CENTRAL REGULATION OF SYMPATHETIC NERVE ACTIVITY BY ...researchbank.rmit.edu.au/eserv/rmit:162285/Habeeballah.pdf · Hamza Habeeb Habeeballah Master of Laboratory Medicine School

CENTRAL REGULATION OF SYMPATHETIC NERVE ACTIVITY BY ADIPOKINES AND INSULIN

A thesis submitted in fulfilment of the requirements for the degree of Doctor of Philosophy

Hamza Habeeb Habeeballah

Master of Laboratory Medicine

School of Health and Biomedical Sciences

College of Science, Engineering and Health

RMIT University

October 2017

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DECLARATION

I, Hamza Habeeballah, declare that the PhD thesis contains no material that has been

submitted previously, in whole or in part, for the award of any other academic degree or

diploma. Except where otherwise indicated, this thesis is my own work. All results in this

thesis are my work performed from the official commencement date of the approved research

program and any editorial work, paid or unpaid, carried out by a third party is acknowledged,

and ethics procedures and guidelines have been followed.

Hamza Habeeballah 21 September 2017

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ACKNOWLEDGEMENTS

Firstly, I would like to express my sincere acknowledge of gratitude to my supervisors,

Professor Emilio Badoer and Dr Martin Stebbing for the continuous support of my PhD study

and related research, for their patience, motivation, and immense knowledge. I am grateful to

Emilio for guiding and encouraging me to nurture my abilities and passion for research and

writing of this thesis. I would also like to thank Emilio for being extremely patient while

teaching me valuable research techniques. Emilio has provided me with invaluable training and

skill sets that I will require for my future endeavours. I also would like to thank him for his

guidance in designing my research at all times. I could not have imagined having a better

advisor and mentor for my PhD study.

Secondly, I would like to thank Naif Alsuhaymi for assisting me with some experiments.

Also, I would like to thank Austin Lai and Omar Al Zahrani for assisting me with performing

the immunohistochemistry experiments. I would also like to thank Dr Trisha Jenkins and the

laboratory (Neuropharmacology and Neuroinflammation Research) members for their help

and support during my PhD study. I would like to thank my friend Khalid Elsaafien for his

frequent help and support.

I thank the Ministry of Higher Education in Saudi Arabia and Saudi Arabian cultural mission

(SACM) for providing me with full funding for my academic and research scholarship. I

thank the Laboratory Medicine Department of the School of Medical Sciences and School of

Graduate Research at RMIT University.

Finally, I would like to thank my father, my mother, my wife and my brothers and sisters for

supporting me spiritually throughout my PhD study and my life in general.

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DEDICATION

I would like to dedicate my hard worked thesis to my loving and supporting parents. Without

the support of my parents, I would have never been able to go through these hard times of

hard work and produce my thesis. I would like to express my sincere gratitude to my father

who always told me “Love what you do, to do what you love”. Indeed, I loved my job, and I

loved what I did during my PhD, and this is what made able to continue all the hard work and

finish my thesis.

To my mother, I would love to say that I have finally fulfilled her dreams of finishing my

PhD and holding the title “Dr”. My mother always told me, “I want to see you a doctor”.

These words were my motivations to continue and fulfil my mother’s dreams and make her

proud of her son.

To all my family, brothers, sisters, and best friends, without your continuous support and

motivation, I would not have been able to go through these hard times easily. My family and

best friends, you were always extreme supporters of me and my passion in research.

Finally, to my beloved wife, thank you for your extreme continuous support during all my

studies. Although I have completed my tertiary studies away from home, my wife always

made feel at home.

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PUBLICATIONS

1- Habeeballah, H., N. Alsuhaymi, M. J. Stebbing and E. Badoer. "Central

Administration Of Insulin Combined With Resistin Reduces Renal Sympathetic

Nerve Activity In Rats Fed A High Fat Diet." (Submitted)

2- Habeeballah, H., N. Alsuhaymi, M. J. Stebbing and E. Badoer (2017). "Effects of

central administration of resistin on renal sympathetic nerve activity in rats fed a high

fat diet; a comparison with leptin." J Neuroendocrinol 29(8). doi:10.1111/jne.12495

3- Alsuhaymi, N., H. Habeeballah, M. J. Stebbing and E. Badoer (2017). "High Fat

Diet Decreases Neuronal Activation In The Brain Induced By Resistin And Leptin."

Frontiers in Physiology, 8. doi:ARTN 867. 10.3389/fphys.2017.00867

4- Habeeballah, H., N. Alsuhaymi, M. J. Stebbing, T. A. Jenkins and E. Badoer (2016).

"Central Administration of Insulin and Leptin Together Enhance Renal Sympathetic

Nerve Activity and Fos Production in the Arcuate Nucleus." Frontiers in Physiology,

7, 672. doi:10.3389/fphys.2016.00672

5- Habeeballah, H., N. Alsuhaymi, M. J. Stebbing, T. A. Jenkins and E. Badoer (2016).

"Central leptin and resistin combined elicit enhanced central effects on renal

sympathetic nerve activity." Exp Physiol 101(7), 791-800. doi:10.1113/EP085723

Thesis

Chapter Publication Title (manuscripts arising from this thesis) Status

Chapter 3 Central Leptin And Resistin Combined Elicits Enhanced Central Effects On

Renal Sympathetic Nerve Activity Published

Chapter 4 Central Administration Of Insulin And Leptin Together Enhance Renal

Sympathetic Nerve Activity And Fos Production In The Arcuate Nucleus Published

Chapter 5 Effects Of Central Administration Of Resistin On Renal Sympathetic Nerve

Activity In Rats Fed A High Fat Diet; A Comparison With Leptin Published

Chapter 6 Central Administration Of Insulin Combined With Resistin Reduces Renal

Sympathetic Nerve Activity In Rats Fed A High Fat Diet Submitted

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COMMUNICATIONS

Communications to scientific meetings during candidature: 2017 Alzahrani, O., E. Alahmadi, H. Habeeballah, E. Badoer and M.J.Stebbing “Brain

inflammation in streptozoocin-treated diabetic rats contributes to the development of

cardiovascular complications and is modulated by oral saline intake”

Neuroscience 2017, Washington, U.S.A

2016 Habeeballah, H., N. Alsuhaymi, M. J. Stebbing, T. A. Jenkins and E. Badoer

“Combined administration of insulin and leptin significantly increased Fos

production in the arcuate nucleus and renal sympathetic nerve activity”

Neuroscience 2016, San Diego, U.S.A


“Renal sympathetic nerve activity is markedly increased by the combination of

insulin and leptin centrally”

Physiology 2016, Dublin, Ireland


“Combined administration of insulin and leptin significantly increased Fos production

in the arcuate nucleus and renal sympathetic nerve activity”

VOC symposium, Melbourne, Australia


“Effects of central administration of resistin on renal sympathetic nerve activity in rats

fed a high fat diet; a comparison with leptin”


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2015 Habeeballah, H., N. Alsuhaymi, M. J. Stebbing, and E. Badoer

“Regulation of renal sympathetic nerve activity by leptin and resistin”

Physiological Society, UK


“Resistin enhances the central effects of leptin on renal sympathetic nerve activity”

HBPRCA, Melbourne, Australia


“Regulation of sympathetic nerve activity by adipokines”

ISN-APSN-ANS Joint Conference, Cairns, Australia

2015 Alsuhaymi, N., H. Habeeballah, Stebbing MJ, Badoer E. "Central neuronal pathways activated by leptin and resistin" ISN-APSN-ANS Joint Conference, Cairns, Australia



CCRCFD, Melbourne, Australia




2014 Badoer, E., S. Kosari, Y. Rathner, H. Habeeballah, N. Alsuhaymi, and M. Stebbing

“Resistin a cytokine from fat tissue”

CCRCFD, Melbourne, Australia

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TABLE OF CONTENTS

DECLARATION ..................................................................................................................... IACKNOWLEDGEMENTS ................................................................................................... IIDEDICATION ...................................................................................................................... IIIPUBLICATIONS .................................................................................................................. IVCOMMUNICATIONS ........................................................................................................... VTABLE OF CONTENTS .................................................................................................... VIILIST OF FIGURES ........................................................................................................... XIIILIST OF TABLES ............................................................................................................. XVIABBREVIATIONS AND ACRONYMS ......................................................................... XVIIABSTRACT .............................................................................................................................. 11 CHAPTER 1: INTRODUCTION ................................................................................... 5

1.1 OBESITY ...................................................................................................................... 51.2 CARDIOVASCULAR DISEASE IN OBESITY ......................................................... 51.3 SYMPATHETIC NERVE ACTIVITY AND CARDIOVASCULAR COMPLICATIONS ............................................................................................................... 61.4 ADIPOKINES AND INSULIN .................................................................................... 7

1.4.1 Leptin ..................................................................................................................... 81.4.1.1 Role of leptin in metabolic function ................................................................. 9

1.4.1.2 Cardiovascular impact of leptin .................................................................... 10

1.4.1.3 Effects of leptin on blood pressure and heart rate ......................................... 10

1.4.1.4 Role of leptin in sympathetic nerve activity ................................................... 11

1.4.2 Resistin ................................................................................................................. 141.4.2.1 Metabolic impact of resistin ........................................................................... 15

1.4.2.2 Resistin in obesity .......................................................................................... 15

1.4.2.3 Cardiovascular impact of resistin .................................................................. 16

1.4.2.4 Effects of resistin on arterial pressure and heart rate ................................... 16

1.4.2.5 Role of resistin on sympathetic nerve activity ................................................ 17

1.4.3 Insulin .................................................................................................................. 181.4.3.1 Insulin in metabolic function ......................................................................... 19

1.4.3.2 Insulin, obesity and cardiovascular function ................................................. 20

1.4.3.3 Effects of insulin on heart rate and mean arterial pressure .......................... 20

1.4.3.4 Role of insulin on sympathetic nerve activity ................................................ 21

1.4.4 Interaction between the hormones ....................................................................... 23

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1.4.4.1 Leptin and resistin ......................................................................................... 23

1.4.4.2 Leptin and insulin .......................................................................................... 23

1.4.4.3 Resistin and insulin ........................................................................................ 24

1.5 SUMMARY ................................................................................................................ 241.6 AIMS .......................................................................................................................... 25

2 CHAPTER 2: MATERIALS AND METHODS ......................................................... 262.1 INSTRUMENTS ........................................................................................................ 26

2.1.1 Manufacture of blood vessel cannulae ................................................................. 262.1.2 Renal dissection instruments ................................................................................ 282.1.3 Manufacture of electrodes .................................................................................... 282.1.4 Blood pressure and heart rate instruments ........................................................... 28

2.1.4.1 Invasive blood pressure and heart rate monitoring ....................................... 28

2.1.4.2 Non-invasive blood pressure and heart rate monitoring ............................... 29

2.1.5 Microinjection instruments .................................................................................. 292.1.6 Perfusion instruments........................................................................................... 292.1.7 Brain removal instruments ................................................................................... 30

2.2 HORMONES/DRUGS ............................................................................................... 302.3 ANIMAL HOUSE, FAT MEASUREMENTS, FOOD AND BLOOD PRESSURE MONITORING .................................................................................................................... 32

2.3.1 Animal type and weight ....................................................................................... 322.3.2 Diet ....................................................................................................................... 322.3.3 Systolic blood pressure monitoring ..................................................................... 332.3.4 Fat measurement .................................................................................................. 33

2.4 SURGICAL PROTOCOLS ........................................................................................ 332.4.1 Femoral vein and artery cannulation .................................................................... 332.4.2 Skull drilling ........................................................................................................ 342.4.3 Renal nerve dissection ......................................................................................... 352.4.4 Intracerebroventricular injection .......................................................................... 372.4.5 RSNA, MAP and HR monitoring ........................................................................ 372.4.6 Perfusion .............................................................................................................. 382.4.7 Tissue collection .................................................................................................. 38

2.5 IMMUNOHISTOCHEMISTRY ................................................................................ 392.5.1 Sectioning of the rat brain .................................................................................... 392.5.2 Immunohistochemical staining procedures .......................................................... 39

2.6 STATISTICAL ANALYSIS ...................................................................................... 402.6.1 Systolic blood pressure, body weight, food intake and calorie intake ................. 402.6.2 RSNA, MAP and HR ........................................................................................... 40

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2.6.3 Fat ........................................................................................................................ 402.6.4 Immunohistochemistry (FOS) ............................................................................. 412.6.5 Invasive blood pressure ........................................................................................ 41

3 CHAPTER 3: CENTRAL LEPTIN AND RESISTIN COMBINED ELICITS ENHANCED CENTRAL EFFECTS ON RENAL SYMPATHETIC NERVE ACTIVITY ............................................................................................................................. 42

3.1 INTRODUCTION ...................................................................................................... 423.2 METHODS ................................................................................................................. 43

3.2.1 Ethical approval ................................................................................................... 433.2.2 General procedures and protocols ........................................................................ 43

3.2.2.1 Renal sympathetic nerve activity ................................................................... 44

3.2.2.2 Microinjections into the lateral brain ventricle (ICV) ................................... 44

3.2.2.3 Experimental protocols .................................................................................. 45

3.2.2.4 Immunohistochemistry for Fos ...................................................................... 45

3.2.3 Statistical analysis ................................................................................................ 463.3 RESULTS ................................................................................................................... 47

3.3.1 Effects of ICV injection of leptin and resistin on RSNA ..................................... 473.3.2 Effects of ICV injection of leptin and resistin on MAP and HR. ........................ 493.3.3 Effects of leptin and resistin, alone and in combination, on Fos-positive cell nuclei… ............................................................................................................................ 52

3.3.3.1 Lamina terminalis .......................................................................................... 52

3.3.3.2 Arcuate nucleus .............................................................................................. 52

3.3.3.3 Paraventricular and supraoptic nuclei .......................................................... 52

3.3.3.4 Dorsal raphe and medulla oblongata ............................................................ 57

3.4 DISCUSSION ............................................................................................................. 594 CHAPTER 4: CENTRAL ADMINISTRATION OF INSULIN AND LEPTIN TOGETHER ENHANCE RENAL SYMPATHETIC NERVE ACTIVITY AND FOS PRODUCTION IN THE ARCUATE NUCLEUS .............................................................. 66


4.2.1 Animals ................................................................................................................ 674.2.2 General procedures and protocols ........................................................................ 67


4.2.2.2 Microinjections into the lateral brain ventricle (ICV) ................................... 68


4.2.2.4 Immunohistochemistry ................................................................................... 69

4.2.3 Statistical analysis ................................................................................................ 70

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4.2.3.1 Physiological parameters .............................................................................. 70

4.2.3.2 Immunohistochemistry ................................................................................... 71

4.3 RESULTS ................................................................................................................... 714.3.1 Effects of ICV injection of leptin and insulin on RSNA ..................................... 714.3.2 Effects of ICV injection of leptin and insulin on MAP and HR. ......................... 724.3.3 Effects of leptin and insulin on Fos-positive cell nuclei ...................................... 76

4.3.3.1 Lamina terminalis .......................................................................................... 76

4.3.3.2 Hypothalamus ................................................................................................ 78

4.3.3.3 Medulla oblongata ......................................................................................... 81

4.4 DISCUSSION ............................................................................................................. 834.5 SUMMARY AND CONCLUSIONS ......................................................................... 86

5 CHAPTER 5: EFFECTS OF CENTRAL ADMINISTRATION OF RESISTIN ON RENAL SYMPATHETIC NERVE ACTIVITY IN RATS FED A HIGH FAT DIET; A COMPARISON WITH LEPTIN ......................................................................................... 88


5.2.1 Ethical approval ................................................................................................... 895.2.2 General procedures and protocols ........................................................................ 89


5.2.2.2 Microinjections into the lateral brain ventricle ............................................. 91


5.2.3 Statistical analysis ................................................................................................ 915.2.3.1 Within high fat diet ......................................................................................... 91

5.2.3.2 Comparisons between high fat and normal chow diets ................................. 92

5.3 RESULTS ................................................................................................................... 935.3.1 Rats fed a high fat diet ......................................................................................... 93

5.3.1.1 Effect on RSNA ............................................................................................... 93

5.3.1.2 Effect on MAP and HR ................................................................................... 93

5.3.2 Comparison of responses to leptin and resistin in rats fed high fat vs normal chow diets ........................................................................................................................ 96

5.3.2.1 RSNA .............................................................................................................. 96

5.3.2.2 MAP and HR .................................................................................................. 98

5.3.2.3 Comparison of systolic BP and metabolic parameters in rats fed high fat vs normal chow diets ....................................................................................................... 102

5.4 DISCUSSION ........................................................................................................... 106

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6 CHAPTER 6: CENTRAL ADMINISTRATION OF INSULIN COMBINED WITH RESISTIN REDUCED RENAL SYMPATHETIC NERVE ACTIVITY IN RATS FED A NORMAL DIET OR A HIGH FAT DIET .................................................................... 111

6.1 INTRODUCTION .................................................................................................... 1116.2 METHODS ............................................................................................................... 112

6.2.1 Ethical approval ................................................................................................. 1126.2.2 General procedures and protocols ...................................................................... 113

6.2.2.1 Renal sympathetic nerve activity ................................................................. 113

6.2.2.2 Microinjections into the lateral brain ventricle ........................................... 114

6.2.2.3 Experimental protocols ................................................................................ 114

6.2.3 Statistical analysis .............................................................................................. 1146.2.3.1 Within diet .................................................................................................... 114

6.2.3.2 Comparisons between high fat and normal chow diets ............................... 115

6.3 RESULTS ................................................................................................................. 1166.3.1 Rats fed a normal diet ........................................................................................ 116

6.3.1.1 Effect on RSNA ............................................................................................. 116

6.3.1.2 Effect on MAP and HR ................................................................................. 116

6.3.2 Rats fed a high fat diet ....................................................................................... 1196.3.2.1 Effect on RSNA ............................................................................................. 119


6.3.3 Comparison of responses to resistin and insulin in rats fed high fat vs normal chow diets ...................................................................................................................... 122

6.3.3.1 RSNA ............................................................................................................ 122

6.3.3.2 MAP and HR ................................................................................................ 122

6.3.3.3 Comparison of systolic BP and metabolic parameters in rats fed high fat vs normal chow diets ....................................................................................................... 127

6.4 DISCUSSION ........................................................................................................... 1317 CHAPTER 7: CENTRAL ADMINISTRATION OF INSULIN COMBINED WITH LEPTIN REDUCES RENAL SYMPATHETIC NERVE ACTIVITY IN RATS FED A HIGH FAT DIET ................................................................................................................. 134

7.1 INTRODUCTION .................................................................................................... 1347.2 METHODS ............................................................................................................... 135

7.2.1 Ethical approval ................................................................................................. 1357.2.2 General procedures and protocols ...................................................................... 136

7.2.2.1 Microinjections into the lateral brain ventricle ........................................... 136

7.2.2.2 Experimental protocols ................................................................................ 137

7.2.3 Statistical analysis .............................................................................................. 137

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7.2.3.1 Within high fat diet ....................................................................................... 137

7.2.3.2 Comparisons between high fat and normal chow diets ............................... 137

7.3 RESULTS ................................................................................................................. 1387.3.1 Rats fed a high fat diet ....................................................................................... 138

7.3.1.1 Effect on RSNA ............................................................................................. 138


7.3.2 Comparison of responses to the combination of insulin and leptin in rats fed high fat vs normal chow diets ................................................................................................ 142

7.3.2.1 RSNA ............................................................................................................ 142

7.3.2.2 MAP and HR ................................................................................................ 142

7.4 DISCUSSION ........................................................................................................... 1448 CHAPTER 8: GENERAL DISCUSSION AND CONCLUSION ............................ 1469 REFERENCES ............................................................................................................. 151APPENDIX I: PERMISSION FOR USING PUBLISHED ............................................. 169APPENDIX II: LETTER OF ETHICS APPROVAL ...................................................... 170

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LIST OF FIGURES

Figure 2.1 PVC tube. ............................................................................................................... 27

Figure 2.2 Wire electrode. ....................................................................................................... 31

Figure 2.3 Screen capture of raw recording of renal sympathetic nerve activity .................... 36

Figure 3.1 Percent changes in RSNA induced by leptin and resistin alone and in combination.

.................................................................................................................................................. 48

Figure 3.2 Changes in MAP and HR induced by leptin and resistin alone and in combination.

.................................................................................................................................................. 50

Figure 3.3 Average number of Fos-positive cell nuclei in OVLT and MnPO induced by leptin

and resistin alone and in combination. ..................................................................................... 53

Figure 3.4 Photomicrographs of Fos-positive cell nuclei in OVLT induced by leptin and

resistin alone and in combination. ........................................................................................... 54

Figure 3.5 Average number of Fos-positive cell nuclei in ARC, PVN and SON induced by

leptin and resistin alone and in combination. ........................................................................... 55

Figure 3.6 Photomicrographs of Fos-positive cell nuclei in PVN induced by leptin and

resistin alone and in combination. ........................................................................................... 56

Figure 3.7 Average number of Fos-positive cell nuclei in DR, RVLM, NTS and RPA induced

by leptin and resistin alone and in combination. ...................................................................... 58

Figure 4.1 Percent changes in RSNA induced by leptin and insulin alone and in combination

in rats fed a normal diet (ND). ................................................................................................. 73

Figure 4.2 Changes in MAP and HR induced by leptin and insulin alone and in combination

in rats fed a normal diet (ND). ................................................................................................. 74

Figure 4.3 Average number of Fos-positive cell nuclei in OVLT and MnPO induced by leptin

and insulin alone and in combination in rats fed a normal diet (ND). ..................................... 77

Figure 4.4 Average number of Fos-positive cell nuclei in ARC, PVN and SON induced by

leptin and insulin alone and in combination in rats fed a normal diet (ND). ........................... 79

Figure 4.5 Photomicrographs of Fos-positive cell nuclei in ARC induced by leptin and insulin

alone and in combination in rats fed a normal diet (ND). ....................................................... 80

Figure 4.6 Average number of Fos-positive cell nuclei in NTS, RPA and RVLM induced by

leptin and insulin alone and in combination in rats fed a normal diet (ND). ........................... 82

Figure 5.1 Percent change in RSNA induced by leptin and resistin alone and in combination

in rats fed a high fat diet (HFD). .............................................................................................. 94

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Figure 5.2 Change in MAP and HR induced by leptin and resistin alone and in combination

in rats fed a high fat diet (HFD). .............................................................................................. 95

Figure 5.3 RSNA response following leptin and resistin alone and in combination in rats fed a

high fat diet (HFD) or normal diet (ND). ................................................................................ 97

Figure 5.4 MAP response following leptin and resistin alone and in combination in rats fed a

high fat diet (HFD) or normal diet (ND). ................................................................................ 99

Figure 5.5 HR response following leptin and resistin alone and in combination in rats fed a

high fat diet (HFD) or normal diet (ND). .............................................................................. 100

Figure 5.6 Basal level of MAP and HR in rats fed a high fat diet (HFD) or normal diet (ND)

prior to hormone administration. ........................................................................................... 101

Figure 5.7 Systolic blood pressure changes over time. .......................................................... 103

Figure 5.8 Fat distribution in rats fed a high fat diet (HFD) or normal diet (ND). ................ 104

Figure 5.9 Effects of body weight, food intake and calorie intake over time. ....................... 105

Figure 6.1 Percent change in RSNA induced by resistin and insulin alone and in combination

in rats fed a normal diet (ND). ............................................................................................... 117

Figure 6.2 Change in MAP and HR induced by resistin and insulin alone and in combination

in rats fed a normal diet (ND). ............................................................................................... 118

Figure 6.3 Percent changes in RSNA induced by resistin and insulin alone and in

combination in rats fed a high fat diet (HFD). ....................................................................... 120

Figure 6.4 Change in MAP and HR induced by resistin and insulin alone and in combination

in rats fed a high fat diet (HFD). ............................................................................................ 121

Figure 6.5 RSNA response following resistin and insulin alone and in combination in rats fed

a high fat diet (HFD) or normal diet (ND). ............................................................................ 123

Figure 6.6 MAP response following resistin and insulin alone and in combination in rats fed a


Figure 6.7 HR response following resistin and insulin alone and in combination in rats fed a


Figure 6.8 Basal level of MAP and HR in rats fed a high fat diet (HFD) or normal diet (ND)

prior to hormone administration. ........................................................................................... 126

Figure 6.9 Systolic blood pressure changes over time. .......................................................... 128

Figure 6.10 Fat distribution in rats fed a high fat diet (HFD) or normal diet (ND). .............. 129

Figure 6.11 Effects of body weight, food intake and calorie intake over time. ..................... 130

Figure 7.1 Percent change in RSNA induced by leptin and insulin alone and in combination


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Figure 7.2 Changes in MAP and HR induced by leptin and insulin alone and in combination


Figure 7.3 Basal level of MAP and HR in rats fed a high fat diet (HFD) or normal diet (ND).

