipose tissue (BAT) (Masuzaki et al., 2001). Compared to controls, adipose tissue corticosterone levels were 30% higher in the aP2-11βHSD1 mice, without changes in circulating corticosterone levels. On a chow diet, the aP2-11βHSD1 mice had a 10% higher food intake, 16% higher body weight, increased visceral fat and reduced BAT UCP1 mRNA

(Masuzaki et al., 2001). On a high salt chow diet, the aP2-11βHSD1 mice had higher blood pressure with elevated plasma angiotensinogen, angiotensin II and aldosterone levels

(Masuzaki et al., 2003).

2.1.2.2.2 Liver-specific overexpression

Using the apolipoproteinE promoter (expressed in the liver, kidney, brain, testes, adipocytes and adrenal glands), 11βHSD1 activity was increased 2- to 5-fold in the liver (Paterson et al.,

2004). Serum corticosterone levels were similar to controls and hepatic corticosterone levels

42 were not reported (Paterson et al., 2004). Although body and adipose tissue weight were the same as control mice, apoE-11βHSD1 mice had higher blood pressure, angiotensinogen levels, hepatic triglycerides and hepatic glucocorticoid receptor numbers (Paterson et al., 2004).

Levels of 11βHSD1 activity in the other tissues, which express the apolipoproteinE promoter, were not reported so the effects of 11βHSD1 expression in this model may not be due solely to increased hepatic 11βHSD1 activity.

2.1.2.3 11BETAHSD1 UNDEREXPRESSION RODENT MODELS

2.1.2.3.1 Whole body knockout

Using mice with different genetic backgrounds, different research groups have produced whole body 11βHSD1 knockout models using the β-actin promoter (which is expressed in all mammalian cells) or the Rosa26-Cre promoter (which is expressed in embryonic stem cells).

In these global knockout mice, basal and stress-related serum corticosterone are elevated, adipose tissue corticosterone is reduced, serum ACTH is elevated, adrenal glands are enlarged, insulin sensitivity and glucose tolerance are increased, high density lipoprotein

(HDL) cholesterol is increased while total cholesterol is decreased. These mice had similar body weight or less weight gain than controls despite higher food intake on HFD (Kotelevtsev et al., 1997, Morton et al., 2001, Morton et al., 2004, Paterson et al., 2004, Paterson et al.,

2007, Harno et al., 2013). These results indicate that reduction in 11βHSD1 expression has beneficial metabolic effects despite elevated circulating corticosterone levels.

11βHSD1 global knockout mice with rescued liver 11βHSD1 expression have similar adrenal gland size as well as similar basal, peak and stress-stimulated corticosterone levels compared

43 to wild type controls (Paterson et al., 2007). Therefore, presumably liver 11βHSD1 expression can produce sufficient quantities of active corticosterone to maintain negative feedback, which is absent in global knockout mice (as evidenced by high ACTH despite high serum corticosterone).

2.1.2.3.2 Liver-specific knockout

11βHSD1 liver knockout mice were generated using an albumin-Cre promoter with hepatic

11βHSD1 mRNA reported to be 60% of controls and no hepatic 11βHSD1 protein detectable by Western blot (Lavery et al., 2012). The whole body 11βHSD1 activity measured in the liver-

11βHSD1 knockout mice was 35-40% of controls, the adrenal glands of the liver-11βHSD1 knockout mice were enlarged while morning circulating corticosterone levels were similar to controls (Lavery et al., 2012). On both low fat and high fat diets, liver-11βHSD1 knockout mice had similar weight gain to controls but had better glucose tolerance (Lavery et al., 2012).

2.1.2.3.3 Combined liver and adipose tissue knockdown

Twice weekly intraperitoneal injection of 11βHSD1 antisense oligonucleotide for twelve weeks reduced hepatic 11βHSD1 mRNA to 10% of controls and no hepatic 11βHSD1 was detectable on Western blot (Li et al., 2011a). In these mice, reduction in hepatic 11βHSD1 reduced food intake and body weight, increased fatty acid oxidation (an important fuel for

BAT thermogenesis), while serum corticosterone levels were unchanged (Li et al., 2011a).

Control mice, which were pair-fed with hepatic 11βHSD1 knockdown mice had higher body weight despite the same food intake (Li et al., 2011a). This suggests hepatic 11βHSD1 knockdown leads to higher energy expenditure. However, peritoneal adipose tissue 11βHSD1

44 expression is likely to have been affected as well, but effects of the injection on adipose tissue

11βHSD1 expression were not reported.

Similarly, twice weekly intraperitoneal injection of 11βHSD1 antisense oligonucleotide for twelve weeks reduced epididymal white adipose tissue 11βHSD1 mRNA and protein to <25% of controls, but no effect was detected in subcutaneous fat and BAT (Li et al., 2012). These adipose-11βHSD1 knockdown mice had reduced food intake on a Western diet, reduced body weight and total fat mass with unchanged lean mass (Li et al., 2012). However, BAT was increased and both energy expenditure and locomotor activity were increased. Additionally, insulin sensitivity and glucose tolerance were higher in the adipose-11βHSD1 knockdown mice (Li et al., 2012). However, similar to the previous study, hepatic 11βHSD1 expression would also have been significantly reduced, but effects on hepatic 11βHSD1 expression were not reported.

2.1.2.3.4 Brain knockout

Brain 11βHSD1 knockout mice were generated using the Nestin-Cre promoter, which is expressed in nerve cells (Harno et al., 2013). 11βHSD1 mRNA in the hypothalamus was reduced to <10% of controls, adipose 11βHSD1 activity was reduced to 60% of controls with no changes detected in liver 11βHSD1 mRNA. 11βHSD1 activity in the brain was <10% of controls, while activity levels in adipose and hepatic tissues were unaffected and serum corticosterone levels were similar to controls (Harno et al., 2013). Compared to controls, brain-11βHSD1 knockout mice had increased food intake on HFD but surprisingly their body weight was similar to controls (Harno et al., 2013). Therefore, the reduction of 11βHSD1 activity in different tissues produces different metabolic phenotypes.

2.1.2.4 11BETAHSD1 INHIBITORS

Following on from the promising results reported above, at least 8 different classes of

11βHSD1 inhibitors have been developed to treat obesity and associated metabolic disorders

(Boyle and Kowalski, 2009). The ability of these inhibitors to penetrate liver, fat and brain

(where 11βHSD1 activity is known to influence metabolism) is likely to affect the efficacy of these medications. Additionally, active glucocorticoids exert negative feedback on pituitary

ACTH, therefore a reduction in active glucocorticoids in the brain (mediated by 11βHSD1 inhibition) may result in increased production of ACTH. Elevated ACTH, in turn, stimulates production of adrenal steroids (both glucocorticoids and androgens), which may produce clinically unwanted effects, especially in women.

2.1.2.4.1 Rodent Studies

Administration of four different 11βHSD1 inhibitors (BVT2733, Compounds 40 and 51,

MK0916 and KR67500) to mice with diet-induced obesity reduces food intake by up to 27%, increases energy expenditure by up to 10%, reduces body weight by up to 10% (with preferential reduction in body fat); with accompanying reductions in insulin, fasting glucose

(in most studies), oral glucose tolerance glucose, leptin, cholesterol and triglycerides (Wang et al., 2012). In apolipoproteinE deficient mice, which develop atherosclerotic plaques on chow diet, Compound 544 reduces atherosclerotic plaque formation (Hermanowski-Vosatka et al., 2005). In low density lipoprotein receptor (LDLR) knockout mice, which also develop atherosclerotic plaques, Compound 2922 administration increases insulin sensitivity and

46 glucose tolerance but has no effect on atherosclerotic plaques (Hadoke et al., 2013). In these studies, plasma corticosterone levels were not affected by 11βHSD1 inhibition.

2.1.2.4.2 Human Studies

Several Phase I and II clinical trials using 11βHSD1 inhibitors have been published. MK0736 administration to obese people with hypertension for 12weeks lowers both systolic and diastolic blood pressure by 4mmHg each, reduces weight by 1.4kg (approximately 1% weight reduction), reduces HDL cholesterol by 6% and LDL cholesterol by 12% (Shah et al., 2011).

Dehydroepiandrosterone (DHEA) was increased but testosterone levels were unchanged and no symptoms of hyperandrogenism were reported by female participants (Shah et al., 2011).

Oral administration of RO5093151 for 12 weeks to patients with non-alcoholic fatty liver disease (NAFLD), a common consequence of obesity which can progress to end-stage liver failure, reduces liver fat content (Stefan et al., 2014). However, some participants experienced gastrointestinal disorders, infections, infestations and nervous system disorders as side effects of treatment with RO5093151 (Stefan et al., 2014). This agent also produces modest weight loss (up to 2%) in obese people with type 2 diabetes (Heise et al., 2014).

11βHSD1 inhibitors have also been used to treat type 2 diabetes. INCB013739 given in conjunction with metformin, a first line treatment of type 2 diabetes, for 12weeks effectively abolishes the conversion of cortisone to cortisol and reduces hepatic glucose production, insulin resistance, fasting glucose, glycated haemoglobin (HbA1c), total and LDL cholesterol and body weight (Rosenstock et al., 2010). This 11βHSD1 inhibitor caused dose-dependent increases in ACTH but serum cortisol was not increased. Higher DHEA, higher testosterone and sex hormone binding globulin levels (so free androgen index was unchanged), nausea,

47 diarrhoea, headaches and upper respiratory tract infections (Rosenstock et al., 2010) were reported. Treatment of obese people with type 2 diabetes with MK0916 reduces HbA1c, increases LDL cholesterol, reduces body weight by 2% (2kg), and reduces systolic and diastolic blood pressure by 8mmHg and 5mmHg respectively (Feig et al., 2011). MK0916 also results in dose-dependent increases in DHEA (which remained within the normal range), without affecting testosterone levels or causing hyperadrogenism in women (Feig et al., 2011).

A summary of the major effects of 11βHSD1 inhibition or reduced expression is presented in

Table 1.

Table 1 – Summary of effects of reduced 11βHSD1 expression and/or activity

INTERVENTION SPECIES EFFECTS VS CONTROLS

Global 11βHSD1 knockout Mice • Lower glucocorticoid receptor mRNA in the PVN • Increased adrenal size and weight • Increased circulating ACTH • Increased basal circulating corticosterone • Lower fasting glucose • Better glucose tolerance and insulin sensitivity • Increased HDL cholesterol • Higher food intake on HFD but less weight gain • Improved performance in watermaze

Combined hepatic and adipose Mice • Increased adrenal size tissue 11βHSD1 knockdown • Serum corticosterone levels unaffected • 12% lower food intake on Western diet • 11% lower body weight • Lower cholesterol • Lower total fat mass • Increased BAT mass • Increased energy expenditure • Better glucose tolerance and insulin sensitivity

Brain 11βHSD1 knockdown Mice • 13% higher food intake on HFD but similar weight to controls • Circulating corticosterone levels were not affected

11βHSD1 inhibitors Mice • Lower food intake • Lower body weight • Lower body fat • Higher energy expenditure • Circulating corticosterone levels were unaffected • Lower fasting glucose and fasting insulin

11βHSD1 inhibitors Humans • Lower fasting glucose • Reduction in HbA1c • Reduction in cholesterol • Reduction in liver fat content • Up to 8mmHg reduction in systolic blood pressure and up to 5mmHg reduction in diastolic blood pressure • 1-2% reduction in body weight • Dose-dependent increases in plasma ACTH • Increased DHEA and/or testosterone in some women but still within normal range and no clinical signs or symptoms of hyperadrogenism

2.1.2.5 11BETAHSD1 OVEREXPRESSION IN THE ARC

Previously in our laboratory, 11βHSD1 expression in the ARC was increased in adult male

Wistar rats via stereotactic bilateral arcuate injection of rAAV-βHSD1 (i.e. recombinant adeno- associated virus containing a plasmid designed to induce expression of 11βHSD1). Following the post-operative recovery period, the food intake and body weight of animals with 11βHSD1 overexpression and control animals (who had received bilateral injection of rAAV-GFP, i.e. recombinant adeno-associated virus with a plasmid designed to induce expression of green fluorescent protein, into the ARC) were measured. Data kindly provided by Dr James Gardiner demonstrated that arcuate overexpression of 11βHSD1 resulted in 6.5% increase in food intake and 6% higher body weight than controls (when animals were fed a standard chow diet for 10 weeks post-arcuate rAAV injection).

2.1.3 SUMMARY

Evidence suggests that local concentrations of active glucocorticoids, determined by local

11βHSD1 activity, play an important role in obesity and metabolic pathology. Published work has focussed on the effects of modulating 11βHSD1 expression and/or activity in whole organs. However, studies investigating the effect of reducing 11βHSD1 expression and/or activity specifically in a key appetite-regulating hypothalamic nucleus, i.e. the arcuate nucleus, have not been reported. Identifying the metabolic effects of reduced 11βHSD1 within the arcuate nucleus will improve our understanding of the hormonal control of

50 appetite and energy homeostasis and may help direct further development of 11βHSD1 inhibitors.

2.2 Hypothesis and aims

2.2.1 HYPOTHESIS

Reduced expression of 11βHSD1 in the ARC will result in reduced levels of corticosterone within the ARC with subsequent effects on food intake and body weight.

2.2.2 AIMS AND OBJECTIVES

1. To determine the effect on food intake and body weight of downregulating 11βHSD1

expression in the arcuate nucleus in rodents on a normal chow diet.

2. To determine the effect on food intake and body weight of down-regulating 11βHSD1

expression in the arcuate nucleus in rodents on a high fat diet.

2.3 Materials and Methods

2.3.1 RECOMBINANT ADENO-ASSOCIATED VIRUS

Recombinant adeno-associated viruses (rAAV) particles (catalogue number iAAV06495002) were purchased from ABM Good (Vancouver, Canada) via NBS Biologicals (Huntingdon, UK). rAAV-siβHSD1 was used to knockdown gene expression of 11βHSD1 and rAAV encoding green fluorescent protein (rAAV-GFP) (catalogue number iAAV01500) were used as controls. ABM

Good confirmed >70% knockdown in gene of interest in vitro. rAAV-GFP has been used extensively by our lab and other groups for control intra-nuclear injections in in vivo experiments and it is not known to have any effects on 11βHSD1 expression, food intake or body weight (Hussain et al., 2015).

2.3.2 IN VIVO METHODS

2.3.2.1 STANDARD CHOW COHORT

Adult male Wistar rats, weighing 180-200g were purchased from Charles River UK Ltd, and were individually housed in the Imperial College Central Biomedical Services animal facility and maintained under a controlled environment (temperature 21-23°C, 12-hour light-dark cycle, lights on at 07:00). All animal procedures were approved under the British Home Office

Animals (Scientific Procedures) Act 1986 (Project Licence no. 70/8068) and an aseptic surgical protocol was followed. Animals were acclimatised to the animal facility for 7 days prior to undergoing any procedures. The day before surgery, animals were weighed and then block randomised into either the control group (rAAV-GFP; n=12, mean weight 244.1±2.2g) or the

52 study group (rAAV-siβHSD1; n=12, mean weight 244.3±2.5g) to reduce bias due to uneven distribution of body weight within groups.

On the day of surgery, the rats were anesthetised with 4% isoflurane 2L.min-1 oxygen, placed in a stereotactic frame and given analgesia (0.12mg.kg-1 subcutaneous buprenorphine). The surgical site was shaved and then cleaned with povidone-iodine. A 1cm rostro-caudal incision was made in the skin overlying the vertex of the skull and the periosteum was removed by sharp dissection to expose the bregma. Bilateral burr holes were drilled using an electric mini- drill and the ARC co-ordinates used were 3.4mm posterior to the bregma, ±0.5mm lateral to bregma and 9.5mm below the skull surface (Hussain et al., 2015). Using a Hamilton® stainless steel injector and infusion pump (World Precision Instruments, Hertfordshire, UK), 1μl (i.e.

0.5µl per side) of rAAV (rAAV-GFP for control animals and rAAV-siβHSD1 for knockdown animals) was injected into each ARC at a rate of 0.2μl.minute-1 over 5 minutes. Following each injection, the cannula and injector were left in situ for 5 minutes to minimise back diffusion and then gradually removed. The scalp incision was closed with a 4.0 polypropylene suture

(Ethicon, New Jersey, USA). 50mg.kg-1 intraperitoneal flucloxacillin and amoxicillin (for prophylaxis) as well as 2.5ml of intraperitoneal 0.9% NaCl were administered (for rehydration). Postoperatively, rats were placed in a warming chamber to facilitate prompt recovery.

Throughout the study, all rats were given ad libitum access to standard chow (RM1 diet: 7.5% fat, 75% carbohydrate, 17.5% protein, Special Diet Services UK Ltd) diet and water, and handled regularly. Starting at 1-week post-surgery and continuing until 10 weeks post- surgery, each rat and its food was weighed (Salter Brecknell, UK) three times a week.

At 9 weeks post-rAAV surgery, hair was shaved from the skin overlying the interscapular fat pad (i.e. the dorsal surface from behind the ears to halfway between the forelegs and the hindlegs) under light isoflurane anaesthesia. Due to the thermogenic-suppressive effect of isolflurane on BAT, infrared thermography was performed 24 hours after isoflurane anaesthesia. A 1-minute recording of thermal images was made of each rat with the cage cover removed and the rats allowed to move freely within their cages using a T440bx thermal imaging camera (Flir Tools, UK). Two stills were taken from each recording and the mean of the highest temperature readings from each still was calculated.

Several days prior to culling, the rats were acclimated to the guillotine. At the end of the study, the rats were killed by decapitation using a guillotine to minimise stress and associated elevations in brain and circulating corticosterone levels. The brain of each animal was carefully dissected, snap frozen in dry ice-cooled isopentane and stored at -80oc. Interscapular

BAT was dissected and all WAT removed from the BAT depot, weighed and frozen in liquid nitrogen. Blood was collected directly from the torso into tubes containing EDTA, 30µl heparin and 200µl apoprotnin, for ACTH, corticosterone, insulin and glucose measurement. Blood samples were centrifuged at 6000rpm for 10mins, plasma was separated and then stored at

-80oC until they were defrosted on ice for 2hours prior to assays being performed. The stomach, small and large intestines were emptied of their contents and the carcasses were weighed and stored at -20oc until body composition analysis was performed.

2.3.2.2 HIGH FAT DIET COHORT

Adult male Wistar rats, weighing 180-200g were purchased from Charles River UK Ltd, were housed in identical conditions to the standard chow cohort. The animals were block randomised into two weight-matched groups (rAAV-GFP group: n=12, mean weight

257.8±4.9g; rAAV-siβHSD1 group: n=12, mean weight 257.8±4.7g) and underwent intra- arcuate injection of rAAV as described in 2.3.2.1 above.

Starting at 1-week post-surgery and continuing until they were culled, each rat and its food was weighed (Salter Brecknell, UK) three times a week until 10 weeks post-surgery. Pre- operatively and for the first 2 post-operative weeks, all rats were given ad libitum access to standard chow (RM1 diet: 7.5% fat 75% carbohydrate 17.5% protein, Special Diet Services UK

Ltd) and water. Starting at 2 weeks post-surgery, and continuing until the animals were culled, standard chow diet was changed to a high fat diet (Open Source Diets® D12451: 45% fat 35% carbohydrate 20% protein, Research Diets Inc., USA). At 9 weeks post-surgery, the skin overlying the interscapular BAT depot was shaved (i.e. from behind the ears to the hindlegs on the dorsal surface) and thermal imaging was performed as described in section 2.3.2.1.

At the end of the study, the animals were culled, and tissues collected and stored as described in section 2.3.2.1.

2.3.2.3 OVEREXPRESSION, UNDEREXPRESSION AND GFP COHORT

Adult male Wistar rats, weighing 180-200g were purchased from Charles River UK Ltd, were housed in identical conditions to the standard chow cohort. The animals were block

55 randomised into three weight-matched groups (rAAV-βHSD1 [overexpression] group: n=10, mean weight 279.6±4.3g; rAAV-siβHSD1 [underexpression] group: n=10, mean weight

279.8±4.6g and rAAV-GFP group: n=13, mean weight 279.6±1.9g) and underwent intra- arcuate injection of rAAV as described in 2.3.2.1 above.

Starting at 1-week post-surgery and continuing until they were culled, each rat and its food was weighed (Salter Brecknell, UK) three times a week until 4 weeks post-surgery. Pre- operatively and post-operatively, all rats were given ad libitum access to standard chow (RM1 diet: 7.5% fat 75% carbohydrate 17.5% protein, Special Diet Services UK Ltd) and water. At the end of the study, the animals (apart from 3) were culled as described in section 2.3.2.1.

Each animal’s brain was carefully dissected and the ventral surface placed on dry ice powder and left until the entire brain was frozen. Frozen brains were transferred into labelled containers and stored at -80oC.

