EXPERIMENTAL STUDIES OF SUBDIAPHRAGMATIC VAGOTOMY AND NICOTINE FOR REDUCING BODY WEIGHT

A Dissertation submitted to the Faculty of the Graduate School of Arts and Sciences of Georgetown University in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Neuroscience

By

Ghazaul Dezfuli, B.A.

Washington, DC August 22, 2014

Copyright 2014 by Ghazaul Dezfuli All Rights Reserved

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EXPERIMENTAL STUDIES OF SUBDIAPHRAGMATIC VAGOTOMY AND NICOTINE FOR REDUCING BODY WEIGHT Ghazaul Dezfuli, B.A.

Thesis Advisor: Richard A. Gillis, Ph.D.

ABSTRACT

Melanocortin neural circuits are important regulators of food intake (FI) and body weight (BW). The hypothesis of the first part of my dissertation is that the dorsal motor nucleus of the vagus (DMV) represents an important output nucleus of central melanocortin signalling. This hypothesis was tested by examining the role of the DMV in the prevention and treatment of obesity caused by a knockout of the melanocortin 4- receptor (Mc4R). The role of the DMV was tested by performing a bilateral subdiaphragmatic vagotomy (SDV) which results in cell loss in the DMV.

In the first study, Mc4R -/- mice were used that had not reached full obesity, while in study 2, Mc4R -/- mice were used that were severely obese. In both studies, complete

SDV blunted the obesity associated with this genotype. The anti-obesity effect of SDV was not associated with a decrease in FI. Complete SDV reduced visceral mesenteric white adipose tissue in both studies. Additionally, Mc4R -/- mice after SDV had increased energy expenditure during the dark cycle and a decreased light cycle respiratory exchange ratio indicating an increase in the utilization of lipids as an energy source.

The second part of my dissertation focused on determining the effectiveness and mechanisms whereby nicotine and drugs that interact with nicotinic receptors (nAChRs) can influence BW in mice. The hypotheses from the second part of my dissertation are that nicotine’s effect on BW is mediated through the melanocortin system at the α4β2 nAChR, and involves inhibition of hypothalamic adenosine 5'-monophosphate (AMP)-

iii activated protein kinase (AMPK). To test these hypotheses, I used Mc4R -/- mice, drugs selective for the α4β2 nAChR, and measured AMPK activity. I found that (1) nicotine causes weight loss independent of the brain melanocortin system; (2) nicotine’s effect to decrease BW is in part due to interaction with the α4β2 nAChR; (3) nicotine’s interaction with the nAChR involves desensitization as opposed to activation; and (4) nicotine reduces BW independent of hypothalamic AMPK inhibition. In summary, my studies revealed that both SDV and nicotine have a robust effect to reduce BW.

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ACKNOWLEDGMENTS

First and foremost, I would like to thank my wonderful mentor Dr. Richard Gillis for whom none of this would be possible. Dr. Gillis has been a constant source of support for me both throughout my dissertation and in life. As a mentor, he has taught me to ask the right scientific questions, design the best possible studies, and be unafraid to question the findings of others. Dr. Gillis' passion for science always served as an inspiration to me. In addition, he always had my professional development in mind and always encouraged me to follow the path that he thought I would the best at. Also, I want to thank Dr. Gillis for the many hours of delightful conversation and the many laughs we had along the way (as we had many), as I will probably miss this the most. It is through

Dr. Gillis that I became excited about science and I am forever indebted to him for all that he has done for me.

I would also like to thank Dr. Niaz Sahibzada who in many ways is my second mentor and has been there for me every step of the way. Dr. Sahibzada, in addition to being on my thesis committee, taught me all the technical aspects of my research. Every time I was ready to panic, he was there to fix the problem. He also taught me how to properly write scientific manuscripts and now I strive to emulate his writing style. I also thank him for the many laughs and jokes we had along the way including the many wasps we counted together (only he will understand this).

I am also grateful for the support that I received from members of the Gillis lab

(past and present) including Janell Richardson Ph.D., who remains a good friend, Mark

Niedringhaus Ph.D., who taught me the ins and outs of surgeries, and Amanda Lewin who has been a great officemate. As well, I am grateful for the help I received by

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Monica Javidnia, Rashmi Venkatesh, Hannah Hathaway, and Yasmine Ibrahim for their help on protein assays and western blotting technique.

As well, I would like to thank the remaining members of my thesis committee,

Dr. Ken Dretchen, Dr. Joe Verbalis, Dr. Ken Kellar, and Dr. Tim Moran. Dr. Ken

Dretchen always supported our lab and met with us regularly to offer direction with my

research. Dr. Joe Verbalis would make eye-opening suggestions that I had previously

not thought of. Dr. Ken Kellar collaborated with us on the nicotine part of my

dissertation, and it is through him that I became interested in further studying nicotine

and nicotinic receptors. Lastly, I would like to thank Dr. Tim Moran for coming down to

Georgetown and serving as my external committee member.

Within the pharmacology department, Dr. Barry Wolfe and Dr. Bob Yasuda always represented to me a constant source of joy. Bob, I always enjoyed our hallway conversations and Barry, thank you for always being up for chatting and teaching me about astronomy. As well, I am thankful for their impromptu help with troubleshooting, sometimes even in the hallways. In addition, I would like to thank Dr. Yingxian Xiao for help with the studies using Sazetidine-A.

I would also like to thank the faculty, staff, and my classmates within the

Interdisciplinary Program in Neuroscience for allowing me to be part of such an enriching graduate program (in particular Dr. Karen Gale and Becky Hoxter).

To my family and friends, you all have been there for me. Special thanks to my

little sister Azal, my parents, my grandparents (especially Mommy), my friends from

Smith College (especially my closest friend Gwendolyn Rayner), and to the new

friendships I made while a graduate student (Monica Javidnia, Shai Chu, Bettina Hager,

Katherine Neuner, and Iain DeWitt).

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Lastly, I would like to thank my boyfriend Stefano whose love and support is unconditional even during all the times I was inattentive to him while writing this dissertation.

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DEDICATION

This dissertation is dedicated to Maman Joopie, my grandmother in Iran, for whom I will

always have the fondest of memories.

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TABLE OF CONTENTS

CHAPTER 1: Experimental Studies of Subdiaphragmatic Vagotomy (SDV) for

Reducing Body Weight 1

I: INTRODUCTION 1

A: The obesity epidemic and treatment 1

B: Rationale for Focusing on SDV as an Approach for Counteracting Obesity: Studies

Indicating that SDV is Effective in Reducing Body Weight and Lowering

Body Fat 4

1: Experimental Studies of the Effect of SDV on Obesity Produced by Hypothalamic

Lesions 4

2: Studies Using Other Obesity Models 10

3: Clinical Studies 12

C: Mechanisms Responsible for the Anti-Obesity Effect of SDV 13

1: Reduction in Food Intake 13

2: Reduction in Serum Insulin 14

3: Increase in Sympathetic Tone 16

4: Changes in Gut Microbiota 16

D: The Advantages and Disadvantages of SDV as a Weight Loss

Intervention 17

E: Strategies Employed in My Studies 18

F: Hypotheses To Be Tested 20

II: MATERIALS AND METHODS 21

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A: Animals 21

B: Surgical Procedure for Bilateral Subdiphragmatic Vagotomy

(SDV) 22

C: Surgical Procedure for Heineke-Mikulicz Pyloroplasty 23

D: Trichobezoars Removal 24

E: Postsurgical Care and Recovery 24

F: Feeding and Weight Measurements 24

G: Neuroanatomical Tract Tracing-Fluorgold for Histological Verification of Completeness of Bilateral Sub-Diaphragmatic Vagotomy 25

H: Fat Pad Excision 25

I: Snout-to-Anus Length 26

J: Metabolic Chamber Recordings of Indirect Calorimetry 26

K: Data Analysis 28

III: RESULTS 30

A: Subdiaphragmatic vagotomy prevents weight gain in Mc4r -/- mouse 30

B: Effect of subdiaphragmatic vagotomy on fat pad weight and on snout-to-anus length in Mc4r -/- 33

C: Histological evidence of complete subdiaphragmatic vagotomy 34

D: Subdiaphragmatic vagotomy treatment alleviates obesity in Mc4r -/- mice 35

E: Effect of subdiaphragmatic vagotomy on parameters of EE in Mc4r -/- mice 38

IV: DISCUSSION 40

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A: Magnitude of Reduction in Body Weight Produced by

Subdiaphragmatic Vagotomy (SDV) 40

B: Mechanisms for SDV-induced reduction in BW 42

C: Efficacy of SDV in Obesity States Other than Mc4R-/- mice, and VMH lesioned rats 45

D: Criterion of using SDV to assess the role of efferent vagus in controlling

BW 45

E: DMV represents one of the key outputs of the central melanocortin neural circuits 47

F: Outcome of Hypotheses Testing, Conclusions, and Future Studies 50

CHAPTER 2: Experimental Studies of Nicotine for Reducing Body Weight 52

I: INTRODUCTION 52

A: Cigarette Smoking and Body Weight 52

B: Review of Studies of Nicotine on Body Weight 53

1: Studies in Rats 54

2: Studies in Mice 60

C: Nicotine Can Intervene at Different Component of the Energy Balance

System to Reduce Body Weight 64

1: Food Intake 64

2: Energy Expenditure 65

D: Mechanisms of Nicotine Mediated Changes in Food Intake and Energy

Expenditure 66

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1: Nicotinic Acetycholine Receptor (nAChR) Subtype 66

2: Role of nAChR Desensitization 69

3: 5’AMP-Activated Protein Kinase (AMPK) and the Melanocortin

System 71

4: Interactions between Homeostatic and Reward Systems 75

E: Hypotheses to be Tested 75

II: MATERIALS AND METHODS 77

A: Animals 77

B: Drugs 78

C: Chronic Nicotine via Daily Intraperitoneal Injection (IP) 78

D: Osmotic Pump Priming for Chronic Delivery of Nicotine and

Sazetidine A (SazA) 79

E: Osmotic Pump Implantation for Chronic Delivery of Nicotine and

Sazetidine A (SazA) 79

F: Chronic Nicotine via Twice Daily Subcutaneous Injection (SC) 80

G: Feeding and Weight Measurements 80

H: Data Analysis 81

I: Micro-Dissection of Hypothalamus and Brainstem Tissue 81

J: Preparation of Membrane Homogenates for Western Blot 82

K: Western Blot Analysis 82

L: Quantification of Western Blot 83

M: Statistical Analyses (for Western Blot) 83

III: RESULTS 84

A: Effects of nicotine administered intraperitoneally (IP) daily to juvenile

xii and adult C57BL6/J and Mc4r (-/-) mice on body weight 84

B: Effect of nicotine infusion by osmotic mini-pump on body weight and food intake of obese Mc4r (-/-) mice: 86

C: Effect of SazA infusion by osmotic mini-pump on body weight and food intake of MC4R(-/-) mice 89

D: Effect of nicotine administered subcutaneous (SC) on body weight, food intake, and AMPK in diet-induced obese mice 91

E: Preliminary Studies of Mice with Knockout of the β2 nAChR subunit 95

IV: DISCUSSION 96

A: Magnitude of reduction in body weight produced by chronic

administration of nicotine using 3 different study designs 96

B: Mechanism for nicotine-induced reduction in the role of body weight gain 97

C: Role of the melanocortin brain circuit in nicotine-induced reduction in the rate of body weight gain 100

D: Subtype of nAChR involved in nicotine-induced reduction in the rate of body weight gain 101

E: Role of desensitization of the nAChR in nicotine-induced reduction in the rate of body weight gain 104

F: Role of AMPK in nicotine-induced decrease in body weight 106

G: The role of endogenously active nAChR in controlling BW 108

H: Outcome of Hypotheses Testing, Conclusions, and Future Studies 109

V: REFERENCES 112

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List of Figures

Chapter 1: Experimental Studies of Subdiaphragmatic Vagotomy (SDV) for Reducing

Body Weight

Figure 1: Weight gain is prevented by subdiaphragmatic vagotomy in not yet fully obese

Mc4r -/-mice 31

Figure 2: Bar graph showing the effect of bilateral subdiaphragmatic vagotomy and pyloroplasty on snout-to-anus length in not yet fully obese Mc4r -/-mice 32

Figure 3: Complete bilateral subdiaphragmatic vagotomy in not yet fully obese Mc4r -/- mice prevents retrograde label in the DMV 34

Figure 4: Subdiaphragmatic vagotomy treatment reduces body weight in severely obese

Mc4r -/- mice. 36

Figure 5: Bilateral subdiaphragmatic vagotomy influences EE in fully obese

Mc4r -/- mice. 37

Figure 6: A simple schematic illustrating how vago-vagal circuitry controlling gastric motility and tone maybe regulated by the hypothalamic-POMC-NTS pathway 49

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Chapter 2: Experimental Studies of Nicotine for Reducing Body Weight

Figure 1: Graph showing the effect of chronically administered nicotine (0.5, 1.0 and 3.0 mg/kg) on % survival rate at the end of a 30-day period. 85

Figure 2: Effect of chronic nicotine treatment on body weight in male Mc4R-/- and

C57BL/6J wildtype mice 87

Figure 3: Effect of chronic SazetidineA (SazA) treatment on body weight in male Mc4R-

/- and C57BL/6J wildtype mice. 89

Figure 4: Effect of chronic Saz-A treatment on FI in male Mc4R-/- and C57BL/6J

wildtype mice 91

Figure 5: Effect of nicotine (0.75mg/kg), Saz-A (6mg/kg) and DHβE (3mg/kg) on net body weight in diet-induced obese male mice 92

Figure 6: Western-blot images showing the effect of 0.75 mg/kg nicotine (NIC) and saline (VEH) administration twice daily on p-AMPKα in NTS-enriched brainstem and whole hypothalamus. 93

Figure 7: Graphs comparing differences in weight gain over an 18-day period in 8-wk

and 15-wk nicotinic receptor β2 knockout mice and age-matched WT controls. 95

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List of Tables

Chapter 1: Experimental Studies of Subdiaphragmatic Vagotomy (SDV) for Reducing

Body Weight

Table 1: Studies employing methods to assess completeness of SDV and its effect on obesity 11

Chapter 2: Experimental Studies of Nicotine for Reducing Body Weight

Table 2: Studies highlighting the role of nAChR in food intake and body weight 69

Table 3: Evidence for Involvement of Feeding-Associated Peptides in Nicotine-Mediated

Effects 73

Table 4: Effectiveness expressed as a percentage and determined by how much drug treatment decreased the weight gain observed in saline treated controls 98

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Chapter 1: Experimental Studies of Subdiphragmatic Vagotomy (SDV) for Reducing Body

Weight

I: INTRODUCTION

A: The Obesity Epidemic and Treatment

Obesity is a major public health problem in America and worldwide that has been steadily growing for the last few decades (Ogden et al., 2012, Ogden et al., 2002, Flegal et al.,

2002). In a study published by the Center for Disease Control (2012), more than one-third of

American adults were reported to be obese including 17% of the youth population. In addition, obesity-related co-morbidities are numerous and have been shown to involve virtually every body system: cardiovascular, pulmonary, hematopoietic, endocrine, gastrointestinal, genitourinary, gynecologic, musculoskeletal and urologic (Schauer & Chand, 2006). Weight loss in obese patients is used as a criterion to reduce these co-morbidities with a therapeutic intervention. The metabolic consequences of obesity are drivers of other life-threatening disorders including dyslipidemia, hypertension, atherogenesis and Type 2 diabetes (Heal et al.,

2009). Several studies have demonstrated the positive effects of weight loss on long-term mortality and morbidity in obese subjects. Christou et al. (2004) report that after bariatric surgery, (where an excess weight loss 67% after 5 years was produced), cardiovascular co- morbidity rates in 1,035 morbidly obese patients was significantly lower than those for the non- surgical group morbidities.

Bariatric surgery, while effective in producing weight loss, is a drastic step to take to resolve the obesity epidemic. Clearly, less invasive and risky measures are needed.

Pharmacotherapy has been used over the years but has been problematic because of questionable efficacy and limiting adverse effects. The poor track record of pharmacotherapy is a reflection of the fact that mechanisms of obesity are not fully understood; hence, there is not a clearly defined

1 target for developing drugs to treat the problem. While it is known that obesity occurs when energy intake exceeds energy expenditure (EE), there are multiple etiologies for this imbalance such as genetics, diet-composition (ingestion of calorically dense foods), hence the rising prevalence of obesity cannot be addressed by focusing treatment on a single etiology.

In terms of pharmacologically treating obesity, up until 2012, only four new drugs have been registered over the last 15 years: dexfenfluramine (Redux), sibutramine (Meridia, Reductil), orlistat (Xenical) and rimonabant (Acomplia). Of these, due to its incidence of side effects, sibutramine has been withdrawn in Europe (European Medicines Agency, EMA, 2010) and the

USA (voluntarily by Abbott). In the Sibutramine Cardiovascular OUTcome (SCOUT) trial, this drug was associated with an increased risk of serious, non-fatal cardiovascular events, such as stroke or heart attack (James et al., 2010).