................................................................................................................................................ 141

Figure 7.4 RSNA, MAP and HR response following leptin and insulin alone and in

combination in rats fed a high fat diet (HFD) or normal diet (ND). ...................................... 143

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LIST OF TABLES

Table 1.1 Summary effects of leptin, insulin and resistin on RSNA on a normal diet and a

high fat diet on RSNA, and the gap that needs to be investigated. .......................................... 25

Table 3.1 Resting MAP and HR. ............................................................................................. 51

Table 4.1 Resting MAP and HR. ............................................................................................. 75

Table 8.1 Summary of the effects of leptin, insulin and resistin on RSNA in rats fed a normal

diet or a high fat diet. ............................................................................................................. 147

xvii

ABBREVIATIONS AND ACRONYMS

ANOVA Analysis of variance ARC Arcuate nucleus BAT Brown adipose tissue BAT SNA Sympathetic nerve activity to brown adipose tissue CNS Central nervous system DAB 3,3′-diaminobenzidine hydrochloride DIO Diet-induced obesity DMH Dorsomedial hypothalamic nucleus ERK Extracellular regulated kinase HFD High fat diet HR Heart rate ICV Intracerebroventricular IML Intermediolateral column IGF Insulin-like growth factor IR Insulin receptor IV Intravenous LSNA Lumbar sympathetic nerve activity MAP Mean arterial pressure MAPK Mitogen-activated protein kinase MCR Melanocortin receptor MnPO Median preoptic nucleus ND Normal chow diet NE Norepinephrine NHS Normal horse serum NTS Nucleus tractus solitaries OVTL Organum vasculosum of the lamina terminalis PAG Periaqueductal grey PBS Phosphate buffered saline PI3K Phosphatidylinositol-3-kinase POMC Pro-opiomelanocortin PVC Polyvinyl chloride PVN Paraventricular nucleus RA Rheumatoid arthritis RPA Pallidus nucleus RSNA Renal sympathetic nerve activity RVLM Rostral ventrolateral medulla SFO Subfornical organ SNA Sympathetic nerve activity SON Supraoptic nucleus VMH Ventromedial hypothalamic nucleus

1

ABSTRACT

Introduction

Leptin, resistin and insulin are hormones that circulate in the blood and cross the blood brain

barrier. In the brain, they are thought to activate specific regions which play important roles

in energy homeostasis and cardiovascular regulation. The plasma levels of these hormones

have been shown to be elevated in overweight and obese conditions, and an increased

sympathetic nerve activity (SNA) is also observed in these conditions. In addition, leptin,

resistin and insulin plasma levels are strongly correlated with increases in blood pressure

suggesting that these hormones may have cardiovascular effects. In high fat diet (HFD), the

effects of leptin on renal sympathetic nerve activity (RSNA) are well known. However, the

effects of insulin are little known, and the effects of resistin are not known. Since leptin,

resistin and insulin levels are increased significantly in obesity and each of these hormones

alone can increase RSNA, the question arises as to whether there is an interaction between

these hormones on cardiovascular regulation.

Thus, the general aim of this thesis was to compare the effects of leptin, resistin and insulin

alone and in combination on RSNA, mean arterial pressure (MAP) and heart rate (HR) in rats

fed a normal chow diet (ND) or HFD.

Methods

Male Sprague-Dawley rats were divided into two groups one fed a ND and the other fed a

HFD for approximately 8-10 weeks. Food intake, caloric intake and body weight were

monitored over the duration of the feeding regime. At the end of the feeding regime, the

whole body fat mass was calculated. On the day of experiments, rats were anaesthetized with

isoflurane gas (initially 5% then 2%) in O2. Anaesthesia was maintained by intravenous (IV)

injection of urethane (1.4-1.6 g/kg) followed by supplemental doses of 0.05 ml (of a 25%

2

solution). The depth of anaesthesia was determined by ensuring the lack of corneal and pedal

reflexes. Saline (5 µl), leptin (7 µg/5 µl), resistin (7 µg/5 µl) and insulin (500 mU/5 µl) were

administered intracerebroventricularly (ICV) and RSNA, MAP and HR were monitored and

recorded before and for 3 hours after hormone administration. Additionally, the potential

central sites of action of the interaction of (leptin + resistin) and (leptin + insulin) were

investigated in rats fed a ND. In these groups, the brains were removed at the end of the

experiments and processed immunohistochemically to detect the protein Fos, a marker of

increased neuronal activation.

Results

In chapter 3, ICV administration of the combination of leptin and resistin in rats fed a ND

produced significant increases in RSNA over time compared to each hormone alone

(P<0.0001). In addition, the central administration of leptin and resistin together increased

Fos production in the arcuate nucleus (ARC) compared to each hormone alone. This finding

suggests that the ARC may be a key central site in which leptin and resistin together induced

greater effects on the RSNA response than either leptin or resistin alone. Therefore, increases

in leptin and resistin levels may contribute to the abnormal elevation in RSNA seen in

overweight/obese conditions.

In chapter 4, in rats fed a ND the increases in RSNA following ICV injection of leptin and

insulin combined were significantly greater than each hormone alone (P<0.0001). The

number of Fos-positive cell nuclei was only significantly greater in ARC following ICV

leptin and insulin together compared to leptin alone, resistin alone or saline. This finding

suggests that sympathetic activity to the kidney is greatly increased when leptin and insulin

levels are elevated. In addition, the ARC might be the site mediating this interaction of leptin

and insulin.

3

In chapter 5, ICV administration of resistin increased RSNA significantly in rats fed a HFD.

This effect was similar to ND group. Moreover, in the HFD group when leptin and resistin

were administered in combination, RSNA increased significantly more compared to each

hormone alone. The greater increases in the HFD following leptin and resistin combined was

not significantly different compared to the response in rats fed the ND. This result suggests

that leptin and resistin elevate the sympathetic activity response to the kidney, and this is not

affected by a diet high in fat.

In chapter 6, in rats fed a ND the administration of resistin and insulin in combination did not

increase the RSNA, and this response was significantly different compared to either hormone

alone. In rats fed a HFD, RSNA fell significantly. These results suggest that HFD markedly

alters the RSNA response and there may be a negative interaction when resistin and insulin

are combined.

In chapter 7, in rats fed a HFD the combination of leptin and insulin produced significant

reductions in RSNA compared with either hormone alone. These results suggest that HFD

can markedly affect the RSNA response of the combination of leptin and insulin compared to

that seen in ND. Therefore, this result suggests that HFD may influence the RSNA response

to leptin and insulin combined.

Summary and Conclusion

The results show that ICV administration of leptin + resistin increases SNA to the kidney in

ND and HFD animals. The effects of leptin and insulin in combination showed that HFD

significantly reduced the RSNA responses induced by these hormones in contrast to the

increases seen in ND. When resistin and insulin were combined, the response to RSNA was

not increased in ND and in HFD, RSNA fell markedly.

4

Together, these data suggest that in HFD the presence of insulin with resistin or leptin may

result in a negative interaction on RSNA. However, the positive interaction in HFD seen with

resistin and leptin might overcome any negative interaction of insulin with resistin or leptin.

Therefore, the interaction between leptin and resistin may contribute to the abnormal

elevation of RSNA, seen in overweight /obese conditions.

5

1 Chapter 1: Introduction

1.1 OBESITY

Weight gain induced by high fat diet (HFD) can lead to obesity, which is becoming a major

health problem worldwide and is rapidly increasing in developed countries (1, 2). According

to the Australian Institute of Health and Welfare, in 2014-15 approximately two thirds of

adult Australians are either obese or overweight (3). Moreover, in the USA, obesity and being

overweight result in approximately 300,000 deaths every year (4). In addition, statistics

indicate that if the current trend towards weight gain continues, by 2025, more than 80% of

the adult population in developed countries will be either obese or overweight (5). Many

studies have shown that weight gain leads to a variety of health complications such as

cardiovascular diseases, metabolic disorders and type-2 diabetes (6, 7).

1.2 CARDIOVASCULAR DISEASE IN OBESITY

Moderate and severe obesity is a well-known risk factor for many cardiovascular

complications such as hypertension. Clinical studies have shown that increased food

consumption and caloric intake leads to increases in blood pressure (6-13). Conversely, other

studies suggest that decreasing weight by a low-calorie diet can lead to reductions in blood

pressure in humans (14, 15). Studies in animal models, including rodents and rabbits, fed a

HFD show increases in blood pressure (1, 16-23). For example, some studies have

investigated the effects of HFD on Sprague-Dawley rats. These rats were fed a HFD and over

time they were observed to develop obesity and arterial hypertension (21, 22). Rats fed a

HFD can be segregated into obese-resistant, which do not develop obesity when fed a HFD,

and obese-prone groups, which do develop obesity when fed a HFD and develop

hypertension (24, 25). Therefore, there is an association of obesity induced by high fat

consumption and elevated blood pressure. Interestingly, studies have shown that one of the

6

common drivers of high blood pressure is an overactivity of the sympathetic nervous system

(7, 26).

1.3 SYMPATHETIC NERVE ACTIVITY AND CARDIOVASCULAR

COMPLICATIONS

Sympathetic activation to the cardiovascular organs can lead to increases in blood pressure by

acting on the heart, kidneys and vasculature. Overactivity of sympathetic nerve activity

(SNA) to the vasculature leads to peripheral vasoconstriction, whereas increased SNA to the

kidneys leads to increased renal tubular sodium reabsorption potential changes in renal

filtrate and function, as well as activation of the renin angiotensin system (7, 26). SNA to the

heart leads to increased heart rate (HR), stroke volume and cardiac output (27). Together

these mechanisms mediate the ability of sympathetic overactivity to increase blood pressure.

In animal models, it possible to measure directly SNA. For example, to measure SNA

contributing to vasoconstriction, the nerves that innervate blood vessels of the hindlimb

muscles can be recorded from lumbar sympathetic nerve activity (LSNA) (28). In addition,

kidneys play a major role in long-term regulation of blood pressure. The nerves can influence

the diameter of the efferent and the afferent arterioles, and the sympathetic nerves can induce

the release of hormones from the renal tissue, such as renin (29). Therefore, recording renal

sympathetic nerve activity (RSNA) gives an indication of sympathetic outflow to an organ of

key importance to cardiovascular regulation. Thus, it is vital to study what hormones

influence the RSNA, as this may underpin the increased level of RSNA associated with high

fat feeding.

The pathophysiology of obesity is complex where activation of the central nervous system

(CNS) is one of the multipotential mechanisms that can lead to obesity-induced hypertension

(30, 31). Body mass index in obesity is correlated with the level of SNA, or norepinephrine

7

(NE) spillover which is a measure of SNA (26), or increased levels of total plasma NE, which

is a general indicator of sympathetic activation (32). Moreover, it has been found that renal

NE spillover accounted for over a half of the total plasma NE in hypertensive patients (33,

34), suggesting that RSNA might be a major contribute to obesity-induced hypertension (35-

37). An increase in SNA to other cardiovascular organs has also been observed in obesity

(35, 38). Several studies have shown that obese patients have increased muscle SNA, a

measure of SNA to the muscle vasculature, compared to normal patients (37).

Furthermore, elevated SNA has been linked to hypertension in animal models of obesity. For

example, it has been shown that in rabbits fed a HFD there are increases in plasma NE, mean

arterial pressure (MAP) and HR (17, 39, 40). By week 3 of feeding these rabbits showed

significant increases in SNA to the kidney (17), supporting the view that activation of SNA in

obesity can lead to hypertension (35, 38, 40, 41).

1.4 ADIPOKINES AND INSULIN

Obesity can be defined by the amount of adipose tissue stored in the body (42). Recent

studies also highlight that adipose tissue has additional roles other than the storage of fat and

those roles include endocrine functions (43, 44). Adipose tissue releases chemical mediators,

which include hormones such as leptin and resistin, which are referred to as adipokines (45).

The level of adipokines such as leptin and resistin are significantly increased with the

increase in weight gain associated with obesity or overweight conditions (46-49). These

hormones can influence metabolic function by regulating the balance between food intake

and energy expenditure (43). They also play a major role in the regulation of cardiovascular

function by influencing SNA (50, 51).

Insulin, although not being an adipokine, is another hormone that has similar effects to leptin

and resistin, in terms of metabolic function (52). It also contributes to cardiovascular

8

regulation by influencing SNA (53, 54). Therefore these hormones may be linked to obesity-

induced cardiovascular complications.

Obesity causes the development of resistance to leptin and insulin, in terms of their effects on

metabolic function (55, 56). However, the effects of leptin on RSNA in obesity or overweight

conditions are not affected, whilst an effect on insulin is controversial (57, 58). The effect of

resistin on SNA with HFD is unknown. Therefore, there is a gap in our knowledge in

understanding the effects of HFD in the response to resistin on SNA, together with any

interactions of leptin, resistin or insulin in combination on SNA.

1.4.1 Leptin

Leptin is a 16kDa polypeptide, which was first discovered in 1994. Leptin is found in adipose

tissue, the primary site of leptin production, although other tissues such as skeletal muscle,

liver and stomach also can produce leptin (59-61). The amount of body fat is the main factor

contributing to the level of leptin in the blood (62). Leptin levels typically increase after a

meal and decrease during fasting, and increases in plasma leptin levels eventually lead to a

loss of body weight (63). But there is also evidence to suggest that leptin is produced by the

brain (64, 65). Leptin has been shown to cross the blood-brain barrier (66). The receptors for

leptin are found to be widely distributed throughout different regions of the hypothalamus,

such as the arcuate nucleus (ARC) and the paraventricular nucleus (PVN) (57, 67). In the

CNS, leptin acts through the hypothalamus by activating its receptors to regulate food intake,

energy expenditure, and cardiovascular regulation (68-71). It has been shown that leptin can

control and regulate appetite (72), energy expenditure through thermogenesis, and

cardiovascular regulation through sympathetic nerve activation (18, 73). Since its discovery,

leptin has been linked to cardiovascular diseases (74-76). In the next section, I discuss the

metabolic and cardiovascular actions of leptin.

9

1.4.1.1 Role of leptin in metabolic function

Leptin plays a major role in the control of feeding behaviour and energy balance. Weight gain

leads to increased plasma leptin levels, and leptin acts centrally in the brain through specific

receptors in the hypothalamus to decrease food intake and increase energy expenditure (62,

65, 69, 70). Typically, leptin levels are observed to be higher at night than during the day, so

that a greater response of leptin occurs to a meal at night time than during the day. It has also

been shown that during fasting periods, plasma leptin levels are reduced (63).

Experimentally, acute and chronic administration of leptin in animals resulted in decreased

body weight (18, 77-79). This occurs through actions in the brain since

intracerebroventricular (ICV) injection of leptin into rats resulted in a reduction in food

intake by approximately 24% (80). Treatment with leptin resulted in a higher body

temperature compared with non-treated animals, suggesting that leptin elicits an increased

thermogenesis resulting in an increased energy expenditure (81). Moreover, leptin treatment

in rats has been shown to reduce visceral fat, suggesting that leptin results in fat redistribution

(82). Finally, leptin treatment in patients has been found to reduce truncal and visceral fat

mass (83, 84). The findings, together, show that leptin plays an important role in a metabolic

function via reducing food intake and increasing energy expenditure in response to feeding.

In overweight and obese conditions, where the amount of fat tissue is elevated, plasma leptin

levels are found to be significantly elevated (79, 85-87). HFD consumption in humans and

animal models increases plasma levels of leptin and also cause weight gain (18, 88).

Moreover, body weight and food intake have been shown to be minimally affected by leptin

in animals fed a HFD (18), suggesting HFD promotes the development of resistance to the

effects of leptin in terms of reducing body weight and increasing energy expenditure (18).

This has been supported by studies, which show that leptin is much less effective in reducing

appetite and food intake in animals fed a HFD (17, 58, 89). In addition, leptin treatment in

10

human overweight patients fails to reduce weight or suppress appetite (90, 91). In fact, leptin

has been shown to play an important role in regulating metabolic function in healthy subjects,

whereas in subjects fed a HFD, leptin’s ability to reduce food intake is markedly attenuated.

1.4.1.2 Cardiovascular impact of leptin

Many previous studies have shown that leptin does not only have actions on metabolic

function, but it also plays an important role in cardiovascular regulation (92-94). Studies

show that mutations in the genes for leptin or leptin receptors in humans and rodents can

prevent hypertension (95, 96). These mutations have been shown to result in hypotension and

a reduced renin-angiotensin system (97). Furthermore, rodents with leptin deficiency have

been shown to have lower blood pressure than wild type rodents (96, 98). This, therefore,

suggests that leptin plays an important role in regulating blood pressure.

1.4.1.3 Effects of leptin on blood pressure and heart rate

Many studies have investigated the effects of leptin administration on blood pressure. For

example, the ICV administration of leptin in male Wistar rats increased MAP by a maximum

of 15% from baseline. However, no statistically significant changes were observed in the HR

(99). In Sprague-Dawley rats, the acute ICV administration of leptin also showed increases in

arterial blood pressure but not HR (100, 101). In rabbits, the acute central administration of

leptin has also been shown to result in increases in MAP (17, 102). This, therefore, shows

that the acute short-term central administration of leptin results in increasing MAP, which

suggests that leptin, plays a central role in regulating blood pressure.

In long-term studies, in male Sprague-Dawley rats, intravenous (IV) infusion of leptin for 12

days increased blood pressure significantly using a high dose of leptin, whereas only slight

increases were observed at a lower dose. HR also increased at both the lower and the higher

11

does (103, 104). The increases in blood pressure and HR were attenuated dramatically upon

cessation of leptin infusion (103, 104). However, short-term IV infusion of leptin for three

hours, did not change MAP (105, 106). Together, the studies show that long-term peripheral

infusion of leptin increases blood pressure, suggesting that leptin may also be playing a role

in regulating blood pressure peripherally.

Surprisingly, in contrast to a metabolic function, leptin has been shown to increase blood

pressure in overweight and obese conditions. In diet-induced obese (DIO) mice, acute and

chronic treatment with leptin has been shown to result in significant increases in MAP and

those increases were similar in both lean and DIO mice (18). Moreover, rabbits on a HFD

have been observed to show significant elevations in MAP following acute leptin

administration (17). In Sprague-Dawley rats, chronic long-term administration of leptin

increased MAP and HR significantly (107). Furthermore, studies in obese leptin-deficient

mice show lower levels of arterial pressure compared to lean mice (98), suggesting the

importance of leptin in maintaining an elevated blood pressure. Thus, leptin maintains its

actions of increasing blood pressure in overweight and obese conditions.

1.4.1.4 Role of leptin in sympathetic nerve activity

The sympathetic nervous system plays an important role in the regulation of the

cardiovascular system by affecting the nerves to the kidney and blood vessels, as well as in

the regulation of metabolic function by influencing sympathetic nerve activity to brown

adipose tissue (BAT SNA). In overweight and obese conditions, the level of NE in human

patients has been observed to significantly increase, suggesting an activation of the

sympathetic nervous system (26, 108). More directly, leptin acts centrally through specific

regions of the brain, including those, which contain many of the premotor sympathetic

12

neurones, which regulate cardiovascular function as well as metabolic function (100). This is

discussed in the following section.

Lumbar sympathetic nerve activity

Leptin has been shown to regulate cardiovascular function through the activation of the

sympathetic nervous system. Acute central administration of leptin in rats results in

significant increases in SNA to the skeletal muscle blood vessels (lumbar) (99, 109).

Moreover, chronic IV administration of leptin in rats also resulted in dose-dependent

increases in LSNA (105).

In DIO and HFD fed animals, the LSNA responses to leptin are attenuated (18, 58). For

example, ICV administration of leptin in DIO mice showed reductions in LSNA compared to

lean mice (18, 58). Moreover, the IV administration of leptin elicited an attenuated LSNA

response in HFD fed mice compared to a normal chow diet (ND) (18, 58). These findings

suggest that in overweight or obese conditions there is resistance to the actions of leptin on

LSNA, just as there is for leptin’s effects on BAT SNA.

Renal sympathetic nerve activity

Leptin has been shown to induce significant increases in RSNA through acting centrally in

the hypothalamus (100). This is seen in rats (99, 100, 109), rabbits (102), and also in mice

(18, 87, 100, 110). Moreover, the IV administration of leptin in rats has been shown to elicit

increases in RSNA (105, 106). These increases in RSNA by central and peripheral

administration suggest that the actions of leptin are elicited centrally. This is confirmed by

microinjection of leptin into many cardiovascular regions such as the PVN of the

hypothalamus, the ventromedial hypothalamic nucleus (VMH), the dorsomedial

hypothalamic nucleus (DMH), the nucleus tractus solitaries (NTS), the rostral ventrolateral

13

medulla (RVLM), and the ARC in rats which elicited increases in RSNA (73, 100, 101, 111,

112).

The sympathetic activation to the kidney in response to leptin was not attenuated by HFD

(18, 58, 79, 87, 110). In DIO mice, the acute administration of leptin after 20 weeks of

feeding on a HFD resulted in increases in RSNA that were similar to lean mice (58).

Similarly, such an effects were seen in mice fed a HFD for 10 only weeks (18). In rabbits fed

a HFD there were significant increases in RSNA after 3 weeks of feeding and the increase in

RSNA was evident within 7 days of the diet consumption (17). In all cases, the elevations in

RSNA induced by leptin in HFD fed animals were similar to the respective response seen in

animals fed a ND (17). Therefore, the data suggests that the central effects of leptin on RSNA

are selectively resistant to the attenuating actions in overweight or obese conditions that have

been observed with LSNA and BAT SNA.

Sympathetic nerve activity to brown adipose tissue

Leptin is well known for its role in regulating food intake and energy expenditure through

thermogenesis. Thermogenesis is a process where the temperature is increased through the

activation of BAT SNA (113). The key area of importance in the brain for regulation of

thermogenesis by leptin is the hypothalamus (114, 115). This is exemplified by studies with

IV and ICV administration of leptin in rats that show an increase in BAT SNA (100, 105,

106). Since injection of leptin directly into the ARC in rats elicited increases in BAT SNA

(100). It would suggest that the ARC is the key site of action for leptin’s effect on BAT SNA.

In overweight and obese conditions, the effects of leptin on BAT SNA are reduced (18, 58).

For example, BAT SNA in DIO mice has been shown to be attenuated following the acute

ICV administration of leptin (18, 58). Moreover, the IV administration of leptin in HFD fed

mice has also failed to increase the BAT SNA (18, 58). Together, these findings suggest that

14

a HFD can lead to obesity-linked induction of resistance to the effects of both systemic and

central administration of leptin on BAT SNA.

Mechanisms mediating the effects on sympathetic nerve activity

The second messenger enzyme Phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K) plays

a major role in regulating RSNA responses induced by leptin (58). Acute administration of a

PI3K inhibitor (LY294002) ICV results in the attenuation of the elevation of RSNA induced

by leptin (58), indicating a key role of central PI3K in mediating the RSNA induced by the

infusion of leptin in the CNS (58). In addition, melanocortin receptors (MCR) in the

hypothalamus also mediates the effects of leptin on RSNA (58, 110). As shown by studies in

which the presence of the MC3/4R antagonist (SHU9119) in the brain significantly attenuates

the increases in RSNA induced by central leptin (58, 110). Together these results suggest that

leptin activates Pro-opiomelanocortin (POMC) neurons resulting in the release of α-MSH that

in turn, activates MC3/4R centrally, and the activation of the second messenger PI3K, to

induce leptin’s effect on RSNA.

1.4.2 Resistin

Resistin is an adipokine, which was discovered in mice in 2001 (47). It was called resistin

because of its ability to induce resistance to the action of insulin (47). Resistin is secreted

mainly by adipose tissue in rodents (47, 116, 117). In humans, resistin is produced mainly by

macrophages, which infiltrate adipose tissue (118-120). In addition to adipose/macrophages

in adipose tissue, resistin has also been found to be expressed in other tissues such as skeletal

muscles and pancreas in humans and rodents (47, 121). Messenger RNA of resistin has been

observed to be expressed in rodent brains in the hypothalamus, and it is also been seen human

cerebrospinal fluid (CSF) (47, 121-125). To date there has not been any studies reporting the

effects of resistin microinjected into specific brain nuclei, nor there has been any studies

15

reporting sites in the brain where resistin can potentially act. Furthermore, resistin receptors

has not been identified yet. The plasma levels of resistin, in normal conditions, are reported to

be approximately 7.3-14.3 ng/ml and 35.7-42.9 ng/ml, in humans and rodents respectively

(48, 126). However, resistin levels were found to be increased significantly in obese and DIO

rodent models (127). In addition, there was a positive correlation between human resistin

plasma levels and obesity (128, 129). Plasma levels of resistin are reduced during fasting; in

contrast, they are increased upon feeding (47, 130, 131). As the levels of resistin change in

relation to food intake, it suggests that resistin regulates energy homoeostasis (132, 133).