2.3.2.4 FIXATION PERFUSION

Fixation perfusion was performed on 3 animals (from the rAAV-GFP group described in

2.3.2.3) 4 weeks post-stereotactic surgery to obtain brain samples for fluorescence microscopy. Each animals was anaesthetised with 4ml phenobarbital (diluted in 0.01M phosphate buffered saline, PBS). Once the animal was unconscious, it was secured to a cork board. Incisions were made to expose the abdominal and thoracic cavities. An incision was made in the right atrium and shortly after 150ml 0.01M PBS was injected into the left ventricle along with 1ml of 5000IU heparin (to prevent clot formation) over 5minutes. Subsequently,

150ml 4% paraformaldehyde was injected into the left ventricle to fix tissues. Brains were carefully dissected and kept in 4% paraformaldehyde at 4oC for 24hours. Brains were then

56 transferred to 40% sucrose and kept at 4oC. After 5days, the sucrose was drained and the brains were stored at -80oC.

2.3.3 SAPONIFICATION OF CARCASSES FOR BODY COMPOSITION ANALYSIS

Frozen carcasses were transferred to a cold room and kept at 4oC for 24 hours. Defrosted carcasses were weighed and placed individually in 1.25L plastic containers. 1ml.g-1 of 3M potassium hydroxide (KOH) in 65% ethanol was added to each container. The containers with the carcasses in KOH solution were kept in an oven at 70oC for 5-7 days (until carcasses were completely dissolved apart from small bone residue). The liquid was strained to remove the bone residue and each carcass liquid was made up to 1L with 100% ethanol. A 25mL aliquot was taken from each 1L carcass liquid, transferred to a clean plastic bottle labelled with the animal number and stored at room temperature until assays were performed to determine body composition.

2.3.4 ASSAYS

2.3.4.1 BRAIN CORTICOSTERONE

The right and left ARC, VMH and PVN were identified using a rat brain atlas (Paxinos and

Watson, 2013) and 1mm diameter punch biopsies of these nuclei were collected using a

Harvard Instruments Neuropunch®. The samples were then placed in microcentrifuge tubes labelled with the animal number and hypothalamic nucleus. 1ml of 90% methanol was added to each microcentrifuge tube and each punch biopsy was homogenised until the methanol-

57 tissue mixture became clear. The mixture was centrifuged for 3 minutes at 13000g. The supernatant was transferred to a clean labelled test tube and dried overnight in a drying centrifuge and vacuum (Savant™ SPD2010 SpeedVac™ Concentrator) at 30oC. The next morning the residual powder was stored at -80oC until the assay was performed.

Brain corticosterone was measured using a competitive enzyme-linked immunosorbent assay

(ELISA) (Cayman Chemical, Ann Arbor, USA), using the manufacturer’s protocol. Each assay was performed in triplicate. On the day of the assay, the powder was reconstituted in 500µl of assay buffer. Tracer (i.e corticosterone-acetylcholinesterase conjugate), antiserum and assay standard or sample were incubated overnight in microplate wells pre-coated with mouse anti-rabbit IgG and proprietary blocking proteins. Following incubation, the plates were washed to remove all unbound reagents and then developed with Ellman’s Reagent. A yellow product, which absorbs light at 412nm, is generated by the reaction and plates were read with a plate reader (SpectraMax® i3x). The absorbance is directly proportional to the amount of tracer bound to the well, which is inversely proportional to the amount of free corticosterone in the sample or standard. The corticosterone in each sample was interpolated from the standard curve.

2.3.4.2 PLASMA CORTICOSTERONE

A competitive ELISA (Cayman Chemical, Ann Arbor, USA) was used to measure corticosterone

(as described in 2.3.4.1 above) in plasma collected as described in 2.3.2.1 above).

2.3.4.3 PLASMA GLUCOSE

A glucose oxidase assay (Randox Laboratories, Ltd, UK) was used to measure glucose in the plasma samples collected as described in 2.3.2.1 above. Each assay was performed in triplicate. Plasma samples were diluted with glass distilled water (GDW) (1:2 for the chow cohort and 1:4 for the HFD cohort) prior to the assay to obtain values within the standard curve. 2.5µl of standard or diluted sample were added to each microplate well followed by

250µl of reagent, and then the plates were left to incubate for 25minutes at room temperature. After incubation, the absorbance was measured at 500nm using a plate reader

(SpectraMax® i3x). The glucose concentrations in the samples were interpolated from the standard curve and multiplied by the appropriate dilution factor to obtain the final values.

2.3.4.4 PLASMA INSULIN

An ultrasensitive rat insulin ELISA assay (Crystal Chem, USA) was used to measure insulin in the plasma samples collected as described in 2.3.2.1 above. Each assay was performed in triplicate using the manufacturer’s protocol. 95µl of sample diluent and 5µl of sample or standard were added to microplate wells coated with guinea pig anti-insulin antibody. The microplate was covered and left to incubate for 2 hours at 4oC. The microplate was washed and then 100µl of anti-insulin enzyme conjugate was added to each well. The microplate was covered and left to incubate for 30minutes at room temperature. The microplate was washed and then 100µl of enzyme substrate was added to each well and left to react for 40minutes at room temperature in a dark room to avoid exposing the microplate to light. 100µl of stop solution was added to each well and the absorbance was measured at 450nm using a plate

59 reader (SpectraMax® i3x). Insulin concentrations in each sample were obtained by interpolation from the standard curve.

2.3.4.5 BODY COMPOSITION PROTEIN

10µl aliquots of liquefied carcasses were diluted to 1:100 with GDW and the protein content of each diluted aliquot was determined using a Modified Lowry Proterin Assay kit (Thermo

Scientific, USA). Each diluted sample was assayed in triplicate. 40µl of standard or diluted sample were added to microplate wells and then 200µl Modified Lowry Reagent were added to each well. After incubation for 10minutes at room temperature, 20µl of Folin-Ciocateu

Reagent was added to each well. The plate was covered and left to incubate for 30minutes and then the absorbance was read at 750nm using a plate reader (SpectraMax® i3x). The protein content in each sample was determined by interpolation from the standard curve, multiplied by 100 then corrected for carcass weight.

2.3.4.6 BODY COMPOSITION GLYCEROL

10µl aliquots of liquefied carcasses were diluted to 1:100 with GDW and the glycerol content of each diluted aliquot was determined using a colorimetric assay kit (Randox Laboratories

Ltd, UK). Each diluted sample was assayed in triplicate. 1M glycerol stock solution was diluted in GDW to obtain the following standard glycerol concentrations: 7.5mM, 3mM, 1.5mM,

0.75mM and 0.3mM. 7.5µl of diluted sample or standard were pipetted into microplate wells and then 250µl of reagent mix was added into each well. The plate was covered and incubated

60 for 10minutes at room temperature and absorbance read at 520nm using a plate reader

(SpectraMax® i3x). Glycerol values were obtained by interpolation from the blank-corrected standard curve, which were then multiplied by 100 and corrected for carcass weight.

2.3.5 STATISTICAL ANALYSIS

A power calculation performed using STATA 14.1, determined that a study with a sample size of 10 animals per group would have 80% power to detect a difference in arcuate corticosterone of 2ng.ml-1 (i.e. similar to changes in corticosterone levels produced by mild stress (Droste et al., 2008)) between the control (i.e. rAAV-GFP) group and siβHSD1 (rAAV- siβHSD1) group at a significance level of 0.05. Therefore, surgery was performed on 12 animals per group due to an expected peri-operative mortality of approximately 10%.

Data are presented as mean ± SEM unless otherwise stated. All analyses were performed using GraphPad Prism 7.0 (GraphPad Software Inc., California, USA), apart from generalized estimating equations (GEE), which were performed using STATA 14.1 (Statcorp LLC, Texas,

USA). T-tests were performed on parametric data, Wilcoxon rank sum tests or Mann Whitney

U tests were performed on non-parametric data and GEEs were performed on longitudinal non-independent datasets. Significance was set at p < 0.05 unless otherwise stated.

2.4 Results

2.4.1 CONFIRMATION OF ARC-SPECIFIC EFFECTS ON CORTICOSTERONE LEVELS FOLLOWING BILATERAL

AAV INJECTION

Bilateral injection of rAAV-siβHSD1 into the ARC resulted in lower corticosterone levels compared to rAAV-GFP injection into the ARC (ARC corticosterone: GFP 31.23±4.08pg.ml-1 vs siβHSD1 22.99±2.08pg.ml-1, p=0.0584) (Figure 1).

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Figure 1: Corticosterone levels in the arcuate nucleus 10 weeks after rAAV-GFP (n=5) or rAAV- siβHSD1 (n=6) injection into the arcuate nucleus. Data presented as mean±SEM. p=0.0584 rAAV-siβHSD1 vs rAAV-GFP (Mann-Whitney U test).

Bilateral rAAV injection into the ARC did not result in significantly different corticosterone levels in the VMH and PVN (Figure 2) between the rAAV-siβHSD1 and rAAV-GFP groups in these nuclei (VMH corticosterone: GFP 29.99±5.75pg.ml-1 vs siβHSD1 29.76±4.28pg.ml-1, p=0.9143; PVN corticosterone: GFP 26.39±5.15pg.ml-1 vs siβHSD1 22.43±2.67pg.ml-1, p=0.6389).

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Figure 2: Corticosterone levels in the ventromedial hypothalamus (VMH) and the paraventricular nucleus (PVN) 10 weeks after rAAV-GFP (n=4-5) or rAAV-siβHSD1 (n=6-7) injection into the arcuate nucleus. Data presented as mean±SEM.

2.4.2 NORMAL CHOW

2.4.2.1 EFFECT OF DECREASING ARC GLUCOCORTICOIDS (VIA REDUCTION OF 11BETAHSD1) ON FOOD

INTAKE AND BODY WEIGHT ON A NORMAL CHOW DIET

Downregulation of ARC 11βHSD1 expression did not affect food intake in rats fed a chow diet as shown in Figures 3 and 4. Total food intake (GFP 1771±164g vs siβHSD1 1788±98g, p=0.7749) and average daily food intake (GFP 28.92±0.97g vs siβHSD1 28.76±1.2g, p=0.7216) were similar between groups.

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Figure 3: Cumulative food intake during the 10-week period following rAAV-GFP (n=11) or rAAV-siβHSD1 (n=10) injection into the arcuate nucleus. Data presented as mean±SEM. p=0.9311 GFP vs siβHSD1 (from day 10 to day 69 post-surgery) using generalised estimating equation.

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Figure 4: Average daily food intake during the 10-week period following rAAV-GFP (n=11) or rAAV-siβHSD1 (n=10) injection into the arcuate nucleus. Data presented as mean±SEM.

Despite very similar food intake to GFP controls, animals with downregulation of ARC

11βHSD1 had lower body weights than controls as shown in Figure 5.

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Figure 5: Body weight pre- and post- rAAV-GFP (n=11) or rAAV-siβHSD1 (n=10) injection into the arcuate nucleus. Data presented as mean±SEM. *p<0.05 GFP vs siβHSD1 (from day 7 to day 69 post-surgery) using generalised estimating equation.

2.4.2.2 EFFECT OF DECREASING ARC GLUCOCORTICOIDS (VIA REDUCTION OF 11BETAHSD1) ON ENERGY

EXPENDITURE ON A NORMAL CHOW DIET

Direct measurements of energy expenditure were not performed. However, surrogate measures of energy expenditure (i.e. BAT weight, BAT UCP-1 RNA expression and temperature of skin overlying interscapular BAT) were carried out, the results of which are detailed below.

There was a trend towards higher BAT weight in the ARC 11βHSD1 knockdown group (GFP

0.93±0.19g vs siβHSD1 1.10±0.18g, p=0.0514) (Figure 6). When BAT weight was corrected for

65 carcass weight (i.e. BAT weight expressed as a percentage of carcass weight) (Figure 7), the values were significantly higher in the ARC 11βHSD1 knockdown group (GFP 0.19±0.04 vs siβHSD1 0.24±0.04, p=0.0205). Thus, ARC 11βHSD1 knockdown increased BAT weight and may therefore have increased energy expenditure.

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Figure 6: Weight of interscapular brown adipose tissue dissected from rat carcasses 11 weeks after rAAV-GFP (n=11) or rAAV-siβHSD1 (n=10) injection into the arcuate nucleus. Data presented as mean±SEM. p=0.0514 using unpaired t-test.

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Figure 7: Weight of interscapular brown adipose tissue dissected from carcass expressed as a percentage of carcass weight 11 weeks after rAAV-GFP (n=11) or rAAV-siβHSD1 (n=10) injection into the arcuate nucleus. Data presented as mean±SEM. *p<0.05 GFP siβHSD1 using unpaired t-test.

BAT releases energy in the form of heat using a specialised protein called UCP-1 (Cannon and

Nedergaard, 2004). The heat produced by active BAT increases the temperature of overlying skin and this elevated skin temperature as measured with infrared thermography is closely correlated with BAT activation (Salem et al., 2016, Law et al., 2017). 9 weeks post-rAAV surgery, infrared thermography of the skin overlying BAT was performed but this did not reveal any difference in the peak skin temperature (and by inference BAT thermogenesis) between the 2 groups (GFP 37.25±0.19oC vs siβHSD1 37.01±0.15oC, p=0.3353).

2.4.2.3 EFFECT OF DECREASING ARC GLUCOCORTICOIDS (VIA REDUCTION OF 11BETAHSD1) ON BODY

COMPOSITION ON A NORMAL CHOW DIET

Body composition, with respect to percentage body protein (GFP 9.86±0.28% vs siβHSD1

10.33±0.38%, p=0.3225) and percentage body triglyceride (GFP 26.65±2.53% vs siβHSD1

29.04±1.89%, p=0.4642), were similar between the ARC 11βHSD1 knockdown and GFP control groups (Figures 8 and 9).

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Figure 8: Percentage protein per carcass of animals culled 11 weeks after rAAV-GFP (n=11) or rAAV-siβHSD1 (n=10) injection into the arcuate nucleus. Data presented as mean±SEM.

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Figure 9: Percentage triglyceride per carcass of animals culled 11 weeks after rAAV-GFP (n=11) or rAAV-siβHSD1 (n=10) injection into the arcuate nucleus. Data presented as mean±SEM.

2.4.2.4 EFFECT OF DECREASING ARC GLUCOCORTICOIDS (VIA REDUCTION OF 11BETAHSD1) ON CIRCULATING

HORMONES AND GLUCOSE LEVELS ON A NORMAL CHOW DIET

As shown in Figure 10 below, selective downregulation of ARC 11βHSD1 did not lead to a significant difference between plasma corticosterone levels in the knockdown and control groups when the animals were culled at the end of the study (plasma corticosterone: GFP

554.3±97.8pg.ml-1 vs siβHSD1 435.0±75.3pg.ml-, p=0.3481).

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Figure 10: Plasma corticosterone in trunk blood collected from male rats culled 11 weeks after rAAV-GFP (n=11) or rAAV-siβHSD1 (n=10) injection into the arcuate nucleus. Data presented as mean±SEM.

Additionally, (fed) plasma glucose (GFP 9.3±0.7mmol.L-1 vs siβHSD1 9.5±0.5 mmol.L-1, p=0.8087) and insulin (GFP 3.98±0.61ng.mL-1 vs siβHSD1 3.57±0.46ng.mL-1, p=0.5972) levels were similar in the downregulation and control groups (Figures 11 and 12).

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Figure 11: Plasma glucose in trunk blood collected from male rats culled 11 weeks after rAAV- GFP (n=11) or rAAV-siβHSD1 (n=10) injection into the arcuate nucleus. Data presented as mean±SEM.

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Figure 12: Plasma insulin in trunk blood collected from male rats culled 11 weeks after rAAV- GFP (n=11) or rAAV-siβHSD1 (n=10) injection into the arcuate nucleus. Data presented as mean±SEM.

2.4.3 HIGH FAT DIET

2.4.3.1 EFFECT OF DECREASING ARC GLUCOCORTICOIDS (VIA REDUCTION OF 11BETAHSD1) ON FOOD

INTAKE AND BODY WEIGHT ON A HIGH FAT DIET

When fed HFD for 8 weeks, animals with ARC 11βHSD1 downregulation, had higher food intake compared to control animals (Figures 13 and 14). The total HFD food intake (GFP vs siβHSD1, p=0.0724 using GEE) and average daily food intake on HFD (GFP 19.93±0.57g vs siβHSD1 21.58±0.64g, p<0.05) were higher in the ARC 11βHSD1 downregulation group.

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Figure 13: Cumulative food intake during the 10-week period following rAAV-GFP (n=12) or rAAV-siβHSD1 (n=12) injection into the arcuate nucleus. Data presented as mean±SEM. p=0.0724 GFP vs siβHSD1 (from day 14, when high fat diet was started, to day 69 post-surgery) using generalised estimating equation.

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Figure 14: Average daily food intake during 8 weeks of high fat diet starting 2 weeks after rAAV-GFP (n=12) or rAAV-siβHSD1 (n=12) injection into the arcuate nucleus. Data presented as mean±SEM. *p<0.05 GFP vs siβHSD1 using Mann-Whitney U test.

Consistent with a higher average daily food intake on HFD, the body weight of animals with

ARC 11βHSD1 downregulation was higher compared to controls, but this difference did not reach statistical significance (Figure 15A). Similarly there was a trend towards higher increase in weight following surgery on a HFD in the ARC 11βHSD1 downregulation group compared with controls (Figure 15B).

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Figure 15: (A) Body weight pre- and post- rAAV-GFP (n=12) or rAAV-siβHSD1 (n=12) injection into the arcuate nucleus in male rats initially fed a chow diet and then fed a high fat diet from 2 weeks post- surgery until the end of the study. Data presented as mean±SEM. p=0.1209 GFP vs siβHSD1 (from day 14 to day 69 post-surgery) using generalised estimating equation. (B) Increase in body weight from pre-surgery baseline in male rats post- rAAV-GFP (n=12) or rAAV- siβHSD1 (n=12) injection into the arcuate nucleus on HFD. Data presented as mean±SEM. p=0.0709 GFP vs siβHSD1 (from day 14 to day 69 post-surgery) using generalised estimating equation.

2.4.3.2 EFFECT OF DECREASING ARC GLUCOCORTICOIDS (VIA REDUCTION OF 11BETAHSD1) ON ENERGY

EXPENDITURE ON A HIGH FAT DIET

Direct measurements of energy expenditure were not performed. However, surrogate measures of energy expenditure (i.e. BAT weight and temperature of skin overlying interscapular BAT) were carried out, the results of which are detailed below.

There was no difference between the weight of interscapular BAT dissected from carcasses of animals with ARC 11βHSD1 downregulation and control animals (BAT weight: GFP

1.13±0.06g vs siβHSD1 1.11±0.05g, p=0.8517). Additionally, BAT weight corrected for carcass weight (i.e. expressed as a percentage of carcass weight) was similar in the two groups (GFP

0.22±0.01% vs siβHSD1 0.20±0.01%, p=0.2837).

As shown in Figure 16, the temperature of skin overlying interscapular BAT was lower in rats with ARC 11βHSD1 downregulation compared to controls (peak skin temperature: GFP

36.98±0.06oC vs siβHSD1 36.61±0.16oC, p<0.01). )

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2.4.3.3 EFFECT OF DECREASING ARC GLUCOCORTICOIDS (VIA REDUCTION OF 11BETAHSD1) ON BODY

COMPOSITION ON A HIGH FAT DIET

Body composition, with respect to percentage body protein (GFP 8.58±0.38% vs siβHSD1

8.25±0.35%, p=0.3777) and percentage body triglyceride (GFP 34.34±2.55% vs siβHSD1

36.67±1.97%, p=0.4774), were similar between the ARC 11βHSD1 knockdown and GFP control groups (Figures 17 and 18).

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Figure 17: Percentage protein per carcass of male rats fed a high fat diet and culled 10 weeks after rAAV-GFP (n=12) or rAAV-siβHSD1 (n=12) injection into the arcuate nucleus. Data presented as mean±SEM.

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Figure 18: Percentage triglycerides per carcass of male rats fed a high fat diet and culled 10 weeks after rAAV-GFP (n=12) or rAAV-siβHSD1 (n=12) injection into the arcuate nucleus. Data presented as mean±SEM.

2.4.3.4 EFFECT OF DECREASING ARC GLUCOCORTICOIDS (VIA REDUCTION OF 11BETAHSD1) ON CIRCULATING

HORMONES AND GLUCOSE LEVELS ON A HIGH FAT DIET

Similar to the normal chow cohort, there were no significant differences in plasma corticosterone (GFP 329.1±57.3pg.ml-1 vs siβHSD1 272.9±65.9pg.ml-1, p=0.3793), plasma glucose (GFP 6.59±0.33mmol.L-1 vs siβHSD1 7.05±0.36mmol.L-1, p=0.5550) and plasma insulin

(GFP 3.64±0.49ng.ml-1 vs siβHSD1 3.63±0.36ng.ml-1, p=0.6864) levels at the time the animals were culled (Figures 19, 20 and 21).