In 2012, two new drugs were proposed for therapy namely Qnexa (a formulation of topiramate and phentermine, now called Qysmia), and lorcaserin (a 5-HT2C serotonin receptor agonist). Their entry into the market suffered setbacks when the Food and Drug Administration

(FDA) appointed expert advisory panel recommended that the products should not be approved for clinical use in the USA. Despite the fact that both drugs met the FDA guidance benchmark for weight loss (i.e., 5%; Poirier et al., 2006; Knowler et al., 2002), nonetheless it occurred over long periods that fell below the expectations of clinicians and consumers (Heal et al., 2012;

Gadde & Allison, 2009; Smith et al., 2010). More importantly, both drugs presented safety concerns, including adverse cardiovascular (CV) effects that have been the bane of establishing successful weight loss pharmacotherapy (Walter et al., 2014). Part of the reasoning behind the

FDA benchmark rule of 5% weight loss is based on data demonstrating that this percentage is associated with favorable changes in biomarkers of CV risk such as decrease in HDL-C, reduction in blood pressure, and improvement in measures of glycemia (Poirier et al., 2006;

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Knowler et al., 2002). However, this has only been adequately tested with one weight loss-drug, sibutramine. This drug, as mentioned before, increased the risk for major adverse cardiac events

(MACE) in a population of older overweight and obese at-risk individuals (James et al., 2010).

In Europe, with respect to Qnexa, the EMA's Committee for Medicinal Products for Human Use

(CHMP) towards the end of 2012 recommended that authorization to market the drug should be withheld. The CHMP noted that despite the fact that Qnexa had shown clinically relevant weight loss (albeit modest), the committee had concerns about the drug’s long-term effects on the heart and blood vessels. Of particular concern was phentermine, which is known to increase the heart rate (Kaplan 2010; Bray 2008; Greenway & Caruso 2005), and whose long-term cardiovascular effects are not clear. They noted in their report that the modest weight benefits of Qnexa did not outweigh its potential harmful cardiovascular effects.

In highlighting the need for more long-term CV outcome studies for anti-obesity pharmacotherapy, it was found that the incidence of cardiac valve disorders was higher with lorcaserin than with placebo in clinical trials (Prescrire Int. 2014). From an average initial weight of 100 kg, patients taking lorcaserin only lost about 3 kg more than those in the placebo groups.

This modest loss in weight compared to its potential for adverse CV effects was considered unjustified for its use as an anti-obesity drug (Prescrire Int. 2014). Other common adverse effects of lorcaserin observed in clinical trials were gastrointestinal (dry mouth, nausea) and neuropsychiatric (dizziness, fatigue, headache, euphoria).

Another example of a failed weight loss drug is the CB1 receptor antagonist, rimonabant that significantly reduced BW in clinical trials (Després et al., 2005; Van Gaal et al., 2005).

Rimonabant's potential for causing severe psychiatric adverse events (e.g., anxiety, depression and suicidal ideation; Topol et al., 2012) has prevented its approval in the US. Hence, due to the

3 poor track record of presently approved drugs for the treatment of obesity, there remains an enormous unmet need for the development of safer and more effective weight-loss measures.

The goal of this dissertation is to seek effective strategies (both surgical and pharmacological) to reduce BW. To this end, two approaches that I have taken to counteract obesity are: (a) subdiaphragmatic vagotomy (SDV), which represents a surgical intervention, and

(b) nicotine and drugs that act at nicotinic acetycholine receptors (nAchRs) as a pharmacological approach. SDV will be discussed in Chapter I of this dissertation, whereas nicotine and related drugs will be discussed in Chapter II.

B: Rationale for Focusing on SDV as an Approach for Counteracting Obesity: Studies indicating that SDV is Effective in Reducing Body Weight and Lowering Body Fat

1: Experimental Studies of the Effect of SDV on Obesity Produced by Hypothalamic

Lesions

High profile studies, beginning in the 1970s, attempted to delineate the role of the vagus nerve in obesity caused by lesions of the ventromedial hypothalamus (VMH) — once dubbed the brain’s “satiety center”. Inspired by the earlier studies of Ridley and Brooks (1965) showing that

VMH lesions not only caused hyperphagia (excessive eating) but also increased levels of fasting gastric acid secretion (mediated by the efferent vagus nerve), Powley and Opsahl (1974) were compelled to study the role of the vagus nerve in this phenomena. They found that SDV (a surgical procedure that severs both vagus nerves below the diaphragm) was found to abolish the resultant obesity due to bilateral electrolytic lesions of the VMH in adult male albino rats

(Powley & Opsahl, 1974). Specifically, SDV performed 40 days post lesion (to allow time for full obesity to develop) lowered the BW of VMH lesioned animals to non-lesioned vagotomized controls when fed a standard laboratory diet. In a follow up study (Inoue & Bray, 1976), SDV

4 performed 2 weeks after VMH lesions in rats (observed for 4 weeks) also completely reversed the VMH obesity syndrome.

To determine if the distribution of white adipose tissue (WAT) was affected by vagotomy in VMH lesioned animals, a controlled feeding strategy (intragastric pair-feeding regimen) was employed by Powly & Cox (Cox and Powley 1981). Compared to previously vagotomized VMH lesioned adult male rats, sham vagotomized VMH rats were shown to have excessive fat deposits. Moreover, SDV in lean rats precluded the typical accumulation of significantly increased levels of WAT in VMH lesioned animals. It is worth noting that in this study, despite the fact that both the lean vagotomized rats with VMH lesions and the obese vagotomized rats with VMH lesions (due to incomplete vagotomies) had equal BW, they differed in their body composition (BC). The leaner animals had normal proportions of fat and lean body mass, while the obese rats had significantly more fat than lean mass. However, the relevancy of BC as an endpoint in evaluating anti-obesity therapy is questionable as there is no indication that the FDA would approve a drug that is unable to reduce BW by at least 5 %, and solely impacted BC.

Merely, changing BC is most likely insufficient for meeting FDA standards.

In evaluating the contribution of the subdiaphragmatic vagus to VMH lesioned (knife-cut) induced obesity, Sawchenko & Gold (1981) performed selective vagotomies in adult male rats with pre-existing VMH lesions. The knife-cut induced obesity involves mechanically destroying the VMH using a knife or a fine blade using stereotaxic coordinates. They report that sectioning the gastric branches of the vagus alone (sparing the hepatic and coeliac branches) was not sufficient to reverse the VMH knife cut-induced obesity. Instead, all 5 abdominal branches of the vagus had to be severed (complete SDV) to reverse the pre-existing VMH lesion induced hyperphagia and obesity. This study demonstrates the importance of a complete SDV for exerting an anti-obesity effect.

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In a more in-depth study (Sawchenko et al., 1981) looking at the contributions of the individual branches of the vagus nerve in obesity, asymmetrical hypothalamic knife cuts in adult male rats that crossed the coronal plane of the paraventricular nucleus of the hypothalamus

(PVN) were used to produce obesity. In these animals that were fed a standard laboratory diet,

PVN lesions accompanied by selective section of the just the coeliac branch of the abdominal vagus nerve produced only 57% of the expected weight gain. Additional section of the gastric branches of the vagus further reduced the knife cut effects. However, it was only complete SDV that reduced BW to below control levels in the hypothalamic lesioned animals (Sawchenko et al.,

1981).

To determine the interaction between SDV and VMH lesions on obesity, King and colleagues (1978) studied obese adult female rats using two different strategies. First, they produced obesity with a VMH lesion and allowed the rats to stabilize. Then, they performed SDV and found that this procedure reduced BW to 85-90% of the body of sham SDV rats that had not been subjected to VMH lesion. Second, these investigators first performed SDV. Next, they subjected the rats to a VMH lesion. After the VMH lesion, the rats increased their food intake

(FI) but it never increased to the level observed in non-SDV rats that were subjected to a VMH lesion. King and colleagues concluded that while enhanced vagal motor activity contributed to

VMH lesion-induced weight gains, intact vagal motor nerves were not necessary for the expression of VMH lesion-induced obesity. However, the methods to test for completeness of

SDV in this study are open to question as King et al., used a test that would only indicate complete sectioning of gastric branches of the vagus.

The reversal of hypothalamic lesion induced obesity by SDV is diet dependent. Gold et al. (1980) found that SDV prevented the weight gain resulting from a parasaggital hypothalamic knife cut (intended to sever fibers going from the VMH to the lateral hypothalamus) in adult

6 female albino rats fed a standard laboratory diet but not in the female rats given highly palatable foods (sweetened condensed milk, shortbread, peanut butter, cheese). Rather the vagotomized animals only exhibited a 1-day delay in the onset of overeating and this occurred only when first exposed to the diet (presumably the delay represents acclimation to the new diet). The study therefore concluded that there existed a two-factor model for hypothalamic obesity based on the composition of the diet. In one situation, the orosensory/oropharyngeal aspect of a high fat diet overpowers the vagus nerve and thus can work independent of the vagus nerve to mediate hypothalamic obesity. In the other situation, the vagus nerve prevails and mediates the hypothalamic obesity in the absence of orosensory/oropharyngeal cues in the diet (as in the case of the more mundane standard laboratory diet). Consistent with the finding of Gold et al. (1981),

Sclafani et al. (1981) found that hyperphagia and obesity induced in adult female rats subjected to VMH lesions (via parasagittal knife cuts) was blocked by SDV when the animals were fed a standard laboratory diet but not when they were offered a highly palatable food diet.

Interestingly, SDV also blocked the overconsumption of a 20% sucrose solution in the lesioned rats (Sclefani et al., 1981).

In another model of hypothalamic obesity, caused by monosodium L-glutamate (MSG) induced excitotoxic lesion of the arcuate nucleus (ARC), SDV reduced visceral WAT but did not significantly affect BW or FI in rats (Balbo et al, 2000; Balbo et al., 2007). However, it is worth noting that in both the Balbo et al., studies and in the studies looking at animals on a highly palatable diet, the completeness of the SDV is questionable. In both studies, residual food remaining in the stomach was used for indication of completeness of SDV.

In summary, the main finding of these hypothalamic lesion studies is that SDV exerts a robust anti-obesity effect characterized by a decrease in BW. In some instances, the anti-obesity effect of SDV persists even when there are no differences in FI or BW (Powley and Opsahl,

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1974; Cox and Powley, 1980; Balbo et al., 2000; Balbo et al. 2007). It is worth noting that there are contradictory studies to these findings, such as those of King et al. (1978). However, these contradictory studies can potentially be attributed to differences in the completeness of SDV.

King and colleagues, in addition to looking at vagally mediated insulin secretion as a test for the completeness of the SDV procedure, also used the neutral red gastric secretion test of Legros and

Griffith (Legros and Griffith, 1969). This test looks for evidence of the neutral red dye in gastric fundus tissue and helps to establish the state (active or quiescent) of parietal cells in secreting gastric acid (Bradford et al., 1950). Vagal sectioning should strongly reduce or eliminate all spontaneous secretion of gastric acid, which would render the neutral red as a faint stain thus indicating a complete SDV (Van Rensburg, 1977). In employing this test, King and colleagues would have only examined vagal fibers innervating parietal cells as opposed to the entire GI tract thus putting into question the completeness of the vagal transection.

The only acceptable method for assessing the completeness of SDV involving the efferent arm is to observe cell loss/degeneration of vagal preganglionic motoneurons in the dorsal motor nucleus of the vagus (DMV). This nucleus contains among others, the cell bodies of origin of the vagus nerves innervating the GI tract, liver and pancreas. It is well known that these motoneurons degenerate after SDV or peripheral axotomy (Laiwand et al., 1987; Szereda-Przestaszewska et al., 1984; Lewis et al., 1972; Navaratnam et al., 1998; Phillips et al., 2003; Rinaman et al., 1991).

In fact, SDV is an accepted surgical procedure intended to produce chromatolysis of preganglionic motoneurons in the DMV, which ultimately leads to extensive motor neuron loss in the DMV (Manaker et al., 1993). Indeed, it has been the preferred methodology in several of the earlier pioneering studies looking at the role of SDV in reversing VMH obesity (Powley and

Opsahl, 1974; Inoue and Bray, 1977; Sclafan et al., 1981; Cox and Powley, 1981). A number of experiments using different species report that within a year of post-vagotomy, approximately 70

8 to 80% of the neurons in the DMV disappear (Aldskogius, 1978; Aldskogius et al., 1980;

Szereda-Przestaszewska, 1985; Laiwand et al., 1987; Navaratnam et al., 1998). Of course, the above studies are not meant to imply that it takes one year for the DMV cells to disappear rather that denervation is a permanent effect. In fact, some early studies indicate that 7 days after cervical vagotomy, DMV neurons have undergone typical retrograde degeneration (Ling et al.,

1987) that is greatly enhanced by postoperative day 14, an early sign of cell death (Ling et al.,

1987). Consistent with this, Aldskogius (1978) demonstrated that in rabbits, cell death among vagal pre-motoneurons of the DMV appears within 3 weeks of vagotomy. Similar time-course for dendritic degeneration of DMV neurons was also reported in monkeys (Ling et al., 1986).

The increased vulnerability of DMV pre-motoneurons to cell death post vagotomy may be due to reasons including but not limited to (a) high sensitivity to inhibitory/repulsive signals produced within the target organs that are unfavorable to DMV neuron survival or (b) the possibility that one or more requisite trophic factor(s) either from the DMV proper or from the adult GI tract are not sufficiently up-regulated after the vagotomy. Consistent with the latter idea, brief pulses of fibroblast growth factor-1 (FGF-1) applied to the vagal trunk have been demonstrated to increase the number of neurons surviving in the DMV after axonal injury (Jacques et al., 1999). This highlights a role for insufficient upregulation of one or more requisite trophic factor(s) necessary for DMV neurons to survive vagotomy or axonal insult. Moreover, it offers a plausible explanation for the increased susceptibility of DMV neurons to neuronal atrophy in contrast to the majority of somatic motor neurons (e.g. in the hypoglossal nerve) that manage to survive axonal injury. In summary, the test for completeness of SDV requires examination of cell loss in the DMV in order to be considered a complete SDV.

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2: Studies Using Other Obesity Models

The Zucker (fa/fa) rat is the best known and most widely used rat model of genetic obesity characterized by a mutation in the long form of the leptin receptor (Phillips et al., 1996).

Opsahl and Powley (1974) showed that SDV in both male and female genetically obese Zucker rats, in comparison to male and female non-obese littermates, did not reverse the obesity characterized by hyperphagia. The authors concluded that the reason SDV did not exert an anti- obesity effect was because the underlying etiology of genetic obesity was different than that of previously studied VMH obesity models where SDV works in reversing the obesity. However, it is worth noting that there are other potential reasons that may have led them to conclude the ineffectiveness of SDV. First, the incompleteness of the vagal transections may be attributable to the inability of SDV to exert an anti-obesity effect. To test for completeness of SDV, the investigators in this study (Opsahl and Powley, 1974) looked at gastric acid secretion response as well as food content of the stomach (i.e. rats were considered to be completely vagotomized only if after 24 hour food deprivation, their stomachs contained significantly more food than those of controls). They did not look at neuronal cell loss in the DMV that I have previously contended is the most important indicator of completeness of the SDV.

Ovariectomies in rats have been shown to increase FI, BW and adiposity (Leshner et al.,

1973; Wade 1976). Estradiol appears to be the principal ovarian hormone affecting BW in rats

(Wade, 1976) and one of the sites of action for estradiol in weight regulation may be in the hypothalamus, since it has been shown that estradiol implants in the VMH suppress FI and BW

(Beatty et al., 1974; Jankowiak & Stern, 1974; Wade & Zucker, 1970). However, the efferent pathways mediating this action of estradiol are not known. Gold et al. (1979) performed SDV in an attempt to determine whether hyperactivity in the efferent vagus nerve could be one of the mechanisms by which ovariectomy-induced obesity was established. They concluded that

10 ovariectomy-induced obesity was not mediated by the vagus nerve based on the failure of SDV to either prevent obesity or to interfere with the BW reducing effects of physiological doses of estradiol benzoate in ovariectomies rats (Eng et al., 1978). It is worth reiterating that in this study (as in the Zucker rat study of Opsahl and Powley, 1974), the ineffectiveness of SDV may have been due to the incompleteness of the vagal transections. Similar to Phillips et al. (1996), the criterion to evaluate completeness of SDV was based on post-mortem anatomical inspection of severed vagal nerves and gross stomach distension due to excessive food retention (Gold et al.,

1979).

In summary, not all studies (see Table 1) used the examination of cell loss in the DMV as a test for completeness of SDV and therefore discrepancies in the effectiveness of SDV to exert an anti-obesity effect (as seen in the studies mentioned above) most likely attributed to this inconsistency in methodology used for SDV verification.

Table 1: Studies employing methods to assess completeness of SDV and its effect on obesity. Study Year Presence of Anti-Obesity SDV Verification Effect of SDV Cox & Powley 1981 Yes -DMV Cell Loss Gold et al. 1979 No -Food content in stomach -Post-mortem inspection Opsahl and 1974 No -Food content in stomach Powley -Gastric acid secretion King et al. 1978 No (obesity reversal); Yes -Neutral red gastric secretion test of (obesity preventitive) Legros & Griffith Balbo et al. 2007 Yes -Food content in stomach Inoue and Bray 1977 Yes -Gastric acid secretion

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3: Clinical Studies

Most of the work on SDV, as an anti-obesity intervention in patients stems from the

work of Dr. J. G. Kral, which spans a 30-year period. In his first report, Kral described

performing ‘truncal’ vagotomy in 3 obese women (Kral, 1978). Each woman, after a 16 to 24

wk period, lost significant weight (i.e., 10, 17 and 19 Kg) “without serious side-effects." As Dr.

Kral notes, “they are eating less than they were preoperatively and report a total lack of hunger.”

This translational aspect of vagotomy was based, in part, on the findings of Powley and Opsahl

(1974) who reported a robust effect of SDV on restoring normal weight to rats with VMH

lesion-induced obesity. Similar reports, attesting to the effectiveness of truncal vagotomies, as a

treatment intervention for obesity, were further published by Kral (1979, 1980; Kral and Gortz,

1981). In all studies, consistent loss of 20 to 30 kg was noted following 6 to 18 months of the

procedure.