1.4.2.1 Metabolic impact of resistin

Resistin has, indeed, been shown to play a role in energy homeostasis (47). The effects of

central resistin administration on food intake have been investigated. ICV administration of

resistin has been shown to have anorectic effects on food intake for approximately 90 minutes

after resistin injection (125). These effects are associated with resistin’s ability to decrease

neuropeptide Y gene expression in the hypothalamus (134). Furthermore, resistin has been

shown to alter energy expenditure. An important component of energy expenditure is

thermogenesis in BAT (135). ICV injection of resistin has been shown to decrease BAT SNA

(136). Therefore, together the evidence suggests that resistin plays an important role in

regulating metabolic function, by suppressing food intake and decreasing energy expenditure.

1.4.2.2 Resistin in obesity

The plasma levels of resistin are increased in obesity. For example, in humans, serum levels

of resistin in obesity were significantly higher compared to lean individuals and were

positively correlated with body mass index (48) and abdominal fat depots (137). Similarly, in

obese animal models, circulating levels of resistin were significantly higher compared with

lean controls (127). In contrast weight loss results in a reduction of resistin plasma levels

16

(138). Thus, studies to date provide evidence of a positive correlation between resistin serum

level and obesity.

As mentioned earlier, resistin has also been shown to induce insulin resistance. This occurs

through attenuation of the phosphorylation of the insulin receptor (139). Several studies have

identified a positive correlation between serum resistin levels and the degree of insulin

resistance in vivo and in vitro (140, 141). This action of resistin has suggested that resistin

may play an important role in the development of diabetes. In humans, it has been shown that

subjects with type-two diabetes show an increase of approximately 20% in serum resistin

levels (142). Furthermore, the infusion of resistin in humans has been shown to decrease

insulin sensitivity (143), and insulin-stimulated glucose uptake (144). Therefore, resistin is

implicated in the complications of obesity and metabolic syndrome by reducing insulin

sensitivity.

1.4.2.3 Cardiovascular impact of resistin

Studies have linked resistin with cardiovascular complications. Increasing serum resistin

levels are associated with high blood pressure and cardiovascular disease (47, 145-149).

Resistin levels may also have a prognostic value since it has been found that in young healthy

patients with a family history of hypertension show elevated plasma resistin levels (150).

Although there appears a strong link between resistin plasma levels and hypertension, the

contribution of resistin to hypertension is still to be clarified.

1.4.2.4 Effects of resistin on arterial pressure and heart rate

Resistin effects on blood pressure are little known. Only one study has investigated the role

of acute resistin administration, and it showed that acute ICV administration of resistin in rats

did not change MAP or HR (136). However, chronic resistin administration showed that in

17

mice the IV injection of resistin for six days significantly elevates MAP and HR (151). This

effect on blood pressure could arise from a sufficiently activated response of the sympathetic

nervous system, as acute administration of resistin has been shown to increase SNA (51,

136). Thus, only two studies have reported the effects of resistin on MAP or HR and these

have been in animals fed a ND. In overweight/obesity conditions, the effects of resistin have

not been investigated yet.

1.4.2.5 Role of resistin on sympathetic nerve activity

The effects of resistin on SNA are not well known. Currently, only tow studies have

examined the effects of resistin on SNA. This will be discussed in detail in the next section.


LSNA is important for the regulation of the hindlimb skeletal muscle vasoconstriction.

Currently only Kosari et al. have investigated the effects of resistin on LSNA (136). In

normal male Sprague-Dawley rats, acute administration of resistin into the lateral cerebral

ventricle significantly increased LSNA when compared with control rats (136). This

sympatho-excitatory effect of resistin is similar to the response seen with leptin (99, 105,

109).


Resistin has been shown to elevate RSNA. Acute administration of resistin in normal rats

results in a significant increase in RSNA within 15 min, with a maximum increase in RSNA

of 40% compared to rats treated with the vehicle alone (51). This study is the only one

performed to date with resistin. The sympatho-excitatory response to resistin is similar to that

seen with leptin (87, 99-102).

18

Sympathetic nerve activity to brown adipose tissue

BAT is important for thermogenesis and energy expenditure. Therefore by regulating BAT

SNA, thermogenesis and energy expenditure is regulated (113, 152). It has been

demonstrated that acute administration of resistin into the lateral cerebral ventricle in normal

rats resulted in significantly decreased BAT SNA (136). This, therefore, suggests that resistin

acting in the brain can reduce thermogenesis and energy expenditure.

Mechanisms mediating changes in sympathetic nerve activity induced by resistin

Extracellular Signal-Regulated Kinase (ERK1/2) is suggested to be the mediator that

regulates the SNA response to resistin in BAT (153). Pre-treatment with an ERK1/2 inhibitor

centrally attenuated the response elicited by resistin on BAT SNA (153). However, inhibition

of ERK1/2 had no effects on the sympatho-excitatory effect of resistin on RSNA.

The increase in RSNA following the administration of resistin is mediated by PI3K in the

brain. The pre-treatment with a PI3K inhibitor attenuated the normal increases in RSNA in

response to resistin (51). This suggests that the intracellular mechanisms by resistin on RSNA

are mediated PI3K. This, too, is similar to the findings with leptin and suggests that resistin

and leptin may share common central pathways with respect to their sympatho-excitatory

responses on RSNA. Interestingly, there are no studies, to date, that have compared resistin

and leptin in the same study.

1.4.3 Insulin

Insulin, a hormone synthesised and released by the beta cells of the pancreas, was first

discovered in 1923 (154). The cognate receptor for insulin is the insulin receptor (IR), which

is expressed in many organs, including the brain (155), liver and pancreas (156, 157). In the

brain, insulin receptors are widely expressed in the hypothalamus; mainly in the ARC, with

19

high levels of expression in the PVN (158). The IR is densely expressed in many brain

regions that are implicated in the control of food intake and the regulation of the autonomic

nervous system (159, 160). As insulin is a major hormone in the control of food intake, the

levels of this hormone are increased with body fat mass (161, 162). As body mass increases,

insulin acts in the brain to reduce weight by decreasing food intake and increasing energy

expenditure (163, 164). In addition to its role in metabolic function, insulin also plays a role

in regulating cardiovascular function (165-167).

1.4.3.1 Insulin in metabolic function

Insulin is a hormone secreted from the pancreas mainly in response to elevated blood glucose

levels (168). It plays an important role in regulating glucose homeostasis, as well as lipid and

protein metabolism (169, 170). It acts on many target tissues, such as the liver and the

muscles, to regulate metabolism. Insulin also plays a role in regulating food intake through

the CNS (163). It is known to act through the ARC to suppress food intake and increase

energy expenditure (171). In the ARC, insulin activates POMC neurones which have an

effect on suppressing food intake and increasing energy expenditure through thermogenesis

(172, 173). It also inhibits neuropeptide Y neurones in the ARC, which are known to inhibit

the actions mediated by POMC neurones (172, 173). Therefore, in healthy conditions, as food

intake increases, insulin is secreted to regulate glucose, suppress food intake, and increase

energy expenditure. Studies show that mice, which lack brain insulin receptors, are generally

obese, with impaired regulation of metabolic function (174-176).

In overweight and obese conditions, resistance to insulin actions on metabolic function

develops (176-178). It is also thought that insulin resistance and hyperinsulinaemia although

caused by obesity is also a key contributor to the development of obesity (179). The

mechanism leading to the increased resistance is not known. Dysfunction of mitochondrial

20

function, inflammation, lipotoxicity in pathways that regulate food and energy expenditure

are believed to be contributors (180-185).

1.4.3.2 Insulin, obesity and cardiovascular function

In overweight and obese conditions induced by HFD, the level of plasma insulin is elevated.

Several clinical studies link hyperinsulinaemia with elevated blood pressure (186-190). HFD

also leads to the development of insulin resistance. As discussed earlier, insulin resistance is

one of the characteristics of obesity and is linked to cardiovascular diseases such as

hypertension. Thus, it is not surprising that there is a strong correlation between obesity and

cardiovascular disease.

1.4.3.3 Effects of insulin on heart rate and mean arterial pressure

Insulin contributes to an elevated blood pressure and heart rate. For example, human studies

reveal that the IV administration of insulin influences blood pressure and HR (191). This is

also reproduced following acute ICV administration of insulin in rats (54), and acute IV

administration in anaesthetised (192) and conscious rats (54, 193, 194). Moreover, studies

also show that the chronic IV infusion of insulin in conscious rats also increases blood

pressure (195, 196) and HR (197).

The relationship of increased insulin in HFD and cardiovascular regulation has not been well

investigated. In rabbits, HFD have been shown to increase blood pressure and insulin may

contribute to the blood pressure elevation (41). Moreover, acute ICV administration of insulin

in rabbits fed a HFD showed increases in MAP and HR (41).

These findings suggest a possible link between increased insulin levels and increases in

cardiovascular complications observed in overweight and obese conditions.

21

1.4.3.4 Role of insulin on sympathetic nerve activity

Insulin resistance and increases in blood pressure have been linked to activation of the

sympathetic nervous system, resulting from chronic hyperinsulinemia (110). IV infusion of

insulin results in significant elevations of NE plasma levels in humans (191, 198), and rats

(192, 196), suggesting that insulin elicits sympathetic activation. More directly, insulin has

been shown to act in the brain to activate the sympathetic nerves supplying many organs (54,

110, 199). This is discussed in the following sections.


Insulin is known to increase SNA to the hindlimb. The IV administration of insulin in rats

resulted in significant increases of LSNA (200-203). This effect is mediated by actions in the

brain since ICV administration of insulin produced a similar response (54, 204, 205). This

has been shown in mice (53, 206) and in rats (207).

In obese conditions, centrally administrated insulin induces increases in LSNA that are

similar to that seen in lean controls (53, 206, 207).


In lean mice, acute ICV injection of insulin causes dose-dependent increases in RSNA (53).

In rats, ICV insulin also increases RSNA (54). But some reports show that the effect of

sympathetic activation on the kidney was absent upon insulin administration (200-202, 204,

205). These variable effects in rats might be due to the different doses that were used.

In obese animals, insulin was also found to activate RSNA. In db/db mice, the increases in

RSNA elicited by ICV administration of insulin were similar compared to control (110).

However, in another study it has been reported that the ICV administration of insulin in obese

mice leads to increased RSNA, but significantly less compared to ND (53). This report

22

suggests that there might be resistance to insulin in overweight and obese conditions, and the

resistance may be selective for metabolic function and selected cardiovascular outputs such

as LSNA but not RSNA.

Sympathetic nerve activity in brown adipose tissue

BAT SNA is important for regulating metabolic function by insulin. Activation of BAT SNA

leads to thermogenesis, increasing energy expenditure, and increasing food intake. ICV

administration of insulin in lean mice and rats results in dose-dependent increases in BAT

SNA (53, 54).

The BAT SNA induced by ICV insulin in obese mice also increases but to a lesser extent

than in lean mice (53). This suggests that the actions of insulin on BAT SNA in overweight

and obese conditions are attenuated.

Mechanisms meditating changes in sympathetic nerve activity by insulin

A number of studies have investigated the cellular transduction pathways mediating the

effects of insulin on SNA. Mitogen-activated protein kinase (MAPK) mediates the increase in

BAT SNA induced by insulin (54). Inhibition of MAPK by PD98059 or U0126 administered

centrally blocked the effects of insulin on BAT SNA in rats (54), but did not affect RSNA or

LSNA (54). However, PI3K plays an important role in mediating the LSNA response to

insulin. In lean mice, pre-treatment with LY294002, a PI3K inhibitor, in the brain attenuates

the increases in LSNA in response to insulin (53, 54, 206). This has been seen in lean, as well

as, obese mice (53). PI3K inhibition does not affect insulin’s ability to increase RSNA (54).

A different mechanism mediates insulin’s RSNA effects. In homozygous and heterozygous

MC-4R knockout mice the effects of insulin on RSNA was absent (110). Together these

findings suggest that the activation of MC-4R is critical for the RSNA response of insulin.

23

Thus, there are distinct central pathways that mediate the sympathetic nervous system

responses to different target organs elicited by insulin.

1.4.4 Interaction between the hormones

1.4.4.1 Leptin and resistin

As discussed above, there is considerable evidence showing leptin has effects on

cardiovascular regulation. However, little is known to date about resistin, and there are no

studies investigating the combined effects of leptin and resistin on cardiovascular regulation.

A few studies suggest there may be an interaction between leptin and resistin. For example,

leptin and resistin act in the brain through the same signalling pathway involving PI3K to

induce increases in RSNA (51, 58, 208). In a separate study in mice overexpressing resistin,

leptin resistance was observed (209). Thus, resistin and leptin may interact to influence

cardiovascular regulation, but this has not been examined to date.

1.4.4.2 Leptin and insulin

There is some evidence showing the potential for leptin and insulin to interact in the brain.

For example, both hormones act through PI3K to induce increases in LSNA (54, 210, 211).

More direct evidence is available from studies in the peripheral cardiovascular system, the

combination of both hormones have additive effects on vascular tone (212). In that study, it

was shown that both hormones combined induce a greater release of nitric oxide compared to

each hormone alone (212). Moreover, in liver cells, insulin alone and leptin alone result in the

activation of JAK2, STAT3/5b, and IRS-1/2. However, the combination of both leptin and

insulin in the liver cell results in a greater activation of JAK2 and STAT5b but no change in

STAT3, IRS-1/2 (213). Therefore, there is evidence that suggests that both insulin and leptin

can act to enhance each other’s actions, and the interaction may be for specific

24

pathway/responses. Since kidney function is important for cardiovascular regulation, whether

there is an interaction between leptin and insulin on RSNA should be investigated.

1.4.4.3 Resistin and insulin

As indicated earlier (1.4.2), resistin was first identified by its ability to induce insulin

resistance (47). This appears to occur through the attenuation of phosphorylation of the

insulin receptor by resistin (139). This is to our knowledge, the only example of an

interaction between the two hormones.

1.5 SUMMARY

Leptin, resistin and insulin are all hormones known to be produced and released in response

to increased food intake in normal conditions. They play important roles in metabolic

function. In overweight and obese conditions induced by HFD, the levels of these hormones

are increased in plasma and SNA is abnormally elevated. Increased SNA is linked to

cardiovascular complications such as hypertension. Therefore, these hormones may be

implicated in the elevated sympathetic activity observed in overweight and obese conditions.

Previous work shows that leptin, resistin and insulin alone increase RSNA in ND. However,

HFD can influence SNA responses to leptin, for example; the LSNA response is reduced but

the RSNA response remains unaffected. In contrast, in HFD the LSNA response to insulin is

unaffected but the RSNA response is reduced. In addition, the effect of resistin on RSNA in

HFD is unknown. Further, the combination of (leptin + resistin), (leptin + insulin) and

(resistin + insulin) on RSNA in both ND and HFD are unknown. This is summarised in in

table 1.1.

25

Table 1.1 Summary effects of leptin, insulin and resistin on RSNA on a normal diet and a

high fat diet on RSNA, and the gap that needs to be investigated.

Hormones Normal Diet - RSNA High Fat Diet - RSNA

Leptin

Insulin ↑

Resistin ?

Leptin + resistin ? ?

Leptin + insulin ? ?

Resistin + insulin ? ?

increase; ↑ smaller increase; ? Unknown

1.6 AIMS

The aims of this study are:

1- To determine the effects on renal sympathetic nerve activity (RSNA) in rats fed a

normal diet chow (ND) by combined administration of (i) leptin + resistin, (ii) leptin

+ insulin, and (iii) resistin + insulin.

2- To determine the effects of resistin on renal sympathetic nerve activity (RSNA) in rats

fed a high fat diet (HFD).

3- To determine whether high fat diet (HFD) influences the effects on renal sympathetic

nerve activity (RSNA) following the combination of (i) leptin + resistin, (ii) leptin +

insulin, and (iii) resistin + insulin.

26

2 CHAPTER 2: Materials and Methods

2.1 INSTRUMENTS

2.1.1 Manufacture of blood vessel cannulae

To cannulate the femoral artery and vein for blood pressure measurement and infusion of

drugs intravenously (respectively), cannulae were manufactured in the following way. Two

different sizes of tubing polyvinyl chloride (PVC) were obtained. The smaller tubing

(catalogue number 530010 – internal diameter of 0.28 mm and outer diameter of 0.61 mm;

Biocorp, VIC, Australia) was cut to a length of approximately 7 cm and inserted to the larger

one (catalogue number 530055- internal diameter 0.80 mm and outer diameter 1.20 mm,

Biocorp, VIC, Australia) which was approximately 15 cm. The two tubing were connected

using glue (Araldite ® Epoxy Resin (Selleys Pty Ltd; NSW, Australia). The glue was inserted

between the tubing to ensure a watertight connection. A small ball of glue was made at the

connection point between the two pieces of tubing, to allow the tying of the vessels using

threads, to secure the catheters after they have been inserted (Figure 2.1).

27

Figure 2.1 PVC tube.

A blood vessel cannula used for cannulating the femoral vessels. The small and large

polyvinyl chloride (PVC) tubing hold together by the ball of glue.

28

2.1.2 Renal dissection instruments

A scalpel blade was used to incise the skin, and scissors were used to blunt dissect and

separate the skin from the muscles. Scissors were used to cut the underlying muscle. Two

cotton tips were used to separate the fat tissue from the muscle to expose the kidney and the

vessels innervating the kidney. Retractors were used to pull and retract the muscles, kidney

and fat tissue aside. An operating microscope was used to identify the renal nerve. After

identification of the nerve, it was placed onto the bared tips of two Teflon-coated silver wire

electrodes. Paraffin oil was used to insulate the nerve electrode junction from surrounding

tissue. An audio amplifier was used so that the renal nerve activity was audible.

2.1.3 Manufacture of electrodes

A Bipolar Electrode was used for sympathetic nerve activity signal recording. These Bipolar

electrodes have two Teflon stainless steel wires (catalogue number: AGT1025, World

Precision Instrument Inc., FL, USA). The tips of the bipolar electrodes were bent to form an

L-shape and were stripped of the Teflon insulation for about 5 mm. To allow amplification

the tips of the electrode were separated by approximately 4mm. The other end of the

electrode had gold plated pins. Each electrode was covered with PVC tube to stabilized and

protect the electrode (catalogue number 530055- internal diameter 0.80 mm and outer

diameter 1.20 mm, Biocorp, VIC, Australia) and to reduce electrical interference (Figure 2.2).

2.1.4 Blood pressure and heart rate instruments

2.1.4.1 Invasive blood pressure and heart rate monitoring

For the invasive blood pressure monitoring, a cannula was inserted into the femoral artery

and was connected to a transducer for the recording of blood pressure using a PowerLab data

29

acquisition system (ADInstruments, NSW, Australia). The heart rate was calculated from the

arterial pressure pulse.

2.1.4.2 Non-invasive blood pressure and heart rate monitoring

For the non-invasive blood pressure monitoring, a tail cuff blood pressure measurement

system (ML125 NIBP Controller and MLT125/R Pulse Transducer/Pressure Cuff for NIBP-

rat) was threaded over the animal’s tail and was connected to a transducer sensor. The sensor

detects the first impulse from the tail artery once deflation occurs after being occluded by the

cuff.

2.1.5 Microinjection instruments

A drill bit was used to carefully drill the dorsal surface of the skull by hand so that the dorsal

surface of the brain was exposed for injections. ICV injections were made using glass

micropipettes. The glass micropipettes were obtained from (Clay Adams, New Jersey, USA).

A micropipette puller (Sutter Instrument Company, Model P-97, CA, USA) was used to make

the fine tipped micropipette injectors. The micropipette puller was programmed so that the

external diameter of the micropipette was approximately 60µm and the internal diameter was

approximately 30µm when the shaft was trimmed down to approximately 12mm in length.

2.1.6 Perfusion instruments

Two bottles were used, one contained the phosphate buffered saline (PBS) (200-220 ml) and

the other contains the fixative (4% paraformaldehyde) (200-220ml). Paraformaldehyde 95%

was purchased from (Sigma Aldrich) Australia.

30

2.1.7 Brain removal instruments

To remove the brain, a large pair of scissors was used to decapitate animals. Rongeurs were

used to remove the neck muscles and the skull bones, and forceps were used to peel off the

dura. A long thin spatula was used to cut the cranial nerves and remove the brain.

2.2 HORMONES/DRUGS

Recombinant rat resistin (lot#L16251/F, L28370,) was purchased from Sigma Aldrich

(Australia), recombinant rat leptin (lot#AQP2411031) was purchased from Sapphire

Biosciences (Australia), human insulin (Actrapid 100 IU/ml) was purchased from a local

pharmacy, and saline (0.9% NaCl, Baxter) was purchased from Baxter (Australia). Urethane

was purchased from Sigma Aldrich (Australia), and pentobarbitone (325 mg) was purchased

from Virbac mental health (Australia).

31

Figure 2.2 Wire electrode.

Bipolar Teflon-insulated stainless steel electrode wire insulated in a polyvinyl chloride (PVC)

tube used for recording from the renal nerve.

Teflon coat

Bent bared tips

PVC Tubing

Gold tip plugs

32

2.3 ANIMAL HOUSE, FAT MEASUREMENTS, FOOD AND BLOOD PRESSURE

MONITORING

2.3.1 Animal type and weight

From the ARC (Animal Resources Center, W.A, Australia) male Sprague-Dawley rats were

obtained. Each animal weighed approximately between 220-240 grams upon they arrival. The

animals were housed in temperature-controlled rooms with a 12:12 hour light / dark cycle

(light off at 7:00 P.M.) in the Animal Facility (RMIT University, Victoria, Australia). These

protocols comply with the Guiding Principles for Research Involving Animals and Human

Beings and the guidelines set by the Australian Code of Practice for the Care and Use of

Animals for Scientific Purposes, 2007 (National Health and Medical Research Council of

Australia). All experiments were approved by the Royal Melbourne Institute of Technology

(RMIT) University Animal Ethics Committee (approval letter is attached in appendix II).

Every attempt was made to minimise animal suffering, discomfort and reduce the number of

animals needed to obtain reliable results.

2.3.2 Diet

In the experiments, rats were acclimatised for seven days prior to transfer to controlled food

experiments. Animals were separated into two groups. One group was allowed free access to

a ND (Fat=4.8%), and the other group was allowed free access to a HFD (SF14-004 (22%

fat), Speciality Feeds, Glen Forrest, WA) for 8-10 weeks. Both groups were also allowed free

access to water. Food intake was monitored by weighting the amount of food each week.

Calorie intake was calculated from the food intake based on (14KJ/g in ND; 18KJ/g in HFD)

of food eaten.

33

2.3.3 Systolic blood pressure monitoring

Blood pressure was monitored using a tail cuff occlusion method, fortnightly during the

experimental period. Firstly the animals were exposed to a heating lamp for approximately 5

minutes and placed in a restrainer. The tail was threaded through the occlusion cuff, and the

occlusion cuff was placed at the base of the tail. Maximum occlusion pressure was set to 250

mmHg, and deflation was set to fifteen seconds. A blood pressure sensor was placed at the

distal end of the cuff to monitor blood pressure. Upon deflation, the pressure at which there

was the first appearance of a pulse was taken as the systolic pressure.

2.3.4 Fat measurement

To measure the percentage of body fat in animals at the end of the feeding period, an

EchoMRI 500 body composition analyser was used. The animal was placed in a tubular

holder and then placed inside the EchoMRI. This analyser utilises magnetic resonance to

allow a precise body composition measurements of fat, lean, and total water mass in awake

animals. This technique takes approximately three minutes to scan and generate values of

body composition.

2.4 SURGICAL PROTOCOLS

2.4.1 Femoral vein and artery cannulation

The femoral artery and the femoral vein were cannulated prior to microinjections. The

cannulation of the femoral artery allows constant monitoring of blood pressure, while the

cannulation of the femoral vein allows intravenous (IV) administration of urethane

anaesthesia to be maintained during the length of the experiment. Initially, rats were

anesthetized using gaseous anaesthesia (initially 5% then 2-2.5% isoflurane in O2), the right

femoral region was shaved and swabbed with 70% alcohol. Following that, a 5-7 mm incision

34

was made through the skin over the femoral neurovascular bundle. The subcutaneous fascia

was cleared away using blunt dissection to expose the underlying bundle. The blood vessels

were separated by dissecting out the femoral sheath using forceps.

The femoral vein was cannulated first. Two lengths of fine thread (silk 3/0, Dynek Pty,

Australia) were inserted under the vessel and clamped by a haemostat. The threads were

pulled to elevate the vessel and temporarily stop the blood flow. A fine needle (26 gauge

needle) was inserted into the vessel, to make an access point for the cannula which was filled

with heparinized saline (50 U/mL). Following the insertion of the needle, the cannula was

inserted into the vessel and the needle was slowly withdrawn from the vessel. After insertion

of the cannula the thread was tied around the cannula, and the other thread was tied around

the ball of glue to secure the cannula in position. The femoral artery was cannulated using a

similar procedure. Upon completion of the cannulation procedures, the incision was closed

using stitches (silk 2/0-USP, Dynek Pty, Australia). The free end of the arterial cannula was

connected to a transducer for recording blood pressure using a PowerLab data acquisition

system (ADInstruments, NSW, Australia). The heart rate (HR) was calculated from the

arterial pressure pulse. The free end of the venous cannula was used for IV infusion. General

anaesthesia was maintained by IV infusion of urethane 0.05 ml/15 sec of a 25% solution total

dose of (1.4-1.6 g/kg). Supplemental doses of 0.05 ml of solution were given to maintain

deep anaesthesia if required. Anaesthesia level was determined by the absence of corneal and

pedal pinch reflexes.