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Figure 20: Plasma glucose levels in trunk blood collected from male rats fed a high fat diet and culled 10 weeks after rAAV-GFP (n=12) or rAAV-siβHSD1 (n=12) injection into the arcuate nucleus. Data presented as mean±SEM.

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Figure 21: Plasma insulin levels in trunk blood collected from male rats fed a high fat diet and culled 10 weeks after rAAV-GFP (n=12) or rAAV-siβHSD1 (n=12) injection into the arcuate nucleus. Data presented as mean±SEM.

2.4.4 COMPARISON OF EFFECTS OF ARCUATE OVEREXPRESSION AND UNDER EXPRESSION OF

11BETAHSD1 ON FOOD INTAKE AND BODY WEIGHT ON STANDARD CHOW DIET

In the 4 weeks following surgery, food intake (GFP vs siβHSD1, p=0.4021 using GEE) and body weight (GFP vs siβHSD1, p=0.2040 using GEE) were similar in the rAAV-GFP and rAAV-siβHSD1

(underexpression) group. However, in Figure 22B the lines depicting the body weight of the two groups had begun to diverge. Therefore, if the measurements were continued over an extended period of time, it is likely that the underexpression group would have a significantly lower body weight compared to the GFP group (i.e. similar to the results presented in Figure

5).

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Figure 22: Food intake (A) and body weight (B) during 4 weeks of chow diet following rAAV- GFP (n=12) and rAAV-siβHSD1 (underexpression group, n=10) injection into the arcuate nucleus. Data presented as mean±SEM.

In the 4 weeks following surgery, food intake (GFP vs βHSD1, p=0.9799 using GEE) and body weight (GFP vs βHSD1, p=0.7379 using GEE) were similar in the rAAV-GFP and rAAV-βHSD1

(overexpression) group.

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Figure 23: Food intake (A) and body weight (B) during 4 weeks of chow diet following rAAV- GFP (n=12) and rAAV-βHSD1 (overerexpression group, n=8) injection into the arcuate nucleus. Data presented as mean±SEM.

2.5 Discussion and Conclusions

2.5.1 SUMMARY OF MAIN FINDINGS AND COMPARISON WITH EXISTING LITERATURE

Long-term downregulation of 11βHSD1 within the ARC of adult male rats via stereotactic rAAV injection resulted in reduced corticosterone within the ARC (but not in neighbouring nuclei) with a less weight gain when animals were fed a normal chow diet despite similar food intake to control rats. This suggests that reduced arcuate corticosterone increases energy expenditure in animals fed a chow diet. These findings are in keeping with studies in which

11βHSD1 inhibitors resulted in increased energy expenditure in rodents (Wang et al., 2006) and increased 11βHSD1 activity resulted in higher body weight than controls (Masuzaki et al.,

2001).

Interestingly, on a HFD, rats with ARC downregulation of 11βHSD1 had significantly higher daily food intake and a trend towards higher body weight (and higher weight gain) compared to controls. Similarly, whole-body as well as brain 11βHSD1 knockout mice have higher food intake on HFD than controls but their body weights are similar (Harno et al., 2013).

Additionally, rats given 11βHSD1 inhibitors which are known to cross the blood brain barrier have similar body weights to controls on HFD (Goldberg et al., 2014). On the other hand, some 11βHSD1 inhibitors are able to reduce both food intake and body weight in mice fed a

HFD (Hermanowski-Vosatka et al., 2005). HFD increases 11βHSD1 expression in the ARC

(Densmore et al., 2006). Therefore, it is feasible that rAAV-mediated knockdown of ARC

11βHSD1 was insufficient to overcome the effect of HFD-enhancement of 11βHSD1 expression.

BAT thermogenesis (as measured with infrared thermography) compared to controls was lower when ARC 11βHSD1 knockdown rats were fed a HFD, and this may have contributed to the trend towards a higher body weight in the knockdown rats. Ingestion of HFD results in persistent activation of ARC AgRP neurons (Wei et al., 2015). Furthermore, HFD feeding increases AgRP expression in 11βHSD1 knockout mice but reduces AgRP expression in wild- type mice (Densmore et al., 2006). AgRP suppresses MC4R-mediated BAT activation (Brito et al., 2007). Therefore, lower BAT thermogenesis in the ARC 11βHSD1 knockdown group during

HFD feeding may be the result of upregulation of AgRP expression.

Plasma corticosterone was unaffected by reduced expression of 11βHSD1 in the ARC in animals fed chow diet and those fed HFD. In contrast, whole-body knockout studies have reported increased circulating corticosterone and/or ACTH (Kotelevtsev et al., 1997) presumably due to reduced negative feedback inhibition of active glucocorticoids on hypothalamic CRH and pituitary ACTH production. However, the PVN is the major site of feedback control of glucocorticoid secretion (Stratakis and Chrousos, 1995) and corticosterone levels were not affected by ARC rAAV injection (Figure 2). Therefore, elevated circulating corticosterone and/or ACTH levels would not be expected following arcuate rAAV injection. The method of culling (i.e. decapitation) did not result in elevated corticosterone levels as the plasma levels in this study are similar to those obtained from unstressed animals

(Siswanto et al., 2008). Therefore, the results are not confounded by stress-mediated increases in corticosterone levels.

Plasma glucose and insulin as well as percentage body fat (in comparison to controls) were unaffected by downregulation of 11βHSD1 in the ARC in animals fed chow diet and those fed

HFD. Since circulating corticosterone levels were unaffected by ARC 11βHSD1

81 downregulation, and culling by decapitation did not increase corticosterone levels in either group, elevated glucose and insulin levels would not be expected. Previous whole-body knockout and some 11βHSD1 inhibitor studies have reported lower fasting glucose and insulin levels in animals with reduced 11βHSD1 activity due to improvements in insulin resistance

(Kotelevtsev et al., 1997, Wang et al., 2012), but this effect is not seen with some brain penetrant 11βHSD1 inhibitors (Goldberg et al., 2014). However, it is not possible to compare the glucose and insulin results obtained in this thesis with the results of previous studies because the animals in this thesis were not fasted and specific tests of insulin sensitivity were not performed. Furthermore, the above studies involved reduction in both central and peripheral 11βHSD1 activity, whilst the reduction in 11βHSD1 activity produced by rAAV- siβHSD1 injection was confined to a specific part of the brain, and this may explain the differences in results.

In contrast to long-term changes in 11βHSD1 activity, short-term modulation of 11βHSD1 expression in the ARC did not result in significant changes (compared to controls) in food intake and bodyweight. Therefore, alterations in energy homeostasis caused by changes in active glucocorticoids in the ARC may require extended periods of time to produce detectable effects on body weight.

2.5.2 STRENGTHS

The reduction in corticosterone (via the reduction in 11βHSD1 expression) was confined to the ARC using stereotactic injection of rAAV. This ensured that the aim of reducing active glucocorticoids specifically in the ARC, without affecting glucocorticoid concentration in other

82 nuclei, were met. AAV serotype 2 was used for the experiments because it produces highly efficient long term gene expression specifically in neurons (Xu et al., 2001). This is essential for long term metabolic studies, like the ones performed in this study. Additionally, AAV do not provoke an inflammatory response (Kaplitt et al., 1994), which may lead to destruction of the AAV and/or cells containing the AAV and confounding of results.

Body composition analysis following saponification of carcasses, provides quantification of the actual protein and fat content of animals, as opposed to imaging-based methods of body composition analysis, which can only provide estimates of these measures. Infrared thermography enabled assessment of BAT thermogenesis (a significant component of energy expenditure in rodents (Cannon and Nedergaard, 2004) while the animals were still alive, which is an advantage over other methods of BAT activity such as post-mortem BAT UCP-1 quantification.

2.5.3 LIMITATIONS

Energy homeostasis consists of a balance between energy intake and energy expenditure.

This work has investigated energy intake (in the form of food intake on chow diet and high fat diet) with some investigation of certain aspects of energy expenditure. The finding that arcuate 11βHSD1 down-regulation results in lower body weight despite matched food intake

(with increased BAT weight), suggests that energy expenditure is increased by arcuate

11βHSD1 down-regulation. However, energy expenditure (in the form of BAT thermogenesis) was measured with infrared thermography, but this did not reveal any differences between groups. This could be due to limited sensitivity of infrared thermography leading to a type 2

83 error or differences in energy expenditure via means other than BAT thermogenesis. In order to investigate other components of energy expenditure, measurements of activity and oxygen consumption should be measured.

The divergent effects of ARC 11βHSD1 down-regulation on food intake and body weight with different diets require further investigation to determine the mechanisms by which this occurs as this will increase our understanding of appetite regulation and may help guide development of more efficacious 11βHSD1 inhibitors. Elucidating these mechanisms would involve assessment of the expression of appetite-regulating hormones within the ARC, such as AgRP, POMC, CART and NPY in response to 11βHSD1 downregulation or inhibition acutely and chronically in animals fed different diets, as well as other approaches.

Selective downregulation of ARC 11βHSD1 produced less weight gain than controls on chow and more weight gain than controls on HFD, and these differences in weight may have altered insulin resistance and glucose tolerance (as reported by previous studies of 11βHSD1 knockdown or inhibition). These parameters were not assessed in this study and therefore the metabolic phenotype produced by selective 11βHSD1 knockdown in the ARC was not fully described.

Another limitation of this study is the absence of plasma ACTH levels. Plasma ACTH may have provided an indication of the effect of downregulation of 11βHSD1 expression in the ARC on feedback regulation of the hypothalamic-pituitary-adrenal axis. ARC 11βHSD1 knockdown did not affect corticosterone levels in the PVN (Figure 2), which is a major site of negative feedback control of corticosterone secretion (Figure 10). Additionally, plasma levels of corticosterone were not affected by ARC 11βHSD1 knockdown (Figure 10 and Figure 20).

Therefore, it is unlikely that feedback regulation of ACTH secretion was affected by ARC

11βHSD1 knockdown.

2.5.4 THERAPEUTIC IMPLICATIONS

Downregulation of 11βHSD1 in the ARC produced some (but not all) of the metabolically beneficial effects of 11βHSD1 inhibitors when rats were fed a chow diet. Thus, the full complement of metabolically beneficial effects of reduced 11βHSD1 activity may require reduced activity in the liver and adipose tissue as well. Furthermore, the finding that ARC

11βHSD1 downregulation results in increased food intake and body weight on HFD may help explain why trials of 11βHSD1 inhibitors (which are brain-penetrant) in humans have had minor effects on body weight. Consumption of excess calories in the form of high fat food contributes to obesity in humans and if reduced 11βHSD1 activity in the ARC (or the human equivalent, the infundibular nucleus) increases food intake on HFD, then this class of drugs are unlikely to have significant weight-reducing effect in humans unless they are modified so that they do not cross the blood-brain barrier.

Active glucocorticoids reduce energy expenditure in rodents, whilst active glucocorticoids increase energy expenditure in humans. Therefore, reducing the levels of active glucocorticoids by reducing 11βHSD1 activity is likely to increase energy expenditure in rodents but reduce energy expenditure in humans. Central control of energy expenditure is likely to have been affected by downregulation of ARC 11βHSD1 in this study as evidenced by the higher BAT weight with lower body weight in the knockdown group despite matched food intake on chow diet. Therefore, determining the effect on energy expenditure on 11βHSD1

85 inhibitors (which act peripherally and centrally) as well as 11βHSD1 inhibitors (which only act peripherally) may lead to the development of more efficacious 11βHSD1 inhibitors.

2.5.5 CONCLUSIONS

Selective down-regulation of 11βHSD1 expression in the ARC of male rats on chow diet results in reduced body weight despite similar food intake to controls. This suggests down-regulation of ARC 11훽HSD1 increases energy expenditure. In contrast, selective down-regulation of

11βHSD1 expression in the ARC of male rats on high fat diet results in higher food intake and a trend towards higher body weight than controls. This finding, whilst surprising, is consistent with increased food intake in mice in knockout studies which include reduction of 11βHSD1 activity in the brain, and may provide an explanation for why brain-penetrant 11βHSD1 inhibitors have less potency as weight-reducing agents in clinical trials compared with their effects in pre-clinical studies. Further work is required to elucidate the mechanisms by which selective down-regulation of 11βHSD1 in the ARC produces divergent effects on food intake and body weight with different diets.

3 Chapter 3 – The effects of kisspeptin on the regulation of

metabolism and energy homeostasis

3.1 Introduction

Reproduction and metabolism are essential for the survival of living things and unsurprisingly, these two fundamental systems are inter-dependent. A minimum weight or degree of adiposity is required for pubertal development to occur (Frisch and Revelle, 1970). Some people who are underweight (Vigersky et al., 1977), obese (Vermeulen et al., 1993) and/or have impaired glucose homeostasis (Grossmann et al., 2008) have hypogonadism. In addition, excessive exercise suppresses LH secretion and/or pulsatility (Loucks et al., 1989), and the orexigenic gut hormone ghrelin (released during periods of fasting or calorie restriction) suppresses LH secretion (Kluge et al., 2007). On the other hand, testosterone replacement in hypogonadal men improves insulin sensitivity (Simon et al., 2001) while use of anabolic androgens in eugonadal men reduces insulin sensitivity (Godsland et al., 1986). Oestrogen- containing contraceptive pills have been found to increase insulin resistance in some studies of pre-menopausal women (Godsland et al., 1992) but not in others (Spellacy et al., 1978), while oestrogen replacement improves insulin sensitivity in post-menopausal women

(Brussaard et al., 1997). However, our knowledge of the hormonal control of and interactions between reproductive and metabolic systems remains poorly understood.

The essential role of the recently discovered hormone, kisspeptin, in the acquisition and maintenance of fertility has been well-characterised (de Roux et al., 2003, Seminara et al.,

2003, Dhillo et al., 2005, Dhillo et al., 2007). Evidence suggesting that kisspeptin may have significant effects on glucose and energy homeostasis in animals has started to emerge, but

87 there is currently no consensus in the literature about whether kisspeptin has a beneficial or detrimental effect on glucose metabolism and energy balance (discussed below). This poses a problem because translational studies exploring the use of kisspeptin to treat reproductive disorders are ongoing, with promising results reported (Jayasena et al., 2014a, Jayasena et al., 2014b). In light of the fact that kisspeptin-based therapies are being developed for use in humans, it is important to adequately characterise the metabolic effects of kisspeptin in humans.

3.1.1 DISCOVERY OF KISSPEPTIN, KISSPEPTIN ISOFORMS AND DISTRIBUTION OF KISSPEPTIN AND ITS

RECEPTOR

Kisspeptin (KISS1) was initially identified as a product of a metastasis suppressor gene (KISS1) in breast cancer and malignant melanoma in 1996 (Lee et al., 1996). KISS1 is expressed in the brain, pituitary, liver, pancreas, adipose tissue, small intestine, placenta, testes, ovaries and uterus (Ohtaki et al., 2001, Horikoshi et al., 2003, Cockwell et al., 2013). Its receptor (KISS1R) is present in the hypothalamus, amygdala, pituitary, the placenta, liver and pancreas (Kotani et al., 2001, Song et al., 2014).

Transcription of KISS1 produces a 145-amino acid peptide, which is subsequently cleaved to yield kisspeptin-10, kisspeptin-13, kisspeptin-14, kisspeptin-52 and kisspeptin-54 (which all have an identical C-terminal 10 amino acid chain) (Lee et al., 1996). Kisspeptin-10 and kisspeptin-54 are the main isoforms found in humans. Kisspeptin-10 has a shorter half-life

(4mins) (Jayasena et al., 2011) than kisspeptin-54 (27.6mins) (Dhillo et al., 2005) and both of these isoforms have similar efficacy when administered intravenously to healthy men

(Jayasena et al., 2015). Throughout this thesis, kisspeptin will be used interchangeably with the kisspeptin family of peptides unless otherwise stated.

3.1.2 BRIEF OVERVIEW OF KISSPEPTIN AND REPRODUCTION

There are direct connections between kisspeptin and gondaotrophin releasing hormone

(GnRH) neurons (Kallo et al., 2012). GnRH neurons express KISS1R (Messager et al., 2005) and kisspeptin stimulates GnRH secretion by acting directly on GnRH nerve terminals (Thompson et al., 2004, d'Anglemont de Tassigny et al., 2008). GnRH release does not occur when kisspeptin-10 is given to Kiss1r knockout mice (d'Anglemont de Tassigny et al., 2008).

Administration of kisspeptin to (non-human) male and female mammals of different species result in increased gonadotrophin secretion via stimulation of GnRH secretion (Thompson et al., 2004, Messager et al., 2005, Caraty et al., 2007). Similarly in men, luteinising hormone

(LH), follicle stimulating hormone (FSH) and testosterone levels are increased by intravenous administration of kisspeptin-10 and kisspeptin-54 in a dose-dependent manner (Dhillo et al.,

2005, Jayasena et al., 2011, Jayasena et al., 2015). In women, both kisspeptin-10 and kisspeptin-54 administration (either intravenously or subcutaneous) produce increases in LH,

FSH and oestradiol but women appear to be less sensitive to the effects of kisspeptin during the follicular phase of the menstrual cycle (i.e. the first 10 days of the cycle starting from the first day of menses) (Dhillo et al., 2007, Jayasena et al., 2011, Narayanaswamy et al., 2016).

Furthermore, kisspeptin has been used to stimulate reproductive hormone release in women with hypothalamic amenorrhea (Jayasena et al., 2014b). In addition, kisspeptin has been used

89 to successfully trigger oocyte maturation in women undergoing in vitro fertilization treatment

(Jayasena et al., 2014a, Abbara et al., 2017).

Mice and humans lacking either functional kisspeptin and/or its receptor do not undergo puberty and are infertile (Silveira et al., 2010, de Roux et al., 2003, Seminara et al., 2003,

Topaloglu et al., 2012), and conversely activating mutations of KISS1R cause precocious puberty (Teles et al., 2008, Silveira et al., 2010). However, there has been one report of female

Kiss1 neuronal knockout mice and Kiss1r neuronal knockout mice, which had smaller ovaries than wild type mice but were fertile when mated with wild type males (Mayer and Boehm,

2011). This suggests that in some situations it is possible to become sexually mature without kisspeptin signalling or as there was incomplete ablation of Kiss1 and Kiss1r (respectively) in these two models (i.e. 3%̴ of kisspeptin-positive neurons were present in the knockout mice), low levels of kisspeptin expression may be sufficient for fertility.

3.1.3 KISSPEPTIN AND GLUCOSE METABOLISM

Kisspeptin has been the subject of intense research in the last decade and unsurprisingly, apart from the pivotal role kisspeptin plays in reproduction, it has become apparent that kisspeptin has effects on non-reproductive systems. Several studies have been published linking kisspeptin to glucose metabolism in animals leading to unresolved controversies in this field, and these are detailed below.

3.1.3.1 IN VITRO STUDIES

Kisspeptin has been shown to increase glucose-stimulated insulin secretion (GSIS) in mouse

(62.5nmol.l-1 to 1µmol.l-1 kisspeptin-10 and 1µmol kisspeptin-54) (Hauge-Evans et al., 2006,

Bowe et al., 2009, Bowe et al., 2012), rat (1µmol kisspeptin-10 and kisspeptin-13) (Bowe et al., 2012), pig (1µmol kisspeptin-10 and kisspeptin-13) (Bowe et al., 2012) and human (1µmol kisspeptin-10 and kisspeptin-13) (Hauge-Evans et al., 2006, Bowe et al., 2012) islets. In contrast, other studies have demonstrated that kisspeptin decreases GSIS from perfused rat pancreases (10nmol to 1µmol kisspeptin-13) (Silvestre et al., 2008), mouse islets (≥0.1nmol kisspeptin-10 and kisspeptin-54) (Vikman and Ahren, 2009, Song et al., 2014) and a mouse insulinoma cell line (1µmol kisspeptin-54) (Hauge-Evans et al., 2006). In most of the above studies, the effect of kisspeptin (albeit stimulatory or inhibitory), was dose-dependent and occurred only when glucose concentrations were ≥9mmol.