To determine how truncal vagotomy was causing weight loss, this procedure was performed by Kral and colleagues in 7 morbidly obese patients (by Gortz et al.,. (1990). Their results showed that truncal vagotomy decreases food intake and liquid consumption. Truncal vagotomy was further studied in patients showing weight loss due to gastroplasty (Kral et al.,

1993). This study asked the question of whether long-term weight loss produced by gastroplasty is further enhanced by vagotomy. Patients with vagotomy that had undergoneunderwent gastroplasty lost 33 +/3 kg compared to 21 + 3 kg in patients with gastroplasty alone (Kral et al.,

1993). In a summary of the results of their vagotomy studies spanning a 30-year period, Kral and colleagues (2009) conclude that the preponderance of evidence indicates that “interrupting abdominal bi-directional vagal transmission demonstrates that the majority of patients report less hunger and lose weight.”

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In 2008, Camilleri and colleagues studied a new medical device capable of producing intermittent vagal blocking in obese patients. This implantable medical device was tested in 31 overweight patients; average excess weight loss was 7.5%, 11.8%, and 14.2% after 1, 3, and 6 months post-implantation of the medical device, respectively. The authors concluded that intermittent, intra-abdominal vagal blocking is associated with significant excess weight loss.

Associated with weight loss was a decrease in hunger between meals and earlier satiation during meals.

Consistent with the findings of Kral and colleagues and Camilleri et al. are data of

Miyata et al. (2012), who took a different approach to examining the role of the vagus in controlling BW. Their concern was to counter the loss in BW in cancer patients who undergo gastrectomy. They report that preservation of the vagus nerve during the gastrectomy operation prevents loss of intra-abdominal fat tissue resulting from the operation.

In summary, the weight of the clinical data examining the effect of suppressing vagal activity on BW is consistent with the findings of the experimental animal studies. Both indicate that suppressing vagal activity results in loss of BW, which is in part due to a reduction in FI. In all the above cited studies, both efferent and afferent vagal nerves are affected by the interventions used to suppress neural activity. What contribution the loss of vagal afferents makes towards the anti-obesity effect is not clear, but since afferent vagal activity provides satiation signals to the brain, it would seem that loss of this sensory input would increase FI and

BW.

C: Mechanisms Responsible for the Anti-Obesity Effect of SDV

1: Reduction in Food Intake

Mordes et al. (1979) found that SDV in rats, in addition to causing a reduction in weight gain, also leads to severe gastric distension. The cause of gastric distension presumably was

13 constriction of the pyloric sphincter, an effect known to occur after vagotomy (Hagiwara et al.,

2000; Mulholland 1997; Thompson 1986). More importantly, the gastric distension would provide satiation signals to the brain (Steinert et al., 2012). The addition of a pyloroplasty

(operation to widen the pyloric opening) to the protocol was found to facilitate the drainage of gastric contents in vagtomized rats. When both procedures (SDV and pyloroplasty) were performed in both male and female rats, the animals maintained a weight that was 14-30% less than control animals over a period extending from 30-300 days. This is especially impressive because it occurred in rats that were not obese but of normal weight. In addition, pair feeding

(i.e., providing the same amount of daily food to the control rats as consumed by rats subjected to

SDV and pyloroplasty) resulted in the same loss of BW as observed in rats subjected to SDV.

These results indicated that SDV was decreasing BW by reducing FI. Finally, these investigators reported that partial vagotomies were ineffective in producing weight loss (Mordes et al., 1979).

Another study indicating a role for reduced FI in mediating the anti-obesity effects of

SDV is the aforementioned study by Inoue & Bray, 1976 where they found that pair-feeding the

VMH lesioned rats without SDV (i.e. providing the same amount of food that was consumed by rats subjected to VMH lesion and SDV) also reversed the obesity suggesting to the authors that

SDV reversed the obesity associated with VMH lesion primarily by decreasing FI.

2: Reduction in Serum Insulin

In the aforementioned study of Inoue & Bray (1976), in addition to reversing the VMH obesity syndrome, SDV also lowered serum insulin levels. This is particularly noteworthy, as hyperinsulinemia is another characteristic of the VMH obesity syndrome; its attenuation indicating a strong role for increased vagal efferent activity (to the pancreas) as the underlying etiology in this obesity model. After the ingestion of food, there is a pre-absorptive rise in the insulin response (cephalic phase of ingestion), which is exaggerated by a VMH lesion (Louis-

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Sylvestre, 1978; Steffens, 1970). It is known that hyperinsulinemia is tethered to insulin resistance. For example, it has been shown that rodents infused with insulin via an implanted osmotic mini-pump become hyperinsulinemic and insulin resistant with impaired glucose tolerance (Destefano et al., 1991). Furthermore, post-absorptive insulin levels and weight gain are highly correlated (Hustvedt & Lovo, 1972). Lesions of the VMH are invariably followed by chronic increases in serum insulin levels (Frohman et al., 1969; Hales & Kennedy, 1964; Steffens et al., 1972). Additionally, the increase (within minutes to hours after surgery) in serum insulin levels after VMH lesion or bilateral hypothalamic lesions (knife cuts) has been prominently mentioned and even considered to be a causative factor in hypothalamic obesity (Bernardis &

Frohman, 1970; Hales & Kennedy 1964; Martin et al., 1974; Steffens et al., 1972; Tannenbaum et al., 1974,). Consistent with a primary role for insulin, it has been shown that when the beta cells of the pancreas are damaged with streptozotocin before any degenerative changes occur in the VMH, obesity and hyperphagia are reduced or prevented (York & Bray, 1972; Goldman et al., 1972). Further evidence suggesting a role for an increase in serum insulin and vagal activity in the VMH obesity syndrome was obtained when female rats with VMH lesions were pair fed with those of non-lesioned rats (bar pressing for food paradigm). The VMH lesioned rats, upon euthanasia contained substantially more carcass lipid than controls and were hyperinsulinemic

(Cox and Powley, 1981). Because these elevations in serum insulin developed in the absence of even minor disturbances in feeding patterns, the data concurs with the view that VMH damage confers an essential metabolic bias favoring increased serum insulin and vagal activity. This corresponds well with the clinical study of Kral (1980) who reported (in addition to a loss in BW) a reduction in plasma insulin levels in severely obese patients after SDV. It was suggested that the reversal of hyperinsulinemia after SDV could hypothetically be expected to influence central components controlling energy homeostasis.

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3: Increase in Sympathetic Tone

The evidence, according to Powley (1977) suggested a prominent role for increased parasympathetic nervous system (PNS) activity (manifested by increase in serum insulin and gastric acidity) in the etiology of VMH obesity. Therefore, if SDV that reduces PNS activity is able to reverse the obesity phenotype, then it was within reason to assume that perhaps the removal of the vagus nerve would result in an adaptation involving unopposed activity of the sympathetic nervous system (SNS) in mediating the anti-obesity effect of SDV. Of course, increase in SNS activity has been observed subsequent to vagotomy (Kumar et al., 1986, Saindon et al., 2001) and rats with VMH lesions display decreased SNS activity (Bray and Nishizawa

1978; Nishizawa & Bray 1978; Niijima et al., 1984; Sakaguchi & Bray 1988;Yoshimatsu et al.

1984; Hogan et al., 1985; Vander et al., 1985; Yoshida & Bray 1984; Monda et al., 1997; Inoue et al., 1977). An important question is to what extent the presumed (unopposed) increase in SNS after vagotomy impacts feeding and BW. This question has not been adequately addressed. It is also worth noting that the SDV procedure will also remove diffuse sympathetic fibers that run alongside the vagus nerves. Regardless of this, SDV still has a robust anti-obesity effect.

Therefore, it is reasonable to assume that removal of SNS activity that occurs with SDV makes a negligible (if any) difference in terms of the effectiveness of the SDV procedure. More importantly, it is worth considering the viewpoint that the remaining SNS fibers could be augmented to increase thermogenic activity to brown adipose tissue (BAT) favoring increase in

EE and reduction in BW (Contreras et al., 2014). This remains to be determined.

4: Changes in Gut Microbiota

Changes in gut microbiota as a result of surgical intervention have been know to have a weight reducing effect on BW and thus should be considered as another possible mechanism by which SDV could be working to reduce BW. Studies in both humans and rats have shown that

16 there is a re-structuring of gut microbiota after Roux-en-Y gastric bypass (RYGB). Currently, this is the most commonly performed and most successful weight loss procedure in the United

States (Li et al., 2011; Furet et al., 2010; Zhang et al., 2009). RYGB results in rapid weight loss and a reduction in adiposity. These beneficial effects cannot be simply attributable to decreased energy intake (Liou et al., 2013). Furthermore, it has been shown that the transfer of the gut microbiota from RYGB-treated mice to non-operated, germ-free mice resulted in weight loss and decreased fat mass in the recipient animals relative to recipients of microbiota from sham animals

(Liou et al., 2013). An explanation for the beneficial effects of gut micriobiota is potentially due to altered microbial production of short-chain fatty acids (Liou et al .2013). However whether or not changes in gut micriobiota resulting in host weight loss is due to SDV is a subject for future study.

D: The Advantages and Disadvantages of SDV as a Weight Loss Intervention

Despite the fact that a complete SDV has been shown to exert robust long-lasting anti- obesity effects, it is worth mentioning some limitations of this surgical approach. The occurrence of gastric distention and pyloric stenosis is probably the most important complication to the SDV approach. These effects of SDV are suggested to be caused by the removal of the relaxation- induced nitric oxide pathway supplied by the vagus nerve (Hagiwara et al., 2000 ). As a result, the pyloric sphincter reliant on the vagal supply of nitric oxide to relax (Hagiwara et al., 2000) goes into spasm causing pyloric stenosis and ultimately uncomfortable gastric disention. In this dissertation, (as described in the Methods section), this problem was circumvented by performing a pyloroplasty, a surgical procedure to widen the opening of the pylorus to allow for drainage of the stomach contents and alleviate the gastric distention. Similarly, total gastric and truncal vagotomies in humans resulting in gastric stasis of solids requires pyloroplasty (or distal gastric

17 resection/gastroenterostomy) to decrease resistance to outflow from the stomach, thereby increasing gastric emptying of solids (Debas 1994).

Another point about SDV is that it will reduce the pH in the stomach. This could potentially affect gut microbiota that play a role in harvesting, storage, and expenditure of energy obtained from dietary sources, which in turn could influence body weight (Angelakis et al.,

2013).

E: Strategies Employed in My Studies

The interest of this dissertation in the anti-obesity effect of SDV stems from the earlier literature documenting a prominent role of SDV in the ability to lower BW in cases of severe obesity such as the VMH obesity syndrome. In addition, the results of the early clinical studies as well as the aforementioned study of patients with gastric cancer (Miyata et al., 2012), give perspective to the claim that inadvertent or incidental vagotomy may explain mechanisms underlying the success of standard bariatric operations in reducing BW. It is entirely plausible that damage or removal to the vagal nerves during standard bariatric procedures (currently the only long-lasting remedy for weight loss) can explain the success of such procedures but whether this is true remains to be determined.

In this dissertation, the strategy employed was to study the anti-obesity effect of complete SDV in a novel model of obesity that had not been previously studied. The novel model of obesity used was that caused by genetic deletion of the melanocortin 4 receptor (Mc4R). The melanocortin circuits in the brain with distinct projection patterns to melanocortin receptors in both hypothalamic and extra-hypothalamic nuclei is viewed as one of the most important, if not the most important mediator of feeding, EE and BW. The Mc4Rs are found throughout the brain and play a key role in weight homeostasis, autonomic and neuroendocrine function (Mountjoy et

18 al., 1994; Harrold et al., 1999; MacNeil et al., 2002; Mountjoy, 2010). Deletion of the Mc4R results in hyperphagia and severe obesity (Huszar et al., 1997, Balthasar et al., 2005).

Tremendous progress has been made in elucidating the neural pathways involving Mc4R in regulating energy homeostasis (Elmquist, 1998; Spiegelman and Flier, 2001; Saper et al.,

2002; Elmquist and Flier, 2004; Skibicka and Grill, 2009; Xu et al., 2011). To provide a brief overview of the role of Mc4R in the melanocortin circuit, one must begin with expression levels of adipokines in the periphery that respond to fluctuations in body fat mass. Leptin, a protein hormone produced by adipocytes crosses the blood-brain barrier and binds to leptin receptors

(OBRb) in two subsets of neurons located in the arcuate nucleus of the hypothalamus (Elmquist et al., 1998; Saper et al., 2002; Williams et al., 2011). One subset of neuron co-express neuropeptide Y (NPY) and Agouti-Related Peptide (AgRP) that are considered the orexigenic arm of the circuit, while the other subset of neurons co-express pro-opiomelanocortin (POMC) and cocaine and amphetamine-regulated transcript (CART); these represent the anorexigenic arm of the circuit. Leptin binding to POMC neurons will depolarize the neuron to release a-melanocyte stimulating hormone (α-elSH), which is the endogenous agonist at the

Mc4R. Subsequently, binding of α-MSH to Mc4R on a second order neuron located in the PVN will exert powerful inhibitory effects on FI and stimulatory effects on pathways promoting EE.

Conversely, leptin binding to OBRb on NYP/AgRP neuron will result in a decrease in the release of AgRP from NPY/AgRP expressing neurons.. Recall that AgRP will bind and antagonize the

Mc4R causing an increase in FI and a decrease in EE. The exact mechanism by which AgRP inhibits melanocortin-receptor signalling is not completely clear. It has been suggested that AgRP binds to the Mc4R and acts as a competitive antagonist of α-he Mc4 In addition to leptin, insulin, and glucose can also influence these two neuronal populations of the ARC to either activate appetite-stimulating pathways or inhibit them (Mercer et al., 2013).

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Previously, I had mentioned that the interest of this dissertation in the anti-obesity effect of SDV was primarily due to the robust effect of SDV in reducing BW in the severe obesity resulting from lesioning the VMH. Further interest in understanding the role of SDV in Mc4R deficient obesity was brought about by the intriguing similarity between VMH obesity and Mc4R deficient obesity. Both obesity phenotypes display hyperphagia, hyperinsulinemia, hyperglycemia, and hypertriglyceridemia. With respect to vagal nerve hyperactivity, both obesity phenotypes exhibit hyperinsulinemia. Furthermore, the translational relevance of studying this particular model of obesity is that the Mc4R mutations are the most frequent monogenic cause of severe early onset obesity in human models (Farooqi et al., 2003).

F: Hypotheses To Be Tested

Taken together, the earlier literature documents an important role for SDV to reverse and prevent obesity in an experimental animal model (VMH obesity) as well as in human morbid obesity. It has also been well documented that SDV exerts an anti-obesity effect in some models of obesity and not in others, which generated an even greater curiosity to study a new model of obesity (Mc4R deficient obesity) that had never been previously studied to see whether SDV would have an anti-obesity effect. Furthermore, the similarities between the VMH obesity phenotype and the Mc4R obesity phenotype, as well as the translational relevancy of studying

Mc4R obesity compelled me to formulate two hypotheses. They are as follows:

Hypothesis 1: SDV will have a robust effect in preventing and reversing the obesity phenotype in the adult male Mc4R-/- mouse.

Hypothesis 2: The mechanism mediating the robust effect of SDV in reversing obesity in the adult male Mc4R-/- mouse is via reduction in FI.

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II: MATERIALS AND METHODS

A: Animals

All experimental procedures were performed on adult male homozygous loxTB Mc4r -/- mice and age-matched C57BL/6J mice. The animals used were obese 3-5-month old male homozygous loxTB Mc4r -/- mice (n=22) and age-matched C57BL/6J (n=6) mice for the first obesity

‘preventative’ study, and severely obese 8-month-old male homozygous loxTB Mc4r -/- mice

(n=15) for the second obesity ‘reversal/treatment’ study. All indirect calorimetry testing was performed on 8-month-old male homozygous loxTB Mc4r -/- mice (n=15). The homozygous loxTB Mc4r -/- animals used in all studies were reared in an in-house breeder colony at

Georgetown University, and were bred from a homozygous breeder pair purchased from Jackson

Laboratories (Bar Harbor, Maine, United States). Homozygous mice exhibit severe obesity due to a loxP-flanked transcriptional blocking (loxTB) sequence that prevents normal endogenous gene transcription and translation from the endogenous locus. As such, homozygous mice are devoid of functional mRNA in all tested regions of the brain that endogenously express Mc4r. The function of this disrupted allele can be restored by the enzymatic activity of Cre-recombinase.

Control C57BL/6J mice, whenever used, were always purchased from Jackson Laboratories. All mice were singly housed in controlled conditions at room temperature (22 °C) and a 12 h light- dark cycle with free access to food and water. Prior to each major surgical procedure

(vagotomy/pyloroplasty/sham), each mouse was placed within a fasting cage and solid food

(pellet form) was withheld overnight (18-24 hr), whereas water was provided ad libitum. Berry- flavored Ensure Enlive nutrition drink was provided in a small petri dish to mice recovering from surgery. The fasting cage consisted of a steel mesh platform that was fitted onto an upside down cage-top. The bottom of a cage (that was inverted) served as the overall enclosure. A hole drilled into the side of this enclosure allowed for access to water ad libitum via an attached water bottle.