2.4.2 Skull drilling

Animals were placed prone, and their heads were anchored in a Stoelting stereotaxic frame,

such that bregma and lambda were positioned on the same horizontal plane. An incision was

made through the dorsal skin of the head. The skin and the subcutaneous tissue and

35

periosteum were cleared to expose the dorsal surface of the skull. Using a hand held drill bit,

a hole (approximately 2 mm diameter) centred 0.7 mm caudal to bregma and 1.4 mm lateral

from midline, was drilled into the skull to expose the dorsal surface of the brain for

microinjections. To prevent drying of the exposed surface, the hole was covered with cotton

wool soaked in normal saline (sodium chloride 0.9%).

2.4.3 Renal nerve dissection

After a flank incision, blunt dissection was used to separate the skin from muscle. Cotton tips

were then used to separate fat tissue from muscle so that the left kidney was exposed

retroperitoneally. With the aid of retractors, the muscle, kidney and fat tissue were pulled

aside. Under microscopic observation, the renal nerve was identified running along the renal

artery. We isolated the nerve without causing any damage to the nerve or blood vessels

supplying it. Saline was used to keep the nerve and the surrounding tissue moist. The distal

end of the nerve was dissected free of surrounding tissue. The distal end was then cut, and

any remaining saline solution was removed. The cut end of the renal nerve was placed on the

bipolar electrodes and covered with paraffin oil to insulate the nerve-electrode junction from

surrounding tissue. To ensure good nerve recording, firstly we confirmed that the firing of the

nerve signals was synchronous with the cardiac rhythm as seen in (figure 2.3). We used audio

to hear clear bursts of the nerve activity signal. Finally, we injected a lethal dose of

pentobarbitone (325 mg IV) at the end of the experiment to obtain the background noise

(figure 2.3).

36

Figure 2.3 Screen capture of raw recording of renal sympathetic nerve activity

Screen capture of raw recording of renal sympathetic nerve activity. (A) Renal sympathetic

nerve activity from an anesthetized rat, horizontal bar = 2 s; vertical bar = 100 mV. (B)

Electrical activity at the end of the experiment, after lethal dose of pentobarbitone (325 mg

IV), and background noise is observed.

37

2.4.4 Intracerebroventricular injection

Injections were made unilaterally using a glass micropipette (50–70 μm tip diameter) inserted

into the lateral brain ventricle (stereotaxic coordinates: 0.7 mm caudal to bregma, 1.4 mm

lateral to midline, and 3.7-3.9 mm ventral to the surface of the dura). Leptin (7μg/5μl),

resistin (7μg/5μl), insulin (500 mU/5μl) and vehicle (saline 5μl) were administered ICV. The

doses of each hormone were chosen on the basis of previous studies (113, 136). The doses

used are larger than those observed in plasma as noted previously (48, 51, 125, 128). After

the microinjections, the micropipette was left in place for 1 min. At the end of the

experiment, a small amount of blue dye was microinjected using the same coordinates to

confirm microinjection into the lateral ventricle.

2.4.5 RSNA, MAP and HR monitoring

The renal sympathetic nerve activity (RSNA), mean arterial pressure (MAP) and HR were

monitored before and for 180 minutes after hormone/saline administration at 15 minutes

intervals. Resting levels before the ICV injections were calculated to obtain the basal levels.

For the RSNA study, the effects of hormone/saline administration were expressed in terms of

percentage change from baseline. RSNA was amplified using a low-noise differential

amplifier (models ENG 187B and 133, Baker Institute, Victoria, Australia), filtered (band

pass 100–1,000 Hz), rectified, and integrated (at 0.5 s intervals for RSNA). The signal was

recorded using a PowerLab data acquisition system (ADInstruments, NSW, Australia).

Background noise was determined by post-mortem measurements after the rat received a

lethal dose of pentobarbitone (325 mg IV). RSNA was obtained by subtracting the

(background noise) from the total activity. Basal levels were recorded as the average of three

measurements over 15 minutes prior to hormone/saline administration.

38

2.4.6 Perfusion

At the end of each experiment, animals were perfused. A 5-6 cm lateral incision through the

integument and abdominal wall just beneath the rib cage was made. The diaphragm was

incised to expose the pleural cavity. The rib cage was then cut on both sides laterally up to

the clavicle. The sternum was then lifted, and a haemostat was used to clamp the rib cage.

Connective tissue and the thymus were cleared to expose the heart. A 15 gauge perfusion

needle was then passed through the left ventricle into the ascending aorta. The perfusion

needle was secured with a clamp, and PBS (200-220 ml) was infused at a pressure of 80-100

mmHg followed by 4% paraformaldehyde (200-220 ml) at the same pressure.

2.4.7 Tissue collection

To collect brains, animals were first decapitated. Muscle layers were dissected out to reveal

the occipital bone and vertebrae. The spongy part of the occipital bone was carefully removed

using rongeurs to reveal the cerebellum. Following the removal of the occipital bone, the

parietal and the frontal bones were then removed using the same procedure, to reveal the

cerebral hemispheres and brainstem. The dura mater was peeled back from the dorsal brain

surface. Using a long thin spatula the cranial nerves were disconnected from the brain, so that

the brain could be removed from the skull.

The brain was placed in 4% paraformaldehyde for three hours and then into PBS (0.1M)

containing 20% sucrose solution and stored at 4 ºC.

39

2.5 IMMUNOHISTOCHEMISTRY

2.5.1 Sectioning of the rat brain

Serial coronal sections (40 μm thick) of the brain were cut using a Leica CM1950 Cryostat

(Leica Microsystems Nussloch GmbH, Nussloch, Germany) and sections were placed in 24-

well plates containing cryoprotectant. Sections were then processed immunohistochemically

to detect Fos protein using standard immunohistochemical procedures.

The following brain areas were examined: the hypothalamus; paraventricular nucleus (PVN),

supraoptic nucleus (SON) and the arcuate nucleus (ARC). The median preoptic nucleus

(MnPO) and the vascular organ of lamina terminalis (OVLT) in the lamina terminalis were

also examined. In the midbrain, the periaqueductal grey (PAG) and finally, in the medulla,

the nucleus of the solitary tract (NTS), rostral ventrolateral medulla (RVLM) and the raphe

pallidus nucleus (RPA) were also examined.

2.5.2 Immunohistochemical staining procedures

To identify increased neuronal activation, immunohistochemistry was performed to detect the

protein Fos, a marker for activated neurons. Sections were washed in PBS (3 times for 5 min

duration). This was also performed between the following incubations. Endogenous

peroxidase activity was destroyed by incubating with 0.5% H2O2. Then, sections were

incubated in 10% normal horse serum (NHS) and 0.5% Triton X-100 to facilitate antibody

penetration. Sections were then incubated in anti-Fos primary antibody (rabbit polyclonal

IgG, c-Fos (K-25): sc-253, Santa Cruz Biotechnology, CA, USA; dilution: 1:5000). Sections

were then left overnight at 4 °C. The next day sections were then incubated with (i)

biotinylated secondary antibody (1:400, Anti-rabbit raised in goat, B8895, Sigma Aldrich,

Australia) and subsequently (ii) Extravidin (1:400, Sigma Aldrich, Australia). Both

40

incubations were at room temperature. Then the sections were incubated in 0.05% 3,3′-

diaminobenzidine hydrochloride (DAB; Sigma Aldrich, Australia). The reaction was initiated

by adding 5 μl of 30% hydrogen peroxide and terminated by washes with fresh PBS. Finally,

the sections were allowed to dry and were coverslipped using Depex mounting medium.

2.6 STATISTICAL ANALYSIS

2.6.1 Systolic blood pressure, body weight, food intake and calorie intake

The changes in systolic blood pressure, body weight, food intake and the average caloric

intake per day during the feeding time were compared between the groups using two-way

ANOVA.

2.6.2 RSNA, MAP and HR

The resting levels of MAP and HR before the ICV injections were compared between groups

using one-way ANOVA. Changes in RSNA, MAP and HR were compared between groups in

each experimental series by using two-way ANOVA with repeated measures.

2.6.3 Fat

The distribution of whole body fat was calculated and expressed as percentages and

compared between groups using Student’s unpaired t-test. Similarly, epididymal fat was

compared between groups using Student’s unpaired t-test.

41

2.6.4 Immunohistochemistry (FOS)

Fos-positive cell nuclei were counted unilaterally in 2 sections per brain region using Image J

software (version 1.7.0). Numbers of Fos positive cell nuclei were compared between groups

using one-way ANOVA.

2.6.5 Invasive blood pressure

The changes in the blood pressure over the feeding time were compared between groups

using two by using two-way ANOVA.

In all the above measures, all results are expressed as means ± SE. P<0.05 was considered to

be statistically significant. Specific details of the statistical analysis for each study are

provided in each specific chapter.

42

3 Chapter 3: Central Leptin And Resistin Combined Elicits Enhanced Central Effects On

Renal Sympathetic Nerve Activity

3.1 INTRODUCTION

Leptin is a well known hormone released from fat tissue and acts centrally to influence

metabolic and cardiovascular functions (18, 89, 98, 214). It can increase renal sympathetic

nerve activity (RSNA) and this action may make an important contribution to the abnormally

elevated RSNA observed in obesity or overweight conditions (17, 50).

Resistin, also a hormone released from fat tissue, has recently been shown to act centrally to

elicit similar effects as leptin on RSNA (51). Both leptin and resistin increase RSNA through

central mechanisms that involve phosphatidylinositol 3 kinase (PI 3-kinase) (51, 208). Thus,

these two hormones appear to act through similar mechanisms to increase RSNA and this

raises the question of whether combined administration of leptin and resistin could elicit

greater increases of RSNA. This could have important implications since plasma leptin and

resistin levels correlate with adiposity (47, 48, 131, 215), and both are elevated in obesity and

overweight conditions. Thus, the first aim of the present study was to measure RSNA in

response to leptin administered in the presence of resistin compared to the responses induced

by leptin and resistin alone.

Although there is considerable knowledge of the brain nuclei activated by leptin, there has

not been a systematic investigation of those activated by resistin. Since similar mechanisms

appear to be mediating the central actions of leptin and resistin, we wondered whether similar

areas in the brain were activated and whether an enhanced effect observed when both were

combined could be correlated with specific brain nuclei. We focused on autonomic brain

nuclei known to influence cardiovascular function. We used the protein, Fos, as a marker of

increased neuronal activity to quantify the number of activated neurons in each brain nucleus

43

and compared their distribution after administration of resistin with that after administration

of leptin given alone and the combination of leptin and resistin.

3.2 METHODS

Please refer to Chapter 2, for a more detailed explanation on methods and materials.

3.2.1 Ethical approval

All the experimental protocols were performed in accordance with the Prevention of Cruelty

to Animals Act 1986 (Australia). The procedures conform to the “Guiding Principles for

Research Involving Animals and Human Beings” and the guidelines set out by the Australian

Code of Practice for the Care and Use of Animals for Scientific Purposes, 2013 (National

Health and Medical Research Council of Australia) and were approved by the RMIT

University Animal Ethics Committee (approval letter is attached in appendix II).

3.2.2 General procedures and protocols

Male Sprague-Dawley rats were obtained from ARC animal resources centre (W.A,

Australia). Rats were housed at 23°C with a 12-h light/dark cycle and allowed free access to

standard rat chow and water for 8-10 weeks. On the day of the experiment, anesthesia was

induced using isoflurane gas (initially 5% then lowered to 2%) in O2. A catheter was inserted

into the femoral vein, so that anesthesia could be maintained with intravenous (IV) urethane

(1.4–1.6 g/kg initially, followed by supplemental doses of 0.05 ml of a 25% solution, as

required). The depth of anesthesia was maintained to ensure the absence of corneal and pedal

reflexes. The femoral artery was cannulated for monitoring arterial blood pressure. Mean

arterial pressure (MAP) and heart rate (HR) were calculated from the arterial pressure pulse

44

using LabChart (ADInstruments, NSW, Australia). Rats were kept on a heating pad for the

duration of the experiments to maintain body temperature at approximately 37.5 oC.

3.2.2.1 Renal sympathetic nerve activity

After a flank incision, the left kidney was exposed retroperitoneally. With the aid of an

operating microscope, the renal nerve was carefully dissected free of surrounding tissue

under mineral oil and placed onto the bared tips of two Teflon-coated silver wire electrodes.

RSNA was amplified using a low-noise differential amplifier (models ENG 187B and 133,

Baker Institute, Victoria, Australia), filtered (band pass 100–1,000 Hz), rectified, and

integrated at 0.5 s intervals. The signal was recorded using a PowerLab data acquisition

system (ADInstruments, NSW, Australia).

3.2.2.2 Microinjections into the lateral brain ventricle (ICV)

Each animal was placed prone, and the head was mounted in a Stoelting stereotaxic frame,

such that bregma and lambda were positioned on the same horizontal plane. For exposure of

the dorsal surface of the brain, a hole (2 mm diameter) centred 0.7 mm caudal to bregma and

1.8 mm lateral to the midline, was drilled into the skull. Pressure ejection was used to make

unilateral microinjections using a fine glass micropipette (50–70 µm tip diameter) inserted

into the lateral brain ventricle (stereotaxic coordinates: 0.7 mm caudal to bregma, 1.8 mm

lateral to midline, and 3.7-3.9 mm ventral to the surface of the dura). The volume injected

was 5µl and was determined by using calibrated markings on the micropipette. After the

microinjection, the micropipette was left in place for 1 min. At the end of the experiment a

small amount of pontamine sky blue was microinjected using the same coordinates to


45

3.2.2.3 Experimental protocols

MAP, HR and RSNA were measured before and for three hours after administration of saline

vehicle (n=5), leptin (7 µg; n=5), resistin (7 µg; n=4) and the combination of both resistin and

leptin (n=6) (leptin was administered 15 minutes after resistin). The doses of each hormone

were chosen on the basis of previous studies (113, 136).

3.2.2.4 Immunohistochemistry for Fos

At the end of the experiment, the animals were overdosed with pentobarbitone (325 mg IV)

and then perfused through the heart with 200 ml of 0.1 M phosphate buffered saline (PBS, pH

7.2), followed by 200 ml of 4% paraformaldehyde in 0.1 M PBS). The brains were then

carefully removed, post-fixed in the same fixative (for 3 hours) and left overnight in 0.1 M

PBS containing 20% sucrose.

Serial coronal sections (40 µm thick) of the brain were cut using a cryostat (Leica, CM1900,

Germany). One in five sections was collected, placed onto gelatine-coated slides, dried for 2

hours at room temperature and then processed immunohistochemically to detect the protein

Fos using standard immunohistochemical procedures. In brief, endogenous peroxidase

activity was destroyed by incubating with 0.5% H2O2 for 30 minutes. The sections were then

incubated in 10% normal horse serum (NHS) for 60 minutes prior to 0.5% Triton X-100 (30

minutes) to facilitate antibody penetration. The sections were then incubated in primary

antibody raised in the rabbit against Fos (1:400, rabbit polyclonal IgG, c-Fos (K-25): sc-253,

Santa Cruz Biotechnology, CA, USA) for 24 hours at room temperature. The sections were

then incubated for 2 hours with biotinylated anti rabbit secondary antibody raised in goat,

(1:400, Sigma Aldrich, Australia) and subsequently with Extravidin – peroxidase complex

(1:400, Sigma Aldrich, Australia) for 1 hour. In between all steps the sections were washed

three times (for 5 minutes each time) with PBS. Then the sections were incubated in 0.05%

46

3,3′-diaminobenzidine hydrochloride (DAB; Sigma Aldrich, Australia) in PBS for 10

minutes. The reaction was initiated by adding 5 μl of 30% hydrogen peroxide and terminated

by washes with fresh PBS. Finally, the sections were allowed to dry and were coverslipped

with Depex.

3.2.3 Statistical analysis

The average resting levels of MAP and HR in the different groups of animals prior to

hormones/saline administration were compared between groups using one-way ANOVA. The

integrated RSNA was calculated over a period of 1-2 minutes at each time point (ie before

and every fifteen minutes after injection) and expressed as a percentage of the resting level

prior to the injections. Changes in MAP, HR and RSNA over time were compared between

groups in each experimental series by using two-way ANOVA. The average of the last 30

minutes of the observation period was used to calculate the maximum changes and these were

compared between groups using one-way ANOVA.

Fos-positive cell nuclei were visualised and counted unilaterally, except in the midline

structures (raphe pallidus, dorsal raphe, median preoptic nucleus and organum vasculosum of

the lamina terminalus), using bright field illumination at 200x magnification. At least two

sections for each brain nucleus were examined. The average number of Fos-positive cell

nuclei per section was calculated for each brain nucleus in each animal and comparisons

between the groups were performed using one-way ANOVA. When an overall significant

difference between groups was detected, comparisons were made between individual groups,

using Holm-Sidak’s test for multiple comparisons. All results are expressed as means± sem.

P<0.05 was considered to be statistically significant. PRISM software was used.

47

3.3 RESULTS

3.3.1 Effects of ICV injection of leptin and resistin on RSNA

Intracerebroventricular (ICV) injection of leptin alone and resistin alone increased RSNA

significantly compared to saline (control). Leptin alone increased RSNA reaching a

maximum of approximately 75% between 2-3 hours after administration (P<0.0001

compared to saline) (figure 3.1). Resistin increased RSNA significantly compared with saline

and followed a similar time course to that of leptin reaching a maximum level of

approximately 50% (P<0.0001 compared to saline). When leptin and resistin were

administered, RSNA increased dramatically to a maximum of approximately 165%, which

was significantly greater compared to the responses of either hormone alone (P<0.0001)

(figure 3.1).

48

Figure 3.1 Percent changes in RSNA induced by leptin and resistin alone and in combination.

Percent changes in renal sympathetic nerve activity (RSNA) from baseline levels over time

after intracerebroventricular injection of leptin (n=5) (7μg/5μl), resistin (n=4) (7μg/5μl),

control (saline, n=5) (5μl) and the combination of leptin and resistin (n=6). (*P<0.0001

compared to control; #P<0.0001 compared to leptin alone or resistin alone).

49

3.3.2 Effects of ICV injection of leptin and resistin on MAP and HR.

Leptin alone induced a small change in MAP of 4+3 mmHg by the end of the observation

period and this was not statistically significantly different from saline control (5+2 mmHg)

(figure 3.2). Following resistin there was a small change in MAP by 11 + 1 mmHg, which

followed a similar time course to that of leptin and was not statistically significantly different

compared to saline (figure 3.2). When leptin and resistin were administered, the response in

MAP was not significantly different to the responses of either hormone alone or control

(figure 3.2).

Similarly, there was no significant difference in the responses in HR between the groups. By

the end of the observation period, neither leptin nor resistin induced a statistically significant

change in HR compared to saline (figure 3.2). When leptin and resistin were administered,

the change in HR was not significantly different to the responses of either hormone alone or

control (figure 3.2).

The average resting MAP and HR prior to hormone/saline administration are shown in table

3.1. One-way ANOVA analysis showed there was no significant differences between the

groups.

50

Figure 3.2 Changes in MAP and HR induced by leptin and resistin alone and in combination.

Changes in mean arterial pressure (MAP) and heart rate (HR) over time induced by the

intracerebroventricular administration of leptin (n=5) (7μg/5μl), resistin (n=4) (7μg/5μl),

Control (saline, n=5) (5μl) and the combination of leptin and resistin (n=6).

51

Table 3.1 Resting MAP and HR.

Resting mean arterial pressure (MAP) and heart rate (HR) prior to intracerebroventricular

injection of saline (n=5), leptin (n=5), resistin (n=4) and the combination of leptin and

resistin (n=6) in anesthetised rats.

Saline Leptin Resistin Leptin+Resistin

MAP (mmHg) 93.5 ± 4.1 79.7 ± 1.6 82.1 ± 3.7 90.5 ± 4.2

HR (beats/min) 360 ± 7 336 ± 11 342 ± 10 323 ± 16

52

3.3.3 Effects of leptin and resistin, alone and in combination, on Fos-positive cell nuclei

3.3.3.1 Lamina terminalis

In the organum vasculosum of the lamina terminalis (OVLT), and in the median preoptic

(MnPO) nucleus, the number of Fos-positive cell nuclei following leptin or resistin alone

were not significantly different compared to control (figures 3.3 and 3.4). However, when

leptin and resistin were administered, there was a significant increase in Fos-positive cell

nuclei by two-fold compared to the saline control (P<0.05) (figures 3.3 and 3.4).

3.3.3.2 Arcuate nucleus

In the arcuate nucleus (ARC), leptin and resistin alone significantly increased the number of

Fos-positive cell nuclei by approximately 50% and 100%, respectively, compared to control

(figure 3.5). When leptin and resistin were administered, the increase in Fos-positive cell

nuclei was significantly greater than in control (P<0.001) and compared to each hormone

alone (P<0.001) (figure 3.5).

3.3.3.3 Paraventricular and supraoptic nuclei

In the hypothalamic paraventricular nucleus (PVN) leptin and resistin alone induced a

significant two-fold increase in the number of Fos-positive cell nuclei compared to control

(P<0.05) (figures 3.5 and 3.6). When leptin and resistin were administered, the increase in the

number of Fos-positive cell nuclei was also significantly greater than control but did not

differ from the result observed with leptin or resistin alone. In the supraoptic nucleus (SON),

the number of Fos-positive cell nuclei following each treatment was not statistically

significant between groups (figure 3.5).

53

Figure 3.3 Average number of Fos-positive cell nuclei in OVLT and MnPO induced by leptin and resistin alone and in combination.

Average numbers of Fos-positive cell nuclei per section in the organum vasculosum of

lamina terminalis (OVLT) and median preoptic nucleus (MnPO) from rats administered

intracerebroventricular saline (control) (n=5) (5µl), leptin (n=5) (7µg/5µl), resistin (n=4)

(7µg/5µl) and leptin combined with resistin (n=5). *P<0.05 compared to control.

54

Figure 3.4 Photomicrographs of Fos-positive cell nuclei in OVLT induced by leptin and resistin alone and in combination.

Photomicrographs showing Fos-positive cell nuclei in the organum vasculosum of the lamina

terminalis (OVLT) of rats following centrally administered saline (5µl; control), leptin (7µg),

resistin (7µg) and leptin combined with resistin. Bar, 200 µm. Dashed lines outline the

OVLT.

55

Figure 3.5 Average number of Fos-positive cell nuclei in ARC, PVN and SON induced by leptin and resistin alone and in combination.

The average number of Fos-positive cell nuclei per section in the arcuate (ARC),

paraventricular (PVN) and supraoptic (SON) nuclei after intracerebroventricular injection of

saline (5µl; control), leptin (n=5) (7µg/5µl), resistin (n=4) (7µg/5µl), and leptin combined

with resistin (n=5). *P<0.05, **P<0.01, ***P<0.001 compared to control; #P<0.001

compared to leptin and resistin alone.

56

Figure 3.6 Photomicrographs of Fos-positive cell nuclei in PVN induced by leptin and resistin alone and in combination.

Photomicrographs showing Fos-positive cell nuclei in paraventricular nucleus (PVN) of rats

following centrally administered saline (5µl; control), leptin (7µg), resistin (7µg) and leptin

combined with resistin. Bar, 200 µm. Dashed lines outline the PVN. Abbreviations, Fx,

fornix.

57

3.3.3.4 Dorsal raphe and medulla oblongata

In the dorsal raphe, the rostral ventrolateral medulla (RVLM), and in the nucleus tractus

solitarius (NTS), there was no significant difference in the number of Fos-positive cell nuclei

between the treatments (figure 3.7). In the raphe pallidus (RPA), following leptin or resistin

alone the average number of Fos-positive cell nuclei was not significantly different from

controls However, when leptin and resistin were administered, the number of Fos-positive

cell nuclei was significantly reduced compared to leptin or resistin alone (P<0.05), but was

not significantly different from control (figure 3.7).

58

Figure 3.7 Average number of Fos-positive cell nuclei in DR, RVLM, NTS and RPA induced by leptin and resistin alone and in combination.

Average number of Fos-positive cell nuclei per section in the dorsal raphe (DR), rostral

ventrolateral medulla (RVLM), nucleus tractus solitarius (NTS) and raphe pallidus (RPA)

following saline (control) (n=5) (5µl), leptin (n=5) (7µg/5µl) resistin (n=4) (7µg/5µl) and

leptin combined with resistin (n=5). *P<0.05 compared to leptin or resistin alone.