3.1.3.2 RODENT STUDIES

A similar pattern of conflicting results has been reported in in vivo rodent studies. Peripheral intravenous administration of 37.5nmol kisspeptin increases GSIS by 400% in male rats (Bowe et al., 2009). On the other hand, central administration of 3.74nmol kisspeptin has no effect on circulating insulin levels in male rats (Bowe et al., 2009). In mice, an intravenous injection of 10nmol kisspeptin-54 reduces circulating insulin concentrations at 3 and 6 hours post- injection (Chen et al., 2014). Interestingly, an intraperitoneal injection of 10nmol kisspeptin-

54 suppresses GSIS in control mice but not in mice that lacked pancreatic kisspeptin receptors

(Song et al., 2014), which suggests that the effects of kisspeptin on GSIS are mediated by

91 pancreatic kisspeptin receptors. Additionally, the opposing effects of kisspeptin on GSIS in vivo in rats and mice indicate that inter-species differences may influence the response to kisspeptin.

3.1.3.3 PRIMATE STUDIES

In both fed and fasted male monkeys, intravenous administration of 38.4nmol kisspeptin-10 enhances glucose-stimulated but not basal insulin secretion (Wahab et al., 2011). This suggests that even though a very high dose of kisspeptin was used in these experiments, insulin secretion was still regulated by glucose concentration.

3.1.3.4 HUMAN STUDIES

One study has reported higher hepatic KISS1 immunoreactivity and higher circulating levels in 3 people with type 2 diabetes compared to 3 non-diabetic controls (Song et al., 2014). In contrast, other researchers have reported lower kisspeptin levels in 16 pregnant women with type 1 diabetes and 20 women with gestational diabetes compared with women with pregnancies which were not complicated by diabetes (Cetkovic et al., 2012). A more recent study reported an inverse relationship (β -0.119, p=0.03) between tertiles of circulating kisspeptin concentrations and oral glucose tolerance test disposition index in 261 people without diabetes (Andreozzi et al., 2017). Interventional studies involving kisspeptin administration and assessment of GSIS would help to clarify the clinical relevance of kisspeptin on GSIS in humans.

3.1.4 KISSPEPTIN AND ENERGY HOMEOSTASIS

Both obesity and states of negative energy balance result in hypogonadotrophic hypogonadism by suppressing gonadotrophin secretion. Kisspeptin is likely to be a major mediator of this regulatory process. Fasting reduces hypothalamic Kiss1 mRNA as well as circulating LH levels in pubertal and adult rats. Conversely, kisspeptin administration to female rats whose pubertal development has been halted by chronic undernutrition, raises reproductive hormone levels and facilitates pubertal progression. Additionally, kisspeptin administration stimulates gonadotrophin and sex steroid hormone in women with hypothalamic amenorrhea due to excessive exercise or low body weight (Jayasena et al.,

2014b) and men with type 2 diabetes-associated hypogonadotrophic hypogonadism (George et al., 2010).

Leptin, a potent anorexic hormone, is produced by adipose tissue and informs the brain about the nutritional status of the individual. As fat mass increases, leptin levels increase and eventually (in obesity) central leptin resistance develops. In prepubertal rats, circulating leptin levels and hypothalamic Kiss1 and Kiss1r mRNA levels are positively correlated (Castellano et al., 2011). Central administration of kisspeptin-10 to overfed (i.e. leptin resistant) and underfed (i.e. with low leptin levels) rodents stimulates gonadotrophin secretion (Navarro et al., 2004, Castellano et al., 2011). Additionally, both ob/ob mice, which are congenitally leptin deficient, and diet-induced obese mice (which are leptin resistant) have significantly lower

ARC Kiss1 mRNA (Smith et al., 2006b, Quennell et al., 2011); and 4 days of twice daily intraperitoneal leptin injections increase arcuate Kiss1 expression in ob/ob mice (Smith et al.,

2006b). Kisspeptin neurons, but not GnRH neurons, express the leptin receptor (Quennell et al., 2009), therefore a leptin-kisspeptin circuit may link nutritional status and reproductive

93 capability. However, there may be intermediateries in this circuit as, in mice, normal pubertal development and post-pubertal fertility are maintained despite selective deletion of the leptin receptor from hypothalamic kisspeptin neurons (Donato et al., 2011).

Kisspeptin itself may directly influence energy homeostasis. Synaptic connections have been identified between hypothalamic kisspeptin and anorexic POMC/CART neurons (Backholer et al., 2010) and between kissppetin and orexigenic NPY/AgRP neurons (Backholer et al., 2010).

CART stimulates kisspeptin neurons (True et al., 2013) while AgRP neurons inhibit kisspeptin neurons (Padilla et al., 2017b). Some studies suggest kisspeptin may have anorectic effects as kisspeptin excites POMC neurons (Fu and van den Pol, 2010) and inhibits NPY neurons (Fu and van den Pol, 2010).

Central administration of kisspeptin reduces food intake in male mice by increasing the inter- meal interval (Stengel et al., 2011) but does not affect food intake in rats (Thompson et al.,

2004, Castellano et al., 2005). However, neither short-term nor long-term peripheral administration of kisspeptin influences food intake in male rodents (Thompson et al., 2006,

Thompson et al., 2004). Additionally, global Kiss1r knockout female (but not male) mice have reduced food intake and higher body weight (and worse glucose tolerance) compared to gonadectomised wild-type mice (Tolson et al., 2014). This raises the possibility that kisspeptin influences food intake and energy expenditure, but these effects of kisspeptin may be sex- specific.

3.1.5 SUMMARY

There is abundant neuroanatomical, in vitro and in vivo evidence for a role for the reproductive hormone kisspeptin in the regulation of glucose and energy homeostasis.

However, the published data is confined to cellular or non-human studies and has yielded conflicting results, so it is difficult to extrapolate published literature to humans. Since kisspeptin-based therapies are being developed to treat reproductive (and other) disorders, it is important to establish the effect of kisspeptin on glucose metabolism and energy homeostasis in humans.

3.2 Hypothesis and aims

3.2.1 HYPOTHESIS

Peripheral kisspeptin administration will affect glucose-stimulated insulin secretion but will not affect food intake in healthy men.

3.2.2 AIMS

1. To determine if kisspeptin influences glucose-stimulated insulin secretion in humans by

performing intravenous glucose and mixed meal tolerance tests in healthy men during

1nmol.kg-1.hr-1 infusions of kisspeptin and rate-matched vehicle infusions (as controls).

2. To determine if kisspeptin affects appetite and food intake in humans during ad libitum

meal ingestion by healthy men during 1nmol.kg-1.hr-1 infusions of kisspeptin and rate-

matched vehicle infusions (as controls).

3.3 Materials and Methods

3.3.1 PARTICIPANTS

Ethics approval for this study was granted by the West London Research Ethics Committee

(16/LO/0391) and this study was carried out in accordance with the Declaration of Helsinki.

Healthy men were recruited using online and print advertisements. Written informed consent was obtained from each participant prior to study enrolment. Exclusion criteria included: body mass index (BMI) <18.5kg.m-2 or >25 kg.m-2, history of medical and psychological conditions, use of prescription or recreational drugs, treatment with an investigational drug within the preceding 2 months, blood donation within 3 months’ of study participation,

96 ingestion or inhalation of nicotine-containing substances, alcoholism, abnormal eating behaviour and history of cancer (Appendix 6.1).

Participants were instructed to abstain from strenuous exercise, alcohol, and caffeine for 24 hours preceding each study visit. Each participant was instructed to choose a meal and eat that same meal at 8pm on the night preceding each study visit, fast overnight and attend the study visit fasted. Each participant received kisspeptin-54 and vehicle infusions in a randomised, single-blinded crossover protocol (Figure 1).

3.3.2 KISSPEPTIN-54

Human kisspeptin-54 was synthesized by Bachem (St Helens), aliquoted into glass vials at a final concentration of 600nM/vial (by Prof Waljit Dhillo and Dr Channa Jayasena, Imperial

College London), freeze-dried and stored at -20oC until they were defrosted prior to administration. Each batch of peptide underwent Limulus amebocyte lysate assay test for pyrogen (performed by Associates of Cape Cod, Liverpool) and culture to confirm sterility

(performed by the Department of Microbiology, Imperial College Healthcare NHS Trust).

3.3.3 INFUSIONS

Kisspeptin infusions were made by dissolving kisspeptin-54 (Bachem, St Helens) in 1ml of 0.9%

NaCl (Braun) and adding the kisspeptin solution to 49ml Gelofusine (Braun, Germany).

Kisspeptin was infused at a rate of 1nmol.kg-1.hr-1, a dose that significantly increases LH levels in men without causing tachyphylaxis (Dhillo et al., 2005). Vehicle infusions consisted of

Gelofusine (Braun, Germany), administered at the equivalent rate to the kisspeptin infusion for each participant. The participants were blinded to the content of the infusions and they received the infusions in a random order.

3.3.4 EFFECTS OF KISSPEPTIN INFUSION DURING INTRAVENOUS GLUCOSE TOLERANCE TESTS (IVGTT)

Using the above kisspeptin infusion protocol, kisspeptin levels peak by T=30mins and remain stable thereafter (Comninos et al., 2017). Therefore, following acclimation and baseline sampling, kisspeptin or vehicle infusion was started at T=0mins and 0.3g.kg-1 of 20% dextrose

(Hameln) was administered intravenously at T=45mins over a maximum of 120 seconds. To obtain glucose and insulin values required for calculation of acute insulin response to glucose

(AIRg) and minimal model insulin sensitivity (Si), a frequent sampling protocol (Tan et al.,

2014) was used as shown below in Figure 1.

-1 0.3g.kg dextrose

INTRAVENOUS INFUSION OF VEHICLE OR KISSPEPTIN

-60 - 30 - 15 0 20 30 45 75 95 115 145 185 225

Time from start of infusion (minutes)

FigureTIME 1: Intravenous glucose tolerance test study visit protocol. Blue arrows indicateTIMEP times (relative to the start of the infusions) at which blood samples were taken. 0.3g.kg-1 of 20% POIN TI TI TI TI TI TI TI TIM TIM OINT TIM dextrose was administered at T=45mins through a separate port of the same cannula as the kisspeptinT or vehicle infusion, while blood samples were taken from a second cannula(minut sited M M EP EP on(minu the opposiMte arm. M M M M EP es) TI tes) EP EP EP EP EP EP EP OIN OI OIN M 98 OI OI OI OI OI OI OI T NT T EP NT NT NT NT NT NT NT (mi (mi (mi OI (m (m (m (m (m nut nut 3.3.5 EFFECTS OF KISSPEPTIN INFUSION DURING MIXED MEAL TOLERANCE TESTS (MMTT)

A taste test was performed prior to the MMTT study visits to determine the study meal

(Waitrose Spaghetti Bolognese 125kcal.100g-1 or Waitrose Mushroom Risotto 124kcal.100g-

1) for each participant. Participants were asked to rate how much they liked each meal using a Taste Test Sheet (Appendix 6.2), and in keeping with standard laboratory practice the meal rated closest to ‘neither like nor dislike’ was used as the study meal to minimise over- or under-eating due to taste preferences.

Following acclimation and baseline sampling, kisspeptin or vehicle infusion was started at

T=0mins and an ad libitum meal was presented to participants at T=45mins. Participants were instructed to eat until comfortably full, all clocks, electronic devices and reading material removed and all other people left the room whilst the participants were eating to reduce distractions which might affect the quantity of food consumed (Blass et al., 2006,

Hetherington et al., 2006). Kisspeptin or vehicle infusion was stopped at T=120mins after the last blood sample was taken (Figure 2). Participants were asked to rate their hunger on visual analogue scales (range 0 to 10cm; Appendix 6.3) 30mins before the infusion was started,

30mins after the infusion was started and 75mins after the infusion was started (i.e. 30mins after they had been given the meal).

MEAL

VEHICLE OR KISSPEPTIN INFUSION

-60 - 30 - 15 0 15 3 0 45 65 75 90 105 120 Time from start of Infusion (minutes)

Figure 2: Mixed meal tolerance test study visit protocol. Blue arrows indicate times (relative to the start of the infusions) at which blood samples were taken. An ad libitum meal was provided at T=45mins. TIME TIME TIME TIME TIME TIME TIME TIME TIME TIME TIME TIME

POIN POIN POIN POIN POIN 99POIN POIN POIN POIN POIN POIN POIN T T T T T T T T T T T T

(minu (minu (minu (minu (minu (minu (minu (minu (minu (minu (minu (minu

tes) tes) tes) tes) tes) tes) tes) tes) tes) tes) tes) tes) 3.3.6 ANALYSIS OF METABOLITES AND HORMONES

3.3.6.1 PRINCIPLES OF RADIOIMMUNOASSAY (RIA)

A fixed quantity of radiolabelled analyte is mixed with a fixed quantity of an antibody to the analyte as well as sample containing an unknown amount of the analyte. Unlabelled analyte competes for antibody binding sites with the radiolabelled analyte. After a period of incubation, the bound analyte is separated from the unbound analyte, by first adding a dextran-charcoal mixture (which traps the unbound, i.e. free, radiolabelled analyte), centrifuging the tubes and transferring the supernatant (which contains bound analyte, i.e. antibody-analyte complexes) into separate tubes. Then the radioactivity in the charcoal pellet

(free analyte) and supernatant (bound analyte) is measured with a gamma counter and the percentage of bound analyte is calculated. Using aliquots of known quantities of analyte, a standard curve is generated by plotting the percentage of bound analyte from the aliquots against the concentrations of the aliquots. The unknown concentrations of analyte in samples can then be determined by interpolation from the standard curve.

Kisspeptin-54, glucagon like peptide-1 (GLP-1) and glucagon were measured using RIAs

(please see 3.3.6.1.1, 3.3.6.1.2 and 3.3.6.1.3).

3.3.6.1.1 Kisspeptin-54

Blood samples for kisspeptin measurement were collected at the timepoints shown in Figures

1 and 2 in lithium heparin tubes containing 5000 kallikrein inhibitor units of aprotinin (200µl

Trasylol, Bayer) to limit enzymatic breakdown of kisspeptin. Immediately after collection, blood samples were centrifuged at 4000rpm for 4mins at room temperature. Plasma was

100 separated and transferred into 1.5ml microcentrifuge tubes (Eppendorf) and stored at -20oC until they were defrosted prior to sample analysis.

Kisspeptin-54 was measured used an established in-house radioimmunoassay (RIA) (Dhillo et al., 2005), the principles of which are outlined above. Antibody GQ2 against synthetic human kisspeptin-54 (Bachem) was raised in sheep, conjugated to bovine serum albumin (BSA). This antibody cross-reacted 100% with human kisspeptin-54, kisspeptin-14 and kisspeptin-10 and cross-reacted <0.01% with other RF amide proteins (Dhillo et al., 2005). The RIA was performed in duplicate using 100µl plasma added to 700µl of 0.06M phosphate buffer (pH

7.3) with 0.3% BSA. After incubation for 3 days at 4oC, free and antibody-bound 125I- kisspeptin-54 label were separated by charcoal adsorption. The lower limit of detection of the assay was 2pmol.L-1, the intra-assay coefficient of variation was 8.3% and the inter-assay coefficient of variation was 10.2% (Dhillo et al., 2005).

3.3.6.1.2 Glucagon-like peptide-1 (GLP-1)

Blood samples for GLP-1 measurement were collected at the timepoints shown in Figures 1 and 2 in lithium heparin tubes containing 2500 kallikrein inhibitor units of aprotinin (100µl

Trasylol, Bayer) to limit enzymatic breakdown of GLP-1. Immediately after collection, blood samples were centrifuged at 4000rpm for 10mins at 4oC. Plasma was separated and transferred into LP4 tubes and stored at -20oC until it was defrosted prior to sample analysis.

Plasma GLP-1 levels were measured used an established in-house radioimmunoassay (RIA)

(Kreymann et al., 1987), the principles of which are outlined above. The antibody against human GLP-17-36 was raised in rabbits and conjugated to BSA. This antibody cross-reacted

100% with GLP11-36, and cross-reacted 0% with glucagon and other gut hormones (Kreymann

101 et al., 1987). The RIA was performed in duplicate using 100µl plasma added to 700µl of 0.06M phosphate buffer (pH 7.3) with 0.3% BSA. After incubation for 4 days at 4oC, free and antibody-bound 125I-GLP-1 label were separated by charcoal adsorption. The intra-assay coefficient of variation was <10% and the inter-assay coefficient of variation was <10%.

3.3.6.1.3 Glucagon

Blood samples for glucagon measurement were collected at the timepoints shown in Figures

1 and 2 in lithium heparin tubes containing 2500 kallikrein inhibitor units of aprotinin (100µl

Trasylol, Bayer) to limit enzymatic breakdown of glucagon. Immediately after collection, blood samples were centrifuged at 4000rpm for 10mins at 4oC. Plasma was separated and transferred into LP4 tubes and stored at -20oC until it was defrosted prior to sample analysis.

Plasma glucagon levels were measured used an established in-house RIA (Ghatei et al.,

1983a), the principles of which are outlined above. The antibody against human glucagon was raised in rabbits and conjugated to BSA. This antibody cross-reacted 100% with pancreatic glucagon, and cross-reacted ≤15% with glucagon fragments, ≤0.5% with proglucagon fragments, <0.1% with gastrointestinal inhibitory peptide and <0.1% with vasoactive intestinal peptide (Ghatei et al., 1983a). The RIA was performed in duplicate using 100µl plasma added to 700µl of 0.06M phosphate buffer (pH 7.3) with 0.3% BSA. After incubation for 4 days at 4oC, free and antibody-bound 125I-glucagon label were separated by charcoal adsorption. The intra-assay coefficient of variation was <10% and the inter-assay coefficient of variation was <10%.

102

3.3.6.2 PRINCIPLES OF CHEMILUMINESCENT IMMUNOASSAY

Samples containing unknown amount of the analyte are mixed with anti-analyte antibody coated with paramagnetic microparticles, and the analyte in the sample binds to the antibodies. The reaction vessel is washed and pre‑trigger and trigger solutions are added to the reaction mixture. The resulting reaction produces light, which is measured as relative light units (RLUs). The RLUs detected is directly proportional to the amount of analyte in the sample.

3.3.6.2.1 LH, FSH and Testosterone

Blood samples for LH, FSH and Testosterone measurement were collected at the timepoints shown in Figures 1 and 2 in tubes containing clot activator (BD Vacutainer). After collection, blood samples were left to clot at room temperature for ≥30minutes and then centrifuged at

3000rpm for 10mins at room temperature. Serum was separated and transferred into LP4 tubes and stored at -20oC until it was defrosted prior to sample analysis. The intra-assay coefficient of variation and the inter-assay coefficient of variation for each assay is as follows:

LH both ≤7%, FSH ≤5% and testosterone ≤8%. These assays were performed using the automated Abbott Architect Analyser (Abbott Diagnostics).

3.3.6.2.2 SHBG

Blood samples for SHBG measurement were collected at the timepoints shown in Figures 1 and 2 in tubes containing clot activator (BD Vacutainer). After collection, blood samples were left to clot at room temperature for ≥30minutes and then centrifuged at 3000rpm for 10mins at room temperature. Serum was separated and transferred into LP4 tubes and stored at -

20oC until it was defrosted prior to sample analysis. The intra-assay coefficient of variation

103 and the inter-assay coefficient of variation for the assay were ≤5.3% and ≤6.6% respectively.

This assay was performed using the automated Siemens Immulite Analyser (Siemens).

3.3.6.2.3 Insulin and C-peptide

Blood samples for insulin and C-peptide measurement were collected at the timepoints shown in shown by blue arrows in Figures 1 and 2 in tubes containing clot activator and serum separator gel (BD Vacutainer). After collection, blood samples were left to clot at room temperature for 10minutes and then centrifuged at 4000rpm for 10mins at room temperature. Serum was separated and transferred into LP4 tubes and stored at -20oC until it was defrosted prior to sample analysis. The intra-assay coefficient of variation and the inter- assay coefficient of variation for each assay is as follows: insulin both ≤7%, and C-peptide ≤4%.

These assays were performed using the automated Abbott Architect Analyser (Abbott

Diagnostics).

3.3.6.3 GLUCOSE

Blood samples for glucose measurement were collected at the timepoints shown by blue arrows in Figures 1 and 2 in tubes containing sodium fluoride and oxalate (BD Vacutainer).

Immediately after collection, blood samples were centrifuged at 4000rpm for 10mins at 4oC.

Plasma was separated and transferred into LP4 tubes and stored at -20oC until it was defrosted prior to sample analysis. Glucose was measured using a colorimetric hexokinase assay using the automated Abbott Architect Analyser (Abbott Diagnostics). The intra-assay coefficient of variation and the inter-assay coefficient of variation for the assay were both

≤2%.

104

3.3.7 STATISTICAL ANALYSIS

3.3.7.1 ANALYSES

Prism 7 software (GraphPad) was used to perform all statistical analyses unless otherwise stated. Statistical significance was set at p<0.05 unless otherwise stated. Data are presented as mean ± SEM unless otherwise stated. Area under the curve (AUC) was calculated using the trapezoidal rule (Matthews et al., 1990). Paired t-tests were performed on parametric data

(as determined by the D’Agostino Pearson normality test) whilst Wilcoxon rank tests were performed on non-parametric data. Generalised estimating equation (GEE) was performed on longitudinal data where subsequent values were not independent of previous values using

STATA 14 software (StataCorp).