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The petri dish filled with Ensure Enlive was taped to the steel mesh platform to avoid being toppled over. To provide adequate ventilation, the fasting cage was elevated 1⁄2 inch off the counter of the laboratory bench on which it was placed. Altogether, the construction of fasting cage (with its meshed platform) was designed to prevent coprophagia and eliminate the animal’s ability to consume bedding, and therefore left the stomach empty of any solid contents prior to surgery. The purpose of the berry-flavored Ensure Enlive drink was to expose the animal to the liquid diet that they would be drinking post-surgery. All experimental protocols conformed to the

National Institutes of Health regulations and were approved by the Georgetown University

Institutional Animal Care and Use Committee.

B: Surgical Procedure for Bilateral Sub-Diaphragmatic Vagotomy

All animals were anesthetized with isoflurane [induction 4%; maintenance 1.5-2% of minimum

alveolar concentration (MAC)], and their body temperature was maintained at 37°C with an

infrared heating lamp. Following adequate depth of anesthesia (as monitored via the toe pinch

reflex), a midline abdominal incision (~1 cm) was made along the linea alba running

approximately 1 cm caudal from the xiphisternum. The liver was retracted with a saline

dampened cotton swab, and gentle traction was applied to the esophagus by lifting the stomach

out of the peritoneal cavity using an umbilical tape wrapped around the gastric antrum. A curved

glass rod (1mm in diameter) was placed carefully under the esophagus to gently lift it and hold it

in an exposed position. All identifiable vagal afferent and efferent fibers running along the

esophagus orad to the esophagogastric junction (~2 cm) were visualized with the aid of a

surgical microscope (Bausch and Lomb, Inc.) and excised with a microsurgical hook having an

interior cutting edge (Circon Corp.) In each case, as long a vagal segment as possible was

exposed and isolated, which was then excised. This consisted of the removal of all four major

components of the abdominal vagus (celiac, hepatic, gastric, and both ventral and dorsal vagal

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trunks). In addition, all neural tissue surrounding the esophagus immediately beneath the

diaphragm was excised with a microsurgical hook to capture any residual vagal branches. In all

animals that received a vagotomy, extreme care was taken to avoid any damage to the

subdiaphragmatic esophagus. As a result, both food intake and defecation were similar in these

animals to those of sham-vagotomized and pyloroplasty alone controls. [Note: Signs of

dysphagia-associated damage to the upper esophageal sphincter or pharynx (abnormal licking,

mastication patterns or abnormal breathing) were not observed in any animals that underwent

vagotomy or sham-vagotomy.] After this procedure, the umbilical tape and glass rod were

removed. For sham-operated animals, the vagus nerves were similarly exposed but not cut, and

the stomach received gentle traction with the umbilical tape.

C: Surgical Procedure for Heineke-Mikulicz Pyloroplasty

In our initial studies of mice that underwent bilateral subdiaphragmatic vagotomy, none of the mice survived more than a few days due to marked gastric distention and pyloric stenosis caused, presumably, by a loss of vagally supplied nitric oxide to the pyloric sphincter (Shiratori 1970;

Huang et al., 1993; Calabuig et al., 1988). To circumvent this problem, we performed a modified

Heineke-Mikulicz pyloroplasty to facilitate gastric drainage and to relieve pyloric stenosis. After bilateral subdiaphragmatic vagotomy, a pyloroplasty procedure was subsequently performed in the anesthetized mice. To accomplish this, the gastric antral-pyloric-duodenal region was gently exposed and lifted with a 22-gauge bent gavage needle and a fine forceps was placed under the pylorus to stretch it. A longitudinal incision (~2-4mm) was made through the pylorus, extending from the distal antrum to the proximal duodenum. Four (5-0 Vicryl) sutures were placed in the pyloric tissue such that by closing the incision transversely, the outlet diameter of the pylorus was increased. A small piece of absorbable gelatin sponge was placed over the pyloroplasty and secured in place with the remaining thread of the last pyloric tissue suture to further ensure

23 against possible leakage of intestinal contents into the peritoneal cavity. The duodenum/stomach were then placed back inside the peritoneal cavity and the abdominal incision closed in a two- step procedure using a 5-0-vicryl suture. No gastric distention was observed in animals undergoing this procedure, indicating that the antro-pyloro-duodenal junction remained patent.

For pyloroplasty only animals, the subdiaphragmatic vagal trunks were similarly isolated and exposed but not excised; only a pyloroplasty was performed.

D: Trichobezoars Removal

Self-grooming rodents often ingest hair that is readily broken down and moved through the gastrointestinal tract. The exposed incision site, at the level of the pylorus, provided easy access to remove any visible residual trichobezoars (hair balls) that may have been left in the stomach after the overnight fast. Using a 22-gauge gavage needle, attached to a water-based aspirator system, the stomach was flushed and aspirated until no residual trichobezoars or solid food was left in the stomach. With the decrease in gastric motility that would be expected to accompany the bilateral subdiaphragmatic vagotomy, removal of the trichobezoars is imperative, as accumulation of hair within the stomach could result in abdominal tenderness, obstruction, bloating, decreased food intake, malnutrition and ultimately death.

E: Postsurgical Care and Recovery

Post-surgical medication consisted of Baytril (5 mg/kg; SC) for infection prevention, saline as prophylactic hydration (1cc; SC), and Rimadyl (5 mg/kg; SC) for analgesia. All animals were monitored for one week for piloerection, ptosis, porphyrin, hunching, mobility and ruffled coat.

Any animal displaying any of these adverse effects was removed from the study. To minimize post-surgery disturbances of intestinal motility, during the post-recovery phase, animals were maintained on Ensure Enlive berry-flavored nutrition drink for one week.

F: Feeding and Weight Measurements

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Mice were acclimated to individual housing for at least 7-15 days before endpoint measurements were taken. Regular mouse chow was weighed and provided to 14-20 wk old mice ad libitum during baseline and after post surgical recovery (where only liquid diet of berry-flavored Ensure

Enlive was supplied ad libitum). Low fat Purina Diet 5001 (23.0 % protein, 4.5% fat, 5.3 % crude fiber, 49% carbohydrate, total digestible nutrient 76%, 3.04 kcal/g metabolizable energy) was used for all groups. Body weight was measured daily for up to 8 weeks post-surgery. Food intake measurements were made at the same time of day (between 3-5 pm) by weighing each pellet manually, taking into account food spillage over the 4 weeks post-surgery period in both the

‘preventative’ and ‘treatment’ studies.

G: Neuroanatomical Verification of Completeness of Bilateral Sub-Diaphragmatic Vagotomy

After the completion of the both studies, each animal received a single intraperitoneal injection of

Fluoro-Gold (0.8 mg/0.4ml saline; Fluorochrome, Denver, CO). One week after injection, all animals were anesthetized with isoflurane and perfused via the ascending aorta with a phosphate buffered saline wash (0.1 M, pH 7.4) followed by a buffered 4% paraformaldehyde fixative (0.1

M, pH 7.4) delivered over a 15 min period. The brains were removed and placed in a cryoprotective fixative (4% buffered paraformaldehyde-20% sucrose) for at least 24 h. After cryoprotection, the brainstem was blocked and cut on a cryostat (Leica, 1500) into 50 µm coronal sections, which were mounted on slides for later imaging.

H: Fat Pad Excision

After the completion of perfusion, fat pads were dissected from the following compartments: mesenteric (fat distributed throughout the gastrointestinal tract), epididymal (abdominal fat surrounding the epididymis), peri-renal, and subscapular brown adipose tissue. After dissection, the wet weight of the fat pad was determined.

I: Snout-to-Anus Length

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After the completion of perfusion, the animal was placed in a supine position and a ruler was used to measure the snout-to-anus length (indicator of animal’s linear length) from the tip of the snout to the anus.

J: Metabolic Chamber Recordings of Indirect Calorimetry

Metabolic parameters were obtained using an indirect calorimetry system (PhenoMaster: TSE

Systems, Bad Homburg, Germany). The TSE LabMaster indirect calorimetry system monitors energy expenditure (EE), locomotor activity, and food and water consumption of mice in individual chambers. Adult male Mc4r -/- mice were acclimated in the metabolic chambers for 3 days before the start of recordings. Up to 4 chambers at a time were used in each round of recordings. Prior to each recording, each mouse was weighed and placed in its individual chamber labeled A, B, C, and D. To minimize the any possible effect of placement (i.e., cage or differential light exposure for example in chamber A vs. D, etc.), animals in each round were rotated between chambers at the start of acclimation to ensure that no single treatment group was always in the same chamber on the laboratory bench in which it was placed. Food and water bottles wereare weighed at the start of acclimation and subsequently monitored for 1-2 days to confirm that mice were eating and drinking. Mice were checked daily during acclimation and subsequent recording. The TSE system allows for sampling of gases (VO2, oxygen consumption and VCO2, carbon dioxide production in ml/hour) and motor activity of each chamber in succession, at 10-minute intervals. Values thus acquired are compared to an unoccupied reference chamber. After acclimation for 3 days, VO2 consumption and VCO2 production were acquired every 10 min for 72 hr via the PhenoMaster software. Metabolic parameters that include energy expenditure (EE kcal/hour) and respiratory exchange ratio (RER=VCO2/VO2) were calculated by algorithms native to the supplied acquisition software.

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Activity was, measured along the long axis of the cage, by an array of infrared beams and sensors. Counts were recorded continuously and reported at 10-minute intervals, with the option of displaying activity in smaller time increments. The system discriminates between ambulatory activity (XA) and fine activity (XF) such as grooming, depending on whether the mouse breaks two adjacent beams in succession, or breaks the same beam twice. Overall, the values for XT

(total activity), XA, and XF were qualitatively similar, hence XT was the only column analyzed for activity. A detailed description and protocol of these can be obtained directly from the manufacturer or found on the website, www.tse-systems.com. Briefly, the software generates 3 types of calorimetric data sets of which the first takes into account the animal’s body weight and second takes into account the lean body mass (LBM calculated from a user-defined power of the animal’s weight in a range of 0.001—0.999). In the third set, neither the animal’s weight or LBM is taken into consideration.

In analyzing the data, a critical question that arose was which subset of the data generated by the

PhenoMaster software would most accurately reflect the calorimetric parameters associated with our experimental conditions. It is well documented that indirect calorimetry to compare EE among animals that differ in body weight has inherent inaccuracies (Arch et al., 2006; Butler and

Kozak 2010; Tschop et al., 2012). First, differences in body weight are usually associated with differences in tissue distribution, making it difficult to calculate their specific contribution. For example, adipose tissue may have low EE in obese animals and although the metabolic rate of fat tissue may be low, it is not zero and may represent a relatively high proportion of the animal’s total mass. Second, adipose tissue releases adipokines, notably leptin, that affect EE in other tissues. Third, bigger animals with their larger body mass are widely accepted to expend more energy (Arch et al., 2006; Felig et al., 1983). Hence, to avoid these potentially confounding variables, many investigators choose to normalize VO2 using body weight or, preferably, lean

27 body mass (LBM), which many investigators feel accurately reflects total EE (Speakman et al.,

2013; Tschop et al., 2012). It is worth noting however, that normalization to LBM has its own inherent flaws in that lean tissue is not distributed evenly in all compartments, and as a result

LBM does not compare isometrically with total body mass (Arch et al., 2006; Speakman 2013).

In addition, normalizing to LBM is only accurate if an EchoMRI body composition analyzer at the beginning and end of the experiment can determine lean mass, which was not done in these studies. In our study, due to substantial differences in the body size of the animals as a result of the powerful effect of bilateral subdiaphragmatic vagotomy on body weight, no ratio-based method for normalizing EE was employed (either to lean body mass or total body mass).

K: Data Analysis

Data are presented as mean ± SEM. In all cases, p < 0.05 was the criterion used to determine statistical significance. Statistical analysis of the fat pads and the snout-to-anus length was performed using a one-way analysis of variance (ANOVA) and unpaired-t- tests, whereas body weight and cumulative food intake was analyzed employing a two-way repeated measure

ANOVA and Bonferroni's/ Tukey’s multiple comparison post hoc tests. To analyze the metabolic data, a linear regression model was employed to determine the relationship of EE (kcal/hour) to body mass within and across groups. EE was averaged with respect to time and the resulting averaged EE values were fitted using a linear regression model with body weight as an independent variable. All parameters (RER, and Activity) were analyzed for both dark (sunset- sunrise) and light cycle (daylight hours) phases using linear mixed effects model (LMM). All post hoc analysis of the data was performed using the Tukey multiple comparison test. [Note:

Light cycle phase data was filtered to only include data points where the animals showed no activity (i.e., animals are presumed to be at rest).] Analysis of the EE data was done in consultation with Dr. Wendy Katz (University of Kentucky, Lexington, KY) who generously

28 offered her time and expertise in the analysis of the TSE chambers EE data. Additional statistical consultation was done with Dr. Valery Korostyshevskiy (Department of Biostatistics,

Bioinformatics and Biomathematics, Georgetown University, Washington DC).

29

III: RESULTS

A: Subdiaphragmatic vagotomy prevents weight gain in Mc4r -/- mouse

As indicated in the methods, bilateral subdiaphragmatic vagotomy (SDV) per se is lethal in mice; therefore, for it to be successful, it has to be performed in conjunction with a pyloroplasty.

Hence, to assess the effect of SDV as a preventative measure for Mc4r -/- mouse obesity, mice were assigned to three groups. These groups underwent the following treatments: Group 1, sham

SDV and sham pyloroplasty (SS, n=7); Group 2, pyloroplasty and sham SDV (PS, n=8); and,

Group 3, pyloroplasty and SDV (PV, n=7). Prior to surgery, (see METHODS), initial baseline weights (obtained over a 2 week period) of the three groups of mice were 44.0 ± 1.1g, 41 ± 1.2g, and 40.0 ± 1.0g, respectively (Figure 1). After surgery, all mice were placed on a liquid diet for

7 days. Their normal diet was re-established on post-surgery Day 8. From thereon, BW was monitored daily and plotted for an 8 wk period (Figure 1). As can be noted, the SS and PS groups, after initially losing weight following surgery, gained it at a fairly constant rate. At the end of the 8 wk period, the SS and PS groups had attained weights of 54 ± 1.4 g and 53 ± 1.3 g, respectively. Mice that underwent both pyloroplasty and SDV (PV) lost weight initially, but then gained it, though at a much slower rate than those mice in the SS and PS groups. Indeed, at the end of the 8 wk monitoring period, the PV group weighed only 39 ± 2.3 g, a value significantly less (p < 0.05) than the corresponding weights of the SS and PS groups. Instead, the PV group of mice just returned to the weight that they had prior to pyloroplasty and SDV (Figure 1A).

The change in weight of the 3 groups over the duration of the post-surgical monitoring period is depicted in Figure 1B. As can be noted, the SS and PS groups gained 10.2 g and 11.6 g respectively, while the PV group lost 0.8 g. Inspection of the data clearly indicates that pyloroplasty was not responsible for the ability of SDV to cause the Mc4r -/- obese mice to gain

30 weight at a much slower rate than the SS and PS groups. Instead, the slower rate of weight gain in the PV group appeared to be due entirely to the SDV procedure.

Examination of the data also raised the question of whether SDV restores the weight of Mc4r -/- mice to normal. To address this question, we studied a fourth group of mice, namely a wild type group (n=6) that underwent both sham pyloroplasty and sham SDV. Their body weights at

Figure 1: Weight gain is prevented by subdiaphragmatic vagotomy in not yet fully obese Mc4r -/- mice. (A) Graph

showing the effect of bilateral subdiaphragmatic vagotomy and pyloroplasty on body weight. Male mice (Mc4r -/ or

wild type; n=6-8 per treatment group) were weighed daily after undergoing: sham subdiaphragmatic vagotomy and

sham pyloroplasty (SS, blue), sham subdiaphragmatic vagotomy and pyloroplasty (PS, orange), subdiaphragmatic

vagotomy and pyloroplasty (PV, green) or wild type sham subdiaphragmatic vagotomy and sham pyloroplasty (WT

SS, red). Data are expressed as the mean ± SEM of the body weight (g). (B) Graph illustrating the effect of

subdiaphragmatic vagotomy and pyloroplasty on average change in weekly body weight of Mc4r -/- mice, relative

to baseline (n= 6-8 per treatment group. (C) Graph showing cumulative weekly food intake in all groups fed on a

normal diet. Statistical significance in weekly food intake was obtained by comparing treatment group PV with

control groups (PS and SS). (D) Bar graphs showing the percent reduction in adipose tissue in PV and PS Mc4r -/-

mice (n=6-8 per treatment group) fed a normal diet. [Note: Values are expressed as mean % change (reduction) in

relation to the wet weight of adipose tissue in the SS controls.] *p<0.05 criterion for statistical significance using a 31 one-way or two-way ANOVA and with post-hoc comparison tests (Bonferoni and Tukey). baseline, during recovery after surgery, and over the 8 wk monitoring period are shown in Figure

1B. The starting average body weight of this group was 27.0 ± 0.4 g. As can be noted from

Figure 1A, except for an initial drop in weight during sham surgery, this group of mice maintained their body weights over the 8 week period of observation, which at the end was 28.3

± 0.6 g. This value was not significantly different from their baseline value, but clearly less than the corresponding value of the PV group (p< 0.05). These data indicate that SDV, while having a robust effect to counter the weight gain observed in the Mc4r -/- knockout mice, did not restore the body weight of these animals to normal.

With regards to the general well being of the PV mice, each mouse appeared in good health and, based on general behavior, could not be distinguished from mice in the other groups; except by body size. Clinical reports from veterinary staff did not record any untoward signs of disease or illness. Moreover, the fecal output of this group was of normal volume and consistency and could not be distinguished from those of other groups; neither could their eating, drinking or urinating.

