The effects of Caralluma Fimbriata on the appetite behaviour and associated neural pathways in ...

315 Pages·2016·10.04 MB·English

by  Joanne Griggs

THE EFFECTS OF CARALLUMA FIMBRIATA

ON APPETITE BEHAVIOUR

AND ASSOCIATED NEURAL PATHWAYS

IN

PRADER-WILLI SYNDROME

By

Joanne Louise Griggs, B. Sc. (Hons.), Dip B. Mgmt., Dip ABS.

Submitted in the month of June 2016
In total fulfilment of the requirements of the degree of

Doctor of Philosophy

Principal Supervisor: Associate Professor Michael Mathai
Co-supervisor: Dr Puspha Sinnayah
College of Health and Biomedicine, Centre for Chronic Disease Prevention and Management,
Victoria University, Melbourne, Victoria 8001, Australia
The effects of Caralluma fimbriata on the appetite behaviour and associated neural pathways in PWS

To Adam

For all the moments you supported me during this long road,
Thank you! I love you, your vision, your passion,
Your awe inspiring curiosity and awareness.
You help me believe anything is possible xxx.

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The effects of Caralluma fimbriata on the appetite behaviour and associated neural pathways in PWS

Contents

I. Doctor of Philosophy Declaration ------------------------------------------------------ 9
II. Acknowledgments ------------------------------------------------------------------------- 10
III. Publications and Presentations --------------------------------------------------------- 11
IV. Prologue ------------------------------------------------------------------------------------- 12
V. Abstract -------------------------------------------------------------------------------------- 14
CHAPTER ONE------------------------------------------------------------------------------------ 19
1. Introduction ----------------------------------------------------------------------------------- 19
1.1 Prader-Willi syndrome and treatment Caralluma fimbriata. ------------------------ 19
I.2 Methodology of Study One, Two and Three --------------------------------------------- 28
CHAPTER TWO ----------------------------------------------------------------------------------- 31
2. Literature reviews ---------------------------------------------------------------------------- 31
2.1 Prader-Willi syndrome--------------------------------------------------------------------- 31
2.2 Progressive hyperphagia and behaviours in Prader-Willi syndrome --------------- 38
2.3 Aetiology in Prader-Willi syndrome ----------------------------------------------------- 45
2.4 Treatments in Prader-Willi syndrome --------------------------------------------------- 59
2.5 Caralluma Fimbriata ---------------------------------------------------------------------- 74
2.6 Key points from literature review -------------------------------------------------------- 87
2.7 Significance ---------------------------------------------------------------------------------- 89
CHAPTER THREE -------------------------------------------------------------------------------- 91
3. Study one -------------------------------------------------------------------------------------- 91
3.1 Abstract -------------------------------------------------------------------------------------- 91
3.2 Methodology --------------------------------------------------------------------------------- 93
3.2.1 Summary of experimental protocol ---------------------------------------------------- 93
3.2.2 Participants, recruitment and inclusion. ---------------------------------------------- 94
3.2.3 Experimental protocol ------------------------------------------------------------------ 100
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The effects of Caralluma fimbriata on the appetite behaviour and associated neural pathways in PWS
3.2.4 Treatment protocol ---------------------------------------------------------------------- 101
3.2.5 Measurement ----------------------------------------------------------------------------- 104
3.3 Results --------------------------------------------------------------------------------------- 109
3.4 Discussion ---------------------------------------------------------------------------------- 115
3.5 Conclusion ---------------------------------------------------------------------------------- 124
3.6 Conflict of interest ------------------------------------------------------------------------- 125
3.7 Acknowledgments -------------------------------------------------------------------------- 125
CHAPTER FOUR --------------------------------------------------------------------------------- 127
4. Introduction ---------------------------------------------------------------------------------- 127
4.1 The animal studies ------------------------------------------------------------------------- 127
4.2. The Snord116 mouse model ------------------------------------------------------------- 129
4.3 Modulation of appetite -------------------------------------------------------------------- 132
4.4 Activity within the central nervous system in animals. ------------------------------- 138
4.5 Aim of the animal studies. ---------------------------------------------------------------- 150