3.3.7.2 IVGTT INDICES OF BETA CELL FUNCTION

Acute insulin response to glucose (AIRg) was calculated as the insulin incremental area under the curve (AUC) from T=45 to 55mins (i.e. 0 to 10mins post-glucose load) using the trapezoid rule (Matthews et al., 1990). IVGTT sensitivity index (IVGTT-Si) was determined by Dr Ian

Godsland using the minimal model (MLAB software) (Bergman et al., 1981) and IVGTT disposition index (IVGTT-DI) was calculated using the following formula: DI = AIRg x Si (Tan et al., 2014). The disposition index is a widely used measure of beta cell function, which quantifies how well beta cells respond to variations in insulin sensitivity and compensate for insulin resistance. DI is closely correlated to indices obtained from hyperinsulinaemic- euglycaemic clamp studies (i.e. the gold-standard in vivo test of beta cell function) (Matsuda

105 and DeFronzo, 1999) and is therefore a reliable and validated measure of beta cell function, which avoids the potential hazards of the hyperinsulinaemic-euglycaemic clamp.

3.3.7.3 MMTT INDICES OF BETA CELL FUNCTION

MMTT insulin secretion index (MMTT-ISI) is the ratio of AUC insulin to AUC glucose. MMTT insulin sensitivity index (MMTT-IS) was calculated using the Matsuda index (with T=45mins values as baseline and mean insulin and glucose values from T=65 to 120mins used in the equation as shown below). MMTT-DI was calculated using the following formula: MMTT-DI =

ISI x IS (Maki et al., 2011).

MMTT-IS = 10000

------

√([insulin45min] X [glucose45min] X [mean insulin65 to 120min] X [mean glucose65 to 120min]

3.3.7.4 POWER CALCULATION

An a priori power calculation was performed using a dataset of IVGTTs performed in 99 healthy men aged 18 to 40 years, kindly provided by Dr Ian Godsland (Wynn Reader in Human

Metabolism, Imperial College London). Using this dataset, a study consisting of IVGTTs

106 performed in 15 healthy men would have 80% power to detect a 25% difference in insulin secretion.

107

3.4 Results

3.4.1 EFFECTS OF KISSPEPTIN DURING IVGTTS IN HEALTHY MEN

3.4.1.1 PARTICIPANTS

Fifteen healthy young men (Table 1) completed two IVGTTs each, one during kisspeptin infusion and one during rate-matched vehicle infusion.

Table 1 – Details of intravenous glucose tolerance test participants (mean±SEM)

Age 25.8 ± 1.4years

Height 1.79 ± 0.02m

Weight 71.35± 2.8kg

BMI 22.2 ± 0.6kg.m-2

3.4.1.2 EFFECT OF ACUTE KISSPEPTIN INFUSION ON GLUCOSE, INSULIN AND C-PEPTIDE LEVELS SECRETION

DURING IVGTT

Mean plasma glucose levels were similar during kisspeptin infusion and vehicle infusion

(mean post-glucose: kisspeptin minus vehicle -0.08mM, 95%CI -0.4 to 0.24, p=0.64 using multilevel linear regression)(Figure 3).

108

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n s ta rt o f I V e n d o f a

e in fu s io n g lu c o s e in fu s io n

0 M

0 5 0 0 0 0 5 7 8 9 0 1 3 5 7 9 1 3 5 7 0 5 5 5 5 5 5 5 3 1 2 3 4 4 4 4 4 5 5 5 5 5 5 6 6 6 6 7 7 8 9 1 4 8 2 - - 1 1 1 2 T im e fro m s ta rt o f k is s p e p tin o r v e h ic le in fu s io n (m in s )

Figure 3: Plasma glucose levels during intravenous glucose tolerance tests performed in 15 male volunteers with kisspeptin infusion and vehicle infusion. Data presented as mean±SEM.

Mean serum insulin levels were similar during kisspeptin and vehicle infusion prior to intravenous glucose administration. However, following the intravenous glucose load, mean insulin levels were 4.1µU.mL-1 (95 CI: 0.9 to 7.3, p=0.01 using multilevel linear regression) higher during kisspeptin infusion compared to vehicle (Figure 4). There was no significant difference in mean circulating C-peptide levels between the two groups from T=45mins to

T=225mins (kisspeptin minus vehicle 56pmol.L-1, 95%CI: -9 to 121, p=0.09 using multilevel linear regression) (Figure 5).

109

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m K is s p e p tin

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( 6 0

n i

l 5 0

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g lu c o s e

m 3 0 in fu s io n in fu s io n

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Figure 4: Serum insulin levels during intravenous glucose tolerance tests with kisspeptin infusion and vehicle infusion. Data presented as mean±SEM.

2 0 0 0 K is s p e p tin

V e h ic le

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)

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Figure 5: Serum C-peptide levels during intravenous tolerance tests performed in 15 male volunteers with kisspeptin infusion and vehicle infusion. Data presented as mean±SEM.

110

3.4.1.3 EFFECT OF ACUTE KISSPEPTIN INFUSION ON ACUTE INSULIN RESPONSE TO GLUCOSE, INSULIN

SENSITIVITY INDEX AND DISPOSITION INDEX DURING IVGTT

Acute insulin response to glucose (AIRg) was not statistically different between kisspeptin and vehicle infusions (Figure 6). Mean AIRg was 18% higher with kisspeptin infusion compared to

-1 vehicle infusion but this was not statistically significant (AIRg µU.mL .min: kisspeptin 422.2 ±

90.77 vs vehicle 357.7 ± 47.15, p=0.4543).

6 0 0

)

m .

1 4 0 0

 (

2 0 0

I A

0 V e h ic le K is s p e p tin

Figure 6: Acute insulin response to glucose during intravenous glucose tolerance tests in 15 male volunteers with kisspeptin infusion and vehicle infusion. Data presented as mean±SEM.

Although the IVGTT insulin sensitivity index (IVGTT-Si) was not significantly different during kisspeptin infusion compared with vehicle infusion, there was a trend towards higher insulin sensitivity index during kisspeptin infusion (IVGTT-Si min-1.µU-1.mL.mmol-1.L.104: kisspeptin

8.11 ± 0.98 vs vehicle 6.85 ± 0.89, p=0.1228) (Figure 7).

111

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L .

1 8

m S

- 6

G m

. 4

I 1

U 

. 2

n i

m 0 ( V e h ic le K is s p e p tin

Figure 7: Insulin sensitivity index during intravenous glucose tolerance tests in 15 male volunteers with kisspeptin infusion and vehicle infusion. Data presented as mean±SEM.

Mean IVGTT-DI was higher during kisspeptin infusion compared with during vehicle infusion

(Figure 8). This indicates that kisspeptin may improve beta cell function in the presence of high glucose concentrations.

4 0 0 0 *

3 0 0 0

)

T 2 0 0 0

I ( 1 0 0 0

0 V e h ic le K is s p e p tin

Figure 8: Intravenous glucose tolerance test disposition index with kisspeptin infusion and vehicle infusion in 15 male volunteers. Data presented as mean±SEM. *p<0.05 using Wilcoxon matched-pairs signed ranked test.

112

There is some evidence that glucagon stimulates hepatic kisspeptin production (Song et al.,

2014), which affects insulin secretion. However, no difference in circulating glucagon concentrations were detect during IVGTTs (Figure 9).

n 3 0 o

g I V a s ta rt o f e n d o f

c K is s p e p tin

in fu s io n g lu c o s e in fu s io n

)

l 1

- 2 0 V e h ic le

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l 1 0

(

a e

M 0 -5 0 -2 5 0 2 5 5 0 7 5 1 0 0 1 2 5 1 5 0 1 7 5 2 0 0 2 2 5 T im e fro m s ta rt o f k is s p e p tin o r v e h ic le in fu s io n (m in s )

Figure 9: Circulating plasma glucagon during intravenous glucose tolerance tests in 15 male volunteers with kisspeptin infusion and vehicle infusion. Data presented as mean±SEM. p=0.9459 using generalised estimating equation from T=-20mins to T=225mins.

GLP-1 is a potent endogenous insulin secretagogue, which is released in response to oral nutrient ingestion. Intravenous glucose is not known to stimulate GLP-1 secretion and in keeping with this, no difference in plasma GLP-1 levels were detected during kisspeptin infusion and vehicle infusion (Figure 10). Therefore, it is likely that the kisspeptin-mediated increase in GSIS occurred via direct action of kisspeptin on beta cells.

113

3 3 0

- 7

1 s ta rt o f I V e n d o f - K is s p e p tin

P in fu s io n g lu c o s e in fu s io n

)

L 1

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a e

M 0 -5 0 -2 5 0 2 5 5 0 7 5 1 0 0 1 2 5 1 5 0 1 7 5 2 0 0 2 2 5 T im e fro m s ta rt o f k is s p e p tin o r v e h ic le in fu s io n (m in s )

Figure 10: Mean circulating plasma GLP17-36 concentrations during intravenous glucose tolerance tests in 15 male volunteers with kisspeptin infusion and vehicle infusion. Data presented as mean±SEM p=0.5912 using generalised estimating equation from T=-20mins to T=225mins.

3.4.1.4 EFFECT OF ACUTE KISSPEPTIN INFUSION ON REPRODUCTIVE HORMONE LEVELS DURING IVGTT

Mean plasma kisspeptin levels were increased by kisspeptin infusion (to a similar level seen in published literature (Comninos et al., 2017), but kisspeptin levels were not affected by vehicle infusion (Figure 11).

114

* * * * s ta rt o f IV e n d o f in fu s io n g lu c o s e in fu s io n

) 1 0

(

t p

e 1 p

s K is s p e p tin

s i

k V e h ic le

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M 0 .0 1 -5 0 -2 5 0 2 5 5 0 7 5 1 0 0 1 2 5 1 5 0 1 7 5 2 0 0 2 2 5 T im e fro m s ta rt o f V e h ic le o r K is s p e p tin in fu s io n (m in s )

Figure 11: Plasma kisspeptin levels during intravenous glucose tolerance tests in 15 male volunteers with kisspeptin infusion and vehicle infusion. Data presented as mean±SEM. ****p<0.0001 using GEE from T=0mins to T=225mins vehicle vs kisspeptin.

In keeping with the rise in kisspeptin levels, serum LH levels (Figure 12) were elevated during kisspeptin infusion but not during vehicle infusion.

* * * *

2 0 s ta rt o f I V e n d o f in fu s io n g lu c o s e in fu s io n

K is s p e p tin

) 1

- V e h ic le L

. 1 5

(

m 1 0

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e M

0 -5 0 -2 5 0 2 5 5 0 7 5 1 0 0 1 2 5 1 5 0 1 7 5 2 0 0 2 2 5

T im e fro m s ta rt o f k is s p e p tin o r v e h ic le in fu s io n (m in s )

Figure 12 – Serum luteinising hormone levels during intravenous glucose tolerance tests with kisspeptin infusion and vehicle infusion. Data presented as mean±SEM. ****p<0.0001 using generalised estimating equation from T=0mins to T=225mins vehicle vs kisspeptin.

115

Mean testosterone levels did not rise during both kisspeptin and vehicle infusions and there was no significant difference between testosterone levels between the two groups (Figure

13). Similar findings have been reported when kisspeptin was infused at the same dose

(Comninos et al., 2017).

4 0 s ta rt o f I V e n d o f in fu s io n g lu c o s e in fu s io n K is s p e p tin

) V e h ic le

3 0

(

r 2 0

s e

t 1 0

0 -5 0 -2 5 0 2 5 5 0 7 5 1 0 0 1 2 5 1 5 0 1 7 5 2 0 0 2 2 5

T im e fro m s ta rt o f k is s p e p tin o r v e h ic le in fu s io n (m in s )

Figure 13: Serum testosterone levels during intravenous glucose tolerance tests in 15 male volunteers with kisspeptin infusion and vehicle infusion. Data presented as mean±SEM. p=0.3833 using generalised estimating equation from T=0mins to T=225mins vehicle vs kisspeptin.

116

3.4.2 EFFECT OF KISSPEPTIN DURING MMTTS IN HEALTHY MEN

3.4.2.1 PARTICIPANTS

15 healthy young men (Table 2) completed two MMTTs each, one during kisspeptin infusion and one during rate-matched vehicle infusion.

Table 2 – Details of mixed meal tolerance test study participants (mean±SEM)

Age 23.7 ± 1.0years

Height 1.79 ± 0.02m

Weight 71.23 ± 2.99kg

BMI 22.2 ± 0.55kg.m-2

3.4.2.2 EFFECT OF ACUTE KISSPEPTIN INFUSION ON GLUCOSE, INSULIN AND C-PEPTIDE DURING MMTT

Mean plasma glucose levels were similar during kisspeptin infusion and vehicle infusion

(mean post-meal glucose: kisspeptin minus vehicle -0.21mmol.L-1, 95%CI -0.56 to 0.14, p=0.25 using multilevel linear regression) (Figure 14).

117

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s K is s p e p tin o

c V e h ic le

u 6

)

l o

m 4

m a

l s ta r t o f m e a l e n d o f

p (

2 in fu s io n in fu s io n

a e

M 0 -3 0 0 3 0 6 0 9 0 1 2 0 T im e fro m s ta rt o f V e h ic le o r K is s p e p tin in fu s io n (m in s )

Figure 14: Plasma glucose concentrations during mixed meal tolerance tests in 15 male volunteers with kisspeptin infusion and vehicle infusion. Data presented as mean±SEM.

In contrast to the IVGTTs, mean serum insulin levels were not significantly elevated by kisspeptin infusion (mean post-meal insulin: kisspeptin minus vehicle 7.7µU.mL-1, 95% CI -1.4 to 16.8, p=0.10 using multilevel linear regression) (Figure 15).

1 0 0 V e h ic le

n i l K is s p e p tin

u 8 0 s

) e n d o f

i - in fu s io n

L 6 0

r U

e 4 0 s

 s ta r t o f

(

n in fu s io n m e a l

a 2 0

e M 0 -3 0 0 3 0 6 0 9 0 1 2 0 T im e fro m s ta rt o f V e h ic le o r K is s p e p tin in fu s io n (m in s )

Figure 15: Serum insulin concentrations during mixed meal tolerance tests in 15 male volunteers with kisspeptin infusion and vehicle infusion. Data presented as mean±SEM.

118

Similarly, mean serum C-peptide concentrations were unaffected by kisspeptin infusion

(mean post-meal C-peptide: kisspeptin minus vehicle -57pmol.L-1, 95% CI -26 to 82, p=0.44 using multilevel linear regression) (Figure 16).

e 3 0 0 0

d V e h ic le

i t

p K is s p e p tin

)

1 -

- 2 0 0 0

u m

r s ta r t o f e n d o f p e 1 0 0 0 ( in fu s io n m e a l

s in fu s io n

a e

M 0 -3 0 0 3 0 6 0 9 0 1 2 0 T im e fro m s ta rt o f V e h ic le o r K is s p e p tin in fu s io n (m in s )

Figure 16: Serum C-peptide concentrations during mixed meal tolerance tests in 15 male volunteers with kisspeptin infusion and vehicle infusion. Data presented as mean±SEM.

3.4.2.3 EFFECT OF ACUTE KISSPEPTIN INFUSION ON INSULIN SECRETION INDEX, INSULIN SENSITIVITY INDEX

AND DISPOSITION INDEX DURING MMTT

MMTT insulin secretion index was slightly higher during kisspeptin infusion but this difference was not statistically different (vehicle 7.20±0.88 vs kisspeptin 8.59±1.37, p=0.2169) (Figure

17).

119

1 2

)

c -

e 8

(

n I

e 4

M M 0 V e h ic le K is s p e p tin

Figure 17: Insulin secretion index during mixed meal tolerance tests in 15 male volunteers with kisspeptin infusion and vehicle infusion. Data presented as mean±SEM.

Insulin sensitivity was not affected by kisspeptin infusion as the insulin sensitivity index was similar during kisspeptin infusion and vehicle infusion (vehicle 146.8±18.0 vs kisspeptin

148.4±23.2, p=0.8040) (Figure 18).

2 0 0

)

S s

I 1 5 0

M n

i 1 0 0

l M

(

d T

n 5 0

M M 0 V e h ic le K is s p e p tin

Figure 18: Insulin sensitivity index during mixed meal tolerance tests in 15 male volunteers with kisspeptin infusion and vehicle infusion. Data presented as mean±SEM.

120

In contrast to the IVGTT-DI, the MMTT-DI was not significantly increased by kisspeptin infusion (MMTT-DI: vehicle 879.8±55.8 vs kisspeptin 937.8±57.4, p=0.4199) (Figure 19). This may be due to the difference in glucose concentrations during IVGTTs (Figure 3) compared with those during MMTTs (Figure 14).

1 2 0 0

n I

9 0 0

)

I 6 0 0

i (

T 3 0 0

M M 0 V e h ic le K is s p e p tin

Figure 19: Mixed meal tolerance tests disposition index in 15 male volunteers during kisspeptin infusion and vehicle infusion. Data presented as mean±SEM.

3.4.2.4 EFFECT OF ACUTE KISSPEPTIN INFUSION ON APPETITE

The change in hunger scores on the visual analogue scale (VAS) from pre-infusion values (i.e. at T=-30mins) to pre-meal values (i.e. at T=+30mins when kisspeptin levels had reached steady state) were very similar during both kisspeptin and vehicle infusions (Figure 20).

121

e )

r 1 0

(

i g

n 6

3 4

e 0

a f

h -2 C V e h ic le K is s p e p tin

Figure 20: Change in hunger reported by 15 male volunteers from pre-infusion (T=-30mins) to pre-meal (T=30mins) during kisspeptin and vehicle infusions. Maximum, minimum and mean values shown on the box and whiskers plot. p=0.8120 kisspeptin vs vehicle using paired t-test.

3.4.2.5 EFFECT OF ACUTE KISSPEPTIN INFUSION ON FOOD INTAKE

There was no significant difference in the number of kilocalories consumed during kisspeptin infusion compared with vehicle infusion (Figure 21). Similarly, there were no significant differences in meal duration (kisspeptin 11.00 ± 0.92mins vs vehicle 11.27 ± 0.69mins, p=0.6102) or volume of water drunk whilst eating (kisspeptin 161.1 ± 40.39ml vs vehicle 182.3

± 42.58ml, p=0.7473) during kisspeptin infusion and vehicle infusion.

122

1 0 0 0

8 0 0

) t

l 6 0 0

d k (

o 4 0 0

o F 2 0 0

0 V e h ic le K is s p e p tin

Figure 21: Food intake (in kilocalories) of 15 male volunteers during an ad libitum meal during kisspeptin and vehicle infusions. Data presented as mean±SEM. p=0.7178 using paired t-test.

3.4.2.6 EFFECT OF ACUTE KISSPEPTIN INFUSION ON REPRODUCTIVE HORMONE LEVELS DURING MMTT

Similar to the infusions during IVGTTs (Figure 11), plasma kisspeptin (Figure 22), serum LH levels (Figure 23) were elevated during kisspeptin infusion but not during vehicle infusion.

Also, serum testosterone levels were not elevated during kisspeptin and vehicle infusions

(Figure 24).

123

* * * * s ta rt o f e n d o f

in fu s io n m e a l in fu s io n n

i 1 0

s s

i 1

k )

K is s p e p tin

M n

m V e h ic le

( s

a 0 .1

a e

0 .0 1 M -3 0 0 3 0 6 0 9 0 1 2 0 T im e fro m s ta rt o f V e h ic le o r K is s p e p tin in fu s io n (m in s )

Figure 22: Plasma kisspeptin levels during mixed meal tolerance tests in 15 male volunteers with kisspeptin infusion and vehicle infusion. ****p<0.0001 using generalised estimating equation from T=0mins to T=120mins vehicle vs kisspeptin.

* * * *

s ta rt o f e n d o f

) 1 2 in fu s io n m e a l in fu s io n

- L

. K is s p e p tin

1 0

U I

( V e h ic le

u r

e 4

n 2

a e

M 0 -3 0 0 3 0 6 0 9 0 1 2 0

T im e fro m s ta rt o f V e h ic le o r K is s p e p tin in fu s io n (m in s )

Figure 23: Serum lutenising hormone (LH) levels during mixed meal tolerance tests in 15 male volunteers with kisspeptin infusion and vehicle infusion. ****p<0.0001 using generalised estimating equation from T=0mins to T=120mins vehicle vs kisspeptin.