59

3.4 DISCUSSION

In the present study we found that when leptin was administered ICV after resistin, there was

a significantly greater increase in RSNA compared to the response elicited by either hormone

alone. We also determined in a more systematic way than has been previously investigated,

the distribution of activated neurons in the brain following resistin. Compared to saline

control we found that resistin significantly increased the number of activated neurons in

arcuate and hypothalamic nuclei, which was similar to leptin. We also found that in the

OVLT, MnPO, ARC, and in the hypothalamic PVN the number of Fos-positive cell nuclei

was increased significantly more when leptin was administered after resistin compared to

control. However, only in the ARC was the increase in the number of activated neurons

significantly greater than when each hormone was administered alone. In the RPA, by

contrast, the number of Fos-positive cell nuclei was significantly reduced when leptin was

administered after resistin compared to that observed after each hormone alone.

In the present study, ICV leptin alone increased RSNA, as did resistin, confirming previous

studies (99, 105). In contrast to leptin alone, the increase in RSNA induced by leptin was at

least doubled when it was administered in the presence of resistin. This finding could have

important implications in conditions in which leptin and resistin are elevated such as in

obesity. In this condition RSNA is abnormally elevated (17), and since leptin is believed to

be an important mediator of the increase in RSNA in that condition (17, 50), the presence of

resistin could contribute to leptin’s key role in elevating RSNA in obesity.

The mechanisms involved in the enhanced response may involve (i) activation of separate

populations of cells; if the number of Fos positive nuclei observed following the

administration of both hormones was the same number as each hormone alone, this would

suggest that both hormones activate the same population of neurons. However, since more

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cells were activated by the combination of leptin + resistin, it suggest there must be a

population of cells that can be activated by leptin or resistin independently, and (ii) a

population of cells that can be activated by both leptin and resistin. In the latter case, the

intracellular transduction pathways resulting in the increased RSNA utilised by each hormone

involves PI3K (51, 208). Thus, it is likely that both possibilities contribute to the present

findings investigating the distribution of Fos.

In contrast to the RSNA response, the maximum changes in MAP and HR following the

combination of leptin and resistin, were not significantly greater than the responses observed

when leptin or resistin were administered alone. This is somewhat surprising given the

dramatic effect on RSNA. It is possible that anaesthesia may have an influence on the control

of MAP and this could affect our ability to detect changes in MAP. Alternatively, the greater

sympatho-excitatory effect is selective for the renal output and may be insufficient to elicit

acute increases in blood pressure. Reflex responses could also contribute to attenuating an

increase in blood pressure.

In the present work we describe the distribution of activated neurons following resistin

administration more extensively than has been performed in the past (125, 136, 216). We

have also, for the first time, compared the distribution with that induced by leptin alone and

following leptin plus resistin in combination. Resistin induced a significant increase in Fos-

positive cell nuclei in the ARC, and PVN in the hypothalamus. In other brain regions

examined we could not detect a statistically significant difference compared to control. In the

PVN and ARC our present data agrees with previous work (136, 216).

In the present study, we found that leptin also significantly increased the number of Fos-

positive cell nuclei in the ARC and PVN, which agrees with previous studies that have

investigated the distribution of activated neurons following leptin administration (217-219),

61

or studies in which leptin was microinjected into specific brain nuclei (220), or studies that

determined the distribution of leptin receptors (221).

We could not detect statistically significant increases in Fos-positive cell nuclei in the

medulla oblongata following leptin administration. This is despite evidence that leptin can

influence cardiovascular and respiratory regulation by acting within nuclei present in the

dorsal and ventral medulla oblongata (222-224). We administered leptin into the lateral

ventricle and it is likely that the hormone spread to more caudal brain regions including the

medulla oblongata. Thus, it is possible that under the present conditions, we did not have the

sensitivity to detect significant increases in Fos-positive neurons in those medullary nuclei,

and we do not exclude actions of the hormones in the medulla.

In contrast to leptin, there have not been studies that have microinjected resistin into brain

nuclei nor has the receptor for resistin been identified. Thus, the distribution of activated

neurons may provide some insight into potential sites in which resistin acts, though it must be

recognised that the protein Fos is a marker of increased neuronal activity rather than a marker

of neurons directly activated by resistin.

When leptin and resistin were administered, the numbers of Fos-positive cell nuclei in the

lamina terminalis, ARC and in the PVN were significantly greater than in controls. We found

that only in the ARC was the increase in the number of Fos-positive cell nuclei significantly

greater than when either hormone was administered alone. This result suggests that in the

ARC the neurons activated by leptin include a population that is separate from the population

activated by resistin. In contrast, a similar increase in the number of Fos-positive cell nuclei

in the PVN was observed following each hormone alone and after leptin and resistin were

combined, suggesting that the same population was activated by leptin and resistin.

62

The PVN receives projections from the ARC (225), so it is perhaps surprising that the

number of Fos-positive cell nuclei was not greater in the PVN following the administration of

leptin and resistin compared to each alone, given the dramatic increase in the ARC following

both hormones combined. This anomaly may be explained when one considers that Fos

protein is a marker of increased neuronal activity but not a measure of the level of increased

activity in a neuron. Thus, in the PVN the level of neuronal activity may have increased

dramatically when leptin and resistin were administered, without a marked increase in the

number of neurons activated. Thus, it is possible that the same neuronal population was

activated by the administration of leptin and resistin but to a much higher level than that

observed after the administration of each hormone alone. Another possibility is that some

neurons activated in the ARC may not project to the PVN.

In contrast to all regions examined, the administration of leptin and resistin resulted in a

significant decrease in the number of Fos-positive cell nuclei in the RPA compared to either

hormone administered alone. This suggests a negative interaction is occurring within this

nucleus. Since the RPA is important in the regulation of sympathetic nerve activity to brown

adipose tissue (BAT SNA) (226), the present findings are in agreement with studies that

show that leptin has sympatho-excitatory actions on BAT, whilst resistin has opposing

inhibitory effects on sympathetic nerves innervating BAT (113, 136). In the present study, we

note that there was no evidence that resistin alone reduced basal Fos production in the RPA.

The physiological responses that may be attributed to the increase in activated neurons in the

ARC, lamina terminalis and the PVN are unknown. The ARC is known for its involvement in

dietary intake and cardiovascular regulation. This nucleus is well recognized as a key brain

nucleus mediating leptin’s anorexigenic actions (227). Resistin administered acutely also

reportedly reduces food intake (125). Thus, the present observation of enhanced neuronal

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activity in the ARC induced by the combination of leptin and resistin could be manifested

functionally as a change in energy balance.

The ARC is also important in eliciting cardiovascular responses (228), and taken together

with the present functional data on RSNA, the enhanced neuronal activity in the ARC could

be related to the greater increase in RSNA observed when leptin and resistin were

administered compared to the responses to each hormone alone. Further studies are needed to

explore these possibilities.

In a study using resistin over-expressing mice crossed with mice lacking the low density

lipoprotein receptor, leptin levels were elevated, suggesting leptin resistance (209). Thus, as

part of their report, the authors investigated the distribution of pSTAT3, a marker of neurons

activated by leptin, in the ARC in vitro following leptin alone and in the presence of resistin.

They reported that pSTAT3 levels induced by leptin were reduced by resistin (209). At first

sight, our present data following the administration of leptin in the presence of resistin

appears to contrast those observations. However, the results may be reconciled in that

pSTAT3 is a marker of neurons that were activated directly by leptin, whilst the presence of

Fos is indicative of neurons whose activity increased (not necessarily by direct effects of the

hormones on the neuron). Nonetheless, our observations on RSNA shows that the

combination of leptin and resistin results in a greater renal sympatho-excitatory actions than

following either hormone alone.

The OVLT and MnPO form part of the lamina terminalis and have been shown to have poly-

synaptic connections to sympathetic nerves innervating the kidneys (229). Those brain nuclei

are also well known to play key roles in osmoregulation and body fluid homeostasis (229).

Whether the marked increase in RSNA observed when leptin and resistin were combined is

related to the greater increase in the number of Fos-positive cell nuclei in the lamina

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terminalis awaits further investigation. Similarly, we are not aware of studies investigating

the effects of leptin or resistin on thirst.

Limitations of the study

One limitation of this study is that we used anaesthetized rats. The presence of Fos is affected

by anaesthesia, where basal levels are usually higher, than in conscious animals (230, 231).

The results in the present study agree with that. Thus, it may be more difficult to detect

changes from control under anesthetized conditions, so we may have underestimated the

brain nuclei activated. Nonetheless, the fact that increases in Fos production were observed

suggests that the areas identified are important.

Fos is a marker of increased neuronal activity, but does not indicate the level of increased

activity. Thus, in areas where the increase in Fos-positive cell nuclei following the

administration of leptin and resistin were not greater than after each hormone alone, does not

necessarily indicate there was not a greater level of activity in that brain nucleus.

Also, in the present work we studied the effects of acute administration of the two hormones.

We observed the responses for three hours and though clearly long enough to see changes in

RSNA, we did not see significant effects on MAP of leptin or resistin. It has been previously

reported that leptin administered acutely increases MAP (100). One difference between the

present work and that study is that the increase in MAP in response to ICV leptin required

more than three hours to be clearly observed, although RSNA was elevated earlier. Thus, it

would also be interesting to observe the effects of leptin and resistin following chronic

administration. This could be particularly relevant to obesity where chronically elevated

plasma leptin levels make an important contribution to the hypertension and the elevated

RSNA associated with obesity (232, 233).

65

Increases in RSNA can induce anti-natriuretic responses (234). In the present study we did

not monitor changes in natriuresis or diuresis. Given the increase in RSNA we observed, one

could expect an anti-natriuretic response. Intravenously administered leptin has natriuretic

actions, which are mediated by peripheral mechanisms (235). Thus, central and peripheral

actions of leptin produce opposing actions on natriuretic responses. Whether this contributes

to the time that it takes to observe pressor responses with leptin is unknown, but it appears to

be insufficient to prevent the chronic pressor action of leptin.

In conclusion, the present study investigated the effects of acute central administration of

leptin and resistin, alone and in combination, on RSNA, MAP and HR. Leptin alone and

resistin alone increased RSNA. When leptin was administered 15 minutes after resistin, there

was a significantly greater increase in RSNA compared to the responses to either hormone

alone. This may be important in conditions like obesity where RSNA is abnormally elevated

and both leptin and resistin levels are increased. The brain nuclei involved in mediating the

enhanced increase in RSNA are not known but may involve the lamina terminalis, ARC and

the hypothalamic PVN.

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4 Chapter 4: Central Administration Of Insulin And Leptin Together Enhance Renal

Sympathetic Nerve Activity And Fos Production In The Arcuate Nucleus

4.1 INTRODUCTION

Insulin is produced by the pancreas and released into the circulation in response to elevated

glucose. In addition to insulin’s critical role in glucose homeostasis, there is now

considerable interest in its role in regulating the autonomic nervous system. Insulin can

induce increases in sympathetic outflow to cardiovascular organs, such as the kidney and

skeletal muscle vasculature, by activating receptors within the central nervous system (53,

58).

Leptin is released from adipocytes and it acts centrally to influence metabolic and

cardiovascular functions (18, 89, 98, 214). It acts within the brain to increase sympathetic

nerve activity to the kidney (54, 57, 99 ). The sympatho-excitatory actions of leptin to the

kidney makes an important contribution to the abnormally elevated renal sympathetic nerve

activity (RSNA) observed in obesity or overweight conditions (17, 50).

Both leptin and insulin can be transported across the blood brain barrier to activate brain

nuclei involved in cardiovascular and metabolic regulation. Pathways involving the activation

of melanocortin 4 receptors (MC4R) is a key common mechanism mediating the increases in

RSNA induced by leptin and insulin (110), suggesting the potential for an interaction on

RSNA, such that increased levels of both hormones could result in an additive action on

RSNA. Interestingly both insulin and leptin can regulate each other’s production and release,

such that an increase in plasma levels of insulin can lead to an increase in leptin levels and

vice versa (236, 237), which further suggests potential interactions of the two hormones.

Thus, in order to explore the possibility that insulin and leptin interact within the brain to

67

induce greater increases in RSNA, the first aim of the present study was to compare RSNA in

response to administration of leptin and insulin alone and in combination.

The second aim was to explore potential sites where leptin and insulin may interact. Thus, we

also quantified the number of activated neurons in brain regions, known to influence the

sympathetic nervous system, and compared their distribution after central administration of

leptin and insulin alone and both combined.

4.2 METHODS


4.2.1 Animals

Experiments were performed to comply with the Prevention of Cruelty to Animals Act 1986

(Australia). All experimental protocols conform with the ‘Guiding Principles for Research

Involving Animals and Human Beings’ and the guidelines set out by the ‘Australian Code of

Practice for the Care and Use of Animals for Scientific Purposes’, 2013 (National Health and

Medical Research Council of Australia) and were approved by the Royal Melbourne Institute

of Technology (RMIT) University Animal Ethics Committee (approval letter is attached in

appendix II). Male Sprague–Dawley rats were obtained from the ARC Animal Resources

Centre (Western Australia, Australia). Rats were housed under a 12:12 h light ⁄ dark cycle at

23° C and allowed free access to standard rat chow and water for 8-10 weeks.


General anaesthesia was induced using isoflurane in O2 (2–5%) on the day of the experiment

to allow catheterisation of the femoral vein and artery. The catheter was inserted into the

femoral vein, so that general anaesthesia could be maintained with intravenous (IV) urethane

68

(1.4–1.6 g ⁄ kg initially, followed by supplemental doses of 0.05 ml of a 25% solution if

required). The depth of anaesthesia was maintained to ensure the absence of corneal and

pedal pinch reflexes. The femoral artery was cannulated for arterial blood pressure

monitoring. Mean arterial pressure (MAP) and heart rate (HR) were calculated from the

arterial pressure pulse using LabChart (ADInstruments, Castle Hill, NSW, Australia). Rats

were kept on a heating pad for the duration of the experiments to maintain body temperature

at approximately 37.5 oC.


A flank incision was made to expose the left kidney retroperitoneally. Using an operating

microscope, the renal nerve was carefully dissected free of surrounding tissue. Under mineral

oil, the renal nerve was then cut and the proximal end was placed onto the bared tips of two

Teflon-coated silver wire electrodes.

The RSNA was amplified using a low-noise differential amplifier (models ENG 187B and

133, Baker Institute, Victoria, Australia), filtered (band pass 100–1,000 Hz), rectified, and

integrated at 0.5 s intervals. The signal was recorded using a PowerLab data acquisition

system (ADInstruments, NSW, Australia).

4.2.2.2 Microinjections into the lateral brain ventricle (ICV)

Animals were placed prone, and their heads were mounted in a Stoelting stereotaxic frame, so

that bregma and lambda were positioned on the same horizontal plane. To expose the dorsal

surface of the brain, a hole (diameter 2 mm) centred 0.7 mm caudal and 1.4 mm lateral from

bregma, was drilled into the skull. After the drilling procedure, the hole was covered with

cotton wool soaked in normal saline to prevent drying of the exposed surface. A

microinjection was made unilaterally using a fine glass micropipette (tip diameter 50–70 µm)

69

inserted into the lateral brain ventricle (stereotaxic coordinates: 0.7 mm caudal to bregma, 1.4

mm lateral to midline, and 3.7-3.9 mm ventral to the surface of the dura). After the

microinjection, the micropipette was left in place for 1 min. At the end of the experiment, a

small amount of pontamine sky blue was microinjected using the same coordinates to



MAP, HR and RSNA were measured prior to intracerebroventricular (ICV) injection of

vehicle (saline 5 μl; n=5), leptin (7 μg in 5ul; n=5) or insulin (500 mU in 5μl; n=4) alone or in

combination (leptin (7 μg in 5μl) was administered 15 min after insulin (500 mU in 5μl)

(n=4)), and the responses were monitored for three hours after the last injection. Apart from

instances in which leptin and insulin were administered in the one animal, only one injection

per rat was performed. The doses of each hormone were chosen on the basis of previous

studies (113, 136). The doses used are larger than those observed in plasma as noted

previously (48, 51, 125, 128). The difference in amounts is likely to be related to the

concentration required at the actual site of action. For saline and leptin alone, the data were

reported in chapter 3, and used in this chapter for comparison only.

4.2.2.4 Immunohistochemistry

At the end of the experiment, the animals were perfused with 200 ml of 0.1M phosphate

buffered saline (PBS, pH 7.2) transcardially, followed with 200 ml of 4% paraformaldehyde

in 0.1 M PBS. The brains were removed carefully and post-fixed for 3-3.5 hours and then

transferred to a solution of 20% sucrose in PBS 0.1M and stored at 4°C.

Brains were sliced into a series of coronal sections (40 µm thick) using a cryostat (Leica,

CM1900). Sections were collected and left in cryoprotectant until processed. One in five

70

sections were then collected and processed immunohistochemically as free floating sections

for the detection of Fos protein. In brief, the process involved placing the sections in 0.5%

H2O2 for 15 min to destroy endogenous peroxidase activity. This was followed by incubation

in 10% normal horse serum (NHS) and 0.5% Triton X-100 for 60 min to facilitate antibody

penetration. Sections were then incubated in the primary antibody raised in rabbit against Fos

(1:5000, rabbit polyclonal IgG , c-Fos (K-25): sc-253, Santa Cruz Biotechnology, CA, USA;)

overnight at 4°C. Sections were then incubated with biotinylated anti rabbit secondary

antibody raised in horse, (1:400, Sigma Aldrich, Australia) for 90 minutes. This was followed

by Extravidin–peroxidase complex (1:400, Sigma Aldrich, Australia) for 45 minutes.

Sections were washed three times in 0.1M PBS (5 minutes each time) between each

incubation. Sections were then placed for 10 minutes in 0.01% 3,3′-diaminobenzidine

hydrochloride (DAB; Sigma Aldrich, Australia) + 0.02% Nickel ammonium + 0.02% Cobalt

chloride in PBS. 5 μl of 30% hydrogen peroxide was added to begin the reaction. To end the

reaction the sections were washed with PBS and then they were placed onto gelatine-coated

slides and left overnight to dry, followed by coverslipping with Depex (Sigma Aldrich

Australia) the next day.


4.2.3.1 Physiological parameters

The resting levels of MAP and HR and RSNA before each injection were averaged from

three 5 minute recordings taken 15-30 minutes prior to the first injection. The average levels

were compared between groups using one-way ANOVA. The integrated RSNA, averaged

over 1-2 minutes, was calculated at 15 minute intervals over the period of 180 min, and

expressed as a percentage of the resting level before the hormone/saline injections. Changes

in MAP, HR, and RSNA were compared between groups in each experimental series by

71

using two-way ANOVA. All results are expressed as the mean ± SE. P < 0.05 was

considered statistically significant.

4.2.3.2 Immunohistochemistry

Under a bright field illumination, Fos-positive cell nuclei were visualised and counted

unilaterally, except in midline structures (organum vasculosum of the lamina terminalis

(OVLT), medium preoptic nucleus (MnPO) and raphe pallidus (RPA)) at 200x magnification.

At least two sections per brain nucleus were examined in each animal. The average number

of Fos-positive cell nuclei per section was calculated for each brain region in each animal and

comparisons between the groups were performed using one-way ANOVA.

For both the physiological and immunohistochemical data, following the detection of an

overall significant difference between groups, comparisons between groups administered the

hormones and control, and between leptin+insulin combined vs leptin or insulin alone, were

made using Holm-Sidak’s multiple comparison test. All results are expressed as means±SE.

P<0.05 was considered to be statistically significant. PRISM software was used.

4.3 RESULTS

4.3.1 Effects of ICV injection of leptin and insulin on RSNA

Following ICV injection of leptin alone RSNA increased significantly by 74+17% (P<0.0001

compared to saline) (figure 4.1). When insulin was administered alone the increase in RSNA

was similar to that of leptin reaching a maximum of 62+13% (P<0.0001 compared to saline)

(figure 4.1). Following the ICV administration of the combination of leptin and insulin the

increase in RSNA was much greater reaching a maximum response of 124+40% by 120

minutes (P<0.0001 compared to control). The marked increase in RSNA was significantly

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greater than that observed after each hormone was administered alone (P<0.0005) (figure

4.1).

4.3.2 Effects of ICV injection of leptin and insulin on MAP and HR.

Compared to saline, there were no marked changes in MAP during the 3 hour observation

period following leptin and insulin administered alone or combined (figure 4.2). However,

HR increased significantly following insulin alone compared to saline (P<0.0001) (figure

4.2). Leptin alone also significantly increased HR compared to saline (figure 4.2). When

insulin and leptin were combined, the HR response was significantly greater than saline

(P<0.0001), but significantly less than that of insulin alone (P<0.001) (figure 4.2). The

response was similar to that of leptin alone. Thus, the presence of leptin attenuated insulin’s

tachycardic actions (figure 4.2).

Resting MAP and HR prior to the ICV injections are shown in table 4.1. No significant

differences were detected between the groups (table 4.1).

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Figure 4.1 Percent changes in RSNA induced by leptin and insulin alone and in combination in rats fed a normal diet (ND).

Percent changes in renal sympathetic nerve activity (RSNA) from baseline levels over time

after intracerebroventricular injection of leptin (n=5) (7μg/5μl), insulin (n=4) (500mU/5μl),

saline (n=5) (5μl) and the combination of leptin and insulin (n=4). (*P<0.0001 leptin alone,

insulin alone and both combined compared to saline; #P<0.001 insulin alone compared leptin

and insulin combined or leptin alone.

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Figure 4.2 Changes in MAP and HR induced by leptin and insulin alone and in combination in rats fed a normal diet (ND).

Changes in mean arterial pressure (MAP) and heart rate (HR) over time induced by the

intracerebroventricular administration of leptin (n=5) (7μg/5μl), insulin (n=4) (500mU/5μl),

saline (n=5) (5μl) and the combination of leptin and insulin (n=4). (*P<0.0001 Leptin alone,

insulin alone and both combined compared to saline; #P<0.0005 leptin and insulin combined

compared to insulin alone).

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Table 4.1 Resting MAP and HR.

Resting mean arterial pressure (MAP) and heart rate (HR) prior to intracerebroventricular

injection of saline (n=5), leptin (7ug, n=5), insulin (500mU, n=4) and the combination of

leptin and insulin (n=4) in anesthetised rats.

Saline Leptin Insulin Leptin +Insulin

MAP (mmHg) 93.5 ± 4.1 79.7 ± 1.6 88.0 ± 6.8 86.2 ± 5.6

HR (beats/min) 360 ± 7 336 ± 11 312 ± 19 318 ± 28

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4.3.3 Effects of leptin and insulin on Fos-positive cell nuclei

4.3.3.1 Lamina terminalis

The numbers of Fos-positive cell nuclei in the OVLT following ICV injection of insulin

alone were not significantly different compared to saline (control) (figure 4.3). However, in

this brain region, leptin alone significantly increased the number of Fos-positive cell nuclei

compared to control (100+6 vs 45+7/section, P<0.01) (figure 4.3). When leptin and insulin

were combined the number of Fos-positive cell nuclei observed almost doubled (91+8)

compared to control and was significantly greater than that observed following insulin alone

(P<0.05) (figure 4.3). The response following combined leptin and insulin was similar in

magnitude to that following leptin alone (figure 4.3). Thus, insulin did not influence the

response to leptin in the OVLT.

In the MnPO, similar effects were observed; insulin alone did not significantly affect the

number of Fos-positive cell nuclei compared to the saline control (figure 4.3) but there was a

significant increase in Fos production after ICV leptin alone (figure 4.3). The administration

of leptin and insulin in combination also significantly increased the number of Fos-positive

cell nuclei compared to control (P<0.01) (figure 4.3). This increase in Fos production was

similar to that observed after leptin alone (figure 4.3). However, the response to the

combination of insulin and leptin was significantly greater that that observed following

insulin alone (P<0.05) (figure 4.3).

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Figure 4.3 Average number of Fos-positive cell nuclei in OVLT and MnPO induced by leptin and insulin alone and in combination in rats fed a normal diet (ND).

Average numbers of Fos-positive cell nuclei per section in the organum vasculosum of

lamina terminalis (OVLT) and median preoptic nucleus (MnPO) from rats administered

intracerebroventricular saline (control) (n=5) (5µl), leptin (n=5) (7µg/5µl), insulin (n=4)

(500mU/5μl), and the combination of leptin and insulin (n=4). (*P<0.01 compared to saline;

#P<0.05 compared to insulin alone).

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4.3.3.2 Hypothalamus

Arcuate nucleus

In the arcuate nucleus (ARC), leptin alone and insulin alone significantly increased the

number of Fos-positive cell nuclei compared to control (P<0.05) (figure 4.4). The combined

administration of leptin and insulin produced a significantly greater increase in the number of

Fos-positive cell nuclei in the ARC compared with either hormone alone (P<0.05) (figures

4.4 and 4.5). Thus, the response to the combined administration of insulin and leptin was

enhanced compared to the responses of the individual hormones.