CHAPTER FIVE ---------------------------------------------------------------------------------- 153
5. Study two ------------------------------------------------------------------------------------- 153
5.1 Abstract ------------------------------------------------------------------------------------- 153
5.2 Introduction -------------------------------------------------------------------------------- 157
5.3. Study two protocols ----------------------------------------------------------------------- 158
5.4 Material and Methods -------------------------------------------------------------------- 160
5.4.1 Animal management -------------------------------------------------------------------- 160
5.4.2 Animals study procedures -------------------------------------------------------------- 163
5.4.3 Jelly Dosing ------------------------------------------------------------------------------ 164
5.4.4 Faecal deposits -------------------------------------------------------------------------- 168
5.4.5 Appetite signalling ---------------------------------------------------------------------- 170
5.4.6 Measurements---------------------------------------------------------------------------- 173
5.4.7 Statistical analysis ---------------------------------------------------------------------- 175
5.5 Results --------------------------------------------------------------------------------------- 178
5.6 Discussion ---------------------------------------------------------------------------------- 195
5.7 Conclusion ---------------------------------------------------------------------------------- 202
CHAPTER SIX ------------------------------------------------------------------------------------ 205
6. Study three ----------------------------------------------------------------------------------- 205
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The effects of Caralluma fimbriata on the appetite behaviour and associated neural pathways in PWS
6.1 Abstract ------------------------------------------------------------------------------------- 205
6.2 Introduction -------------------------------------------------------------------------------- 207
6.3 Methods and materials -------------------------------------------------------------------- 211
6.3.1 Preparation ------------------------------------------------------------------------------ 211
6.3.2 Perfusions -------------------------------------------------------------------------------- 213
6.3.3 Immunohistochemistry ------------------------------------------------------------------ 214
6.3.4 Statistical analysis ---------------------------------------------------------------------- 219
6.4 Results: Immunohistochemistry --------------------------------------------------------- 220
6.5 Discussion ---------------------------------------------------------------------------------- 245
6.6 Limitations ---------------------------------------------------------------------------------- 246
6.7 Further study ------------------------------------------------------------------------------- 247
6.8 Conclusion ---------------------------------------------------------------------------------- 249
CHAPTER SEVEN ------------------------------------------------------------------------------- 251
7. 1 Concluding remarks ---------------------------------------------------------------------- 251
7.2 A special mention. ------------------------------------------------------------------------- 252
References --------------------------------------------------------------------------------------- 253
Appendixes: ---------------------------------------------------------------------------------------- 282
Appendix A. Inclusion criteria --------------------------------------------------------------- 282
Appendix B. Exclusion criteria --------------------------------------------------------------- 284
Appendix C. Hyperphagia questionnaire --------------------------------------------------- 285
Appendix D. Investigator Training ---------------------------------------------------------- 287
Appendix E. Rodent Health Screen Panel -------------------------------------------------- 288
Appendix F. Generational Mating and Birth Charts -------------------------------------- 289
Appendix G. Jelly Volume, Dose, Equipment and Recipe. ------------------------------- 290
Appendix H a. Timeline of Animal Experiment -------------------------------------------- 293
Appendix H b. Time table Colour Code ----------------------------------------------------- 294
Appendix I. Animal Characteristics --------------------------------------------------------- 295
Appendix J a. Mean and Standard deviation study two ---------------------------------- 302
Appendix J b. Appetite and thirst stimulant and deprivation t-tests -------------------- 303
Appendix K. Perfusions ----------------------------------------------------------------------- 309
Appendix L. Neuronal cell counts mean and SD. ------------------------------------------ 311
Appendix M. Mia’s story and history ------------------------------------------------------- 312
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The effects of Caralluma fimbriata on the appetite behaviour and associated neural pathways in PWS