124

3 0 V e h ic le

)

1 -

L K is s p e p tin

o m

m 2 0

(

s e s ta r t o f

m e a l n

n in fu s io n

o a

r e n d o f e

e 1 0 t

M in fu s io n

e t 0 -3 0 0 3 0 6 0 9 0 1 2 0

T im e fro m s ta rt o f V e h ic le o r K is s p e p tin in fu s io n (m in s )

Figure 24: Serum testosterone levels during mixed meal tolerance tests in 15 male volunteers with kisspeptin infusion and vehicle infusion. Data presented as mean±SEM. p=0.4072 using generalised estimating equation from T=0mins to T=120mins vehicle vs kisspeptin.

125

3.4.3 DISCUSSION AND CONCLUSIONS

3.4.3.1 SUMMARY OF RESULTS AND COMPARISON WITH EXISTING LITERATURE

An acute intravenous infusion of 1nmol.kg-1.hr-1 kisspeptin-54 (but not vehicle infusion) caused predictable increases in kisspeptin and LH, but did not increase testosterone during the infusion. During IVGTT supraphysiological glucose concentrations were obtained and under these conditions 1nmol.kg-1.hr-1 kisspeptin infusion enhanced post-glucose load insulin secretion compared to vehicle infusion (without affecting glucose levels).

Furthermore, the IVGTT disposition index was higher during kisspeptin infusion compared to vehicle infusion, with a trend towards higher insulin sensitivity index during kisspeptin infusion.

The IVGTT-DI during kisspeptin infusion was higher than vehicle infusion. DI is a composite index of βcell function which correlates highly with insulin secretion data from hyperinsulinaemic-euglycaemic clamp studies (Matsuda and DeFronzo, 1999), and lower baseline IVGTT-DI predicts conversion from normal glucose tolerance or impaired glucose tolerance after 5 years (Lorenzo et al., 2010). Therefore, kisspeptin administration may have beneficial effects on βcell function.

In healthy men, 1nmol.kg-1.hr-1 kisspeptin infusion did not affect pre- and post-meal glucose, insulin and C-peptide levels. MMTTs provide a more physiological stimulus to pancreatic

βcells due to the mixed nutrient composition of the meals, as opposed to the glucose only stimulus provided by IVGTTs. The plasma glucose levels achieved during the MMTTs were lower than those achieved in the IVGTTs and therefore kisspeptin enhancement of GSIS may only be detectible when the prevailing glucose concentrations are supraphysiological. In

126 keeping with this, kisspeptin increased GSIS in cultured human islet cells incubated with

17mmol glucose but had no effect on insulin secretion when the islet cells were incubated with 3mmol glucose (Izzi-Engbeaya and Comninos et al. 2017, in review at Diabetes – Section

7.1). Similar in vitro findings have been obtained by a number of investigators using rat, mouse, porcine and human islets (Hauge-Evans et al., 2006, Bowe et al., 2009, Bowe et al.,

2012), but inhibition of GSIS from mouse islets by kisspeptin has been reported by other investigators (Vikman and Ahren 2009, Song et al., 2014).

Acute kisspeptin infusion did not affect appetite or food intake. These results are in keeping with rodent studies in which central and peripheral kisspeptin administration had no effect on food intake and body weight in male rodents (Thompson et al., 2004). In contrast, central kisspeptin administration reduces food intake in male mice by increasing inter-feeding intervals (Stengel et al., 2011). However, stimulation of ARC kisspeptin neurons does not suppress feeding in male mice (Fenselau et al., 2017). Functional neuroanatomical connections have been discovered between hypothalamic kisspeptin neurons and appetite- regulating neurons with kisspeptin stimulating POMC/CART neurons and kisspeptin inhibiting

AgRP/NPY neurons (Backholer et al., 2010). Furthermore, peripherally administered kisspeptin can cross the blood brain barrier (Comninos et al., 2017). Therefore, the absence of an effect of kisspeptin on appetite and food intake in the MMTTs suggests that even though peripherally administered kisspeptin is able to access central compartments, it is unable to influence food intake at the administered dose. Alternatively, appetite reduction by kisspeptin may be species-specific as reduction in food intake with kisspeptin has only been reported with central administration of kisspeptin to mice. Furthermore, there may be sex-

127 specific effects as female (but not male) Kiss1r knockout mice have reduced food intake compared with gonadectomised controls (Tolson et al., 2014).

3.4.3.2 STRENGTHS

Efforts were made to reduce sources of variability, which could have confounded the results obtained in this study. All volunteers were male, had similar ages and BMIs, had stable weights for 3 months prior to enrolment into the study and during the study period, and followed the same instructions regarding physical activity, food and caffeine intake prior to each study visit. Additionally, study visits were performed in a randomised order with participants blinded to the contents of intravenous infusions.

Raw data as well as well-established indices of beta cell function were used to determine the effect of kisspeptin on GSIS, which ensured robust conclusions could be drawn from the results. GSIS was assessed when circulating glucose levels were elevated (i.e. during the

IVGTTs) and during normal post-prandial glucose excursions (i.e. during the MMTTs). This ensured the effects of kisspeptin on insulin secretion in response to differing glucose concentrations could be assessed.

MMTTs facilitated the assessment of kisspeptin on appetite and food intake as well as GSIS in the same cohort. Thus the effect of kisspeptin on several metabolic parameters could be assessed simultaneously, ensuring more efficient use of resources and reducing inconvenience to volunteers.

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3.4.3.3 LIMITATIONS

This study was carried out in healthy young men with a narrow BMI range and therefore it may not be generalisable to other populations, such as women, people aged above 50years and people who are either underweight or overweight. Additionally, the effects of kisspeptin on metabolic parameters was measured during acute kisspeptin infusions, and different results might be obtained with longer kisspeptin administration.

The dose of kisspeptin used in this study produced plasma kisspeptin levels which are exponentially higher than those found in men and non-pregnant women, therefore the physiological role of kisspeptin on metabolism in men and non-pregnant women cannot be directly inferred from the results obtained but it might provide insight into the metabolic roles of kisspeptin in pregnant women.

3.4.3.4 THERAPEUTIC IMPLICATIONS

Kisspeptin infusion did not change baseline (i.e. pre-glucose load and pre-meal) and physiological range (i.e. post-meal) glucose and insulin levels, which provides reassurance of the safety of kisspeptin-based medications, which are being developed for treatment of malignant and reproductive disorders. Additionally, the enhancement of GSIS and DI when circulating glucose concentrations were high, raises the possibility that kisspeptin-based therapies could be investigated for the management of hyperglycaemic conditions. Since the data presented in this thesis is the first human study of the metabolic effects of kisspeptin, further studies are required to confirm the results and explore the effects of kisspeptin in other population groups.

129

3.4.3.5 CONCLUSIONS

An acute infusion of 1nmol.kg-1.hr-1 kisspeptin-54 increases glucose-stimulated insulin secretion in healthy young men during IVGTTs, increases IVGTT disposition index and does not affect appetite or food intake. This novel data contributes to our understanding of the interactions between reproductive systems and metabolic systems mediated by kisspeptin, and help guide the development of kisspeptin-based treatments.

130

4 Chapter 4 – General Discussion and Conclusions

4.1 Discussion

Energy homeostasis requires a delicate balance between energy intake and energy expenditure. Numerous factors influence energy intake and energy expenditure, and hormones produced centrally and/or acting centrally play a major role in the control of energy homeostasis. In this thesis, the metabolic effects of two major neuroendocrine systems (i.e. glucocorticoids and kisspeptin) were investigated.

Glucocorticoids are secreted by the adrenal glands and are converted to their active forms via catalysis by the enzyme 11βHSD1. Published reports suggest that tissue specific activity of

11βHSD1 is responsible for the adverse metabolic effects of glucocorticoids. Since glucocorticoids are required for normal development, post-pubertal rats were used in the experiments, which investigated the effects of reduced active glucocorticoids in the ARC. Long term feeding studies demonstrated that a reduction in ARC active glucocorticoids (via rAAV- mediated knockdown of 11βHSD1) resulted in less weight gain than controls on normal chow diet but resulted in higher average daily food intake and a trend towards higher weight gain than controls on HFD. These results highlight that fact that appetite regulation and therefore the control of bodyweight is complex as different diets may produce different phenotypes.

This may be due to the fact that ingestion of HFD results in stimulation of central orexigenic pathways (Wei et al., 2015).

Energy expenditure is the other key component of energy homeostasis and glucocorticoids decrease BAT thermogenesis in rats (Strack et al., 1995). A reduction in active glucocorticoids in the ARC may have increased energy expenditure on a normal chow diet as the 11βHSD1 131 knockdown group had matched food intake to controls but less weight gain. However, direct measures of different components of energy expenditure are required to conclusively determine the effect of ARC 11βHSD1 on energy expenditure in rats. Infrared thermography was used to determine if there were differences in BAT thermogenesis between the 11βHSD1 knockdown and control groups. However no differences were detected in this component of energy expenditure in the normal chow cohort. This might be due to insufficient sensitivity of this method, or BAT activation in both knockdown and control rats due to stress caused by moving them from the room they were housed in to a procedure room prior to infrared thermography (Gartner et al., 1980) (although they remained in their cages). However, a difference in BAT thermogenesis using infrared thermography was detected in the HFD cohort, which make the above explanations less likely.

Elevated glucocorticoids increase adiposity and therefore a reduction in active glucocorticoids would be expected to result in lower adiposity. However, no differences in percentage body fat were detected between ARC 11βHSD1 knockdown and control animals fed a normal chow diet or a HFD, despite a difference in body weight between the two groups. This may be due to the fact that selective knockdown of 11βHSD1 (and therefore selective reduction in active glucocorticoids) in the ARC may not be sufficient to produce differences in body composition, and knockdown within adipose tissue may be required to reduce adiposity.

Kisspeptin is produced in the hypothalamus, amygdala, liver, placenta and pancreas. Its effects on the reproductive system have been extensively investigated. As detailed in Chapter

3, it has been recognised that metabolic derangement (e.g. obesity, underweight, starvation, hyperglycaemia) suppresses kisspeptin production and kisspeptin itself influences metabolic processes including appetite, body weight and insulin secretion in rodents. In this thesis, the

132 effect of kisspeptin administration in humans on GSIS under hyperglycaemic and euglycaemic conditions were investigated. The results demonstrated that supraphysiological kisspeptin levels increase GSIS under hyperglycaemic (but not euglycaemic) conditions. Therefore, kisspeptin-based therapies may have metabolically beneficial effects and are unlikely to cause hypoglycaemia, as elevated glucose levels are required for kisspeptin-mediated augmentation of insulin secretion. Additionally, the plasma levels of kisspeptin produced by kisspeptin administration in this thesis are similar to plasma kisspeptin levels in human pregnancy (Horikoshi et al., 2003) and kisspeptin levels are lower in pregnant women with diabetes compared to pregnant women who do not have diabetes (Cetkovic et al., 2012).

Therefore, the high kisspeptin levels in pregnancy may serve to enhance insulin secretion and thus be a component of glucose homeostasis during pregnancy.

Kisspeptin enhancement of GSIS appears to occur via a direct action on pancreatic β-cells as pancreatic β-cells have kisspeptin receptors (Hauge-Evans et al., 2006) and kisspeptin increases GSIS in vitro in human islets (Hauge-Evans et al., 2006). Additionally, incretins such as GLP-1 did not contribute to GSIS in this study. GLP-1 is produced by intestinal L-cells in response to alimental nutrient ingestion and kisspeptin receptors are found on intestinal cells.

However, levels of the endogenous insulin secretagogue GLP-1 did not increase during the

IVGTTs performed in this project.

Glucagon, is another gut hormone, which has been shown to stimulate hepatic kisspeptin production, which in turn reduces GSIS in mice (Song et al., 2014). However, in this study in humans, glucagon levels were not altered by kisspeptin administration and therefore, glucagon may not be involved in kisspeptin-mediated GSIS in humans.

133

Animal studies have demonstrated that central kisspeptin administration reduces appetite in male rats while peripheral kisspeptin administration does not affect appetite in male rats.

Furthermore, female kisspeptin receptor knockout mice have reduced food intake compared to ovariectomised controls, while male kisspeptin receptor knockout mice have similar food intake to gonadectomised controls (Tolson et al., 2014). In this thesis, acute peripheral kisspeptin administration did not influence appetite or food intake in healthy men. Kisspeptin may influence appetite differently in different sexes, and this is worthy of further explanation.

4.2 Summary of Future Work

ARC-specific knockdown of 11βHSD1 in adult rats produces lower body weight on normal chow diet despite matched food intake compared to control rats. To determine whether this effect occurs via increased energy expenditure, the experiments should be repeated with measurement of activity, oxygen consumption and respiratory quotient using comprehensive laboratory animal monitoring systems. Additionally, it will be necessary to collect and analyse faeces to determine whether the lower body weight in the knockdown group is due to increased excretion of calories.

In contrast to chow diet, ARC-specific 11βHSD1 knockdown animals had a higher average daily intake of food on a HFD with a corresponding trend towards increased body weight compared to control animals. It is important to determine the mechanism by which this occurs, as this may indicate why 11βHSD1 inhibitors have limited efficacy with regards to weight reduction

134 in human trials. RNA sequencing of the ARCs of the underexpression/overexpression/GFP cohort is planned in collaboration with Dr Giles Yeo (Cambridge University), who has significant expertise of laser-dissection and RNA sequencing. This will help determine which appetite-regulating hormonal RNA is modulated following ARC 11βHSD1 alteration at a timepoint prior to when significant divergence in body weight between groups occurs. It may also provide other valuable information about changes in RNA expression produced by ARC

11βHSD1 underexpression and/or overexpression. Additionally, this experiment may be repeated, with animals fed a HFD, to compare the differences in RNA expression produced by different diets following changes in ARC 11βHSD1 expression.

Other experiments can be performed to determine if the difference in body weight produced by 11βHSD1 knockdown in the ARC is produced via altered sensitivity to the effects of ARC neuropeptides known to influence energy expenditure. To do this, at the time of stereotactic surgery, an indwelling cannula will need to be implanted into the ARC. One month post- surgery α-MSH can be injected into the ARC and food intake as well as energy expenditure measured. At 2-weekly intervals, this can be repeated using AgRP, leptin and other appetite modulators.

Kisspeptin increases GSIS in healthy men only when the circulating glucose concentrations are elevated. Therefore, it would be important to assess the effect of kisspeptin on GSIS on populations, which have elevated glucose levels such people with impaired glucose tolerance, impaired fasting glycaemia, gestational diabetes and people with diabetes mellitus. This may provide data, which may lead to additional therapeutic applications for kisspeptin-based treatments.

135

In this study, an acute infusion of kisspeptin was used to assess the effect of this hormone on

GSIS. The next phase of investigation would involve longer periods of kisspeptin administration using subcutaneous pump delivery of kisspeptin to enable the volunteers carry on with their usual activities and not be confined to a clinical research facility. It would be important to determine if the enhancement of GSIS persists after chronic kisspeptin administration. Additionally, although in this thesis kisspeptin did not affect appetite in men, in light of the results from studies in female rodents, the effects of kisspeptin on appetite in women should be investigated using similar protocols.

136

4.3 Conclusions

In this thesis, key aspects of the neuroendocrine control of appetite, food intake, energy expenditure, body weight and glucose metabolism have been investigated using a variety of techniques. Reduction of active glucocorticoids in the ARC has been shown to significantly influence food intake and body weight, both on normal chow and HFD, in male rats. In humans, acute kisspeptin administration has been shown to increase glucose-stimulated insulin secretion during IVGTTs, whilst glucose levels, insulin secretion, appetite and food intake are unaffected by acute kisspeptin administration. Thus, this work has provided greater insights into the influence of these hormones on mammalian control of energy homeostasis.

Further work is required to elucidate the mechanisms by which these hormones exert their metabolic effects.

137

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6 Appendix

6.1 Screening Proforma

Screen Number:

Screen Date:

Issues:

ADMINISTRATION: Photocopy consent form:  Enrolled Yes /No: File original consent form with this screening proforma in hospital notes:  Place photocopy of consent form in Trial Master File:  Study ID: Send letter to GP:  Screening performed by:

PRINT NAME: ......

SIGNATURE: ......

DATE: ......

FOR COMPLETION ONCE RESULTS ARE AVAILABLE: This subject meets all inclusion and exclusion criteria: Yes No Eligibility confirmed by: PRINT NAME: ......

SIGNATURE: ......

DATE: ......

159

Occupation: Phone numbers:

Address: E-mail address:

GP Name/Address/Telephone No:

Ethnicity: WHITE BLACK OR BLACK BRITISH British Caribbean Irish African Any other White background Any other Black background

ASIAN OR ASIAN BRITISH OTHER ETHNIC GROUPS Indian Chinese

Pakistani Any other ethnic group Bangladeshi Please specify: Any other Asian background I do not wish to disclose my ethnicity group MIXED

White & Black Caribbean

White & Black African

White & Asian

Any other mixed background

Bank account in own name? Yes/No Availability? Previous research participation including dates: Weight change in last 3 months (give details)? Donated blood during the last 3 months or intention to do so before the end of the study? Yes/No 160

Details of any problems with libido/erectile dysfunction? Current medical conditions (please circle all that apply and write details of all conditions):

Asthma/hayfever /eczema Diabetes Epilepsy Heart Anorexia/Bulimia Claustrophobia Pregnancy/Breastfeeding Past medical history: Gonadectomy Chemotherapy Radiotherapy Pituitary conditions/surgery Psychiatric history: Drugs (inc. Prescription, OTC, vitamins, OCP, alternative/traditional/herbal medicine): Hormonal contraception Hormone replacement Steroids ______Allergies:

Smoker? (exclude if nicotine in ≤3months) Never: Yes/No Current: Yes/No Smokes what? ...... months/years since giving up Electronic cigarettes? Yes/No Nicotine replacement therapy Yes/No

Alcohol & caffeine intake: (exclude if alcohol ≥40units/week; caffeine intake ≥6cups or 2cans/day) Alcohol: units / week Tea: cups/day Chinese tea: cups/day Coffee: cups/day Caffeine-containing energy/soft drinks: cans/day Recreational drug use? Yes/No (exclude recreational drug users) Description: Cannabis Benzos

161

Opiates Cocaine Meth Ecstasy Amphetamines Legal highs Family history: IHD Stroke Epilepsy

Review of systems (please circle all that apply): General: CVS: Respiratory: GI tract: GU: Neuro: weight chest pain cough abdo pain dysuria fits change fatigue S.O.B. sputum diarrhoea discharge faints appetite palpitations wheeze constipation haematuria falls sleep ankle haemoptysis vomiting nocturia headache swelling night haematemesis migraine sweats aches & melaena visual loss pains stiffness PR blood diplopia rashes dysphagia weakness paraesthesia numbness

162

Please circle the response that applies:

Do you make yourself sick because you feel Yes No uncomfortably full?

Do you worry you have lost control over how much Yes No you eat?

Have you recently lost more than one stone (6.35 kg) in a Yes No 3-month period?

Do you believe yourself to be fat when others say you Yes No are too thin?

Would you say that food dominates your life? Yes No

Score

Investigator Comments: ……………………………………………………………………………………………

Exclude if more than 2 Yes answers

163

1. If you have put on weight, do you eat less than you usually do? Never Seldom Sometimes Often Very Often 2. Do you try to eat less at mealtimes than you would like to eat? Never Seldom Sometimes Often Very Often 3. How often do you refuse food or drink offered because you are concerned about your weight? Never Seldom Sometimes Often Very Often 4. Do you watch exactly what you eat? Never Seldom Sometimes Often Very Often 5. Do you deliberately eat foods that are slimming? Never Seldom Sometimes Often Very Often 6. When you have eaten too much, do you eat less than usual the following days? Never Seldom Sometimes Often Very Often 7. Do you deliberately eat less in order not to become heavier? Never Seldom Sometimes Often Very Often 8. How often do you try not to eat between meals because you are watching your weight? Never Seldom Sometimes Often Very Often 9. How often in the evening do you try not to eat because you are watching your weight? Never Seldom Sometimes Often Very Often 10. Do you take into account your weight with what you eat? Never Seldom Sometimes Often Very Often

Total Average

Investigator Comments: …………………………………………………… Exclude if average score >2.5

164

Height: ...... metres Pulse: ...... bpm

Weight: ...... kg BP: ...... mmHg

BMI: ...... kg/m2 Temp: ...... °C

Attach body composition printout

CVS: HS: I II I Peripheral pulses:

JVP:

Respiratory: Abdomen:

CNS: Not formally assessed. Grossly intact? 