Paraventricular and supraoptic nuclei

In the hypothalamic paraventricular nucleus (PVN) ICV administration of leptin alone

induced a significant increase in the number of Fos-positive cell nuclei compared to control

(figure 4.4). However, insulin alone or the combination of leptin and insulin did not attain

statistically significant increases compared to control (figure 4.4).

In the supraoptic nucleus (SON), although a significant difference between the treatment

groups was detected overall (P<0.05), the analysis of the individual comparisons between a

treated group (ie leptin, insulin alone, or combined) compared to control did not identify a

statistically significant difference (figure 4.4).

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Figure 4.4 Average number of Fos-positive cell nuclei in ARC, PVN and SON induced by leptin and insulin alone and in combination in rats fed a normal diet (ND).

The average number of Fos-positive cell nuclei per section in the arcuate (ARC),

paraventricular (PVN) and supraoptic (SON) nuclei after intracerebroventricular injection

saline (control) (n=5) (5µl), leptin (n=5) (7µg/5µl), insulin (n=4) (500mU/5μl), and the

combination of leptin and insulin (n=4). (*P<0.01 compared to saline; #P<0.05 compared to

insulin alone).

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Figure 4.5 Photomicrographs of Fos-positive cell nuclei in ARC induced by leptin and insulin alone and in combination in rats fed a normal diet (ND).

Photomicrographs showing Fos-positive cell nuclei in the arcuate nucleus of rats following

centrally administered saline (5µl; control), leptin (7µg), insulin (500mU) and leptin

combined with insulin. Bar=100 µm. Dashed lines outline the arcuate nucleus. The third

ventricle is to the right of each photomicrograph.

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4.3.3.3 Medulla oblongata

Fos-positive cell nuclei were examined in the nucleus tractus solitarius (NTS), (RPA), and

rostral ventrolateral medulla (RVLM) in the medulla oblongata. In the NTS and in the RPA

there were no significant differences between groups overall. However, in the RVLM there

was a significant difference between the groups (P<0.05). There was a significant increase in

the number of Fos-positive cell nuclei following the administration of leptin alone (P<0.05)

but not with insulin alone (figure 4.6). Following the combination of leptin and insulin the

increase in Fos-positive cell nuclei was similar to that of leptin and two-fold greater than in

control (P<0.05 compared to control) (figure 4.6). Thus, insulin did not affect the increase

induced by leptin in the RVLM.

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Figure 4.6 Average number of Fos-positive cell nuclei in NTS, RPA and RVLM induced by leptin and insulin alone and in combination in rats fed a normal diet (ND).

Average number of Fos-positive cell nuclei per section in the nucleus tractus solitarius

(NTS), raphe pallidus (RPA) and rostral ventrolateral medulla (RVLM), following saline

(control) (n=5) (5µl), leptin (n=5) (7µg/5µl), insulin (n=4) (500mU/5μl), and the combination

of leptin and insulin (n=4). (*P<0.05 compared to saline).

83

4.4 DISCUSSION

This report is the first to demonstrate that the ICV administration of leptin combined with

insulin results in significantly greater increases in RSNA compared with either hormone

administered alone. The sympatho-excitatory effect did not appear to be a generalised

response since the combined administration of leptin and insulin did not result in an increased

HR response, rather the tachycardic effects elicited by insulin alone were attenuated by the

presence of leptin. We also investigated brain nuclei involved in autonomic regulation to

determine whether there was a correlation with those responses and increased neuronal

activation (detected by the presence of Fos-positive cell nuclei) when leptin and insulin were

administered. In the regions we investigated, we found that in the ARC, OVLT, MnPO and

RVLM the number of Fos-positive cell nuclei were significantly greater following ICV

injection of leptin and insulin together compared to control. However, only in the ARC was

the increase significantly greater than insulin or leptin administered alone.

The present work is the first to show a greater sympatho-excitatory response when leptin and

insulin are present together compared to the responses elicited by each alone. The present

work confirms previous studies that have shown significant increases in RSNA following the

central administration of leptin alone or insulin alone (53, 54, 58, 99). The significance of

our current work is that it highlights the potential for a much greater sympatho-excitatory

effect on RSNA in conditions in which leptin and insulin are elevated, such as metabolic

syndrome and obesity (17, 50, 55, 56). In those conditions, RSNA is abnormally elevated and

leptin is believed to make a critical contribution to the increase (233). Our findings suggest

that the considerable contribution of leptin to this elevated RSNA may be, in part, due to the

presence of increased levels of insulin.

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In the present study we found that central administration of insulin increased heart rate,

however, the response was prevented when leptin was present. Since the changes in HR

elicited by central insulin are mediated by increases in sympathetic nerve activity (238), the

inhibition of this effect when leptin was present contrasts dramatically with the observed

effect on RSNA, and suggests that the central interactions between leptin and insulin on the

sympathetic nervous system are complex and may reflect differences in the output to

different organs such that effects on the renal output is an additive response whilst that to the

heart is an antagonistic interaction.

There was no effect on MAP following the ICV administration of leptin or insulin

administered alone or in combination, suggesting that increases in RSNA are not sufficient to

acutely increase MAP. The present findings are consistent with previous reports that found

that acute ICV administration of leptin did not increase MAP over the time frame of our

observations (99, 100). Similarly, central administration of insulin has also been reported to

have little effect acutely on blood pressure (54, 239, 240).

In contrast, effects on MAP following chronic systemic or central administration, for 3-10

days, of leptin or insulin alone have been more consistent reporting significantly increased

MAP responses (103, 107, 241). Since renal function is an important determinant in the

regulation of chronic blood pressure, the possibility exists that if the effect of the acute

combination of leptin and insulin on RSNA could be reproduced with chronic infusions, then

increases in MAP in conditions in which both hormones are chronically elevated may occur,

but this awaits further investigation.

Previous work on interactions between leptin and insulin has focussed on metabolic

functions, with little investigation into interactions on cardiovascular function. In particular,

there is a distinct gap investigating an interaction on the cardiovascular responses arising

from leptin and insulin acting within the brain, despite the fact that the central mechanisms

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that mediate the increase in RSNA induced by leptin or insulin alone have a common

pathway that involves the activation of MC4 receptors (110). This strongly suggests that an

interaction is likely, and the present study now provides evidence to support a central

interaction on RSNA.

The central interaction of insulin and leptin may not be restricted to the renal output.

Evidence indicates that increases in lumbar sympathetic nerve activity induced by centrally

administered insulin involves phosphatidylinositol 3 kinase (PI3K) as is the case with leptin,

whilst the increase in sympathetic nerve activity to brown adipose tissue (BAT SNA) induced

by leptin or insulin involves activation of Extracellular Signal-Regulated Kinase (ERK1/2)

(54). Thus, an interaction between leptin and insulin influencing at least three sympathetic

outputs is a distinct possibility.

In the present study we investigated the distribution of activated neurons using the protein

marker, Fos, in cell nuclei in brain regions known for their contribution to cardiovascular

regulation. Only in the ARC was the number of activated neurons significantly greater

following the combination of leptin and insulin than the increase seen after either hormone

alone. This result is in agreement with reports that show leptin and insulin act within the

ARC to increase sympathetic nerve activity (SNA) (53, 54, 100, 110, 203, 227, 242-244).

Thus, the activation of neurons in the ARC would be in accord with the increase in RSNA

induced by leptin or insulin. The greater number of activated neurons in the ARC after the

administration of leptin and insulin in combination correlates with the greater increase in

RSNA compared to each hormone alone that we observed.

We found a significant increase in Fos-positive cell nuclei in the lamina terminalis (OVLT,

MnPO), hypothalamic PVN and in the RVLM following leptin administration. We did not

detect a statistically significant increase following insulin in those same regions. Although,

the PVN and RVLM are believed to be involved in mediating the sympatho-excitatory effects

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of hyperinsulinemia, microinjections of insulin directly into those brain nuclei did not induce

an increased SNA suggesting there is no direct activation of insulin receptors in those brain

nuclei (200, 201, 245). Since we found that insulin alone increased RSNA, it was perhaps,

surprising that we did not detect an increase in Fos in those regions. Whether the type of

anesthesia used (eg urethane in this study and alpha chloralose in those mentioned above)

contributes to this anomaly is unknown. We also acknowledge that the numbers of animals

may be a limitation to detect small changes. In contrast, no anomaly appears to exist with

leptin since it significantly increased Fos production in the PVN and RVLM. Other factors

that could contribute to the greater basal Fos may include duration of the stimulus and the

surgical intervention that has occurred. Additionally, it should be noted that subtle influences

or changes in the nuclei that affect sympathetic outflow may not be easily visualised by the

use of the immunohistochemical detection of Fos.

In the OVLT, MnPO, and RVLM the combined administration of leptin and insulin

significantly increased Fos-positive cell nuclei but the effects were similar to leptin alone.

Thus, in those brain nuclei, the results suggest that the increase in Fos-positive nuclei were

due primarily to leptin and that that there was no evidence of an additive interaction between

leptin and insulin in those brain nuclei. These findings contrast with the observations we

made in the ARC, suggesting that interactions between leptin and insulin in the brain may

occur in selective brain nuclei.

4.5 SUMMARY AND CONCLUSIONS

In summary, following ICV administration of leptin and insulin together there was a

significantly greater increase in RSNA than either hormone alone. Insulin alone increased HR

significantly but this was prevented by the presence of leptin. MAP was not significantly

different between groups. Of the brain regions examined, only in the ARC did the

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combination of leptin and insulin together increase the number of Fos-positive cell nuclei

significantly more than the increases following leptin or insulin alone. In contrast, in the

lamina terminalis and RVLM, leptin and insulin combined increased Fos-positive cell nuclei

to levels similar to leptin alone. The results suggest that where leptin and insulin are elevated,

there may be greater increases in RSNA. This may contribute to cardiovascular

complications. The ARC may be a common site of cardiovascular integration for leptin and

insulin.

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5 Chapter 5: Effects Of Central Administration Of Resistin On Renal Sympathetic Nerve

Activity In Rats Fed A High Fat Diet; A Comparison With Leptin

5.1 INTRODUCTION

Resistin is a member of the resistin-like molecule (RELM) hormone family and is so named

because of its ability to induce insulin resistance. In obese and diabetic humans and rats

resistin levels in plasma are elevated (47, 48, 131). Resistin levels correlate with an increased

risk of hypertension and major cardiovascular events like stroke and myocardial infarction

(246-249). Resistin acts in the brain, and mRNA for resistin has been detected in several

brain nuclei of rodents suggesting there is also endogenous production of resistin in the brain

(250, 251).

Resistin acts centrally to increase renal sympathetic nerve activity (RSNA) (51). These

effects are similar to those observed with another well-known adipokine, leptin (99, 100).

Both leptin and resistin increase RSNA through central mechanisms that involve

phosphatidylinositol 3 kinase (PI3K) (51, 58, 208), and this may explain why the combined

administration of leptin and resistin elicits greater increases of RSNA than either alone.

Increased dietary intake of fat leads to increased adipose tissue and increased circulating

levels of leptin and resistin (47, 48, 131). Cerebrospinal fluid levels of leptin in humans

correlate with circulating levels of leptin and adiposity (252). Cerebrospinal fluid levels of

resistin also correlate with circulating levels in males but not females and this has been

interpreted as an impairment in the uptake process of resistin into the central nervous system

(CNS) in obese women (124). Diets high in fat lead to a reduction in the sensitivity to the

anorexigenic actions of leptin (253, 254). However, the sympatho-excitatory action of leptin

on RSNA is not affected by high fat diets (HFD). This has led to the view of a selective

89

resistance to leptin (18, 79, 87, 255) and could be a key mechanism contributing to obesity

induced hypertension (17, 20).

Since resistin, like leptin, increases RSNA, we investigated whether a HFD could influence

the sympatho-excitatory effects induced by resistin. Furthermore, we also explored whether a

HFD would influence the increase in RSNA induced by the combination of resistin and

leptin, since the combined effect of resistin and leptin in normal chow fed (ND) rats results in

significantly greater sympatho-excitatory actions on RSNA, as shown in chapter 3 (256).

5.2 METHODS




to Animals Act 1986 (Australia). The procedures conform to the ‘Guiding Principles for

Research Involving Animals and Human Beings’ and the guidelines set out by the Australian





Male Sprague–Dawley rats were obtained from ARC animal resources centre (Canning Vale,

WA, Australia). Upon arrival rats were acclimatised for one week, before a controlled

feeding period began. Rats were divided into two groups, where one group was exposed to a

ND (4.8% fat) and the other to a HFD (SF14-004 (22% fat), Speciality Feeds, Glen Forrest,

WA). Rats were housed in groups of two at 23°C with a 12 h–12 h light–dark cycle and

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allowed free access to rat chow and water. The feeding period lasted for 8-10 weeks, and

body weight, food intake, and caloric intake were monitored weekly. In addition, systolic

blood pressure was monitored fortnightly during the feeding period, using a non-invasive tail

cuff occlusion method. At the end of the feeding period, the percent whole body fat was

determined using an EchoMRI 500 (Houston Texas, USA) in a sample of animals of each

dietary treatment.

On the day of the experiment, anaesthesia was induced using isoflurane (2-5% in O2,

followed by femoral artery and vein cannulation as previously described in chapter 2 (256).

The femoral vein was cannulated so that anaesthesia could be maintained with intravenous

(IV) of urethane (1.4–1.6 g/kg; IV with supplemental doses of 0.05 ml of a 25% solution, if

required). The depth of anaesthesia was maintained to ensure the absence of corneal and

pedal reflexes. The femoral artery was cannulated to monitor mean arterial pressure (MAP)

and heart rate (HR) was determined from the arterial pressure pulse using LabChart

(ADInstruments, NSW, Australia). Rats were kept on a heating pad for the duration of the

experiments to maintain body temperature at approximately 37.5 oC. The rats were not fasted

prior to the experimental day.


After a flank incision, the left kidney was exposed retroperitoneally and dissected clear of

surrounding tissue, placed onto the hooked, bared-ends of Teflon-coated bipolar silver

electrodes and insulated from the surrounding tissue by covering them with paraffin oil as

previously described in chapter 2 (51, 256). RSNA was amplified using a low-noise

differential amplifier (models ENG 187B and 133, Baker Institute, Victoria, Australia),

filtered (band pass 100–1,000 Hz), rectified, and integrated at 0.5 s intervals. The signals was

recorded using a PowerLab data acquisition system (ADInstruments, NSW, Australia) (256).

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The background electrical noise levels were determined post mortem and subtracted from the

nerve activity obtained in the anesthetised rat.

5.2.2.2 Microinjections into the lateral brain ventricle

Rats were placed prone, with the head mounted in a stereotaxic frame, such that bregma and

lambda were positioned on the same horizontal plane. To expose the dorsal surface of the

brain, a hole (2 mm diameter), centred 0.7 mm caudal to bregma and 1.4 mm lateral to the

mid-line, was drilled into the skull. Unilateral microinjections (5 µl) were made using a fine

glass micropipette (50–70µm tip diameter) inserted into the lateral brain ventricle (stereotaxic

co-ordinates: 0.7 mm caudal to bregma, 1.4 mm lateral to the mid-line and 3.7–3.9 mm

ventral to the surface of the dura). The micropipette was left in place for 1 min, after the

microinjection. At the end of the experiment, a small amount of Pontamine Sky Blue was

microinjected using the same co-ordinates to confirm microinjection into the lateral ventricle.


The MAP, HR and RSNA were measured before and for 180 min after administration of

saline vehicle (n = 5; 5µl), leptin (7 µg in 5µl; n = 4), resistin (7 µg in 5µl; n = 5) and the

combination of both resistin and leptin (n = 6; leptin was administered 15 min after resistin).

The doses of each hormone were chosen on the basis of previous studies (113, 136, 256). At

the end of the experiment, the epididymal fat was removed and weighed.


5.2.3.1 Within high fat diet

The average resting levels of MAP, HR and integrated RSNA prior to hormone

administration were compared between groups using one-way ANOVA. The integrated

RSNA, averaged over 1-2 minutes, was calculated at 15 minute intervals over the period of

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180 min, and expressed as a percentage of the resting level before the intracerebral injections.

Changes in MAP, HR and RSNA over time following hormone/saline administration were

compared between groups by using two-way ANOVA. When an overall significant

difference between groups was detected, comparisons were made between individual groups,

using Holm-Sidak’s test for multiple comparisons. The maximum responses in each variable

were calculated by averaging the last 30 min of the observation period in each rat.

5.2.3.2 Comparisons between high fat and normal chow diets

The changes in RSNA, MAP and HR over time following leptin alone, resistin alone and the

combination of leptin and resistin were compared between HFD and ND using two-way

ANOVA. Data for the ND fed rats used for this comparison has been reported previously in

chapter 3 (256). The calculated maximum responses were compared between the dietary

groups using Student’s unpaired t-test.

The body weight, average food intake, average caloric intake, and systolic blood pressure

during the feeding period were compared between dietary groups using two-way ANOVA.

Systolic blood pressure was measured via indirect tail cuff plethysmography. Due to a

technical issue, systolic blood pressure was recorded before and for 6 weeks after the start of

feeding. The percentage of whole body fat and the epididymal fat were compared between

the dietary groups using Student’s unpaired t-test.

All results are expressed as means ± SEM. A value of P<0.05 was considered to be

statistically significant. PRISM software was used (GraphPad San Diego, California, USA).

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5.3 RESULTS

5.3.1 Rats fed a high fat diet

5.3.1.1 Effect on RSNA

The intracerebroventricular (ICV) injection of resistin alone and leptin alone increased RSNA

significantly compared with the saline control (P<0.0001, figure 5.1). Resistin alone

increased RSNA, which followed a similar time course to that of leptin and reached a

maximum of 62±4%. Leptin alone increased RSNA, to a maximum of 71±16%. When

resistin and leptin were combined, there was a significantly greater increase in RSNA

(maximum change of 195±41%) than that observed following resistin or leptin alone

(P<0.0001) (figure 5.1).

5.3.1.2 Effect on MAP and HR

There were no significant differences in the changes in MAP between the groups over the

duration of observation (figure 5.2).

There was a significant difference between the groups in the HR responses over time

(P<0.0001). However, by the end of the observation period, neither resistin nor leptin alone

or both in combination induced a statistically significant change in HR compared to saline

(figure 5.2).

The resting levels of both MAP and HR prior to hormone administration did not show any

significant differences between groups (see figure 5.6).

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Figure 5.1 Percent change in RSNA induced by leptin and resistin alone and in combination in rats fed a high fat diet (HFD).

Percent change in renal sympathetic nerve activity (RSNA) from baseline levels over time in

rats fed a high fat diet (HFD) after intracerebroventricular injection of leptin (n=4) (7μg/5μl),

resistin (n=5) (7μg/5μl), control (saline, n=5) (5μl) and the combination of leptin and resistin

(n=6). *P<0.0001 leptin alone, resistin alone and the combination of both hormones

compared to saline; #P<0.0001 combination of leptin and resistin compared to leptin alone or

resistin alone.

95

Figure 5.2 Change in MAP and HR induced by leptin and resistin alone and in combination in rats fed a high fat diet (HFD).

Change in mean arterial pressure (MAP) and heart rate (HR) over time in rats fed a high fat

diet (HFD) induced by the intracerebroventricular administration of leptin (n=4) (7μg/5μl),

resistin (n=5) (7μg/5μl), control (saline, n=5) (5μl) and the combination of leptin and resistin

(n=6). *P<0.0001 leptin alone, resistin alone and the combination of both hormones

compared to saline.

96

5.3.2 Comparison of responses to leptin and resistin in rats fed high fat vs normal chow diets

5.3.2.1 RSNA

There was a no statistically significant difference overall in the RSNA response elicited by

resistin over time or in the maximum response in rats fed a HFD compared to the response in

rats fed a ND (figure 5.3). Similarly, the RSNA response to leptin was not significantly

different between rats fed the different diets (figure 5.3).

When resistin and leptin were combined, the increase in RSNA appeared to be slower in

onset in the HFD rats and there was an overall statistically significant difference between

groups (P<0.005 two-way ANOVA) (figure 5.3), but comparisons between individual time

points failed to show a significant difference at any time point. Despite the apparent slower

start, the maximum increase in RSNA was not significantly different by the end of the

observation period from the response seen in rats fed ND (figure 5.3).

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Figure 5.3 RSNA response following leptin and resistin alone and in combination in rats fed a high fat diet (HFD) or normal diet (ND).

Renal sympathetic nerve activity (RSNA) responses between rats fed a normal chow (ND)

and a high fat diet (HFD), elicited by intracerebroventricularly administered resistin (n=4

ND, n=5 HFD) (7 μg/5 μl), leptin (n=5 ND, n=4 HFD) (7μg/5μl), the combination of leptin

and resistin (n=6 ND and HFD). Left panels show changes in RSNA over time; Right panels

show the average maximum change in RSNA. *P<0.005 compared to ND.

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5.3.2.2 MAP and HR

There was a statistically significant difference detected in the change in MAP induced by

resistin alone over time between rats fed the HFD and ND (P<0.05) (figure 5.4). The

maximum change in MAP in the rats fed a HFD was significantly less than that seen in the

ND fed rats (P<0.005) (figure 5.4). However, there were no significant differences in the

changes on MAP over time or in the maximum responses in rats fed the HFD compared to

the ND when leptin was administered alone or when leptin and resistin were combined

(figure 5.4).

The HR response over time and the maximum change in HR induced by resistin alone in the

HFD rats were significantly smaller than that observed in the rats fed a ND (figure 5.5). The

HR response over time induced by leptin alone was also significantly smaller in the HFD rats

(P<0.05), however, the maximum change in HR was not significantly different between the

rats fed the different diets (figure 5.5). When leptin and resistin were combined there were no

significant differences in the HR responses over time, or in the maximum HR changes

observed in rats fed the two different diets (figure 5.5).

Basal MAP and HR prior to hormone administration in rats fed a HFD were not significantly

different from those observed in rats fed a ND (figure 5.6).

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Figure 5.4 MAP response following leptin and resistin alone and in combination in rats fed a high fat diet (HFD) or normal diet (ND).

Comparison of the mean arterial pressure (MAP) responses between rats fed a normal chow

(ND) and a high fat diet (HFD), elicited by intracerebroventricularly administered resistin

(n=4 ND, n=5 HFD) (7 μg/5 μl), leptin (n=5 ND, n=4 HFD) (7μg/5μl), the combination of

leptin and resistin (n=6 ND and HFD). Left panels show changes in MAP over time; Right

panels show the average maximum change in MAP. *P<0.005 compared to ND.

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Figure 5.5 HR response following leptin and resistin alone and in combination in rats fed a high fat diet (HFD) or normal diet (ND).

Comparison of the heart rate (HR) responses between rats fed a normal chow (ND) and a

high fat diet (HFD), elicited by intracerebroventricularly administered resistin (n=4 ND, n=5

HFD) (7 μg/5 μl), leptin (n=5 ND, n=4 HFD) (7μg/5μl), the combination of leptin and resistin

(n=6 ND and HFD). Left panels show changes in HR over time; Right panels show the

average maximum change in HR. #P< 0.0001 compared to ND; *P < 0.05 compared ND.

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Figure 5.6 Basal level of MAP and HR in rats fed a high fat diet (HFD) or normal diet (ND) prior to hormone administration.

Basal levels of mean arterial pressure (MAP) and heart rate (HR) in rats fed normal chow diet

(ND; open bar) and high fat diet (HFD; black bar) before intracerebroventricular

administration of resistin (n=4 ND, n=5 HFD) (7 μg/5 μl), leptin (n=5 ND, n=4 HFD)

(7μg/5μl) and the combination of both hormones (n=6 ND and HFD) (7 μg/5 μl).

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5.3.2.3 Comparison of systolic BP and metabolic parameters in rats fed high fat vs normal chow diets

Systolic blood pressure changes during the feeding period

Systolic blood pressure in the HFD fed rats rose over time but this was not sustained and was

not statistically different from that observed in rats fed the ND (figure 5.7).

Metabolic parameters

The percent whole body fat mass was approximately 40% greater in the rats fed the HFD

compared to the rats fed a ND (P<0.05) (figure 5.8). Similarly, epididymal fat was

significantly greater, by approximately 50%, in the HFD compared to the ND fed group

(P<0.005) (figure 5.8).

Body weight, food and caloric intake

Over time, body weight increased in both the HFD and ND fed groups of rats, with a small

but significantly greater increase seen in the HFD group (P<0.005) (figure 5.9). This occurred

despite the significantly lower average daily food intake in the HFD rats (P<0.0001) (figure

5.9). However, the average daily caloric intake was significantly greater in rats fed the HFD

(P<0.0001) (figure 5.9).

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Figure 5.7 Systolic blood pressure changes over time.

Systolic blood pressure changes over time in rats fed a normal chow diet (ND) (n=11) and

high fat diet (HFD) (n=11).

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Figure 5.8 Fat distribution in rats fed a high fat diet (HFD) or normal diet (ND).