List of tables
Table 1 Current Genomic Definitions of Prader-Willi syndrome. ........................................32
Table 2 Aetiology in PWS .....................................................................................................50
Table 3. Treatments in PWS cohort of 1-4 ............................................................................68
Table 4 Treatments in PWS cohorts of 5 and above .............................................................70
Table 5 Questionnaire Allocation ........................................................................................106
Table 6 Anthropometric Characteristics ..............................................................................110
Table 7 Results Study One .................................................................................................113
Table 8 Dose of Stimulants .................................................................................................172
Table 9 Study Two Animal Groups .....................................................................................174
Table 10: Results for the Eight-Week Chronic Treatment Trial .........................................178
Table 11 Body Weight Results ............................................................................................180
Table 12 Appetite Stimulation Study Two ..........................................................................184
Table 13 Comparisons of Stimulants ...................................................................................185
Table 14 Between Treatment and Strain Effects of Appetite Stimulation ..........................189
Table 15 Results for Faecal Sample Testing in Animals .....................................................194
Table 16 Stimulant Groups for the Immunohistochemistry Study Three ...........................212
Table 17 Nikon A1 Desktop Confocal Settings ..................................................................216
Table 18 Group Comparisons during Stimulation in Arcuate Nucleus ..............................222
Table 19 Paraventricular Cell Count Comparisons .............................................................233
Table 20 Comparisons of Strain and Treatment in the Median Preoptic ............................239
Table 21 Inclusion Criteria Study One ................................................................................282
Table 22 Exclusion Criteria for Study One .........................................................................284
Table 23 Training ................................................................................................................285
Table 24 Health Screen Panel ..............................................................................................287
Table 25 First Generation Snord116del and WT mice ........................................................288
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The effects of Caralluma fimbriata on the appetite behaviour and associated neural pathways in PWS
Table 26 Treatment Dose Calculations ...............................................................................289
Table 27 Volume Per Animal ..............................................................................................290
Table 28 Animal Group Time Table ...................................................................................293
Table 29 Mean and Standard Deviation Appetite Signalling ..............................................302
Table 30 Mean and Standard Deviation Water Appetite .....................................................303
Table 31 Saline Food and Fluid ...........................................................................................304
Table 32 2-Deoxy-Glucose Four-Hour Food and Fluid Study ............................................305
Table 33 Beta-Mercaptoacetate Four Hour Food and Fluid study ......................................306
Table 34 5-HT2C Antagonist Four-Hour Food and Fluid study .........................................307
Table 35 Four day 50% Deprivation study .........................................................................308
Table 36 Immunohistochemistry Cell Counts ....................................................................311

List of figures
Figure 1. PWS Critical Region……………………………………………………………..33
Figure 2. Eating Phenotype: Phases in PWS……………………………………………….39
Figure 3 Appetite Interactions…………………………………………………………….. 48
Figure 4 Caralluma fimbriata Molecular Structure………………………………………...76
Figure 5. The PWS Phenotype ………………………………………………..……………78
Figure 6. Hodia Gordonii Active Molecule…………………...………………………….. 93
Figure 7. Study One Protocol …………………….………………………………………. 98
Figure 8 Individual Outcomes Study One…………….……………………….…….….…114
Figure 9 Variations in Size of Mouse Models…………………………………………….132
Figure 10 Adiposities’ Role in Appetite………………………………………………..…133
Figure 11 Hypothalamic Signalling Activity…………………………………………..… 136
Figure 12 Glucose and glucose Inhibition ……………………………………………..…140
Figure 13 Blood Glucose Serum Pathway………………………………………...………142
Figure 14 Serotonin and a 5-HT2C Antagonist……………………………..…....….…….146
Figure 15 selective Serotonin Reuptake Inhibiter…………………………..…..………....148
Figure 16 CFE’s Proposed Pathway of Activity …………………………...…………......149
Figure 17 Jelly Dosing…………………………………………………..……………….. 165
Figure 18 Appetite Stimulant Study Two Histograms………………………..…...........…186
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The effects of Caralluma fimbriata on the appetite behaviour and associated neural pathways in PWS
Figure 19 Water Appetite During the Stimulation Stud………………………….……….190
Figure 20Food and Water after Four Days of 50% Food Deprivation……………………192
Figure 21 Areas of Interest in The Hypothalamus……………………………….………. 218
Figure 22 Comparisons of Activity in the Arcuate Nucleus………………..……………..223
Figure 23 Immunohistochemistry c-Fos Labelling……….………………….…………….224
Figure 24 Immunohistochemistry Cell Counting….……….……………………………... 225
Figure 25 Cell c-Fos Activity in The Hypothalamus……..………..….……………….…...226
Figure 26 Neuro Transmitter Co-Localization ………...…………..…….……………...….227
Figure 27 Secondary Antibody Co-Localization……...………...…………………….……228
Figure 28 Mouse Model c-Fos Activity …………….……….……………….…………….229
Figure 29 Neurotransmitter Cell Activity in The Hypothalamus……..…………...……….230
Figure 30 Staining of Fibrous Tissue………………………................………….…………231
Figure 31 Group comparisons of Arcuate Nucleus c-Fos Activity...……...……….…….…232
Figure 32 Paraventricular Comparisons of treatment and Strains ……………..…….…….234
Figure 33 Group Cell Activity in the Paraventricular…………………………..………….235
Figure 34 C-Fos Activity Paraventricular Comparison……………..…….……………….236
Figure 35 PVN, C-Fos, NPY and α-MSH Activity Labelling……..…….……….………...237
Figure 36 Median Preoptic Comparisons of Animal Groups Cell Co-Localization….....….240
Figure 37 median preoptic c-Fos and Neuropeptide –Y in the Snord 116 Strain …..….…..241
Figure 38 median preoptic c-Fos and Neuropeptide –Y in the Wild Type ………..…..…...242
Figure 39 Close ups of c-Fos and Fibrous Non-Labelling……………………….…………243
Figure 40 Paraventricular & Median preoptic co-labelling MA Stimulant………...…........244
Figure 41 Hyperphagia Questionnaire………...……………………………………………285