SKIN:

VEINS (a green cannula is required in each antecubital fossa for this study.) Antecubital fossae? Right: Left: Dorsum of Hand? Right Left:

1Access2-LEAD will ELECTROCARDIOGRAM: be (please circle all that apply): EASY / POSSIBLE / DIFFICULT Heart PR QRS QT QTcB rate interval duration interval interval (bpm) (msec) (msec) (msec) (msec)

165

CLINICAL LABORATORY TESTS: Date:

WBC · Sodium RBC · Potassium · Hb · Chloride Haematocrit · Urea · MCV · Creatinine MCH · Bicarbonate · MCHC · ALT RDW · ALP Platelets Bilirubin MPV · Calcium · Corr Neutrophils · · Calcium Lymphocytes · Phosphate · Monocytes · Albumin Total Eosinophils · Protein Basophils · AST HbA1c . Gamma GT Prolactin . Urate · LH . TSH · FSH . fT3 · E2 . fT4 · Progesterone . Total Chol . SHBG . LDL . Testosterone . HDL . TG . Glucose . Insulin . HbA1c .

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6.2 Taste Test

Food tasted

Spaghetti Mushroom

Bolognese Risotto

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6.3 Visual Analogue Scale

How hungry do you feel right now?

NOT AT ALL EXTREMELY

How sick (or nauseous) do you feel right now?

NOT AT ALL EXTREMELY

How pleasant would it be to eat right now?

NOT AT ALL EXTREMELY

How much do you think you could eat right now?

NOTHING A LARGE AMOUNT

How full do you feel right now?

NOT AT ALL EXTREMELY

How sleepy do you feel right now?

NOT AT ALL EXTREMELY

AFTER MEAL ONLY: How tasty was the meal?

NOT AT ALL EXTREMELY

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7 Publications and Communications

7.1 Izzi-Engbeaya and Comninos et al. - Manuscript under review at Diabetes

The Effects of Kisspeptin on β-cell Function, Metabolite Profile and Appetite in

Humans

Short Running Title: Effects of Kisspeptin on Metabolism in Humans

Chioma Izzi-Engbeayaa,1, Alexander N. Comninos,a,1,2, Sophie Clarke1, Anne Jomard1, Lisa

Yang1, Sophie Jones1, Ali Abbara1, Shakunthala Narayanaswamy1, Pei Chia Eng1, Debbie

Papadopoulou1, Julia K. Prague1, Paul Bech1, Ian F. Godsland3, Paul Bassett4, Caroline

Sands5, Maria Gomez Romero5, Jake T. M. Pearce5, Matthew R. Lewis5, Elaine Holmes5,

Jeremy K. Nicholson5, Tricia Tan1, Risheka Ratnasabapathy1, Ming Hu6,7, Gaelle Carrat6,7,

Lorenzo Piemonti8,9, Marco Bugliani10, Piero Marchetti10, Paul R. Johnson11,12,13, Stephen J.

Hughes11,12,13, Guy A. Rutter6,7, Waljit S. Dhillo1,*

aThese authors contributed equally.

*Corresponding author.

1Section of Endocrinology and Investigative Medicine, Division of Diabetes, Endocrinology

and Metabolism, Department of Medicine, Imperial College London, London, W12 0NN,

UK.

2Department of Endocrinology, Imperial College Healthcare NHS Trust, London, W12 0HS,

UK.

3Section of Metabolic Medicine, Imperial College London, St Mary’s Hospital, London, W2

1NY, UK.

169

4Statsconsultancy Ltd, 40 Longwood Lane, Amersham, Buckinghamshire, HP7 9EN, UK.

5The MRC-NIHR National Phenome Centre and Imperial BRC Clinical Phenotyping Centre,

Division of Computational, Systems and Digestive Medicine, Department of Surgery and

Cancer, South Kensington Campus, London, SW7 2AZ, UK.

6Section of Cell Biology and Functional Genomics, Division of Diabetes, Endocrinology and

Metabolism, Department of Medicine Imperial College London, London, W12 0NN, UK.

7Imperial Pancreatic Islet Biology and Diabetes Consortium, Hammersmith Hospital,

Imperial College London, London, UK.

8Diabetes Research Institute (SR-DRI), IRCCS San Raffaele Scientific Institute, Milan, Italy

9Vita-Salute San Raffaele University, Milan, Italy.

10Department of Clinical and Experimental Medicine, Islet Cell Laboratory, University of

Pisa, 56126 Pisa, Italy.

11Nuffield Department of Surgical Sciences, University of Oxford, Oxford, UK.

12Oxford Centre for Diabetes, Endocrinology, and Metabolism, University of Oxford,

Oxford, UK.

13National Institute of Health Research Oxford Biomedical Research Centre, Churchill

Hospital, Oxford, UK.

Correspondence to:

Prof Waljit S. Dhillo,

Section of Endocrinology and Investigative Medicine,

Division of Diabetes, Endocrinology and Metabolism,

170

Department of Medicine,

Imperial College London,

London,

W12 0NN, UK.

+44 (0)20 3313 3242

[email protected]

Abstract: 193 words, Manuscript: 3548 words, References: 50, Tables: 0, Figures: 4

171

Abstract

Reproduction and metabolism are inter-dependent, but our understanding of the hormones which regulate both these systems is currently limited. Kisspeptin has well-established crucial roles in mammalian reproduction, and recent reports in animal models suggest kisspeptin may have additional roles in metabolism. However, the effects of kisspeptin on metabolism in humans are as yet unknown. We therefore designed a study to address this for the first time in humans.

In 15 healthy men (age 25.2±1.1years; BMI 22.3±0.5kg.m-2), we compared the effects of

1nmol.kg-1.hr-1 kisspeptin versus vehicle administration on glucose-stimulated insulin secretion, metabolites, gut hormones, appetite and food intake. In addition, we assessed the effect of kisspeptin on glucose-stimulated insulin secretion in vitro in human pancreatic islets.

Kisspeptin administration to healthy men enhances insulin secretion following an intravenous glucose load, and modulates serum metabolites. In keeping with this, kisspeptin dose- dependently increases glucose-stimulated insulin secretion from human islets in vitro. In addition, kisspeptin administration does not alter gut hormones, appetite or food intake in healthy men. Collectively, these data demonstrate a beneficial role for kisspeptin on insulin secretion, which has important translational implications for the ongoing development of kisspeptin-based therapies for reproductive (and potentially metabolic) conditions.

172

Reproduction and metabolism are fundamental aspects of mammalian physiology, which are intricately linked. However, our understanding of the hormonal links between these two fundamental biological systems is currently limited. Recent evidence in animals suggests that the recently discovered hormone kisspeptin may link reproduction and metabolism, however until now human in vivo studies have not been performed.

Kisspeptin sits at the apex of the hypothalamo-pituitary-gonadal axis, controlling downstream reproductive hormone secretion. Kisspeptin has pivotal roles in fertility (Pineda et al., 2010), which are capitalised on by kisspeptin-based therapies now in development for common reproductive disorders (MacLean et al., 2014, Jayasena et al., 2014a, Jayasena et al., 2014b,

Millar et al., 2017, Comninos et al., 2017). Interestingly, kisspeptin (KISS1) and its receptor

(KISS1R) are also expressed in the hypothalamus, pancreatic β-cells, liver and adipose tissue, suggesting roles in key metabolic processes (Ohtaki and Niwa, 2001, Muir et al., 2001, Hauge-

Evans et al., 2006, Brown et al., 2008, Silvestre et al., 2008). Kisspeptin has recently been reported to increase glucose-stimulated insulin secretion (GSIS) in rats and monkeys (Bowe et al., 2009, Wahab et al., 2011), and this is supported by in vitro islet studies which demonstrate enhancement of GSIS by kisspeptin (Hauge-Evans et al., 2006, Bowe et al., 2009, Bowe et al.,

2012, Song et al., 2014). Interestingly, it has been reported that kisspeptin can also inhibit GSIS at lower kisspeptin (Song et al., 2014) and glucose concentrations (Vikman and Ahren, 2009), but stimulate GSIS at high kisspeptin concentrations (Song et al., 2014).

Furthermore, there are neuroanatomical and functional connections between kisspeptin and hypothalamic neuropeptides known to be important in appetite regulation (Backholer et al.,

2010, Fu and van den Pol, 2010, Padilla et al., 2017a). Intracerebroventricular (ICV) administration of kisspeptin has been reported to alter food intake in mice (Stengel et al., 2011) and chicks (Khan et al., 2009), whilst other studies report no effect on appetite in rodents

(Castellano et al., 2005, Thompson et al., 2006). In addition, impaired kisspeptin signalling

173 disrupts metabolism and promotes glucose intolerance and obesity in mice (Tolson et al.,

2014).

Thus, data in animals strongly suggests that kisspeptin plays a role in metabolism, but the effects of kisspeptin on metabolic parameters in vivo in humans are currently unknown. There is therefore an important clinical need to elucidate the metabolic effects of kisspeptin in humans, to further our understanding of the physiology of human metabolism, as well as to inform the ongoing development of kisspeptin-based therapies.

In this study, we demonstrate that in humans, administration of kisspeptin stimulates GSIS in vivo and in vitro and increases disposition index, as well as modulating serum metabolites.

Furthermore, kisspeptin administration did not affect appetite or food intake. These data provide new conceptual insights into the metabolic actions of the reproductive hormone kisspeptin in humans with significant translational implications relating to glucose homeostasis.

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Research Design and Methods

Human Studies

STUDY PARTICIPANTS

This study was reviewed and approved by the West London Research Ethics Committee

(16/LO/0391), and was carried out in accordance with the Declaration of Helsinki. Healthy men (aged 18-40 years) were recruited using online and print advertisements. Written informed consent was obtained from each participant prior to study enrolment. Exclusion criteria included: body mass index (BMI) <18.5 or >25kg.m-2, history of medical and psychological conditions, use of prescription, recreational or investigational drugs within the preceding 2 months, blood donation within 3 months of study participation, ingestion or inhalation of nicotine-containing substances, alcoholism, and history of cancer.

Participants were instructed to abstain from strenuous exercise, alcohol, and caffeine for 24 hours preceding each study visit. Each participant was instructed to choose a meal and eat that same meal at 8pm on the night preceding each study visit, fast overnight and attend the study visit fasted. Each participant underwent two intravenous glucose tolerance tests (IVGTTs, one with kisspeptin and one with vehicle administration) and/or two mixed meal tolerance tests

(MMTTs, one with kisspeptin and one with vehicle administration).

INFUSIONS

Infusion order was randomised using a random number generator, and participants were blinded as to the infusion identity. Kisspeptin infusions were prepared by dissolving kisspeptin-

54 (Bachem, St Helens) in 1ml of 0.9% NaCl (Braun, Sheffield) and adding the kisspeptin solution to 49ml Gelofusine (Braun, Sheffield). Kisspeptin was infused at a rate of 1nmol.kg-

1.hr-1, a dose established to be bioactive (Comninos et al., 2017). Vehicle infusions consisted

175 of Gelofusine (Braun, Sheffield), administered at the equivalent rate to the kisspeptin infusion for each participant.

IVGTT

On arrival at the Clinical Research Facility (CRF), and after a period of acclimatisation, two intravenous cannulae (one in each antecubital fossa) were inserted (one for blood sampling and the other for intravenous infusion administration). Following baseline sampling, kisspeptin or vehicle infusion was started at T=0mins and infused until T=225mins. 0.3g.kg-1 of 20% dextrose (Hameln, Gloucester) was administered intravenously over 120 seconds starting from

T=45mins (when kisspeptin levels had reached steady state, Figure 1A and 1B). To obtain glucose and insulin values required for calculation of acute insulin response to glucose (AIRg) and minimal model insulin sensitivity index (Si), an established frequent sampling protocol

(Tan et al., 2014) was used as shown in Figure 1A. AIRg was calculated as the incremental

AUC using the trapezoid rule (Matthews et al., 1990) of insulin from T=45mins to T=55mins

(i.e. 0 to 10mins post-glucose load). Si was determined using the minimal model (MLAB software) (Bergman et al., 1981) and the disposition index (IVGTT-DI) was calculated as the product of AIRg and Si (Tan et al., 2014). Blood was taken for measurement of glucose, insulin, kisspeptin, luteinising hormone (LH), testosterone, glucagon-like peptide-1 (GLP-1) and glucagon.

MMTT

A taste test was performed prior to the MMTT study visits to determine the study meal

(Spaghetti Bolognese 125kcal.100g-1 or Mushroom Risotto 124kcal.100g-1; both from

Waitrose, Bracknell) for each participant. These meal options were selected as they provided

176 similar kcal.weight-1. Following acclimatisation and baseline sampling, kisspeptin or vehicle infusion was started at T=0mins and continued until T=120mins (Figure 4A).

Visual analogue scales to measure participants self-reported hunger were performed at T=-

30mins, T=30mins and T=75mins. Forty-five minutes into the infusion (i.e. when kisspeptin levels had reached steady state) (Figure 4B) an ad libitum meal was given to participants. They were instructed to eat until comfortably full, with all clocks, electronic devices and reading material taken away whilst the participants were eating, and researchers left the room (to minimise distractions which are known to influence meal size and duration). Blood was taken for measurement of glucose, insulin, kisspeptin, LH and testosterone.

Calculations were made using established methods as follows: MMTT-ISI as the ratio of AUC insulin to AUC glucose, MMTT-IS using the Matsuda index (with T=45mins values as baseline and mean insulin and glucose values from T=65-120mins used in the equation), and MMTT-

DI as the product of MMTT-ISI and MMTT-IS (Maki et al., 2011).

METABOLIC PROFILING

Blood samples for metabolic profiling were taken at pre-infusion (T=-15mins) and at steady state prior to intravenous glucose or meal ingestion (T=45mins), as shown in Figures 1A and

4A. Serum sample handling (sorting, formatting, aliquotting) and preparation were performed as previously described (Lewis et al., 2016). Modification from the reported sample aliquotting and preparation protocols can be found in the supplemental information. Serum was analysed using ultra-performance liquid chromatography coupled to mass spectrometry (UPLC-MS). A reverse-phase chromatographic separation tailored to complex lipid retention and separation was used to profile the complex lipid species present in the sample, while hydrophilic interaction liquid chromatography (HILIC) was used to retain and separate polar metabolites

177

(Lewis et al., 2016). High resolution orthogonal acceleration time-of-flight mass spectrometry

(oaTOF-MS) operating in the positive ion mode was used for both assays.

All UPLC-MS analyses were performed on Acquity UPLC instruments, coupled to Xevo G2-

S oaTOF mass spectrometers (Waters Corp., Manchester, UK) via Zspray electrospray ionisation (ESI) source. Further details of the analytical methods used can be found in the supplemental information. Feature extraction and retention time alignment were performed in

Progenesis QI (Waters Corp. Milford, MA, USA). In-house scripts (Python software) were developed for the elimination of potential run-order effects and noise filtering. Linear mixed effect models were generated using the lmer4 R software package (Bates et al., 2015) according to the formula: model <- Feature ~ Time*Class + (1|SubjectID) + (1|Challenge). A model was generated for each feature including fixed effects for interaction between class (kisspeptin or vehicle alone) and time (T=-15mins and T=45mins), and allowing for both participant and challenge-specific random effects (owing to the presence of multiple challenges per participant). Features showing significant differences between the two time-points for vehicle and/or kisspeptin classes were identified by false discovery rate correction of the appropriate model estimates using the locfdr package (Efron et al., 2015). Subsequently assignment of significant features in the kisspeptin class alone (FDR α=0.05) was performed. Where significant features were found to be adduct or fragment ions, the full spectrum (including the protonated molecule) was deduced and used in molecular assignment. Chemical identity was assigned by matching accurate mass and tandem mass spectrometry (MS/MS) fragmentation

(of the protonated molecule) measurements to reference spectra using LIPID MAPS online tools (for lipid species) (Fahy et al., 2007) or an in-house database constructed from analysis of authentic reference materials (for 5'-Methylthioadenosine). Where authentic reference materials were commercially available, they were used to generate definitive molecular identification by direct matching of chromatographic and spectral qualities (including accurate 178 mass, MS/MS spectra, and isotopic distribution) to those observed in the profiling data and subsequent targeted MS/MS experiments.

BIOCHEMICAL ANALYSES

Plasma kisspeptin and gut hormone levels were measured using established in-house radioimmunoassays (kisspeptin intra-assay CV 8.3% and inter-assay CV 10.2%, GLP-1 intra- assay & inter-assay CV: ≤10%, glucagon intra-assay & inter-assay CV: ≤10%) (Kreymann et al., 1987, Ghatei et al., 1983b, Dhillo et al., 2005). Serum insulin, plasma glucose, serum LH and serum testosterone were measured in the Clinical Chemistry Laboratory of Imperial

College Healthcare NHS Trust on the automated Abbott Architect® platform.

Chemiluminescent immunoassays were used to measure serum insulin (intra-assay & inter- assay CV: ≤7%), serum LH (intra-assay & inter-assay CV: ≤5%) and serum testosterone (intra- assay & inter-assay CV: ≤8%). Plasma glucose was measured with a colorimetric hexokinase assay (intra-assay & inter-assay CV: ≤2%).

In vitro Studies

HUMAN ISLET CELL CULTURE

Preparations from four different donors (see Table S1) were used to perform insulin secretion experiments. Islets (10/well) were incubated in triplicate for each condition in a 12-well non- treated cell culture plate. As previously described (Hodson et al., 2013), insulin secretion assays were performed in Krebs-Ringer-Hepes-Bicarbonate (KRHB) buffer (10mM Hepes, 2mM

NaHCO3, 140mM NaCl, 3.6mM KCl, 0.5mM MgSO4, 0.5mM NaH2PO4, 1.5mM CaCl2) saturated with 95% O2/5% CO2 and adjusted to pH 7.4. Islets were pre-incubated in a 37˚C water bath under agitation for 1hour in 3mM glucose KRHB, then prior to the secretion assay

179

(for 30mins) in KRHB, 3mM or 17mM glucose, in presence of different concentrations of kisspeptin-54 (0nM, 2.7nM or 1000nM). The 2.7nM dose was selected as this is similar to plasma kisspeptin levels produced by 1nmol.kg.hr-1 kisspeptin administration in our in vivo study (i.e. 2-3nM during kisspeptin steady state – see Figure 1B). The 1000nM dose was selected as this was similar to the dose of kisspeptin used in other in vitro studies (1000nM)

(Hauge-Evans et al., 2006, Bowe et al., 2009, Song et al., 2014). The supernatant was collected and the islets were lysed in 1mL of acidified ethanol (75% ethanol, 23.5% H2O, 1.5% 1M HCl,

0.1% Triton) and sonicated two times for 10s, to extract total islet insulin content. Insulin concentration was measured using an ultrasensitive HTRF kit (Cisbio Bioassays, Codolet), and secreted insulin was normalised as percentage of total insulin content.

Power calculation, quantification, and statistical analysis

Using STATA, an a priori power calculation was performed using a dataset of IVGTTs performed in 99 healthy men aged 18-40years, provided by IFG. Using this dataset, a study consisting of IVGTTs performed in 15 healthy men would have 80% power to detect a 25% difference in insulin secretion.

Statistical analyses were performed with the assistance of a statistician (PBa). Unless otherwise stated, statistical analysis was performed using Prism (GraphPad, California) and data are presented as mean±SEM. Paired t-tests were performed on parametric data, Wilcoxon matched-pairs signed rank tests were performed on non-parametric data and 2-way ANOVA with Dunnett’s multiple comparison tests were performed for comparison of >2 groups. Multi- level linear regression modelling was performed on insulin and glucose curves. Generalised estimating equation (GEE) was performed on other non-independent longitudinal data using

STATA (Statacorp, Texas). Statistical significance was set at p<0.05.

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Results

Elevated circulating kisspeptin enhances insulin secretion during intravenous glucose challenges in healthy men

Kisspeptin administration resulted in elevated circulating kisspeptin levels (Figure 1B) which, as expected, resulted in elevated luteinising hormone (LH) levels, confirming peptide bioactivity (Figure S1A). Consistent with previous studies, testosterone levels did not rise during the time-period of kisspeptin administration (Figure S1B) (George et al., 2013,

Comninos et al., 2017).

A 0.3g.kg-1 intravenous glucose load was administered at T=45mins, a time point at which circulating kisspeptin levels had reached steady state (Figure 1B). In response to the intravenous glucose load, kisspeptin administration significantly enhanced GSIS compared to vehicle (Figure 1C). Two-level linear regression modelling confirmed that mean post-glucose load insulin levels were 4.1µU.mL-1 higher (95% CI: 0.9 to 7.3; p=0.01) during kisspeptin compared with vehicle administration. Glucose levels were elevated similarly in both groups following the intravenous glucose load (Figure 1D) and insulin sensitivity index (Si) was similar between groups (Si: kisspeptin 8.11±0.98 vs vehicle 6.85±0.89, p=0.1228).