Upper panel: Effect of high fat diet (HFD (n=13) on the whole body fat mass compared to

rats fed a normal chow diet (ND) (n=12). *P<0.05. Lower panel: Effect of high fat diet

(HFD (n=16) on the epididymal fat mass compared to rats fed a normal chow diet (ND)

(n=15). #P<0.005 between groups.

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Figure 5.9 Effects of body weight, food intake and calorie intake over time.

Effect of high fat diet (HFD; n=17-19) on the body weight, food intake per day and average

calorie intake per day over time compared to rats fed a normal chow diet (ND) (n=13).

#P<0.005, *P<0.0001 between groups.

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5.4 DISCUSSION

The key findings of the present work are that in the HFD rats, resistin and leptin (alone and in

combination) act in the brain to induce significant increases in RSNA and that combining

resistin and leptin elicited significantly greater effects than either hormone alone. Further,

MAP and HR responses induced by resistin, leptin and both combined were not significantly

different from the control group. Comparing the responses induced by the hormones in the

HFD rats to those observed in the rats fed ND showed that the HFD had no effect on the

maximum increases in RSNA. The results suggest that with high fat feeding, the increase in

RSNA induced by resistin is not reduced.

The increase in RSNA induced by centrally administered resistin in the HFD rats was

significantly greater than in the control group, and was similar to that observed in rats fed a

ND. Thus, the sensitivity to the sympatho-excitatory effects on RSNA induced by resistin

was not affected by high fat feeding. This was similar to our observations with leptin. Our

findings with leptin are in agreement with studies in the rodent that show high fat feeding

does not reduce the sympatho-excitatory RSNA responses to leptin (18, 58, 257). In the

rabbit, however, the RSNA response to leptin was greater following high fat feeding (17).

Since the effects of leptin on sympathetic outputs like lumbar and brown adipose tissue

(BAT) are reduced by HFD, as is the effect on dietary intake (18, 58), the evidence indicates

that the RSNA response elicited by leptin may be selectively resistant to the actions of high

fat feeding (79, 87, 255, 258). Our present findings suggest that the excitatory effects of

resistin on RSNA are not influenced by HFD and this could be interpreted to mean that

resistin’s actions on RSNA are also resistant to the desensitising effects of HFD. The

influence of HFD on responses in lumbar sympathetic nerve activity (LSNA) and brown

adipose tissue sympathetic nerve activity (BAT SNA) elicited by resistin have not been

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investigated to date. In rats fed ND resistin increases LSNA which also occurs with leptin

(99, 136). In contrast, resistin reduces BAT SNA, and hence thermogenesis, which is

opposite to leptin (113, 136). This could contribute to the conclusion that resistin induced

resistance to leptin’s thermogenic effects (209).

In the HFD rats, the combined administration of leptin and resistin produced a greater

response in RSNA compared to each hormone alone. A similar effect was observed in ND

fed rats (256). The magnitude of the enhancement was similar by the end of the observation

period irrespective of the dietary regime. This also suggests that the sensitivity of the RSNA

response elicited by resistin and leptin is not markedly affected by HFD.

There did appear to be a slower onset in the RSNA response elicited by the combination of

resistin and leptin in the HFD fed rats compared to ND fed rats. This suggests there may be

some influence of diet on the development of the sympatho-excitatory RSNA response,

though not on the magnitude of the response. The influence of the HFD, on the development

of the RSNA response, however, was not observed when either hormone was administered

alone. The renal sympatho-excitatory effects of resistin and leptin involves PI3K (51, 208).

Thus the present findings suggest that this mechanism is not influenced by high fat feeding.

This contrasts to the anorexigenic effect of leptin where the evidence suggests that

intracellular transduction pathways involving SOCs3, PTP1B and PKA mediate the

decreased sensitivity to leptin receptor activation seen with HFD (259-261).

It has been argued that changes in receptor densities with different diets could also influence

sensitivity to leptin and resistin. The receptor for resistin has not been identified, hence, there

are no studies investigating receptor distribution for resistin. Leptin receptors have been

investigated and the receptor density in the brain does not appear to change markedly with

high fat feeding (262). However, the receptor protein levels were reduced with high fat

feeding suggesting this could contribute to the decreased sensitivity observed to some of the

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actions of leptin (262). The increased circulating levels of leptin and resistin that are

associated high fat feeding and increased adiposity would therefore be less effective if the

receptor protein levels are reduced. However, the present study suggests this does not appear

to influence the RSNA response elicited by centrally administered leptin or resistin.

The MAP responses to resistin alone, leptin alone and both combined were not significantly

different compared to control in the HFD fed rats. Compared to the responses seen in the rats

fed a ND, the maximum changes in MAP induced by leptin alone, or leptin combined with

resistin were not significantly different to those in the HFD fed rats. Interestingly, the

maximum change in MAP elicited by resistin alone was lower in the HFD fed group

compared to the ND fed group.

Previous studies with leptin in HFD rodents have shown that the acute effect of leptin on

MAP was not influenced by the diet (18). In rabbits fed a HFD, however, acute ICV

administration of leptin resulted in an increased MAP and this response was similar to that

seen in rabbits fed a ND (17, 102). In longer-term studies, the MAP response induced by a

chronic infusion of leptin was not increased by HFD in rodents (18).

To date there are no reports of the effects of chronic long-term infusion of resistin on blood

pressure. Thus, it remains to be determined whether chronic infusion of resistin can increase

blood pressure and whether the response can be influenced by a HFD.

In the HFD rats the HR responses to each hormone alone or in combination were not

significantly different compared to control. The comparisons of the maximum changes in HR

seen in the rats fed a ND or a HFD showed there were no significant differences following

leptin alone or in combination with resistin. However, the maximum change in HR with

resistin alone was lower in the HFD fed group, as was seen with MAP. The physiological

relevance of the differences in the HR and MAP responses between the diets following

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resistin alone are difficult to interpret. We suspect the differences are probably a statistical

anomaly since the changes in MAP and HR elicited by resistin alone within the HFD group

were not significantly different from the saline control.

In the present study, systolic blood pressure over the duration of the dietary feeding regime

was not significantly elevated by the HFD compared to ND feeding. This is similar to

previous studies using Wistar rats fed a HFD for 10-20 weeks which did not show an

elevation in blood pressure (263). Sprague-Dawley rats may segregate into obese prone and

obese resistant rats but only those prone to obesity are reported to have increased blood

pressure following 8-10 weeks of a HFD (264), or 13 weeks of a HFD (19). In the present

study we used Sprague Dawley rats fed for up to 10 weeks and we suspect the difference may

be that we did not see any evidence of segregation. We did see a small but significantly

greater increase in body weight over time in the HFD fed rats though this was smaller than

that observed in the obese prone rats previously reported (19, 264). By contrast we have

observed that Long-Evans rats fed a western diet (60% fat) had a small but significant

increase in blood pressure compared to ND fed rats (265). In mice it has been found that

when they are fed a HFD for 10 weeks they become obese with an elevated blood pressure

when measured directly using radiotelemetric probes (18). In the present study we measured

blood pressure via tail cuff plethysmography which will not detect small differences

accurately. In rabbits, high fat feeding for only 4 weeks appears sufficient to induce

hypertension (17). Thus, elevations in blood pressure in rodents fed HFD seems to be

variable and may depend on various factors including the strain, amount of fat in the diet and

the duration of feeding, whereas rabbits appear to be particularly susceptible to obesity-

induced hypertension.

There was a small increase in body weight over time with the HFD. This was due to the

increased intake of calories by rats on the HFD, even though the rats reduced their average

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daily intake of the HFD. We did not observe any segregation of rats into obese-sensitive or

obese-resistant groupings in this study, although this has been reported (19). Nonetheless,

there was a clear redistribution of body fat in rats fed the HFD such that there was a 40%

increase in percent body fat and a larger increase in the amount of epididymal fat present.

Thus, although we did not observe an overt obesity in the HFD fed rats, a clear redistribution

of body fat was seen.

Perspective views

In HFD fed rats ICV administration of resistin or leptin alone increased RSNA. The

combination of both hormones resulted in a greater increase in RSNA compared to each

hormone alone. The responses were similar in magnitude to those observed in rats fed a ND,

suggesting that feeding a HFD sufficient to markedly alter visceral fat distribution, but elicit

only a small, but statistically significant, increase in body weight, does not diminish the

greater increase in RSNA. Thus, we find no evidence of a reduced sensitivity of the RSNA

response elicited by centrally administered resistin. Furthermore, as both hormones act via

PI3K to increase RSNA, these results may suggest that the stimulatory effect is not

desensitized following a HFD feeding.

Furthermore, our study suggests that in conditions in which both resistin and leptin are

elevated, such as in obesity and diabetes, there may be greater sympatho-excitatory actions

on RSNA, compared to that elicited by each hormone alone. This may contribute to the long-

term cardiovascular complications associated with those conditions, however, this requires

further investigation.

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6 Chapter 6: Central Administration Of Insulin Combined With Resistin Reduced Renal

Sympathetic Nerve Activity In Rats Fed A Normal diet Or A High Fat Diet

6.1 INTRODUCTION

Insulin is a hormone produced by the pancreas, in response to elevated blood glucose (110).

Insulin is released into the circulation and can cross the blood-brain barrier. Insulin receptors

are widely distributed in the central nervous system (CNS) (158, 266, 267), and their

activation by insulin can influence body weight control, glucose homeostasis and sympathetic

nerve activity to the kidney, hindlimb and brown adipose tissues (53, 54, 85, 110, 163, 200,

202, 203, 268-271).

Resistin is an adipokine, which is produced by adipose tissue, and has been shown to play a

role in promoting insulin resistance (151, 272, 273). Recent studies have demonstrated that

resistin acts in the brain to elicit increases in sympathetic nerve activity to the kidney (RSNA)

and hindlimb (51, 136), which is similar to central actions of insulin. By contrast, resistin

reduces sympathetic nerve activity to brown adipose tissue (BAT SNA) (136).

The sympatho-excitatory effects of resistin and leptin are similar to the sympatho-excitatory

actions of centrally administered leptin (51, 73, 100, 256). Furthermore, we have recently

found that the combined administration of resistin and leptin into the lateral brain ventricle

induced a greater increase in RSNA than that induced by either hormone alone, as has been

shown in chapter 3 (256). Similarly, in a more recent study, we have found that the combined

administration of leptin and insulin produced a greater increase in RSNA compared to either

leptin or insulin alone, as has been shown in chapter 4 (274). Therefore, one could

hypothesise that the combined administration of insulin and resistin may also produce a

greater increase in RSNA. However, resistin is known to induce resistance to insulin’s

metabolic action (151, 272, 273). Thus, in this study we investigated the effect of combined

112

administration of insulin and resistin on RSNA in rats fed a normal chow diet (ND). This

interaction may be important in conditions in which both hormones are elevated, for example

in conditions of high adiposity.

Consumption of high fat diets (HFD) is known to reduce the dietary effects of leptin, as well

as insulin (53, 78, 79, 176, 177). The increase in RSNA induced by leptin, however, remains

unaffected by HFD. Similarly, the excitatory action of resistin on RSNA is not affected by a

HFD (275). The RSNA responses to insulin, however, are controversial, and have only been

performed in two studies in mice (53, 110). Thus, insulin effects may be influenced by HFD

and this could impact on interactions with resistin. The effects of insulin in HFD has not been

examined in rats previously.

Therefore, the main aims of the present study were to investigate (i) the central effects of

insulin in rats fed a HFD, and (ii) the central effects of combined administration of resistin

and insulin on RSNA in rats fed a ND, and whether HFD was able to influence the responses.

6.2 METHODS




to Animals Act 1986 (Australia). The procedures conform to the ‘Guiding Principles for

Research Involving Animals and Human Beings’, and the guidelines set out by the Australian




113


Male Sprague-Dawley rats were obtained from ARC animal resources (Canning Vale, WA,

Australia). Rats were acclimatised for 7 days after arrival and were housed at 23°C with a 12

h light–dark cycle. The rats were allowed free access to water and food; one group were fed

with ND (4.8% fat), and the others were fed a HFD (SF14-004 (22% fat), Speciality Feeds,

Glen Forrest, WA) for 8-10 weeks. Body weight, food intake and caloric intake were

monitored weekly. Using a non-invasive tail cuff occlusion method, systolic blood pressure

was monitored fortnightly during the feeding period. The percent whole body fat was

determined at the end of the feeding period, using an EchoMRI 500 (Houston Texas, USA) in

a sample of animals of each dietary treatment.

On the day of the experiments, rats were anaesthetized using isoflurane gas (2-5% in O2). The

femoral vein and artery were cannulated as previously described in chapter 2 (Habeeballah

2016). The femoral vein was cannulated to maintain anaesthesia using intravenous (IV)

urethane (1.4-1.6 g/kg, supplemented with 0.05 ml of a 25% solution if required). The depth

of anaesthesia was maintained to ensure the absence of corneal and pedal reflexes. The

femoral artery was cannulated for monitoring the arterial pressure. The mean arterial pressure

(MAP) and heart rate (HR) were determined from the arterial pressure pulse using LabChart

(ADInstruments, NSW, Australia). To maintain body temperature at approximately 37.5 oC

rats were kept on a heating pad during the experiments.


After a flank incision, the left kidney was exposed retroperitoneally as previously described

in chapter 2 (51, 256). RSNA was amplified using a low-noise differential amplifier (models

ENG 187B and 133, Baker Institute, Victoria, Australia), filtered (band pass 100–1,000 Hz),

114

rectified, and integrated at 0.5 s intervals. The signal was recorded using a PowerLab data

acquisition system (ADInstruments, NSW, Australia) (256).


Using a stereotaxic frame, rats were placed prone, and bregma and lambda were positioned

on the same horizontal plane. A hole (2 mm diameter), centered 0.7 mm caudal to bregma

and 1.4 mm lateral to the mid-line, was drilled into the skull, to expose the dorsal surface of

the brain. Unilateral microinjections (5 µl) were made using a fine glass micropipette (50–

70µm tip diameter) inserted into the lateral brain ventricle (stereotaxic co-ordinates: 0.7 mm

caudal to bregma, 1.4 mm lateral to the mid-line and 3.7–3.9 mm ventral to the surface of the

dura). The micropipette was left in place for 1 min, after the microinjection. At the end of the

experiment, a small amount of Pontamine Sky Blue was microinjected using the same

coordinates to confirm microinjection into the lateral ventricle.


MAP, HR and RSNA were measured before and for 180 min after administration of saline

(control) (n=5 HFD and ND), resistin (7 µg; n=4 ND, n=5 HFD), insulin (500 mU; n=6 ND,

n=5 HFD) and the combination of both resistin and insulin (n=7 ND, n=5 HFD; insulin was

administered 15 min after resistin). The doses of each hormone were chosen on the basis of

previous studies (113, 136, 256). At the end of the experiment, the epididymal fat was

removed and weighed.


6.2.3.1 Within diet

The average resting levels of MAP and HR before hormone administration were compared

between groups using one-way ANOVA. The integrated RSNA averaged over 1-2 minutes,

115

was calculated at 15-minute intervals over the period of 180 min and expressed as a

percentage of the resting level before the injections. Changes in MAP, HR and RSNA over

time following hormone/saline administration were compared between groups by using two-

way ANOVA. When an overall significant difference between groups was detected,

comparisons were made between individual groups, using Holm-Sidak’s test for multiple

comparisons.


The changes in RSNA, MAP and HR over time following resistin alone, insulin alone and the

combination of insulin and resistin were compared between HFD and ND using two-way

ANOVA. Data for resistin alone and saline (control) in ND and HFD has been reported by

our lab previously in chapter 3 and 5 (256, 275) and were used in this study for comparison.

For the insulin alone, the data in the ND rats includes data previously published and reported

in chapter 4 (274), but we have increased the amount of data by adding N=2 more rats.

The body weight, average food intake, average caloric intake, and systolic blood pressure

during the feeding period were compared between dietary groups using two-way ANOVA.

Systolic blood pressure was measured via indirect tail cuff plethysmography. Due to a

technical issue, systolic blood pressure was recorded before and for 6 weeks after the start of

feeding. The percentage of whole body fat and the epididymal fat were compared between

the dietary groups using Student’s unpaired t-test.

All results are expressed as means ± SEM. A value of P<0.05 was considered to be

statistically significant. PRISM software was used for statistical analysis and graphical

representation (GraphPad San Diego, California, USA).

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6.3 RESULTS

6.3.1 Rats fed a normal diet


When insulin and resistin were given in combination, there was no increase in RSNA and this

was not significantly different from the saline (control) group (figure 6.1). However, this

response was significantly different compared to insulin or resistin alone (P<0.0001) (figure

6.1). Insulin alone increased RSNA significantly by 81±28% (P<0.0001 compared to saline).

Similarly, RSNA increased significantly following the ICV injection of resistin alone (Figure

6.1).


There was an overall statistically significant difference between groups in the change in MAP

(P<0.0001) (figure 6.2). Following the administration of resistin and insulin in combination,

there was no significant differences in the change in MAP compared to either hormone alone.

However, there was a small but statistically significant difference from the saline (control)

group (P=0.0001) (figure 6.2). ICV administration of insulin alone had no significant effect

on MAP compared to saline (figure 6.2).

There was also an overall significant difference in the HR responses between the groups

(P<0.0001) (figure 6.2). Compared to saline, insulin alone and the combination of insulin

with resistin significantly increased HR (P<0.0001) (figure 6.2).

The resting levels of both MAP and HR before hormone administration were not significantly

different between groups.

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Figure 6.1 Percent change in RSNA induced by resistin and insulin alone and in combination in rats fed a normal diet (ND).

Percent changes in renal sympathetic nerve activity (RSNA) from baseline levels over time in

rats fed a normal chow diet (ND) after intracerebroventricular injection of insulin (n=6) (500

mU/5μl), resistin (n=4) (7μg/5μl), control (saline, n=5) (5μl) and the combination of resistin

and insulin (n=7). *P<0.0001 insulin alone and resistin alone compared to saline; #P<0.0001

combination of resistin and insulin compared to resistin alone or insulin alone.

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Figure 6.2 Change in MAP and HR induced by resistin and insulin alone and in combination in rats fed a normal diet (ND).

Change in mean arterial pressure (MAP) and heart rate (HR) over time in rats fed a normal

chow diet (ND) induced by the intracerebroventricular administration of insulin (n=6) (500


and insulin (n=7). *P<0.0001 between groups.

119



In rats fed a HFD, there was a significant increase in RSNA following intracerebroventricular

(ICV) administration of insulin alone compared to saline (P<0.0001) and reached a maximum

of 46±16% (figure 6.3). Similarly, resistin alone increased RSNA significantly over time and

reached a maximum of 64±4% (P<0.0001 compared to saline).

When resistin and insulin were administered in combination, RSNA fell by over 35% and

was significantly different compared to saline (P<0.0001) (figure 6.3). The reduction in

RSNA following the combined administration of resistin and insulin was significantly

different from the increases observed following resistin alone (P<0.0001) and insulin alone

(P<0.0001) (figure 6.3).


There was an overall significant difference in the MAP response between groups (P<0.01)

(figure 6.4). The comparison between individuals showed that only the response to insulin

was significantly different compared to saline (P<0.005) (figure 6.4).

In the HR response, the comparison between individual groups showed that resistin alone

(P<0.0001), insulin alone (P<0.05) and the combination (P<0.05) were significantly different

from saline.

When resistin and insulin were combined the HR response was significantly different from

resistin alone and insulin alone (P<0.0001).

The resting levels of both MAP and HR prior to hormone administration did not show any

significant differences between groups.

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Figure 6.3 Percent changes in RSNA induced by resistin and insulin alone and in combination in rats fed a high fat diet (HFD).


rats fed a high fat diet (HFD) after intracerebroventricular injection of insulin (n=5) (500


and insulin (n=5). *P<0.0001 insulin alone and resistin alone compared to saline; #P<0.0001

combination of resistin and insulin compared to resistin alone or insulin alone or saline.

121

Figure 6.4 Change in MAP and HR induced by resistin and insulin alone and in combination in rats fed a high fat diet (HFD).


diet (HFD) induced by the intracerebroventricular administration of insulin (n=5) (500


and insulin (n=5). *P<0.01; #P<0.0001 between groups.

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6.3.3 Comparison of responses to resistin and insulin in rats fed high fat vs normal chow diets

6.3.3.1 RSNA

In rats fed a HFD the increase in RSNA following insulin was significantly less compared to

rats fed a ND (P<0.01) (figure 6.5). However, the response to resistin was not significantly

different between dietary groups (figure 6.5).

When both hormones were combined, there was a statistically significant difference in the

RSNA response between the diets. In the HFD RSNA fell markedly compared to the ND

(P<0.01) (figure 6.5).

6.3.3.2 MAP and HR

The response to MAP over time induced by insulin alone or in combination with resistin was

not significantly different between the diets (figure 6.6). However, the response to resistin

was significantly less in rats fed a HFD compared to ND (P<0.001) (figure 6.6).

The HR response to insulin was significantly less in rats fed a HFD compared to rats fed a

ND (P<0.0001) (figure 6.7). Similarly, resistin alone also induced a smaller HR response in

the HFD group (P<0.0001) (figure 6.7). When resistin and insulin were administered in

combination, there was an increase in the HR that was not significantly different between the

rats fed a HFD or ND (figure 6.7).

The basal level of the MAP and HR prior the hormones administration were not significantly

different between the dietary groups (see figure 6.8).

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Figure 6.5 RSNA response following resistin and insulin alone and in combination in rats fed a high fat diet (HFD) or normal diet (ND).

Renal sympathetic nerve activity (RSNA) responses over time between rats fed a normal

chow (ND) and a high fat diet (HFD), elicited by intracerebroventricularly administered

resistin (n=4 ND, n=5 HFD) (7 μg/5 μl), insulin (n=6 ND, n=5 HFD) (500 mU/5μl) and the

combination of resistin and insulin (n=7 ND, n=5 HFD). *P<0.01 compared to ND.

124

Figure 6.6 MAP response following resistin and insulin alone and in combination in rats fed a high fat diet (HFD) or normal diet (ND).

Mean arterial pressure (MAP) responses over time between rats fed a normal chow (ND) and

a high fat diet (HFD), induced by intracerebroventricularly administered of resistin (n=4 ND,

n=5 HFD) (7 μg/5 μl), insulin (n=6 ND, n=5 HFD) (500 mU/5μl) and the combination of

resistin and insulin (n=7 ND, n=5 HFD). *P<0.05 compared to ND.

125

Figure 6.7 HR response following resistin and insulin alone and in combination in rats fed a high fat diet (HFD) or normal diet (ND).

Heart rate (HR) responses over time between rats fed a normal chow (ND) and a high fat diet

(HFD), induced by intracerebroventricularly administered of resistin (n=4 ND, n=5 HFD) (7

μg/5 μl), insulin (n=6 ND, n=5 HFD) (500 mU/5μl) and the combination of resistin and

insulin (n=7 ND, n=5 HFD). *P<0.0001 compared to ND.

126

Figure 6.8 Basal level of MAP and HR in rats fed a high fat diet (HFD) or normal diet (ND) prior to hormone administration.

Basal levels of mean arterial pressure (MAP) and heart rate (HR) in rats fed normal chow diet


administration of resistin (n=4 ND, n=5 HFD) (7 μg/5 μl), insulin (n=6 ND, n=5 HFD)

(500mU/5μl) and the combination of both hormones (n=7 ND, n=5 HFD).

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6.3.3.3 Comparison of systolic BP and metabolic parameters in rats fed high fat vs normal chow diets

Systolic blood pressure changes during the feeding period

There was no significant difference observed in the systolic blood pressure over time between

rats fed a HFD and rats fed a ND (figure 6.9).

Metabolic parameters

There was a significant increase in the percent of whole body fat mass in rats fed a HFD

(P<0.005) (figure 6.10). Similarly, epididymal fat was increased significantly in the HFD

groups (P<0.05) (figure 6.10).

Body weight, food and caloric intake

In rats fed HFD, body weight increased over time and it was slightly but significantly greater

compared to ND (P<0.005) (figure 6.11). Caloric intake was also greater in the HFD group

(P<0.0001), but the average daily food intake in the HFD fed rats was significantly lower

than in the ND group (P<0.0001) (figure 6.11).

128

Figure 6.9 Systolic blood pressure changes over time.

Systolic blood pressure over time in rats fed a normal chow diet (ND) (n=11) and high fat

diet (HFD) (n=14).

129

Figure 6.10 Fat distribution in rats fed a high fat diet (HFD) or normal diet (ND).

Upper panel: Effect of high fat diet (HFD) (n=12) on the whole body fat mass compared to

rats fed a normal chow diet (ND) (n=10). *P<0.005 between groups.

Lower panel: Effect of high fat diet (HFD) (n=14) on the epididymal fat mass compared to

rats fed a normal chow diet (ND) (n=12). #P<0.05 between groups.

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Figure 6.11 Effects of body weight, food intake and calorie intake over time.