Solid FI data recorded for 4 weeks for the 3 Mc4r -

/- groups are presented in Figure 1C. Cumulative weekly FI of the PV group of mice was significantly less (p<0.05) than that of the SS and

PS groups in the first week after post-surgery recovery and significantly less (p<0.05) that that of Figure 2: Bar graph showing the effect of bilateral the PS group in the second week after recovery. subdiaphragmatic vagotomy + pyloroplasty on snout-

-/- For the ensuing weeks, there was no difference in to-anus length in not yet fully obese Mc4r (n=6-8 per treatment group) fed a normal diet. *p<0.05 the FI between the PV group and the SS and the PS criterion for statistical significance using a one-way groups (Figure 1C). ANOVA and Tukey post-hoc comparison test.

32

B: Effect of subdiaphragmatic vagotomy on fat pad weight and on snout-to-anus length in

Mc4r -/- mice

I also determined whether SDV would prevent white adipose tissue (WAT) accumulation in

Mc4r -/- mice. I weighed the fats pads taken from the visceral compartments of the 3 groups of mice and found that the WAT in the PV group as a percentage of the WAT present in SS controls

(mesentery = 2.18 ± .13g; peri-renal=1.34 ±.09g; epididymal = 2.7 ± .21g; brown adipose tissue=.72 ± .10g) was significantly less in both the mesenteric (MES) and peri-renal (PR) sub- compartments of visceral WAT (p<0.05; Figure 1D). In contrast, the weights of the fat pads taken from the PS was not different from that of the SS group. Sub-scapular brown fat (BAT) and epididymal fat pad (EPI) in the PV group was not statistically significant from that of the other two groups (Figure 1D). In addition, I measured the snout-to-anus length, as an indicator of body growth, and known to increase in Mc4r -/- mice obese mice (Huszar et al., 1997; Balthasar et al., 2005)). SDV was found to significantly blunt this end point, whereas pyloroplasty, per se, had no effect on linear growth (p<0.05; Figure 2).

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C: Histological evidence of complete subdiaphragmatic vagotomy

To test for the completeness of SDV, the brains of both the PV (n=7) and the SS (n= 7) groups

Figure 3: Complete bilateral subdiaphragmatic vagotomy in not yet fully obese Mc4r -/- mice prevents retrograde label in the DMV. (A and B) Representative photomicrographs showing the presence and absence of Fluoro Gold label in DMV neurons of a SS sham (A) and a PV vagotomized (B) Mc4r -/- mouse. (C & D) Representative associated weight curves following sham surgery in A and vagotomy + pyloroplasty in B, respectively. Abbreviations: XII, hypoglossal nucleus; AP, area postrema; CC, central canal; DMV, dorsal motor nucleus of the vagus; NTS, nucleus tractus solitarius. Scale bar = 100µm that had received Fluro-Gold were examined at the end of the post-surgical weight-monitoring period (see Methods). In the PV group, no retrograde Fluoro-Gold label could be seen in neurons of the DMV, indicating the success of SDV (n=7). This was not the case in the SS group; DMV neurons were densely labeled, thus attesting to the intact vagal nerves in the sham animals

(Figure 3).

34

D: Subdiaphragmatic vagotomy treatment alleviates obesity in Mc4r -/- mice

My purpose was to determine whether SDV could reverse the established obesity in the Mc4r -/- mouse. Mice had already become obese before SDV treatment was performed. Prior to surgery, initial baseline weights (obtained over a 1 week period) were 51 ± .33g, (SS) 52 ± .34g, (PS) and

55 ± .30g (PV), respectively (Figure 4A). After surgery, all mice were placed on a liquid diet for

7 days. Their normal diet was re-established on post-surgery day 8. From thereon, body weights were monitored daily and plotted for a 4-week period. As can be noted, the SS and PS groups initially lost weight after surgery, but gained it back over time (Figure 4A). At the end of the 4 week period, the SS and PS groups had attained weights of 51 ± 3.6g, 55 ± 3.4g, respectively.

Obese mice that underwent both pyloroplasty and SDV (PV) lost weight initially, and continued to lose weight over the 4-week period of study (Figure 4A). Indeed, at the end of the 4-week monitoring period, the PV group weighed only 38.4 ± .81g, a value significantly less (p<0.05) than the corresponding weights of the SS and PS groups. The change in weight of the 3 groups over the duration of the post-surgical monitoring period is depicted in Figure 4B. As can be noted, at the end of the 4-week monitoring period, the SS and PS groups had returned to their baseline weights while the PV group lost 17 g.

Solid food intake (FI) recorded weekly for the 4 Mc4r -/- groups are presented in Figure 4C.

Similar to the prevention study (see Figure 1C), FI in the PV group of mice was significantly less (p<0.05) than that of the SS and PS groups at post-surgical week 1 (week 3 of the overall study). In addition, FI of the PV group was reduced relative to the SS groups during week 2.

Although FI for the PV group was not significantly reduced relative to the SS group during weeks 3 and 4, it was significantly less (p<0.05) than the PS group (Figure 4C).

35

I also determined whether SDV would reverse white adipose tissue (WAT) accumulation in Mc4r

-/- mice. Fat pads were taken from the visceral components in the 3 groups of mice and weighed.

The WAT content in PV group as a percentage of that present in SS controls tissue (mesentery =

1.8 ± .41g; peri-renal=1.59 ±.50g; epididymal = 2.02 ± .17g; brown adipose tissue =.31 ± .05g) was significantly less in the mesenteric (MES) sub-compartment (Figure 4D; p<0.05). In the

Figure 4: Subdiaphragmatic vagotomy treatment reduces body weight in severely obese Mc4r -/- mice. (A) Graph

showing the effect of bilateral subdiaphragmatic vagotomy and pyloroplasty on body weight. Male mice (Mc4r -/-,

n=4-5 per treatment group) were weighed periodically after undergoing: sham subdiaphragmatic vagotomy and sham

pyloroplasty (SS, blue), sham subdiaphragmatic vagotomy and pyloroplasty (PS, orange), or subdiaphragmatic

vagotomy and pyloroplasty (PV, green). Data are expressed as the mean ± SEM of the body weight (g). (B) Shows the

effect of subdiaphragmatic vagotomy and pyloroplasty on mean weekly change in body weight, relative to baseline (n=

4-5 per treatment group. (C) Graph showing cumulative weekly food intake in all groups fed on a normal diet.

Statistical significance in weekly food intake was obtained by comparing treatment group PV with control groups (PS

and SS). (D) Bar graphs showing the percent reduction in adipose tissue in PV and PS Mc4r -/- mice (n=4-5 per

treatment group) fed a normal diet. [Note: Values are expressed as mean % change (reduction) in relation to the wet

weight of adipose tissue in the SS controls.] *p<0.05 criterion for statistical significance using a two-way ANOVA and

Tukey post-hoc comparison test. [Note: an unpaired t-test was used for fat pad analysis.].

36 peri-renal (PR) and epididymal (EPI) sub-compartments, WAT in the PV mice was not significantly different from that of the SS group, as was the sub-scapular brown fat (BAT), which was similar to those of the PS and SS groups (Figure 4D). Additionally, no significant differences were found between the weights of the fat pads in the PS group and the SS group.

The snout-to-anus length, reflective of body growth, was also unaffected by SDV in these fully obese animals (data not shown).

In summary, SDV used as both as a prevention and a treatment strategy against Mc4r -/- mouse obesity demonstrates a robust and lasting anti-obesity effect. The mechanisms for the anti-obesity

Figure 5: Bilateral subdiaphragmatic vagotomy influences EE in fully obese Mc4r -/- mice. (A) Graph showing the relationship of

EE (expressed in kcal/hour) to body mass (n=4-5 per treatment group). Data are presented as a linear regression plot. (B-C) Time course plots of the respiratory exchange ratio (RER) during the light and the dark cycles. RER data was sampled at consecutive ten- minute intervals over the course of the monitoring period. (D) Time course plot of total activity (combination of axial and fine movements) during the dark cycle acquired over a 48-hr period that was sampled at ten-minute intervals. *=p<0.05 criterion for statistical significance. 37 effect initially include a reduction in FI (Figures 1C and 4C). However, the reduction in FI did not persist, which suggested to me that the SDV effect on body weight maybe due to an increase in energy expenditure (EE).

E: Effect of subdiaphragmatic vagotomy on parameters of EE in Mc4r -/- mice

Metabolic parameters associated with EE were analyzed (see Methods) in relationship to body weight in the same 3 groups of fully obese adult Mc4r -/- mice that were used in the ‘treatment’ study from Chapter I (SS, PS, and PV). I particularly focused on this relationship during the dark cycle, which is correlated with increased activity in rodents. While there was no significant correlation between body mass and EE in the SS control group, animals undergoing SDV (PV group) had a strong inverse linear relationship (p = 0.014, slope = -0.009, 95% confidence interval = [-0.014, -0.003], R2 = 0.81; Figure 5A). Thus, the decrease in body weight in the PV group was associated with an increase in EE. This contrasts with the relationship of EE to body mass in animals subjected to pyloroplasty and sham vagotomy (PS group). In this group (see

Figure 5A), there was an indication of a positive correlation between EE and body weight indicating that as they became heavier, they appeared to consume more energy (p =0.07, slope

=0.01, 95% confidence interval= [0.002, 0.026], R2 = 0.71). Despite the fact that there is no significant correlation in either control group, it is worth noting that both groups exhibit positive relationship between body mass and EE indicating that as they get heavier they seem to expend more energy. This is in contrast to the PV group that exhibits a negative relationship between the two variables. I also employed linear regression analysis to the aforementioned 3 groups during the light cycle. No significant differences were observed in the relationship of EE to body mass between the 3 groups (data not shown).

Data for respiratory exchange ratio (RER) during the light cycle for the 3 groups was also analyzed. As can be noted (Figure 5B), RER tended to be lower in the PV group in comparison

38 to SS group at specific time intervals (i.e. from 0 to 750 min). Moreover, the overall RER was significantly lower in PV mice as compared to those in the PS group (p<0.05; Figure 5A) in the light cycle. Comparison of RER values during the dark cycle revealed no significant differences between the 3 groups (Figure 5C).

Locomotor activity, (a combination of axial/ambulatory and fine movements) during the dark cycle was also obtained for all the groups. There were no significant differences between the 3 groups (Figure 5D).

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IV: DISCUSSION

A: Magnitude of Reduction in Body Weight Produced by Subdiaphragmatic Vagotomy

The most impressive finding of my studies was the efficacy of SDV to reduce BW.

When SDV was used as a 'preventative' to determine if it would prevent the obesity of Mc4R-/- mice, their weights were maintained at levels prior to SDV. These mice, initially weighing 40.1

+ 1.0 g, were monitored over an 8-wk period. After SDV, vagotomized mice with a pylroplasty dropped weight initially after SDV then remained below that ofand found to have significantly no change in their initial baseline weights throughoutBW after the 8-wk monitoring periodprocedure

(i.e., 39 + 2.3. g, a 2.7% decrease; see Figure 1B). ). In contrast, mice undergoing sham vagotomy and sham pyloroplasty gainedput on about 10 g of weight over this 8-week period.

Their weights increased from 44.1 + 1.2 g to 54.0 + 1.4 g (an overall increase of 23 % in BW).

Likewise, mice undergoing concurrent sham vagotomy and pyloroplasty gained about 12 g of weight over the 8-week period. Their weights increased from 41.0 + 1.2 g to 53.0 + 1.3 g (a 29 % increase in BW).

When SDV was used as a treatment in the obese Mc4R-/-, it was equally impressive.

Sham animals, weighing 51.5 + 4.9 g prior to SDV, were 4-wk later found to weigh 51.0 + 3.6 g.

Similarly, mice that underwent concurrent sham vagotomy and pyloroplasty also did not significantly differ in their weights four weeks later (before, 52.5 + 5.7 g; after 54.8 + 3.8 g). In comparison, mice that received SDV weighed 38.4 + 0.8 g, a decrease of about 31% in BW.

This powerful weight loss effect observed in my studies is similar to that reported by others when

SDV was used to treat obesity produced by VMH lesions. For example, Powley and Opsahl

(1974) reported that SDV completely reversed the weight gain produced by VMH lesions in rats.

In their study, weights increased after VMH lesions from approximately 370-512 g to 540 g over a 40 daydays period, subsequent to which SDV or sham vagotomy was performed. Forty and

40 seventy-five days later, the sham vagotomy rats weighed 610 and 804 g, respectively (an increase of 16 and 53 %). Rats that underwent SDV weighed only 411 and 552 g, respectively (-22 and +5

%). Another example can be found in the publication of Inoue and Bray (1977). Rats weighed approximately 250 g and were subjected to VMH lesions. Over the next two weeks, rats grew to be approximately 350 g. At this time SDV was performed in some of the VMH lesioned rats while others received sham vagotomy. VMH lesioned rats subjected to sham vagotomy grew to an average weight of 485 g over the next 4 weeks (a 38 % increase in BW). Rats subjected to

SDV were restored to the same weight, i.e., restored to approximately 250 g, the weight at the time of VMH lesion.

Taken together, these effects of SDV are robust. A key question is whether such robust effects observed in rodents can be obtained in obese humans? A recent publication by Shikora and colleagues (2013) suggests that this may indeed be possible. These investigators prospectively evaluated the effect of intermittent vagal blocking on weight loss in obese subjects with Type 2 diabetes mellitus. At 12 months of vagal blocking, mean excess weight loss in percentage was 25 + 4 %. In addition, there was a decrease in hemoglobin A1C of 1.0 + 0.2 %, indicating an improvement of glycemic control. This effect of suppressing vagal activity on BW is of a higher magnitude than those clinical studies with approved weight loss drugs for obesity.

Lorcaserin, a serotonin receptor agonist, is one such drug where clinical trials (BLOSSOM;

BLOOM) have shown that from an initial average BW of 100kg, obese patients lost on average

5.8 kg after 52 weeks on the drug (Fidler et al., 2011). Other studies report similar findings showing only weight loss of at least 5% (O Neil et al., 2011). In the obesity ‘reversal’ study reported in Chapter I, obese Mc4R-/- mice on average lost 22% weight after the 4-wk period. In the obesity ‘preventative’ study done in the not yet fully obese Mc4R-/- mice, an average weight loss of 5.6 % was seen after 8-wk and a prevented weight gain of 23%. Overall the effect seen

41 with SDV on BW is much moreone of the most robust than current (if not the most robust effect) on BW compared to weight loss drugs on the market.

B: Mechanisms for SDV-induced reduction in BW

How does SDV reduce BW? I will focus on components of the energy balance system

(i.e., the tightly regulated processes of FI and EE) to determine which component SDV was acting upon to reduce BW, and how my results compare with other investigators. In my studies, I focused on the time course of weight loss in the animals taking cumulative FI into consideration in both the preventative and the treatment studies. During the first week after surgery, when animals were put back on a solid food diet, there was a reduction in FI in the vagotomized Mc4r -

/- mice across both models (preventative and treatment) compared to controls (SS and PS groups). However, the reduction in FI did not persist throughout the post-surgery monitoring period. It is worth noting however that despite the fact that the reduction in FI did not persist, vagotomized Mc4r -/- mice did not exhibit any rebound hyperphagia (after the first week of reduced FI). Instead, vagotomized Mc4r -/- mice normalized their FI to that of controls in the subsequent weeks. Hence, I measured EE in mice after cessation of the ‘treatment’ study; once a robust loss in weight had occurred in the PV group (that had undergone vagotomy). EE in this study was calculated via indirect calorimetric methods where EE is measured in kcal/hour and is calculated by using V02 (oxygen consumption) and VC02 (carbon dioxide production) as well as physiological constants native to the data acquisition software (CV02 = 3.941 CVC02 = 1.106).

Using these values, the software is then able to calculate the energy production rate in a steady- state condition (constant uniform preset temperature & 02 / C02 levels constant at any given time), which is equal to the EE rate. The non-normalized form of the EE data (i.e., not normalized to animal body weight) is then plotted against animal body weight and analyzed via linear regression analysis to look at patterns of energy production in relation to body weight

42 within each of the three treatment groups (PV, SS, PS). RER is calculated as the ratio of VC02 /

V02. Results from this study via the use of linear regression modeling indicate that as weight is decreased in the PV group as a consequence of SDV, EE increases without any concomitant increase in activity levels.

RER was also measured in PV animals and found to be significantly lower than PS treated controls in the light cycle. Additionally, RER tended to be lower in the PV group in comparison to SS group at specific time intervals. RER, which normally displays a circadian rhythm, is a function of macronutrient utilization, molecular interconversion (e.g., lipogenesis) and blood chemistry. Observed RER can be compared to the respiratory food quotient, which is a theoretical RER (dimensionless number) based on the macronutrient composition of the diet

(Toubro et al., 1998). The decrease in the RER in the PV group, relative to the control groups (SS and PS), in our study indicates that in addition to carbohydrates, the PV group was also utilizing lipids as an energy source. This is further highlighted by the reduction in mesentery WAT in this group compared to the controls.

Tschop et al. (2012) advise against the use of comparing mean EE data (i.e., presentation of group effects as histograms) but rather recommend that the analysis of EE data should be made on the raw data expressed per mouse. Additionally, it is recommended that analysis of co- variance or general linear modeling be used to analyze EE data (Tschop et al., 2012). This statistical approach to plot EE in relation to body weight or lean body weight is considered to be the most appropriate as it accommodates discrete (genotype) and continuous (body mass) traits that can be obscured by the normalization method. According to Tschop et al., mean values can be misleading because differences directly attributable to an effect (for instance genotype) can

“cancel out” differences attributable to imbalances of body mass.