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The effects of Caralluma fimbriata on the appetite behaviour and associated neural pathways in PWS

I. Doctor of Philosophy Declaration

“I Joanne L. Griggs, declare that the PhD thesis entitled the effects of Caralluma fimbriata
on Appetite Behaviour and associated Neural Pathways in Prader-Willi syndrome, is no
more than 100,000 words in length including quotes and exclusive of tables, figures,
appendices, bibliography, references and footnotes. This thesis contains no material that has
been submitted previously, in whole or in part, for the award of another degree or diploma.
Except where otherwise indicated, this thesis is my own work”.

Joanne Griggs Date 03/06/2016

College of Health and Biomedicine, Centre for Chronic Disease Prevention and
Management, Victoria University, Melbourne, Victoria 8001, Australia
Student number: s3878226
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The effects of Caralluma fimbriata on the appetite behaviour and associated neural pathways in PWS
II. Acknowledgments
Thank you, to my daughter Mia for your living example of persistence without complaint.
You are teaching us all so much. To my husband Adam you are the other half of this work.
I’m so grateful for your efforts, your support and your daily reminders of magic. Ruben your
compassion, humour and encouragement are so precious to us all. Your smile, and creative
talent always balances our journey towards the vision of full potential for all.
Thank you too, to our family and our extended community of friends and supporters. To
Associate Professor Michael Mathai, principal researcher, for giving me the opportunity and
your life altering questions. Dr Puspha Sinnayah, for your side by side supervision,
friendship and expertise and Dr.Xiao Su - my associate supervisor from the honours study -
thank you for your quick, calm responses. A very special thank you to the committed
participants and their parents and the Prader-Willi Association of Victoria, Australia and
New Zealand for their involvement in recruitment.
Thank you to Dick Denton and Michael McKinley for their initial support and belief in my
motherly wish; an important step towards becoming a researcher. Thank you to Maria Mouse
at the Florey Neuroscience Institute… what don’t you know about mice? Melinda, Robin,
Dave, Song and Tony, thanks for your generosity and “in the lab” example of scientific
enquiry. Angela your expert knowledge has added colour to my thesis. Thanks to my
compatriots and students at the National Ballet School…you certainly kept me fit. To my
mentor Dr. John Demartini; your example is forever uplifting. Mum and Dad, thank you,
who would have thought it… from ballet to the lab… it is always interesting! Lastly; I want
to thank the powers that be for giving me the chance to take this leap of faith… I will
continue to live in gratitude and service.
Funding Disclosure Statement
The Student Investigator received the following relevant financial backing:
Gencor Pacific Ltd. - Top up scholarship of $8,000 per year over the animal study period of
two years, plus funding of $34,000 towards the animal studies equipment and procedures.
The investigators are also grateful to Gencor Pacific Ltd. for the in kind gift of treatments
and placebo and the in kind support by Professor Herbert Herzog from the
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