Effect of high fat diet (HFD; n=18-19) on the body weight, food intake per day and average

calorie intake per day over time compared to rats fed a normal chow diet (ND) (n=18).

#P<0.005, *P<0.0001 between groups.

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6.4 DISCUSSION

In this study, the key findings are that in rats fed a ND, the combination of resistin and insulin

did not increase RSNA which was significantly different to the sympatho-excitatory effects

seen with resistin alone or insulin alone. In rats fed the HFD, insulin induced a significant

increase in RSNA, but this was smaller than the increase observed in the group fed a ND.

Surprisingly in the HFD group, when resistin and insulin were combined, RSNA did not

increase but, rather, fell by 35%.

In the present study, we show for the first time that in rats fed a HFD insulin acts centrally to

induce increases in RSNA. However, the increase was significantly less than that observed in

rats fed a ND. This result is in agreement with reports in agouti obese mice compared to lean

mice (53). However, in another report insulin increased RSNA in ob/ob obese mice and the

response was not less than in control lean mice (110). Thus, the present study would lend

weight to the suggestion that HFD decreased the sensitivity to insulin to elicit an increase in

RSNA.

In rats fed ND, both resistin and insulin alone elevate RSNA, therefore, when combined we

expected a greater effect than each alone, as we observed previously with the combination of

resistin and leptin and also the combination of leptin and insulin (see chapter 3 and 4) (256,

274). Surprisingly, our present results show that the combination of resistin and insulin did

not increase RSNA in ND rats, and in the HFD rats RSNA fell significantly. Thus, the results

suggest a centrally-mediated resistance to the sympatho-excitatory effects when resistin and

insulin are combined and this negative interaction is exacerbated by HFD.

Previous studies show that resistin induces resistance to the metabolic functions of insulin

(151, 272, 273). Our present results suggest that this may also occur for RSNA. However,

such an explanation is unlikely to fully explain the marked reduction in RSNA observed in

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the HFD, and the absence of any increase in ND following the combined administration of

resistin and insulin. There are differences in the central mechanisms used by the two

hormones to elicit an increase in RSNA. Resistin utilises phosphatidylinositol 3 kinase

(PI3K) whereas it is not involved in the RSNA response to insulin (51, 54). Whether such

differences contribute to our present observations is unknown. Clearly, the interaction

between resistin and insulin needs further exploration, as this may be important in conditions

in which both hormones are elevated.

The dramatic central interaction between resistin and insulin that resulted in the marked

decrease in RSNA in the HFD in the present study differs markedly to our previous findings

of resistin combined with leptin. In that study, a greater increase in RSNA than that induced

by either hormone alone was observed (see chapter 5) (275). Thus, two opposing effects

occur involving interactions with resistin despite the fact that insulin, leptin and resistin alone

induce increases in RSNA (17, 18, 51, 53, 58, 87, 110). The mechanisms contributing to the

complex interactions of these important hormones needs to be investigated in future studies.

In the present study, we did not see a significant difference between the HFD and ND in the

MAP responses to insulin alone or combined with resistin. This contrasts with the RSNA

responses. This suggests that in the HFD, the marked reduction in RSNA after acute

administration of insulin combined with resistin was not sufficient to induce a fall in MAP, or

compensatory mechanisms come into play.

In HFD, the HR response induced by insulin was significantly less than in the ND, and this

difference between diets was also seen with resistin. However, when insulin and resistin were

combined, there was no significant difference in the HR responses between the diets. This is

a very different interaction compared to that seen with the RSNA response, suggesting that

there is little evidence for resistin inducing resistance to insulin in the HR response. This

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suggests that resistance to insulin may not be a generalised effect of resistin but may target

selected outputs.

Body weight increased over time and the increase in HFD was significantly greater than in

the ND. This was associated with a greater increase in caloric intake in the HFD animals but

a lower total food intake compared to the ND rats. In the HFD there was a clear redistribution

of body fat. There was no difference in systolic blood pressure over the feeding period

between the groups. These findings are in agreement with our previous observations in

chapter 5 (275). These metabolic changes highlight that marked interactions between insulin

and resistin occur within the brain even in the absence of overt obesity.

Perspective

In rats fed a HFD centrally administered insulin or resistin alone increased RSNA, but the

response to insulin was significantly attenuated compared to ND. No such attenuation was

seen with resistin. Surprisingly, combining insulin and resistin did not increase RSNA in ND,

and resulted in a marked fall in RSNA in HFD rats.

In obese and overweight conditions both insulin and resistin levels are elevated. The results

of the present study, suggest that this may not be detrimental to RSNA and therefore to renal

function. However, in overweight/obese conditions RSNA is abnormally elevated, and it

contributes to the associated hypertension (35, 41). This suggests that the mechanisms

responsible for increasing RSNA overcome the inhibitory action of the combination of

resistin and insulin. In a previous study we found that resistin combined with leptin resulted

in a greater increase in RSNA than that produced by each hormone alone (see chapter 5)

(275). Since leptin is also elevated in obesity, it is conceivable that the presence of resistin

acts in combination with leptin to contribute to the sympatho-excitatory RSNA actions and

this overcomes the sympatho-inhibitory response of the combination of resistin and insulin.

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7 Chapter 7: Central Administration Of Insulin Combined With Leptin Reduces Renal

Sympathetic Nerve Activity In Rats Fed A High Fat Diet

7.1 INTRODUCTION

Insulin is a hormone produced by the pancreas (154, 276). It plays an important role in

glucose homeostasis and autonomic nervous system regulation by acting through its

receptors, which are widely distributed throughout the central nervous system (CNS) (163,

169, 170, 277, 278). Insulin acts within the brain to induce increases in sympathetic nerve

activity to the kidney, hindlimb and brown adipose tissue (53, 54, 202, 203, 205).

Leptin is a hormone produced by adipose tissue. It is well known to play important roles in

metabolic regulation and cardiovascular function (63, 68-71). Leptin acts centrally to increase

the sympathetic nerve activity to kidney, hindlimb and brown adipose tissue (50, 73, 99, 100,

105). Thus, leptin and insulin have similar effects on these sympathetic nervous system

outputs.

Indeed, leptin and insulin increase the sympathetic drive to the kidney through a common

pathway within the brain mediated by activation of melanocortin 4 receptors (MC-4R) (110).

This suggests these hormones may augment each other’s actions on renal sympathetic nerve

activity (RSNA). In fact, we have found previously, in chapter 4, that combined central

administration of leptin and insulin induced greater increases in RSNA, than each hormone

alone, in rats fed a normal diet (ND) (274). This augmented sympatho-excitatory response in

RSNA, following the combination of leptin and insulin, may be implicated in the abnormally

elevated RSNA seen in overweight /obesity, conditions in which leptin and insulin are

elevated (79, 85-87, 161, 162).

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High fat diet (HFD) significantly attenuates the metabolic action of leptin and insulin (78, 79,

178). However, leptin’s effect on RSNA is not attenuated by HFD (17, 53, 79, 110), whilst

the effects of HFD on the RSNA response to insulin is still controversial (53, 110).

Thus, HFD can induce resistance to the metabolic actions of leptin and insulin but with the

RSNA response only the actions of insulin may be reduced by HFD.

Therefore, in this study, we investigated whether HFD induced resistance to the effects of

leptin and insulin combined on RSNA.

7.2 METHODS



Experiments were performed to comply with the Prevention of Cruelty to Animals Act 1986

(Australia). All experimental protocols conform with the ‘Guiding Principles for Research

Involving Animals and Human Beings’ and the guidelines set out by the ‘Australian Code of

Practice for the Care and Use of Animals for Scientific Purposes’, 2013 (National Health and

Medical Research Council of Australia) and were approved by the Royal Melbourne Institute

of Technology (RMIT) University Animal Ethics Committee (approval letter is attached in

appendix II). Male Sprague–Dawley rats were obtained from the ARC Animal Resources

Centre (Western Australia, Australia). Rats were housed under a 12:12 h light ⁄ dark cycle at

23° C and allowed free access to standard rat chow and water.

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Upon arrival, rats were acclimatised for one week then were allowed free access to food and

water. One group was exposed to ND (4.8% fat) and the other to a HFD (SF14-004 (22% fat),

Speciality Feeds, Glen Forrest, WA) for 8-10 weeks. On the day of experiments, isoflurane

gas (2-5%) in O2 was used to induce general anaesthesia. The femoral vein was cannulated

for intravenous (IV) infusion of urethane (1.4–1.6 g/kg; IV with supplemental doses of 0.05

ml of a 25% solution, if required) to maintain anaesthesia. In addition, the femoral artery was

cannulated to measure and record blood pressure and heart rate. Mean arterial pressure

(MAP) and heart rate (HR) were calculated from the arterial pressure pulse using LabChart

(ADInstruments, Castle Hill, NSW, Australia). Rats were kept on a heating pad for the

duration of the experiments to maintain body temperature at approximately 37.5oC.

The left kidney was exposed using a retroperitoneal approach. The renal nerve was identified

and carefully dissected free of surrounding tissue. Under mineral oil, the renal nerve was cut

and the proximal end was placed onto the bared tips of two Teflon-coated silver wire

electrodes as previously described in chapter 2.


Animals were placed prone, and their heads were mounted in a Stoelting stereotaxic frame, so

that bregma and lambda were positioned on the same horizontal plane. A hole (diameter 2

mm) was drilled into the skull (centred 0.7 mm caudal and 1.4 mm lateral from bregma). The

microinjections was made unilaterally using a fine glass micropipette (tip diameter 50–70

µm) inserted into the lateral brain ventricle (stereotaxic coordinates: 0.7 mm caudal to

bregma, 1.4 mm lateral to midline, and 3.7-3.9 mm ventral to the surface of the dura). After

the microinjection, the micropipette was left in place for 1 min. At the end of the experiment,

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a small amount of pontamine sky blue was microinjected using the same coordinates to



In these experiments, MAP, HR and RSNA were recorded before and for 3 hours after the

intracerebroventricular (ICV) administration of control (saline; 5 μl) (n=5), leptin (7 µg; 5 μl)

(n=4), insulin (7 µg; 500 mU) (n=5) and the combination of both insulin and leptin (n=5) (5

μl of each hormone; leptin was administered 15 min after insulin). The resting level was

recorded for approximately 15 minutes before the ICV administration of hormone/saline. The

variables were calculated every 15 minutes prior to and for 3 hours after the injection.


7.2.3.1 Within high fat diet

Resting levels of MAP and HR before hormone administration were compared between

groups using one-way ANOVA. The integrated RSNA was calculated and averaged over 1-2

minutes at each time point over the period of 180 minutes, and expressed as a percentage of

the resting level before the ICV injections. Changes in MAP, HR and RSNA over time

following ICV administration were compared between groups by using two-way ANOVA.

When an overall significant difference between groups was detected, comparisons were made

between individual groups, using Holm-Sidak’s test for multiple comparisons.


The changes in RSNA, MAP and HR over time following the hormones were compared

between dietary groups using two-way ANOVA. For comparisons shown in the figures using

the hormones alone and with animals fed a ND we have used data from previous chapters

(256, 274, 275). All results are expressed as means ± SEM. A value of P<0.05 was

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considered to be statistically significant. PRISM software was used (GraphPad San Diego,

California, USA).

7.3 RESULTS



ICV administration of leptin and insulin combined resulted in a reduction in the RSNA so

that by two hours after administration the RSNA was reduced by 37 ± 12% (figure 7.1). This

contrasts with responses observed following saline, or either hormone alone (figure 7.1).

Leptin or insulin alone increased RSNA (figure 7.1).


When insulin and leptin were combined there was a small increase in MAP (figure 7.2),

which was significantly different compared to saline and to the administration of the

hormones alone (P<0.0001 between the groups) (figure 7.2).

With respect to the HR response, the combination of leptin and insulin induced a small

increase, which was significantly less than the saline (control) (P<0.0001) (figure 7.2).

The resting levels of MAP and HR prior to hormone administration were not significantly

different between groups (figure 7.3).

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Figure 7.1 Percent change in RSNA induced by leptin and insulin alone and in combination in rats fed a high fat diet (HFD).


rats fed a high fat diet (HFD) after intracerebroventricular injection of leptin (n=4) (7μg/5μl),

insulin (n=5) (500 mU/5μl), control (saline, n=5) (5μl) and the combination of leptin and

insulin (n=5). *P<0.0001 between groups.

140

Figure 7.2 Changes in MAP and HR induced by leptin and insulin alone and in combination in rats fed a high fat diet (HFD).


diet (HFD) induced by the intracerebroventricular administration of leptin (n=4) (7μg/5μl),

insulin (n=5) (500 mU/5μl), control (saline, n=5) (5μl) and the combination of leptin and

insulin (n=5). *P<0.0001 between groups.

141

Figure 7.3 Basal level of MAP and HR in rats fed a high fat diet (HFD) or normal diet (ND).

Basal levels of mean arterial pressure (MAP) heart rate (HR) in rats fed normal chow diet


administration of leptin (n=4) (7μg/5μl), insulin (n=5) (500 mU/5μl) and the combination of

both hormones (n=5).

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7.3.2 Comparison of responses to the combination of insulin and leptin in rats fed high fat vs normal chow diets

7.3.2.1 RSNA

In rats fed a HFD, the response to RSNA induced by the ICV administration of insulin and

leptin combined was in stark contrast to that observed in rats fed a ND. In HFD, the reduction

in RSNA was significantly different to the increase in RSNA induced by the combination of

leptin and insulin in rats fed a ND (P<0.0001) (figure 7.4).

7.3.2.2 MAP and HR

The MAP response over time induced by the combination of insulin and leptin was

significantly higher in rats fed a HFD compared to rats fed a ND (P<0.0001) (figure 7.4).

However, the response to the combination of insulin and leptin on the HR over time was not

significantly different between rats fed the different diets (figure 7.4). In both groups the time

courses for HR over time were similar.

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Figure 7.4 RSNA, MAP and HR response following leptin and insulin alone and in combination in rats fed a high fat diet (HFD) or normal diet (ND).

Renal sympathetic nerve activity (RSNA), mean arterial pressure (MAP) and heart rate (HR)

responses between rats fed a normal chow (ND) and a high fat diet (HFD), induced by

intracerebroventricularly administered leptin (n=5 ND, n=4 HFD) (7 μg/5 μl), insulin (n=6

ND, n=5 HFD) (500 mU/5μl) and the combination of leptin and insulin (n=4 ND, n=5 HFD).

*P<0.0001 compared to ND.

144

7.4 DISCUSSION

The main finding of this study is that the ICV administration of leptin and insulin combined

in rats fed a HFD significantly decreased RSNA in comparison to either hormone alone or

saline. MAP, however, was significantly increased compared to either hormone alone.

In HFD, leptin alone induced an increase in RSNA, this was reported in our previous work in

chapter 5 and also by others (17, 18, 58, 79, 87, 275). Similarly, insulin alone also induced

increases in RSNA and is in agreement with work in mice (53). In ND, similarly, leptin or

insulin alone induced increases in RSNA. This is in agreement with others (53, 54, 73, 99,

100, 102, 110, 256, 274).

Leptin or insulin alone acts centrally to increase RSNA through a similar mechanism

involving activation of melanocortin 4 receptors (MC4R) in the brain (110). Therefore, one

could hypothesise an interaction where insulin and leptin combined induced a greater

sympatho-excitatory action on RSNA. Indeed in ND, we found previously in chapter 4, that

leptin and insulin combined induced greater increases in RSNA compared to either hormone

alone (274).

Surprisingly, in the present study in HFD rats, leptin and insulin in combination did not

induce a sympatho-excitatory effect on RSNA. Therefore, HFD can influence the RSNA

response to leptin and insulin combined such that, a sympatho-excitatory response (seen in

ND) is converted to a sympatho-inhibitory response by HFD.

In contrast to the effects on RSNA, in rats fed a HFD, blood pressure was elevated slightly

but significantly following the combination of leptin and insulin. This suggests that leptin and

insulin may be elevating blood pressure through outputs/mechanisms other than RSNA.

145

This present study found that acute central administration of insulin and leptin combined

increased HR. This response was similar to ND groups, indicating that the HR response was

not altered by HFD, in contrast to RSNA. Thus, the present findings suggest that HFD can

selectively influence different outputs following the combination of leptin and insulin. A

possible explanation could be that HFD is having an effect on baroreflex sensitivity of RSNA

during the MAP increases, resulting in inhibiting RSNA. However, It has been shown that

HFD can decrease the sensitivity of the RSNA baroreflex response (279), suggesting that this

is an unlikely explanation for our observation. Thus, the exact mechanisms mediating this

interesting observation, however, are unknown, and further investigation is needed.

In conclusion, the main finding of the present study is that the combination of both insulin

and leptin significantly decreased RSNA when administered centrally in rats fed a HFD.

146

8 Chapter 8: General discussion and conclusion

In this thesis, we investigated the effects of central administration of leptin, resistin and

insulin alone, and in combination on renal sympathetic nerve activity (RSNA), mean arterial

pressure (MAP) and heart rate (HR). The findings of this thesis show, for the first time, that

resistin acts centrally to influence the RSNA in rats fed a high fat diet (HFD), and this effect

was similar to the response in rats fed a normal chow diet (ND). In addition, I showed that

leptin acts centrally to increase RSNA in ND and HFD, confirming previous work in the

literature (17, 18, 58, 79, 87). It was also shown that in rats fed a ND or HFD, leptin

combined with resistin induced greater increases in RSNA compared to each hormone alone.

In this thesis, I found for the first time in rats fed a HFD that central administration of insulin

increased RSNA significantly, but this effect was less than in rats fed a ND. Furthermore,

leptin and insulin combined in rats fed a ND induced greater increases in RSNA compared to

either hormone alone. In contrast, in rats fed a HFD leptin and insulin combined reduced

RSNA.

Finally, I also investigated the effect of resistin and insulin combined. In ND rats there was

no increase in RSNA, but in HFD fed rats RSNA fell significantly. In table 8.1, I summarise

the main findings of this present work, and it shows that the knowledge gaps highlighted in

the aims in table 1.1 have now been filled.

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Table 8.1 Summary of the effects of leptin, insulin and resistin on RSNA in rats fed a normal

diet or a high fat diet.

Hormones Normal Diet - RSNA High Fat Diet - RSNA

Leptin Insulin ↑ Resistin Leptin + resistin Leptin + insulin Resistin + insulin ▬

↑smaller increase; increase; greater increase; ▬ no change; greater decrease.

Prior to the current work, no previous studies investigated the cardiovascular effects of

resistin in HFD conditions. In this thesis, it was found that resistin increased RSNA in HFD.

This effect was similar to ND, suggesting that the effects of resistin on RSNA are not

attenuated by HFD. Similarly, the RSNA response to leptin was not attenuated in ND and

HFD.

In rats fed a ND, the combination of leptin and resistin induced a greater sympatho-excitatory

on RSNA response when compared to each hormone alone. This response was also observed

in rats fed a HFD. This key finding suggests that HFD does not influence the sensitivity of

the responses to leptin and resistin combined. Thus, in conditions such as overweight/obesity,

where these hormones are elevated, the sympatho-excitatory responses of the interaction of

leptin and resistin may be important and may contribute to the cardiovascular complications

observed in those conditions.

In this study, for the first time, it was shown that insulin acts centrally to increase RSNA in

rats fed a HFD. However, this effect was less than that seen in ND. Therefore, our result

suggests that the HFD attenuates the RSNA response. This finding is in agreement with the

148

recent work from Rahmouni’s laboratory in mice (53), but both studies contrast with earlier

work on mice that showed no attenuation by HFD (110).

Insulin and leptin alone increased RSNA. Since both hormones share a common pathway to

elicit this response (110), it is possible that an interaction between these hormones may occur,

as seen with the combination of leptin and resistin. Furthermore, since HFD attenuated the

RSNA response following insulin, it was of interest to see whether HFD affected the RSNA

response when insulin and leptin were combined.

I found in rats fed a ND, that the central administration of leptin and insulin combined

increased RSNA compared to leptin or insulin alone. This was similar to the interaction seen

with leptin and resistin combined in ND. In both cases, the responses were at least additive.

However, in HFD rather than an increase in RSNA, there was a reduction following the

combination of leptin and insulin. This suggests that HFD in some way converts a sympatho-

excitatory effect to a sympatho-inhibitory response.

Similarly, the combination of resistin and insulin also reduced RSNA in rats fed a HFD, but

in ND fed rats, there was no change in RSNA following resistin and insulin combined. This

suggests that insulin and resistin combined mutually inhibit their sympatho-excitatory

actions, and HFD in some way enables the expression of a sympatho-inhibitory response. The

implication of these complex interactions are discussed further below.

In this thesis, the changes in MAP and HR did not correlate with the clear changes in RSNA

observed. This may be due to the fact that this work investigated acute responses of these

hormones, whilst chronic studies suggest more consistent changes in blood pressure and HR

particularly with leptin (103, 104, 193, 195).

149

Perspective

Plasma levels of leptin, resistin and insulin are increased in overweight/obese conditions (79,

85-87, 127, 128, 161, 162). These hormones can increase RSNA (51, 53, 54, 58, 99, 100,

110). In overweight/obese conditions, RSNA is known to be increased abnormally (6, 7, 11,

23, 35). In the present work leptin and resistin combined increased RSNA in HFD, whereas

the combination of leptin or resistin with insulin significantly reduced RSNA in HFD fed

rats. Therefore, our results seem to suggest that HFD affects the sensitivity of the interaction

of leptin or resistin with insulin on RSNA. Since RSNA is abnormally elevated in obesity our

present results suggest that the sympatho-excitatory effects of leptin and resistin combined

dominate over any sympatho-inhibitory actions induced by leptin or resistin combined with

insulin. Future experiments in which the central effects of leptin, resistin and insulin

combined together could be useful in clarifying this suggestion.

Limitations and Future directions

One of the main confounding factors that may implicate the results of the present work is the

use of anesthetics. Anesthetics may affect the regulation of blood pressure and the autonomic

nervous system. Thus, performing these experiments in conscious animals may be

advantageous. Another limitation is the use of short-term acute administration of the

hormones in the present study. Although, we did see changes in RSNA with the use of short-

term acute administration, the use of chronic long-term infusion of the hormones would be an

excellent extension of the present work that could provide valuable insight into the chronic

regulation of RSNA and MAP.

Additionally, we have injected the hormones directly into the CSF, and this method is

important to investigate the central action of the hormones. It is important to note that these

hormones also circulate in the blood and their access to the brain may differ compared to ICV

150

injection. Thus, futures studies could be directed to comparisons of intravenous (IV) vs ICV

administration.

Finally, we discovered novel interactions between (leptin + insulin), (resistin + insulin) and

(leptin + resistin). Therefore, future studies investigating the effects of these three hormones

together could be valuable. Extending the sympathetic nerve activity outputs to include

nerves to other organs may also provide insight into whether the observations we made with

RSNA are selective or more generalized to other outputs.

151

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APPENDIX I: PERMISSION FOR USING PUBLISHED

Statements of thesis/publication contribution

Do you give me the permission to add these data and paper to my thesis? Habeeballah, H., N. Alsuhaymi, M. J. Stebbing, T. A. Jenkins and E. Badoer (2016). "Central leptin and resistin combined

elicit enhanced central effects on renal sympathetic nerve activity." Exp Physiol 101(7): 791-800.

Hamza Habeeballah Contribution

• Performs the experiments • Analysis the data • Produce the figures • Contribute to the manuscript

*NA was contributed equally in this paper

Habeeballah, H., N. Alsuhaymi, M. J. Stebbing, T. A. Jenkins and E. Badoer (2016). "Central Administration of Insulin and

Leptin Together Enhance Renal Sympathetic Nerve Activity and Fos Production in the Arcuate Nucleus." Front Physiol 7:

672.


• Performs the experiments • Analysis all the data • Produce all the figures • Contribute to the manuscript

Habeeballah, H., N. Alsuhaymi, M. J. Stebbing and E. Badoer (2017). "Effects of central administration of resistin on renal

sympathetic nerve activity in rats fed a high fat diet; a comparison with leptin." J Neuroendocrinol.


• Performs all experiments • Analysis all data • Produce all figures • Contribute to the manuscript

Habeeballah, H., N. Alsuhaymi, M. J. Stebbing and E. Badoer. "Central Administration Of Insulin Combined With Resistin

Reduces Renal Sympathetic Nerve Activity In Rats Fed A High Fat Diet." (submitted)


• Performs all experiments • Analysis all data • Produce all figures • Contribute to the manuscript

Mr.Naif Alsuhaymi Dr.Trisha Jenkins Dr.Martin Stebbing

Date/ Signature Date/ Signature Date/ Signature

Senior Supervisor

Professor. Emilio Badoer

Date/ Signature

170

APPENDIX II: LETTER OF ETHICS APPROVAL

171

Documents

CENTRAL REGULATION OF SYMPATHETIC NERVE ACTIVITY BY ...researchbank.rmit.edu.au/eserv/rmit:162285/Habeeballah.pdf · Hamza Habeeb Habeeballah Master of Laboratory Medicine School