43

With respect to the data pertaining to the brown adipose tissue (BAT), no change due to SDV was seen in either the ‘preventative’ or ‘reversal’ studies. However, it should be noted that despite the fact that the actual physical amount (wet weight) of the BAT did not significantly change in either study, I did not measure any thermogenic markers in the tissue such as uncoupling protein 1 (UCP-1). Measurement of these markers indicates changes in thermogenic quality of the BAT, irrespective of its wet weight.

In summary, the indirect calorimetry EE data indicate that the weight-reducing effect of

SDV is strongly correlated with an increase in energy production in melanocortin-deficient mice.

This offers insight into the mechanisms by which vagotomy may be working to exert an anti- obesity effect. However, the exact biochemical, hormonal and metabolic pathway (s) by which vagotomy works to increase EE remains to be determined.

In addition, results of the ‘preventative’ study (anti-obesity effect of SDV), which I observed was that a decreased snout-to-anus length (indicator of animal linear growth) was absent in animals in the SDV ‘treatment study’. This is noteworthy, as there are reports in the literature that show that mutations in the Mc4r gene are associated with accelerated linear growth, which is disproportionate to the degree of the obesity present (Martinelli et al., 2011).

Moreover, deficiency in Mc4r has also been observed to result in increased pulsatile and total growth hormone secretion, as well as elevated insulin levels in humans (Martinelli et al., 2011).

In rats, vagotomy decreases the basal levels of both growth hormone (Al-Massadi et al., 2011) and insulin levels (Lee & Miller 1985) suggesting that in my study, vagotomy 'blunted' the increase in the growth in mice in part by attenuating both growth hormone and insulin levels.

In summary, my data suggests that the primary mechanism whereby SDV decreases BW is an increase in EE, and not a reduction in FI. Powley and Opsahl (1974) appear to come to a similar conclusion regarding the lack of an important role for reduced FI in the anti-obesity effect

44 of SDV in VMH lesioned rats. In contrast, Inoue and Bray (1977) suggest that SDV reversed the obesity of VMH lesioned rats largely by decreasing FI. This evidence was based on pair feeding of VMH lesioned rats without vagotomy reversing the obesity (pair-feeding rats received the same amount of food taken by the rats subjected to SDV).

C: Efficacy of SDV in Obesity States Other than Mc4R-/- mice, and VMH lesioned rats

There are publications that describe the ineffectiveness of SDV to counter obesity. These are studies where SDV is tested in rat models of obesity produced by highly palatable foods

(Sclafani, 1981), ovariectomy (Eng et al., 1979) and genetically obese Zucker rats (Opsahl and

Powley 1974). A concern that I have about the inability of SDV to counter obesity in these models is that the “gold standard” for demonstrating completeness of vagotomy was not used, namely, loss of vagal nerve cell bodies in the dorsal motor nucleus of the vagus (Powley et al.,

1987). Tests that were used included stomach examinations of residual food content greater than control stomachs and gastric acid response to vagal stimulation (Opsahl and Powley 1976). These tests only assess loss of vagal innervation of the stomach and not the pancreas, liver and intestines.

The data obtained from clinical studies where obesity may be due to different etiologies but appear to all respond to suppression of vagal activity (Kral, 1978; Kral 1979; Kral 1980; Kral and Gortz 1981; Gortz et al., 1990; Kral et al., 1993; Camilleri et al., 2008; Shikora et al., 2013) suggests that SDV may be effective in may types of obesity.

D: Criterion of using SDV to assess the role of efferent vagus in controlling BW

While SDV is an accepted method for evaluating the role of the efferent vagus in controlling BW (Manaker et al., 1993; Powley and Opsahl, 1974; Inoue and Bray, 1977; Sclafan et al., 1981; Cox and Powley, 1981), it is nevertheless not an ideal one as it also eliminates afferent vagal fibers. These are well known to affect BW (Berthoud 2008), as their loss per se

45 would diminish satiation signals from the periphery and therefore lead to an increase in food intake (FI; Rogers et al., 1980; Rossi et al., Schwartz 2000) and presumably an increase in BW- effects opposite to what I observed with SDV. However, my use of the SDV model to assess the role of vagal efferents in weight control is valid as our findings are consistent with those of vagal de-afferentation studies (Fox et al., 2001; Schwartz et al., 1999). With respect to the vagal de- afferentation studies, loss-of-function mutations to produce a loss of vagal sensory neurons alongside selective vagal rhizotomies to produce selective de-afferentation in rodents have been shown not play a role in chronic body weight regulation, rather disruptions in short-term satiety.

Additionally, data in the first part of this dissertation is consistent with vagal regeneration studies

(Phillips et al., 2003). In these studies, afferent fibers showed regeneration (albeit imperfect) after

18 weeks subsequent to SDV, whereas the same was not evident for efferents. Moreover, the

SDV animals continued to lose weight further establishing the importance of vagal efferents in long-term BW control. These findings together with my data, makes it evident that parasympathetic preganglionic neurons in the brainstem contribute to orexigenic aspects of energy homeostasis. I am cognizant that the ideal design of my study would have been to examine the effect of selective bilateral lesion of the DMV as opposed to performing SDV. This would have selectively eliminated the efferent vagal fibers innervating peripheral organs such as the GI tract, liver, and pancreas. However, this is technically very challenging if not impossible due to the close proximity of DMV to the NTS and area postrema (McCann and Rogers 1992) structures, which also influence GI, liver, and pancreatic function (Gillis et al., 1989; Woods and

Porte 1974).

As mentioned earlier, there are other limitations of the SDV approach, namely the occurrence of gastric distention and pyloric stenosis. Performing a pyloroplasty circumvented this problem. Similarly, total gastric and truncal vagotomies in humans resulting in gastric stasis

46 of solids requires pyloroplasty (or distal gastric resection/gastroenterostomy) to decrease resistance to outflow from the stomach, thereby increasing gastric emptying of solids (Zhang et al., 2009; Khan et al., 2007; Masquisi & Velanovich 2007). Another concern of SDV is that it will change pH in the stomach. This could potentially affect gut microbiota that play a role in harvesting, storage, and expenditure of energy obtained from dietary sources, which in turn could influence BW (Angelakis et al., 2013). However, the exact manner by which SDV may influence gut microbiota is a subject for future study.

E: DMV represents one of the key outputs of the central melanocortin neural circuits

In showing that SDV prevents the obesity associated with the loss of Mc4Rs, it suggested to me that vagal preganglionic cell bodies located in the DMV serves as an important output of the melanocortin system. The DMV, a hindbrain nucleus contains primarily cells of origin of the preganglionic vagal neurons that innervate organs involved in body weight (BW) control such as the GI tract (McCrea, 1924; Jackson, 1949; Berthoud et al., 1991; Berthoud &

Powley, 1992; Berthoud & Patterson, 1996; Powley & Phillips, 2011), liver (Sawchenko et al.,

1979) pancreas (Woods et al., 1974) and adipose tissue (Kreier et al., 2007). Specifically, based on findings in Chapter I of this dissertation, I suggest that activity in the anorexigenic facet of melanocortin neural circuits eventually inhibits DMV neurons, which results in the suppression of vagal efferent output to the above organs. Conversely, interruption of melanocortin neural circuits should increase efferent vagal activity, as is evident by the increase in hyperinsulinemia in Mc4r -/- mice (Huszar et al., 1997). Indeed, elimination of efferent vagal activity by bilateral

SDV has been shown in a model of obesity (produced by VMH lesions) to restore body weight to normal (Powley and Opsahl 1974).

If the DMV serves as an important output of the melanocortin system, then SDV should prevent the obesity associated with its disruption, namely, the loss of Mc4r. Hence, in my first

47 model, the effects of SDV on weight was assessed in adult Mc4r -/- mice that were significantly heavier than their wild type cohorts (WT), but had not yet reached full obesity. This obesity preventative strategy inhibited mice from gaining additional weight. However, it did not restore their weight to the level of the WT mice. In my second model, I used adult Mc4r -/- mice that were fully obese to assess SDV as an obesity reversal strategy. SDV produced a robust reversal of established obesity in these mice. This anti-obesity effect of SDV in Mc4r -/- mice corresponds well with my view that the DMV serves as an important conduit of central melanocortin circuitry by which the brain conveys information to the periphery.

My present findings are consistent with the view that the release of α-MSH in the NTS via activation of a hypothalamic-POMC-NTS circuit causes amplification of satiety signals arriving from the stomach (Zheng et al., 2010). Although how this is accomplished is speculative, my view is that α-MSH in the NTS inhibits DMV projection neurons (Richardson et al., 2013) by suppressing the high GABAergic tone present in this nucleus (Herman et al., 2010;

Herman et al., 2012; Herman et al., 2012). This in turn allows NTS inhibitory neurons that project to the DMV to be excited by glutamatergic vagal afferents, thereby suppressing vagal output (Figure 6).

The role of the DMV in the regulation of feeding and body weight, as it pertains to Mc4r stimulation, has also been explored. Administration of the Mc4r agonist, Melanotan-II (MT-II), into the fourth ventricle or into the DVC suppresses food intake; but this is unaffected by SDV

(Williams et al., 2000). This led the authors to conclude that suppression of food intake due to stimulation of Mc4rs in the DVC did not require an intact vagus nerve. However, several explanations can be offered to account for these negative findings. One is that α-MSH was not studied; instead, the Mc4r agonist, MT-II, was investigated. As described recently by us

(Richardson et al., 2013), there are significant differences between the effects of α-MSH and

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MT-II. The direct effect of α-MSH on DMV output neurons is excitatory whereas that of MT-II is inhibitory (Richardson et al., 2013). The inhibitory effect of MT-II on parasympathetic DMV neurons has also been observed by Elmquist and colleagues (Sohn et al., 2013). As regards to

Figure 6. A simple schematic illustrating how vago-vagal circuitry controlling gastric motility and tone maybe regulated by the hypothalamic-POMC-NTS pathway. Release of α-MSH in the NTS by Hypothalamic-POMC-efferents (1) in the NTS inhibits high GABAergic tone due to interneurons in this nucleus. This in turn allows NTS inhibitory neurons (2) that project to the DMV to be excited by glutamatergic vagal afferents (3), thereby suppressing vagal output to the stomach (4). microinjection of α-MSH into the NTS, the effect observed is inhibition of DMV neurons presumably because α-MSH is inhibiting a GABA interneuron in the NTS that is tonically inhibiting NTS GABAergic neurons projecting to the DMV (Lewin et al., 2013).

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Rossi et al. (Rossi et al., 2011), using a transgenic approach, assessed the effects of genetic restoration of melanocortin receptors in DMV neurons of Mc4r -/- mice. Restoration of Mc4rs in this nucleus proved to be ineffective in influencing either hyperphagia or low energy expenditure, characteristics that are present in these mice. This was not the case in the PVN and amygdala, brain areas that have been documented to contribute to energy balance (Balthasar et al., 2005).

Here, restoration of Mc4r in the PVN and amygdala countered the hyperphagia in Mc4r -/- mice

(Balthasar et al., 2005). When Mc4rs were reintroduced into sympathetic preganglionic neurons of the intermediolateral cell column, the lower energy expenditure characteristic of the Mc4r -/- mice was counteracted (Rossi et al., 2011). The inability of re-expressed Mc4rs in the DMV to decrease hyperphagia or increase energy expenditure is not surprising given our recent findings

(Richardson et al., 2013), which indicate that activation of these receptors in the DMV by α-

MSH increases vagal output. Instead, the CNS location where re-expression of Mc4r would be expected to counter obesity is in the NTS. Here, I have shown that activation of the Mc4r in NTS results in inhibition of the DMV and consequently vagal outflow (Richardson et al., 2013).

In summary, my data are consistent with the function of the DMV as one of the key outputs of the central melanocortin neural circuits. Not only did SDV in Mc4r -/- obese mice ameliorate the obesity that develops in these animals over time, but it also reduces the established obesity in this mouse model. My data indicate that this anti-obesity effect has 2 components.

Initially, there is a reduction in FI, while later there is an increase in EE as mice lose weight. The increase in EE is associated with excess burning of fat stores. Furthermore, these findings fit with my view that Mc4r -/- mice lack tonic α-MSH stimulation in the NTS responsible for suppressing the orexigenic/anabolic signals of vagal efferent output to the periphery, which is responsible for weight gain.

F: Outcome of Hypotheses Testing, Conclusions, and Future Studies

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The outcome of my studies from Chapter I allow me to conclude that SDV is an incredibly effective weight-reducing intervention. In terms of my hypotheses, my data supportI was able to prove Hypothesis# 1 that SDV haswill have a robust effect in preventing and reversing the obesity phenotype in the adult male Mc4R-/- mouse. In terms of Hypothesis # 2, my data do not support the hypothesisI was unable to prove that the mechanism mediating the robust effect of SDV in reversing obesity in the adult male Mc4R-/- mouse was a reduction in FI.

Rather, I showed that it was an increase in EE as FI normalized in vagotomized animals and their weights did not revert back to that of controls. In terms of future directions, I would like to further characterize biochemical mechanisms of how SDV is a able to exert a weight-reducing effect that include (1) assessing thermogenic markers in the BAT, (2) markers indicative of activation of lipolytic pathways, and (3) measuring hormone levels (insulin, ghrelin, leptin).

Another future direction is to assess the role of SDV in a high fat diet model since previous studies have only looked at this with inconclusive tests for completeness of SDV. Additionally, in view of the clinical findings (Shikora et al., 2013) showing that suppressing vagal activity improves glycemic control in Type 2 diabetes mellitus, I am eager to determine whether SDV will reverse obesity and diabetes in a rodent model of this disease (homozygous leptin-resistant db/db obese mouse).. Finally, in advancing my idea that the DMV represents one of the final outputs of the melanocortin circuit, I am also eager to exploit the CRE-Lox technology to enable me to restore Mc4R receptor expression back in brain nuclei of the Mc4R-/-mouse, particularly in the mNTS and evaluate the effect on BW. I predict that this intervention will attenuate neural activity in the DMV, which should reduce BW.

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Chapter II: Experimental Studies of Nicotine for Reducing Body Weight

I: INTRODUCTION

A: Cigarette Smoking and Body Weight

Compared to SDV, there are many more studies documenting weight loss with nicotine, the primary psychoactive substance in tobacco smoke. Indeed, the public perception is that smoking is associated with loss of BW (Chen et al., 2012) and is used as a means to regulate BW by adolescents and females (Camp et al., 1993; Wiseman et al., 1998; Fulkerson & French,

2003). Smoking and BW have been examined extensively and numerous cross-sectional studies indicate that BW and body mass index (BMI; in kg/m2) are lower in cigarette smokers than in non-smokers (Williamson et al., 1991, Shimokata et al., 1989; Flegal et al., 1995; Huot et al.,

2004;) Adult smokers of both genders tend to weigh less than nonsmokers (Albanes et al., 1987;

Grunberg et al., 1992; Klesges et al., 1991; Klesges et al., 1989; Rasky et al., 1996, USDHHS

2001). Furthermore, evidence also indicates that moderate smokers may tend to have lower body weights than do non-smokers and heavy smokers (Albanes et al., 1987; Rasky et al., 1996). A 6- month study looking at the safety and efficacy of transdermal nicotine therapy on cognitive performance in nonsmokers with mild cognitive impairment (MCI) found that there was a significant decline in BW (~2.5 kg) in the nicotine treated group by day 182 compared to placebo treated controls (Newhouse et al., 2012). Consistent with smoking (and nicotine) causing a loss in

BW is the finding that weight gain is reported to be a consequence following smoking cessation among adults (USDHSS 2001; Flegal et al., 1995; Klesges et al., 1997b; Nides et al., 1994;

O’Hara et al., 1998; Perkins, 1993; Williamson et al., 1991, USDHHS, 2001). In summary, a plethora of studies in human smokers show a robust anti-obesity effect of smoking. However, it is not readily appreciated as the effect of smoking at reducing abdominal fat mass is “marginal”

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(Chen et al., 2002). Furthermore, the finding that it promotes loss of lean body mass (LBM) in animal studies (Chen et al., 2012) would limit it as a therapeutic measure to treat obesity.

Focusing on nicotine, studies show that nicotine is widely known to decrease BW (Jo et al., 2002;

Li & Kane 2003), which is accompanied by a decrease in abdominal fat (Mangubat et al., 2012).

While nicotine and cigarette smoking both cause weight loss, nicotine and smoking are not equivalent. Mangubat and colleagues (2012) state "nicotine has beneficial effects on body weight control, food intake, energy expenditure, as well as improving cognition and preventing inflammation, whereas cigarette smoking is associated with increased risk factors for cardiovascular diseases and other morbidities and mortalities."

In addition to nicotine being widely documented and accepted as a weight reducing agent

(Chiolero et al., 2008; Eisen et al., 1993; Albanes et al., 1987; USDHHS 2001), its efficacy or power has been shown to be considerable. For example, Mineur and colleagues (2011) report that

3 mg/kg nicotine (free base) administered daily for 30 days to mice adds only approximately 0.5 grams of weight over that time-span. This is in contrast to vehicle treated mice, which put on approximately 5.5 grams of weight over the same period (Mineur et al., 2011). Impressed with the wide acceptance that nicotine causes loss of BW and that the magnitude of that loss is very robust, I focused my attention on this pharmacologic intervention as Chapter II of my thesis research.

B: Review of Studies of Nicotine on Body Weight

There is a large body of scientific literature describing nicotine’s effect on BW in experimental animals. Most of the studies have been performed in rodents, and while data obtained for the most part indicate a reduction in BW, there are some exceptions. In addition, data reported raise issues about how these types of studies should be conducted.