The disposition index (DI) is a well-established method for assessing β-cell function as it is comprised of measures of both insulin secretion and insulin sensitivity (Lorenzo et al., 2010).

Kisspeptin elicited a significantly higher IVGTT-DI compared to vehicle (IVGTT-DI: kisspeptin 2768±484 vs vehicle 2061±255, p<0.05, Figure 1E).

Additionally, kisspeptin administration did not alter glucagon-like peptide-1 (GLP-1) (Figure

S1C) or glucagon secretion during IVGTT (Figure S1D).

Kisspeptin dose-dependently stimulates GSIS in human islets 181

In donor human islets, kisspeptin elicited dose-dependent increases in insulin secretion (Figure

2), providing key mechanistic evidence that kisspeptin can stimulate GSIS by direct action on

β-cells. Of note, kisspeptin stimulated human islet insulin secretion (in the presence of elevated glucose levels, Figure 2) at similar kisspeptin concentrations to the circulating kisspeptin levels obtained in our human in vivo IVGTT studies above (i.e. 2-3nM; Figure 1B).

Kisspeptin alters the metabolic profile in humans

Linear mixed effect modelling (Bates et al., 2015) was used to identify metabolites significantly modulated between baseline (T=-15mins) and steady-state (T=45mins) following kisspeptin but not vehicle administration. Annotated metabolites which significantly changed during kisspeptin administration included lysophosphatidylinositol and sphingomyelins (Figure 3 and

Table S4), which have been previously shown to be associated with insulin dynamics (Metz,

1986, Li et al., 2011b).

Kisspeptin does not affect appetite or food intake in humans

During kisspeptin administration, plasma kisspeptin levels were elevated (Figure 4B), which resulted in elevated LH levels (Figure S2A) and no change in testosterone (Figure S2B), similar to the IVGTTs.

Self-reported hunger on a visual analogue scale (VAS, range 0-10cm) were not significantly different during kisspeptin or vehicle administration (hunger VAS scores from baseline to

30mins after the start of the infusion: kisspeptin 1.88±0.63cm vs vehicle 1.69±0.40cm, p=0.8120) (Figure 4C). Hunger VAS scores from T=30mins (pre-meal) to T=75mins (post- meal) decreased similarly during kisspeptin and vehicle administration (change in VAS: kisspeptin -6.02±0.56cm vs vehicle -5.6±0.49cm, p=0.5183). 182

Furthermore, there was no difference in the mean kilocalories (kcal) consumed during the ad libitum meal during kisspeptin and vehicle administration (kisspeptin 844.7±160.9 kcal vs vehicle 834.4±174.8 kcal, p=0.7178) (Figure 4D).

During ad libitum meal ingestion, mean glucose levels achieved were lower than during post- glucose IVGTT as expected (mean glucose level: IVGTT 9.72±0.49 vs MMTT 6.62±0.20, p<0.05), with no associated effect of kisspeptin administration on insulin levels (Figure S2C), glucose levels (Figure S2D) or MMTT-DI (kisspeptin 696.3±159.1 vs vehicle 671.9±145.6, p=0.6286).

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Discussion

This is the first study investigating the effect of kisspeptin on β-cell function and appetite in humans in vivo. We demonstrate that the reproductive hormone kisspeptin enhances GSIS in humans in vivo without affecting insulin sensitivity. Our in vitro data shows a direct effect of kisspeptin on β-cells, which are known to possess abundant kisspeptin receptors (Hauge-Evans et al., 2006). Furthermore, kisspeptin modulates the metabolite profile in humans, but does not influence appetite or food intake. These data indicate that kisspeptin plays a role in glucose homeostasis in humans, and therefore is a hormonal mediator linking reproductive and metabolic systems.

Kisspeptin administration increased insulin secretion and disposition index during IVGTT but not during MMTT. The glucose concentrations were more markedly elevated as expected during the IVGTT (mean peak glucose 14.01mM, Figure 1D) compared to the MMTT (mean peak glucose 7.48mM, Figure S4D). Therefore, this data suggests that kisspeptin increases insulin release when ambient glucose concentrations are high in humans in vivo. In keeping with this, our data shows that in human islets in vitro kisspeptin increases insulin secretion at

17mM but not at 3mM glucose (Figure 2). This is also consistent with previous data in human islets in vitro, which shows that kisspeptin stimulates insulin release at higher ambient glucose concentrations (20mM vs 3mM) (Hauge-Evans et al., 2006, Bowe et al., 2012). However, there are differing reports of the effects of kisspeptin on insulin secretion from animal islets (Hauge-

Evans et al., 2006, Vikman and Ahren, 2009, Bowe et al., 2012, Song et al., 2014). This may reflect methodological differences in the experiments and/or species differences, which are often observed between animal and human islet preparations (Bosco et al., 2010).

Testosterone has also been shown to increase insulin secretion from isolated islets (Grillo et al., 2005). However, the effects of kisspeptin administration on metabolism during our study

184 were not confounded by altered testosterone levels as serum testosterone did not rise during the time-period of this study, because more prolonged kisspeptin administration is required to produce elevations in testosterone levels (George et al., 2013).

The gut hormones, GLP-1 and glucagon have key roles in glucose homeostasis. In addition, in rodent studies, GLP-1 has been shown to alter hypothalamic kisspeptin expression and neuronal activity (Oride et al., 2017, Heppner et al., 2017) and glucagon stimulates hepatic kisspeptin production to alter GSIS (Song et al., 2014). In our study, there was no difference in circulating GLP-1 or glucagon levels following intravenous glucose between kisspeptin and vehicle groups (Figure S1C & D). This is consistent with previous data which shows that intravenous glucose administration alone does not activate the incretin response mediated by gut hormones (Perley and Kipnis, 1967). In addition, the above studies suggest that GLP-1 and glucagon act upstream of kisspeptin, whereas in our study we directly administered kisspeptin. To address this, future studies could assess the effects of GLP-1 and glucagon administration on kisspeptin secretion in humans.

To provide further insights into the metabolic effects of kisspeptin, we compared the metabolite profiles of serum samples taken pre-kisspeptin administration (T=-15mins) to those taken when plasma kisspeptin levels had reached steady-state (T=45mins) and equivalent vehicle timepoints. Importantly, samples were collected before the glucose loads to prevent this from confounding the results. We demonstrate for the first time that kisspeptin modulates serum metabolites. Several of these metabolites (lysophosphatidylinositol and sphingomyelins) are known to be associated with insulin dynamics (Metz, 1986, Kim et al., 2011), and so this provides additional evidence of kisspeptins modulation of human metabolism, which (with further study) may elucidate additional pathways based on these profiles.

185

In light of emerging evidence for neuroanatomical and functional connections between kisspeptin and key appetite-regulating neurones in the hypothalamus (Backholer et al., 2010,

Fu and van den Pol, 2010), and animal data which suggests a role for kisspeptin in energy homeostasis (Tolson et al., 2014), we investigated the effect of kisspeptin on appetite in healthy men. Our data demonstrates kisspeptin had no effect on appetite and food intake in men. This is in keeping with rodent data showing that kiss1r knockout male mice have unaltered food intake (Tolson et al., 2014).

It is of interest to consider the potential pharmacological and physiological implications of our findings. During IVGTTs, pharmacological kisspeptin administration increased insulin secretion and disposition index (IVGTT-DI). IVGTT-DI quantifies the ability of the β-cell to counter insulin resistance (Lorenzo et al., 2010), with lower baseline IVGTT-DI values independently predict conversion from normal glucose tolerance or impaired glucose tolerance to type 2 diabetes within 5 years (Lorenzo et al., 2010). Therefore, our finding that kisspeptin increases GSIS and IVGTT-DI demonstrates metabolically beneficial effects of kisspeptin.

This is especially important as kisspeptin-based treatments are currently being developed to treat reproductive disorders (MacLean et al., 2014, Jayasena et al., 2014a, Jayasena et al.,

2014b, Millar et al., 2017, Comninos et al., 2017) and such treatments may therefore have additional metabolic therapeutic applications. For example it has been shown that in animals

(Castellano et al., 2006) and humans (George et al., 2013), kisspeptin administration can improve the secondary hypogonadism associated with diabetes. Our finding that kisspeptin administration also improves GSIS suggests that kisspeptin could have a dual therapeutic role in patients with diabetes to improve hypogonadism as well as enhance insulin release, specifically only when hyperglycaemia occurs (thereby potentially avoiding the risks of hypoglycaemia associated with other diabetes treatments). Further studies are needed to test this hypothesis in patients with diabetes and related hypogonadism. 186

Our data demonstrating that pharmacological elevation of circulating kisspeptin levels results in a significant increase in GSIS in humans in vivo may also have potential physiological relevance. Similar high circulating kisspeptin levels (1-10nM) (Horikoshi et al., 2003) are observed in humans physiologically during pregnancy due to placental kisspeptin production

(Horikoshi et al., 2003). Based on these data, it could be hypothesised that the higher circulating kisspeptin levels during normal pregnancy enhance insulin secretion to protect the mother and foetus from increasing glucose levels and the development of gestational diabetes mellitus. In keeping with this hypothesis, pregnant women without diabetes have higher kisspeptin levels than pregnant women with diabetes (Cetkovic et al., 2012). In addition, previous rodent work has shown that selective ablation of kiss1r from pancreatic islet β-cells in pregnant mice results in impaired glucose tolerance and reduced insulin secretion (Hill et al., 2017). Together this data suggests that elevated kisspeptin during pregnancy may play a physiological role in glucose homeostasis and may protect against the development of gestational diabetes mellitus.

Further studies are needed in pregnant patients with diabetes to determine if kisspeptin administration can improve their glucose homeostasis.

Conclusion

Reproduction and metabolism are fundamental and interdependent aspects of mammalian physiology. This study, employing multiple methodological approaches, demonstrates that administration of the reproductive hormone kisspeptin to humans significantly increases GSIS in vivo and in vitro via actions on pancreatic islet cells, with alterations in the metabolite profile.

We also show that kisspeptin does not affect appetite or food intake in healthy men.

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These data provide a conceptual advance in our understanding of the interaction between metabolic and reproductive systems through kisspeptin, as well as informing the ongoing development of kisspeptin-based treatments for common reproductive (and potentially metabolic) disorders.

188

Author Contributions

Conceptualization: C.I-E., A.N.C. and W.S.D.; Methodology: C.I-E., A.N.C. and W.S.D.;

Investigation: C.I-E., A.N.C, S.C., A.J., L.Y., S.J., A.A., S.N., P.C.E., D.P., J.K.P., P.Be., R.R.,

M.H.,G.C., L.P., M.B., P.M., P.R.J. and S.J.H. Formal Analysis: C.I-E., A.N.C., P.Ba., I.F.G.,

M.R.L., J.T.M.P., C.S., M.H. and G.C.; Writing – Original Draft: C.I-E., A.N.C. and W.S.D.;

Writing – Review & Editing: all authors.; Funding Acquisition: W.S.D.; Resources: E.H.,

J.K.N., G.A.R. and W.S.D.

Guarantor Statement

W.S.D. is the guarantor of this work and, as such, had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.

Conflict of Interest Statement

The authors declare they do not have any conflicts of interest.

Funding and Acknowledgments

This article presents independent research supported by the Imperial College Academic Health

Sciences Centre, National Institute of Health Research Clinical Research Facility and

Biomedical Research Centre (NIHR CRF and BRC) at Imperial College Healthcare NHS Trust.

The Section of Endocrinology and Investigative Medicine is funded by grants from the Medical

Research Council, Biotechnology and Biological Sciences Research Council, NIHR, an

Integrative Mammalian Biology Capacity Building Award, an FP7- HEALTH- 2009- 241592

EuroCHIP grant, and is supported by the NIHR BRC Funding Scheme. The National Phenome

189

Centre is supported by the MRC and NIHR (grant number MC_PC_12025). The views expressed are those of the authors and not necessarily those of the MRC, the NHS, the NIHR or the Department of Health. C.I-E. and R.R. are recipients of MRC Clinical Research

Training Fellowships. A.N.C. is supported by the NHS and BRC. S.C. and P.C.E. are supported by funding from an NIHR Research Professorship. A.A. is supported by an NIHR Clinical

Lectureship. L.Y. is a recipient of an Imperial College Healthcare Charity Fellowship. D.P. is supported by NIHR CLRN funding. T.T. is supported by grants from the MRC and Wellcome

Trust. W.S.D is supported by an NIHR Research Professorship (Grant number RP-2014-05-

001). G.A.R. is supported by MRC Programme (MR/J0003042/1; MR/N00275X/1;

MR/L020149/1 [DIVA]), Wellcome Trust Senior Investigator (WT098424AIA) and Diabetes

UK Project (BDA11/0004210; BDA/15/0005275) grants.

The authors thank Drs Philippe Ravassard (Université Pierre et Marie Curie, Paris, France) and

Raphael Scharfmann (INSERM U1016, Cochin Institute, Paris, France) for the provision of

EndoC-βH1 cells as well as Stephane Camuzeaux, David Berry, and Ash Salam for their technical assistance in the identification of metabolites.

190

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Figure 1: Kisspeptin administration enhances β-cell function during IVGTT in humans

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A: Following an overnight fast, 15 healthy men (age 25.2±1.1years; BMI 22.3±0.5kg.m-2) were administered 1nmol.kg-1.hr-1 kisspeptin or rate-matched vehicle for 225mins, in random order.

At T=45mins 0.3g.kg-1 of 20% dextrose was infused intravenously over 2mins. Blood sampling was performed regularly (black arrows), with samples for metabolic profiling collected at T=-

15mins and T=45mins (green stars).

B: Mean plasma kisspeptin levels during intravenous glucose tolerance test (IVGTT), were higher during kisspeptin compared to vehicle administration. Data presented as mean±SEM.

****p<0.0001 kisspeptin vs vehicle using generalised estimating equation (GEE).

C: Mean serum insulin levels during IVGTT were higher during kisspeptin compared to vehicle administration. Data presented as mean±SEM. **p=0.01 kisspeptin vs vehicle (multi-level linear regression).

D: Mean plasma glucose levels during IVGTT were similar during kisspeptin and vehicle administration. Data presented as mean±SEM. p=0.64 kisspeptin vs vehicle (multi-level linear regression).

E: Mean intravenous glucose tolerance test disposition index (IVGTT-DI) was higher during kisspeptin administration. Data presented as mean±SEM. p<0.05 kisspeptin vs vehicle (paired t-test).

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Figure 2: Kisspeptin enhances GSIS in cultured human islets

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216

Figure 3: Kisspeptin modulates the metabolic profile in healthy men

Manhattan plot showing -log10(Q) X sign of direction of change for each of the 5200 mass spectrometry reversed-phase chromatography with positive ion mode detection (MS LPOS) derived features, where Q represents the FDR corrected value of the appropriate linear mixed effect model estimates. Statistical significance for association with kisspeptin administration was determined based on a Q value threshold of 5% and statistically significant features are coloured red and blue if levels are increased or decreased with time respectively. Features successfully annotated are indicated on the plot with abbreviations as follows: LPI: lysophosphatidylinositol; LPC: lysophosphocholine; PC: phosphocholine; SM: sphingomyelin

(see also Table S4).

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Figure 4: Kisspeptin administration does not affect appetite or food intake in healthy men

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218

A: Following an overnight fast, 15 healthy men (age 25.2±1.1years; BMI 22.3±0.5kg.m-2) were administered 1nmol.kg-1.hr-1 kisspeptin or rate-matched vehicle for 120mins, in random order.

At T=45mins 0.3g.kg-1 participants were given an ad libitum meal (which was eaten over a maximum of 20mins). Blood sampling was performed regularly (black arrows), with samples for metabolic profiling collected at T=-15mins and T=45mins (green stars). Visual analogue scales (VAS) for volunteer-reported hunger were performed at T=-30mins, T=30mins and

T=75mins.

B: During mixed meal tolerance test (MMTT), mean plasma kisspeptin levels were higher during kisspeptin compared to vehicle administration. Data presented as mean±SEM.

****p<0.0001 kisspeptin vs vehicle (GEE).

C: Mean change in pre-meal volunteer-reported hunger scores from T=-30mins to T=30mins, as measured with VAS, were similar during kisspeptin and vehicle administration. Data presented as mean±SEM. p=0.8120 kisspeptin vs vehicle (paired t-test).

D: Mean number of kilocalories (kcal) ingested were similar during kisspeptin and vehicle administration. Data presented as mean±SEM. p=0.7178 kisspeptin vs vehicle (paired t-test).

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7.2 Association of Physicians of Great Britain and Ireland Annual Meeting 2018,

Manchester – Abstract accepted for oral presentation

Kisspeptin Increases Glucose Stimulated Insulin Secretion and Changes the Metabonomic

Profile in Humans

C. Izzi-Engbeaya1, A.N. Comninos1, S. Clarke1, A. Jomard1, L. Yang1, S. Jones1, A. Abbara1, S.

Narayanaswamy1, P.C. Eng1, D. Papadopoulou1, J. Prague1, P. Bech1, I.F. Godsland2, P. Bassett3,

C. Sands4, M. Gomez Romero4, J. T. M. Pearce4, M. R. Lewis4, E. Holmes4, J. K. Nicholson4, T.

Tan1, R. Ratnasabapathy1, M. Hu5,6, G. Carrat5,6, L.Piemonti7, M. Bugliani8, P. Marchetti8, P. E.

MacDonald9, J. E. Manning Fox9, P. R. Johnson10,11,12, S. J. Hughes10,11,12, G. A. Rutter5,6, W.S.

Dhillo1

1Section of Endocrinology and Investigative Medicine, Division of Diabetes, Endocrinology and Metabolism, Department of Medicine, Imperial College London, London, W12 0NN, UK 2Section of Metabolic Medicine, Imperial College London, St Mary’s Hospital, London, W2 1NY, UK 3Statsconsultancy Ltd, 40 Longwood Lane, Amersham, Buckinghamshire, HP7 9EN, UK 4National Phenotyping Centre, Imperial College London, Hammersmith Hospital, London, W12 0NN, UK 5Section of Cell Biology and Functional Genomics, Division of Diabetes, Endocrinology and Metabolism, Department of Medicine Imperial College London, London, W12 0NN, UK 6Imperial Pancreatic Islet Biology and Diabetes Consortium, Hammersmith Hospital, Imperial College London, London, UK 7Diabetes Research Institute (HSR-DRI), San Raffaele Scientific Institute, Via Olgettina 60, 20132 Milan, Italy 8Department of Clinical and Experimental Medicine, Islet Cell Laboratory, University of Pisa, 56126 Pisa, Italy 9Alberta Diabetes Institute Islet Core and Department of Pharmacology, University of Alberta, Edmonton, AB T6G 2E1, Canada 10Nuffield Department of Surgical Sciences, University of Oxford, Oxford, U.K. 11Oxford Centre for Diabetes, Endocrinology, and Metabolism, University of Oxford, Oxford, U.K.

220

12National Institute of Health Research Oxford Biomedical Research Centre, Churchill Hospital, Oxford, U.K.

Aims/Objectives

Reproduction and metabolism are closely intertwined. Recent animal data suggests the essential reproductive hormone, kisspeptin, may have roles in metabolism. However, the effects of kisspeptin on human metabolism are unknown, but may be important for understanding links between reproduction and metabolism, and for ongoing development of kisspeptin-based therapies.

We sought to determine the effect of kisspeptin on glucose-stimulated insulin secretion (GSIS) and appetite in humans.

Methods

Intravenous (IVGTTs), oral glucose tolerance, and mixed meal tests were performed in 19 healthy men (age 26±1y, BMI 22.6±0.4) on two occasions (once with 1nmol.kg-1.hr-1 kisspeptin infusion, once with vehicle infusion) using a blinded randomised crossover design. Blood samples for metabonomic analyses were collected pre- and during infusions. Static incubation experiments were performed with the human pancreatic βcell line EndoC-βH1 (n=3) and human donor islet cells (n=4) to assess in vitro effects of kisspeptin on GSIS.

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Results

During IVGTT, insulin secretion was higher with kisspeptin infusion (mean insulin concentration kisspeptin–vehicle: 4.1µU.mL-1 [95%CI: 0.9 to 7.3], p=0.01; disposition index: kisspeptin 2768±484 vs vehicle 2061±255, p<0.05). Kisspeptin elicited 2.6 and 1.5-fold increases in insulin secretion in EndoC-βH1 cells (p<0.001) and islets (p<0.001) respectively.

Kisspeptin infusion elicited changes in serum sphingomyelins and phosphocholines without affecting appetite.

Discussion and Conclusions

Our study examines, for the first time, the metabolic effects of kisspeptin in humans. We demonstrate that kisspeptin increases GSIS with specific changes in metabonomics. Our observations suggest novel kisspeptin-mediated links between reproduction and metabolism, which could be of clinical importance for the development of kisspeptin-based therapies.

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