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1: Studies in Rats

One of the earliest seminal studies reported was that of Schechter and Cook (1976) conducted in adult female rats. Treatments were as follows: (a) three daily intraperitoneal (IP) injections of saline vehicle, (b) three daily IP injections of nicotine 0.4 mg/kg (free base), and, (c) three daily IP injections of nicotine 0.8 mg/kg (free base). All daily injections were made at 8 hr time intervals over a five-wk period. While the 0.4 mg/kg dosing was not significant in lowering

BW compared to vehicle controls, the 0.8 mg/kg dosing effect proved to be statistically significant. Controls added approximately 21 g of BW whereas the nicotine treated rats lost approximately 3 g over the 5 wk testing period. No significant effect of nicotine was noted on food consumption. Pretreatment with the nAChR channel blockers hexamethonium (10mg/kg;

BID, IP) or mecamylamine (5mg/kg; BID, IP) had no effect on nicotine-induced reduction in BW and no effect per se, suggesting that ongoing activity in nAChRs blocked by these drugs plays no role in controlling normal BW (Shechter & Cook, 1976).

One of the most cited investigators to have studied the effect of nicotine on BW in experimental animals is Neil E. Grunberg, whose research extends over a 20 year period. In one of his earliest studies, Grunberg (1982) examined the effect of nicotine administration on total food consumption, sweet tasting food consumption, and BW in adult male rats. Spontaneous activity was also monitored. Nicotine (2.5, 5.0, and 10.0 mg/kg/day) was administered subcutaneously (SC) by osmotic mini-pumps for one to two weeks. The saline vehicle group exhibited significantly (p< 0.05) greater increases in BW than did all three nicotine groups. The nicotine treatment groups had decreased caloric intake, which was due to significant decreases in sweet tasting foods (specifically decreases in consumption of a sucrose solution). Activity was only increased with the 10-mg/kg/day dose of nicotine. As a follow-up study, Grunberg and colleagues (1984) determined whether SC administered nicotine infused by osmotic pumps

54 would reduce BW when rats were consuming bland food and water. While BW was reported to decrease, there were no changes in bland food or water consumption. Further studies to examine the role of activity in nicotine's effects on BW showed that the reduction in weight was not due to an increase in physical activity (Grunberg & Bowen 1985).

To establish if there were sex differences in the effect of nicotine on BW, Grunberg and colleagues (1986) using osmotic pumps administered the same three doses of nicotine

(mentioned earlier) in adult female rats. As with males, nicotine decreased normal BW, but this change was associated with reduction in bland food and water consumption, effects not observed in male rats. The investigators concluded that females are more sensitive than males to the weight reducing effect of nicotine.

In the above studies, it was not clear what form of nicotine (free base or salt) was tested in adult rats. However, in the study reported by Winders and Grunberg (1990) nicotine dihydrochloride was tested. This study examined the effects of nicotine (6 and 12 mg/kg/day via osmotic mini-pumps) in adult male rats on BC in addition to BW and food consumption.

Nicotine “attenuated body weight gains”, which occurred concurrently with decreases in fat composition, but with no change in body water or protein. Thus, nicotine appears to reduce BW by selectively reducing fat stores.

To explain the underlying mechanism whereby nicotine administration resulted in a reduction in BW, Grunberg and his colleagues (1994) proposed that nicotine causes a fall in plasma insulin levels. To test their hypothesis, plasma insulin levels were measured in both male and female adult rats that were implanted with osmotic pumps that delivered nicotine (6 or 12 mg/kg calculated as the base). In addition to BW, nicotine reduced plasma insulin in both male and female rats.

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Many of the studies of nicotine in experimental animals used doses of nicotine, which result in plasma nicotine levels that exceed that obtained by heavy smokers. This includes the studies of Grunberg described above and many other studies (Blaha et al., 1998; Miyata et al.,

2001; Faraday et al., 2001; Grunberg et al, 1986; Grunberg et al, 1987; Levin et al., 1987, Levin et al., 1993; Bishop et al., 2004). This is the case where doses of nicotine range from 5-12 mg/kg/day (see Murrin & Ferrer 1987; Li et al, 2000a). Another concern expressed about the experimental studies of Grunberg and others is that nicotine is administered chronically for 24 hours per day using osmotic mini-pumps and therefore does not mimic the intermittent use of nicotine in smokers. Additionally, constant infusion of nicotine may obviate the initial excitatory effect since, with inhalation a brief high concentration of drug is achieved quickly in the blood and brain (Benowitz et al., 2010) and is assumed to produce excitation of nAChRs. With the mode of administration using osmotic pumps, only the receptor desensitizing effect of nicotine may be present. Furthermore, constant administration of nicotine may induce up-regulation of nAChRs (Schwartz and Kellar 1983, 1985; Marks et al.,1983,1985; Flores et al., 1992; Moretti et al., 2010; Marks et al., 2011). Also, a constant administration regimen might reduce compensatory events such as increase in meal frequency (when drug-induced decreases in meal size occur) during the “off phase” of nicotine administration. Lastly, an important consideration with studies using the osmotic pump, is that most researchers focus on plasma levls. However, steady state levels of nicotine will be achieved in the brain in addition to the plasma. In other words, one needs to consider that steady state levels of nicotine will be achieved in the brain during chronic administration regardless of route of administration.

Finally, another suggested flaw in experimental design is administering nicotine to rodents during the light phase when they are not normally active (Bellinger et al., 2003a). To avoid this, Bellinger and colleagues (Bellinger et al., 2003a) using “pulsed” administration of a

56 lower range of nicotine doses. In their study, nicotine (salt form) was administered 5 times a day during the animal’s activity period (i.e., during the dark cycle) at 5 equally divided doses (2 to 4 mg/kg/day; IP/ 2 hr intervals) while monitoring FI, meal patterns, water intake, and BW loss/gain in male adult rats. The first effect noted was a reduction in 24 hr meal size that occurred on day

1, which was restricted to the dark phase. Meal number was unchanged until the fifth day of treatment, at which time it increased during the dark phase and remained increased during the rest of the of 14 day treatment period. BW gains of the nicotine treated group, relative to the vehicle treated control group, were significantly suppressed by day 6 and remained suppressed throughout the 14-day treatment period. Only the 4-mg/kg/day-nicotine dose produced statistically significant changes in meal pattern and BW. The effect of nicotine on BW was robust; vehicle treated rats gained approximately 48 g of weight over 14 days whereas nicotine treated rats gained approximately 28 g of weight over this time-period. It should be noted that even though meal frequency increased significantly over 5 days of nicotine treatment and maintained, gains in BW of the nicotine treated rats remained significantly suppressed. This suppression occurred in spite of a normalization of the 24 hour decrease in FI after day 9. The increase in meal frequency that occurred at day 5 was interpreted as due to a homeostatic compensation for the nicotine-induced reduction in meal size. These findings were confirmed by a subsequent study (Bellinger et al., 2003b) and were found to be independent of sex (Bellinger et al., 2005). Moreover, it was found that suppression of FI by nicotine was larger in rats fed a high fat diet (HFD) than those maintained on a normal diet (Wellman et al., 2005). However, the usual increase in meal number noted in rats (on normal diet) after 5 days initiation of nicotine treatment was not observed in rats on the HFD. Additionally, the ability of nicotine to reduce BW was greater in the HFD group than the normal diet group (Wellman et al., 2005).

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As noted, in the previous studies of Bellinger and colleagues, BW of rats was initially reduced because of nicotine-induced decreases in FI. Over time, FI normalized but BW remained suppressed compared to vehicle treated controls. Hence, it was proposed that nicotine must also act to increase EE and/or lower the BW set-point. To test this hypothesis, “pulsed” administration of nicotine was employed (Bellinger et al., 2010). Specifically, male adult rats were injected IP with nicotine (1.4 mg/kg/day; free base) in 4 equally spaced intervals during the dark phase for

14 days while measuring FI, BW, and EE. The nicotine dose was derived from a dose-response study of the drug (0.75 mg/kg/day and 1.4 mg/kg/day, free base) given IP at 4 separate intervals during the dark phase (Kramer et al., 2007). BW was reduced starting on day 3 of treatment and remained reduced during the study-period; FI was reduced at day 7 of the 14 days of nicotine administration. No effect of nicotine was observed on EE. In addition, a pair-fed group of rats was studied in parallel with the vehicle treated and nicotine treated rats, each receiving equal amount of food. At the end of the study, both the pair-fed and nicotine treated groups had similar

BW gain values but were less than the vehicle treated groups. This latter experiment shows that the BW loss of nicotine treated rats was due to lowered FI. The authors make the interesting point that during FI suppression, animals react by breaking down energy stores, which would contribute to their weight loss. Consistent with this point was the finding in the authors’ study that the daily dark and light phase respiratory quotient (RQ) of the nicotine treated rats was significantly reduced. Also, consistent with this point are data of others showing that nicotine increases plasma free fatty acids in rats, which is dependent on the presence of the adrenal medulla (Bizzi et al., 1972). Focusing on the EE data of Bellinger and colleagues (Bellinger et al., 2010), nicotine given during the dark phase for 14 days did not affect the 24 hour EE compared to the vehicle treated rats. EE is used interchangeably with the term metabolic rate, and the authors state that the results “strongly suggest that the significant weight loss experienced by

58 the nicotine injected rats can be attributed to the suppression of FI by nicotine and not by a significant increase in metabolic rate. ” (Bellinger et al., 2010). Consistent with these observations are the data of others that show a lack of effect of nicotine on metabolic rate

(Schwid et al., 1972; Wager-Srdar et al., 1984; and Bishop et al., 2004). However, a recent study in adult male rats indicates that nicotine treatment does increase metabolic rate, i.e. EE (Martinez de Morentin et al., 2012). In this study, nicotine given (2mg/kg/ 12 hr, expressed as the salt; SC) over a 48 hr period increased EE and reduced RQ thus creating a state of negative energy balance compared with vehicle-treated rats. Associated with these metabolic changes were reduced FI and increased locomotor activity. Other notable effects were a short-term respiratory depressant effect after the nicotine injection, and a rise in body temperature (~ 0.7 ° C) at the 48 hour time- point after initiation of nicotine treatment. This rise in body temperature, to me, is unexpected because in surveying the scientific literature on temperature effects of nicotine, all papers report data showing the opposite effect, i.e. hypothermia (13 papers surveyed).

In a second study, by the above same group of investigators (Seoane-Collazo et al.,

2014), nicotine at the same dose was tested in adult male rats that were fed a HFD. Data obtained indicated that nicotine reduced BW, FI, and fat mass in diet induced obese rats (DIO). There were no changes in lean body mass (LBM).

In summary, early studies of nicotine in adult rats indicate that nicotine administration causes a loss in BW, which in male rats is usually not related to a decrease in FI or an increase in physical activity (Schechter & Cook 1976; Grunberg & colleagues series of studies). The reduction in BW is associated with a reduction in fat stores and plasma insulin; the reduction in fat stores may be related to the reduction in insulin. Pretreatment with the nAChR antagonists, mecamylamine or hexamethonium does not block the nicotine effect on BW, while the antagonists per se had no effect on BW. Doses of nicotine found to be effective were 0.8 mg/kg

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(free base) given IP 3 times a day and constant infusions of nicotine in a range of 6-12 mg/kg/day. Later studies, highlighted by the series of investigations of Bellinger and colleagues over a span of about 10 years, emphasized the importance of good experimental design in assessing nicotine’s effect on BW. In their view, the best design was to employ IP doses of nicotine that mimic the pattern of blood concentrations observed in smokers. To do this, they used “pulsed” administration of nicotine during the period of highest animal activity (dark phase) while monitoring BW and meal patterns. Data from their studies revealed: (1) BW gains of nicotine treated rats relative to the vehicle treated control group were suppressed; (2) suppression of BW was completely explainable by nicotine's effect on reducing meal size; and, (3) these effects of nicotine also occur in DIO rats. In the most recent studies in rats, Lopez and colleagues

(Martinez de Morentin, et al., 2012; Seoane-Collazo et al., 2014) also describe that repeated doses of nicotine (2 mg/kg/12 hour of the salt; SC) decreases FI but also increases EE (an effect not seen by Bellinger and colleagues). Of note, in the first study reported by Lopez and colleagues, toxicity was observed with initial dosing of nicotine.

2: Studies in Mice

All of the data described thus far have been obtained in adult rats. Focusing on data obtained in mice, clearly the most impressive data on the efficacy or power of nicotine to decrease BW is found in the paper published by Picciotto and colleagues (see Figure 1 of Mineur et al., 2011). Adolescent male mice, when treated with saline vehicle (controls), grew from a BW of ~20.7 g to ~26.4 g over a 30 day period (an increase ~ 5.7 g). In contrast, mice injected daily with nicotine (3 mg/kg of the base; IP) grew from ~ 20.3 g to ~20.8 g (an increase of ~0.5 g).

Thus, nicotine administration essentially prevented any additional weight from being added during a one-month period. According to data from Lopez’s group (Martinez de Morentin et al.,

2012), 2 mg/kg nicotine (as the salt ) given SC causes toxicity in rats. No mention of toxicity is

60 alluded to in the Mineur et al., paper. It should be noted that nicotine is metabolized much faster in the mouse than in the rat (Matta et al., 2007) and therefore it is possible that the mouse can tolerate a higher dose of nicotine than the rat without exhibiting adverse effects. It should be noticed that in the Mineur et al., study, the non-selective nAChR antagonist mecamylamine was also evaluated on BW in adolescent male mice. Mecamylamine (1 mg/kg; IP) given for 30 days had no significant per se effect on BW compared to vehicle control animals. This is consistent with the earlier finding of Schechter and Cook (1976) in the rat.

Picciotto’s group also published a second study using mice (Fornari et al., 2007). Eight- week-old male mice were placed on either a low-fat diet (10% fat, usually referred to as a control diet) or a HFD (60% fat). The effect of orally administered nicotine (given in the drinking water) was tested on BW and FI over a period of 16 weeks in both groups of mice. Mice on low fat diet

(LF) exhibited a significant nicotine-induced decrease in BW gain over the 16-wk period compared to vehicle controls. Initial weight and final BW of vehicle controls were 22.1 + 0.5 g and 32.4 + 0.6 g, respectively. Initial weights and final BW of nicotine treated mice were 22.5 +

0.5 g and 29.9.4 + 0.5 g, respectively. Nicotine's effect on FI was not provided. However, when nicotine was tested in mice on the HFD, surprising and unexpected findings were reported. Mice on the HFD showed the expected increase in BW with respect to LF fed mice, and nicotine treatment initially accelerated this process. In addition, FI in nicotine treated HFD mice was significantly increased in relation to both the LF fed mice and vehicle treated HFD mice in the first week of treatment. This surprising accelerated increase in weight gain and FI that was observed initially in nicotine treated mice did not persist. At the 11 wk time-point, the BW and FI of nicotine treated mice fed a HFD compared to their vehicle treated cohorts did not differ. As will become clearer later, in this review, nicotine induced acceleration of weight gain, while FI increased has never been reported. In addition, in all studies reviewed, the BW reducing effect of

61 nicotine is always observed in rodents fed a HFD. Indeed, the effect of nicotine on reducing BW has been reported to intensify on a HFD (Hur et al., 2010, Mangubat et al., 2012). Specifically,

Mangubat and colleagues state that decreased caloric intake accounted for all the weight loss seen in animals fed a standard laboratory diet (non-fat) and only 66% of the weight loss in animals on a HFD was due to a decreased FI (Mangubat et al., 2012). This suggested to the authors that the intensified weight reducing effect of nicotine on animals on a HFD involves potentially increasing EE in addition to lowering FI, creating a greater weight reductive effect of nicotine on animals fed a HFD.

The nicotine related increase in FI of mice fed a HFD as reported by Picciotto’s group

(Fornari et al., 2007) is not supported by others. Hur et al. (2010), studying the effect of nicotine in mice on HFD, report that FI together with BW is significantly decreased compared to vehicle controls. In their study, adult male mice on HFD (44.9% fat) were treated with nicotine (1.5 mg/kg of the free base; SC) twice daily (total nicotine 3 mg/kg/day) for 2 wk. However, significant changes in FI or BW were not observed when nicotine was administered to mice on a normal fat diet (10.0 % fat; NFD). Neither were changes observed when nicotine (1.5 mg/kg; SC) was administered once per day. Hence, nicotine not only reduced BW in mice on the HFD, but also its BW reducing effect was intensified compared to its non-effect in mice on a NFD.

Interestingly, as in the case of Bellinger and colleagues’ results in rats, the effect of nicotine on

FI was observed at the initiation of treatment and waned after this early effect. EE was also measured in this study and was significantly increased following nicotine treatment for 14 days in the HFD group compared to mice in the non-fat control group; this was not the case in mice in the NFD group. Moreover, no effect of nicotine on RQ was observed in either the HFD or the

NFD groups. The authors concluded that the nicotine effect to reduce BW in obese mice resulted from both a decrease in FI and the increase in EE.

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The most recent study of nicotine on BW in mice is that of Friedman’s group (Mangubat et al., 2012). Male adult mice were placed on either a normal fat diet (NFD; 5% fat) or a HFD

(34.9 % fat). Both groups received twice daily nicotine injections of either 0.5 mg/kg or 1.5 mg/kg (liquid form of nicotine; IP). In addition, an escalating dose of nicotine starting at 0.25 mg/kg per day, given twice daily and increased to 4.5 mg/kg/day by an increment of 0.5 mg/kg/day every 3 days was administered. Mecamylamine (2 mg/kg/day; IP; a non-selective nicotinic antagonist) and varenicline (2 mg/kg/day; IP; an α4β2 nAChR partial agonist) were also tested concurrently with nicotine in the mice on a HFD. The 1.5 mg/kg nicotine dose in contrast to vehicle controls decreased the weight gain in mice on both diets. However, the decrease in weight gain due to nicotine was greater in the HFD group compared to mice in the NFD group.

Both the 1.5 mg/kg and the escalating doses of nicotine exhibited decrease in weight gain. No effect was observed with the 0.5 mg/kg nicotine dose. Mecamylamine completely and varenicline partially antagonized the weight reducing effect of nicotine on mice fed a HFD. The 1.5 mg/kg dose but not the escalating dose of nicotine significantly decreased FI. Additionally, nicotine significantly decreased visceral fat in the mice on the HFD.

In summary, nicotine effects in mice are similar to those reported for rats in that the drug decreases BW (Mineur et al., 2011; Fornari et al., 2007; Mangubat et al., 2012) and FI (Mineur et al., 2011; Mangubat et al., 2012) in mice on a normal diet. In 2 of the 3 studies, nicotine was reported to evoke a more robust effect in mice on a HFD (Hur et al., 2010; Mangubat et al., 2012) and FI was also reduced. In one of the 3 studies, no decreases in BW or FI were observed

(Fornari et al., 2007). This was the study where nicotine was administered in the drinking water

(Fornari et al., 2007). In 2 studies, doses of nicotine that were ineffective were revealed. These doses were 1.5 mg/kg given SC once a day (Hur et al., 2010) and 0.5 mg/kg IP given twice a day

(Mangubat et al., 2012). Finally, studies with mecamylamine indicated that this drug prevents

63 nicotine-induced effects on BW and FI (which is in contrast to what has been reported in the rat studies of Schechter and Cook, 1976). But like the finding of Schechter and Cook in the rat, mecamylamine, per se, has no effect in the mouse.

C: Nicotine Can Intervene at Different Components of the Energy Balance System to Reduce

Body Weight

In general terms, intervening at several different levels can regulate energy balance. In this section, I will focus on mechanisms that include nicotine-induced changes at the level of FI

(i.e., energy intake) that depends on meal size (MS) as well as meal frequency (MF). As well, I will draw attention to energy expenditure (EE), another level where nicotine could be working to influence changes in energy balance. EE includes changes in basal metabolic rate (BMR), adaptive thermogenesis and locomotor activity.

1: Food Intake

In laboratory rodents fed a standard diet of 5% fat, both acute and chronic nicotine treatments result in a decrease in BW that is associated with hypophagia (decrease in FI) predominantly due to a decrease in MS or an increase in satiation (Bellinger et al., 2003a;

Bellinger et al., 2005; Kramer et al., 2007a; Bellinger et al., 2009; Blaha et al., 1998; Bray 2000;

Grunberg et al., 1986). Bellinger et al. (2003a) specifically showed, using computerized meal pattern analyses (MPA), that adult rats treated for two weeks with either saline or nicotine (2 or 4 mg/kg/day) spread over five equal amounts during the dark phase exhibit consistent changes in meal patterns. MPA analyses revealed the FI reduction on the first day of nicotine treatment causes a lasting decrease in dark phase MS. Several days later (day 5) of nicotine treatment, the rats compensated by significantly increasing the number of meals they took, which tended to normalize the dark phase FI. Congruently, dark phase inter-meal-interval was also decreased.

More importantly, these changes in meal pattern persisted for two weeks upon cessation of

64 nicotine treatment. Interestingly, other investigators have also shown that even after FI had returned to normal in nicotine treated animals that had initially displayed hypophagia, their BW remains significantly lower than the saline group after FI is normalized (Levin et al., 1987; Arai et al, 2001; Bellinger et al 2003a; Guan et al., 2004; Bellinger et al., 2005; Kramer et al., 2007).

2: Energy Expenditure

Although numerous rodent studies have provided convincing evidence for the inverse association between nicotine exposure and food intake (Martinez de Morentin et al., 2012;

Bellinger et al., 2009; Grunberg et al., 1987; Levin et al., 1987), there are, however, studies showing that nicotine either has no effect or only a transient effect on FI despite claims of lowering BW (Schechter & Cook 1976; Grunberg et al., 1984; Levin et al., 1987; Arai et al.,

2001; Bellinger et al., 2003a; Guan et al., 2004; Bellinger et al., 2005; Kramer et al, 2007a).

Therefore, mechanisms underlying EE would be expected to come into play in these studies.

In terms of parameters of EE, the literature is also unsettled on the effects of nicotine on basal metabolic rate (BMR) with numerous studies showing that nicotine administration either has no effect or decreases it (Perkins et al., 1992b; Dill et al., 1934; Hiestand et al., 1940; Hadley 1941;

Goddard & Voss 1941; Evans & Stewart 1943; Ilebekk et al., 1975; Wager-Srdar et al., 1984;

Robinson & York 1986; Stamford et al., 1986; Perkins et al., 1990; Schwid et al., 1992; Collins et al., 1994; Bishop et al., 2004).

In a study by Martinez de Morentin et al. (2012), nicotine was found that in addition to lowering feeding, nicotine treated rats over a 24 and 48 hr period, showed a significant increase in body temperature associated with higher EE compared with vehicle treated rats. Specifically, data from this study showed that nicotine treated rats displayed a marked increase in skin temperature surrounding the interscapular brown adipose tissue (BAT) indicating an increase in thermogenesis. Consistent with this, nicotine treatment for 48 hr increased the expression of

65 thermogenic markers in the BAT such as Uncoupling Protein 1 (UCP-1). In addition, they found that nicotine treated animals had an increase in dark phase physical activity.

D: Mechanisms of Nicotine Mediated Changes in Food Intake and Energy Expenditure

1: nAChR subtype

The relevant nAChR subtype involved in nicotine mediated reduction in FI, can be best understood in the context of the previously mentioned study of Mineur et al. (2011). In their study, they report that the mechanism responsible for the ability of nicotine to reduce FI in a mouse model is through central activation of the nAChR subtype, α3β4. This nAChR subtype was in their view located on POMC neurons within the arcuate nucleus of the hypothalamus

(ARC). To demonstrate this, Mineur et al. (2011) tested the nicotine related drug cytisine (a full agonist at α3β4 nAChR but has weaker effects at other nAChRs; (Luetje & Patrick 1991; Papke

& Heinemann 1994). They found that cytisine decreased weight gain over time, and the hypophagia mediated by it was blunted by pretreatment with the noncompetitive nicotinic antagonist mecamylamine. Furthermore, Mineur et al. showed that knockdown of the β4 nAChR subunit in the ventral hypothalamus blunted the ability of cytisine to decrease FI but they did not show that this was also true for nicotine. As it is well known, cytisine is selective for α3β4, hence, the β4 knockdown does not provide any information as to how nicotine works but rather confirms that cytisine works through α3β4 to cause hypophagia. Using various methods, Mineur et al. (2011) present data to determine how nicotine decreases FI in the arcuate nucleus (ARC).

Specifically, they state that when nicotine reaches the ARC (an area devoid of blood brain barrier), activity of POMC neurons increases through activation of α3β4 nAChRs located on these neurons. This subsequently activates an Mc4R located on a second order neuron in the

PVN. This effect in turn results in inhibition of FI. To demonstrate the role of Mc4R in nicotine-

66 mediated hypophagia, the receptor was knockdown by AAV-shRNA specifically in the PVN, which significantly counter-acted the effect of nicotine to reduce FI.

Furthermore, the study by Martinez de Morentin et al. (2012), discussed in more detail later on, state that the nicotine’s action to increase EE (increase thermogenesis) via hypothalamic pathways is also mediated by α3β4 nAChR, which would also be consistent with the findings of

Mineur et al. who used the more selective α3β4 nicotinic agonist (cytisine). However, it is critical to note that in both studies, the evidence that nicotine and cytisine are working through

α3β4 nAChRs is based on co-treatment with mecamylamine, a non-selective antagonist at α3β4 receptors. Thereby it is conceivable that because mecamylamine is non-selective, the blunted nicotine induced weight loss seen after mecamylamine reported by Martinez de Morentin et al., could well be due to another subtype of nAChR.

Despite the fact that the previously mentioned studies implicate a role for the α3β4 nAChR in mediating nicotine mediated anorexigenic effects, it is worth noting that the nAChR most closely associated with nicotine addiction is the α4β2* subtype, as it predominates within brain reward circuits that are the targets of drugs of abuse (Marks et al., 1992; Zoli et al., 1992;

Picciotto et al., 1998)The α4β2 nAChR subtype is also involved in mediating nicotine-stimulated dopamine release (Rapier et al., 1988; Rowell & Wonnacott 1990; Grady et al., 1992; Grady et al., 1994).. Consistent with this line of thinking, Hussmann et al. (2014) in a recent study showed that in a 2-phase treatment design using Alzet osmotic mini-pumps, rats that were previously on nicotine during the 1st phase of the treatment gained significantly less weight than the animals on saline. When the nicotine treated animals were switched over to Sazetidine A (a partial to full agonist at α4β2* nAChR depending on receptor subtype stoichiometry) during phase 2 of treatment, they gained weight at a slower rate than controls and ended up weighing 11% less than

67 the controls at the end of the 4-wk treatment period (Hussmann et al., 2014). Additionally, Saz-A potently and selectively desensitizes α4β2* nAChR (Xiao et al., 2006).

Another study emphasizing a role for the α4β2 nAChR, as well as the α7 nAChR, is the study by Huang et al., (Huang et al., 2011) using voltage and current clamp whole-cell recording techniques to study the actions of nicotine on POMC and NPY neurons in hypothalamic slices.

They show that the depolarizing effect of nicotine on the membrane of POMC neurons is significantly decreased by methyllycaconitine citrate (MLA) an antagonist at the α7 nAChR.

Antagonism was also observed with dihydro-β-erythroidine hydrobromide (DHβE), an antagonist at the α4β2 nAChR suggesting that both α7 and α4β2 exist on these neurons and are potentially mediating the nicotine induced depolarization. The authors go on to suggest that depolarizing actions could explain the mechanisms of the weight loss caused by nicotine.

Marrero and colleagues (2010) probed the role of the α7 homomeric nAChR in controlling BW. These investigators tested the effects of a highly selective novel α7 nAChR agonist (TC-7020) given orally on body mass, glucose levels, lipid metabolism and pro- inflammatory cytokines in a mouse model of type 2 diabetes (leptin-resistant db/db obese mouse). Specifically, Marrero et al. (2010) found that weight gain was significantly reduced in the α7 agonist-treated db/db obese mice after 7 weeks of drug treatment compared to vehicle treated controls. This effect was characterized by a reduction in FI. These changes were reversed by the α7-selective antagonist methyllycaconitine, confirming the involvement of α7 nAChRs

(Morrero et al., 2010). Oral administration of TC-7020 also reduced elevated glucose levels; glycated hemoglobin levels, lowered elevated plasma levels of triglycerides, and suppressed the pro-inflammatory cytokine tumor necrosis factor-α. In summary, α3β4 and α4β2* heteromeric nAChRs and the homomeric α7 nAChR have all been implicated in the ability of nicotine to

68 exert BW reducing effects on FI and EE. The studies implicating pertinent subtypes of nAChR involved in nicotine-influenced changes in energy balance are listed in Table 2.

Table 2: Studies highlighting the role of nAChRs in food intake and body weight. nAChR Subtype Year Study Evidence

α3β4 2011 Mineur et al., -Cytisine, Mecamylamine 2012 Martinez de Morentin et al., -Mecamylamine α4β2* 2011 Huang et al., -DHβE 2014 Hussman et al., -SazA α7 2011 Huang et al., -Methyllycaconitine citrate (MLA) 2010 Marrero et al., -TC-7020, MLA

2: Role of nAChR Desensitization

As mentioned previously, the studies of Grunberg and colleagues used the osmotic pump method to chronically deliver nicotine. This method allows for precise and controlled drug delivery with a pre-determined amount of drug continuously delivered every hour. While the nicotine build up is slow and steady with this route, it does not mimic the initial excitatory effect seen with nicotine inhalation where a brief high concentration of drug is achieved quickly in the blood and brain (Benowitz et al., 2010). Additionally, with this mode of administration, nicotine effect may extend only to receptor desensitizing whereas the faster IP or intravenous routes

(would most likely mimic inhalation of nicotine in a smoker), and would be expected to produce nAChR activation. As is generally accepted, nicotine can both activate and desensitize nAChRs

(Del Castillo & Katz 1955; Katz &Thesleff 1957), and an ongoing debate in the field has always been to what extent receptor desensitization is involved in various behavioral processes such as

FI and BW regulation. During exposure to nicotine, neuronal nAChR desensitize rapidly, becoming unresponsive for a period of time, and is seen in virtually all neuronal nAChR that 69 have been examined. In the desensitized state, the nAChR have a much higher affinity for agonists than they do in their resting/activatable state (deCosta and Baenziger 2009). Moreover, in the prolonged presence of agonist such as with chronic nicotine exposure via the osmotic pump method, receptors are recruited into the desensitized state. Of course, the differences in the potency of nicotine to desensitize nAChRs compared to activating them depends on the receptor subtype (Kellar and Xiao, 2007). For example, it has been shown that in a human embryonic kidney cell line, nicotine is more potent in desensitizing than in activating α3β4 nAChRs (Meyer et al., 2002). In mammalian cells, nicotine was more potent in desensitizing than in activating

α3β4 and this potency was much higher at the α4β2 (Paradiso and Steinbach 2003). Because nicotine and most other nAChR agonists have a higher affinity for α4β2 than α3β4 nAChR subtypes, behaviors mediated by the α4β2*, which are the predominant subtype in the CNS, are more likely to be effected by desensitization than the α3β4. The rate of onset of desensitization versus activation by agonists is concentration dependent and also varies among receptor subtype.

In vivo, nicotine is 5 times more potent in desensitizing nicotine-induced prolactin release in the rat than in stimulating the response (Hulihan-Giblin et al., 1990).

More importantly, Lu and colleagues (Lu et al., 1999) have shown that nicotine agonists will stimulate the release of GABA from synaptosomes at concentrations that are less than those required to activate the nAChRs (sub-activating concentrations of agonist). Due to slow build up of nicotine via certain routes of administration such as that with the osmotic pump or the doses taken in by a chronic smoker, it is possible that sub-activating concentrations of nicotine may be responsible for chronically keeping the receptor in a chronic desensitized state. The α4β2* nAChR subtype at the nicotine doses taken in by a chronic smoker is probably desensitized all the time except for the overnight abstinence from smoking where a significant fraction would be expected to recover function. It is also worth noting that during the periods of α4β2*

70 desensitization, the α3β4* receptors that desensitize less readily (or recover more quickly) may offer compensatory function in terms of modulating neurotransmitter release. Depending on where this desensitized nAChR is in the CNS will impact how the flow of endogenous neurochemical transmission is disrupted and/or altered. Therefore, having the receptor kept in a chronically desensitized state, could have serious implications for nicotine mediated effects on feeding and BW regulation.

3: AMP-Activated Protein Kinase (AMPK) and the Melanocortin System

While the previous section focused on the role of different subtypes of nAChR involved in the anorexigenic response to nicotine, as well as the role of nAChR desensitization, this section attempts to delineate the precise neuronal populations and cellular mechanisms involved in the ability of nicotine to lower BW. Previously, I had mentioned that the study by Mineur et al.

(2011) showed an important role for the melanocortin system in the ability of nicotine to reduce

FI. Knockdown of the melanocortin Mc4R receptor blunts the nicotine-mediated hypophagia

(Mineur et al., 2011). With the prominent role that the melanocortin system plays in feeding and

EE, and the consistency of studies showing an inverse association between cigarette smoking and

BW, many investigators sought to elucidate the effect of both chronic and acutely administered nicotine on the expression profiles of various feeding associated peptides working through the melanocortin system. Peptides extensively looked at in the literature include (but not limited to) the orexigenic peptides NPY, AgRP, POMC, CART and leptin. As shown in Table 3, the majority of studies report that nicotine administration results in an increase in POMC expression.

Also, as can be seen from the lists of studies presented in Table 3, the literature is unclear on what effect nicotine has on the expression profiles of NPY and AgRP. The discrepancies in the data related to these neuropeptides could be due to differences in nicotine dose and/or method of nicotine administration, or general limitations inherent to expression profile studies.

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If one accepts the fact that nicotine increases activity in POMC neurons to exert a weight reducing effect, the question now becomes how does nicotine increase POMC gene expression

(i.e., activate the melanocortin system?) In other words, what is the missing link between nicotine and the melanocortin system? The answer may lie in the cellular energy sensor 5' AMP-activated protein kinase or AMPK.

The previously mentioned Martinez de Morentin et al. (2012) study demonstrated that nicotine induced weight loss is associated with inactivation of hypothalamic AMPK. This is a cellular energy sensor whose activity is regulated by the ratio of adenosine monophosphate/adenosine triphosphate (AMP/ATP) inside cells and by upstream kinases.

AMPK activity can also be regulated by physiological stimuli, independent of the energy change of the cell, including hormones such as leptin, adiponectin, resistin, ghrelin and cannabinoids

(Kahn et al., 2005; Kola et al., 2005; Hardie et al., 2008). The diversity of biological effects mediated by AMPK includes coordinating context-specific metabolic responses in many tissues, as well as playing a role in both physiological (feeding) and pathophysiological (ischemic) states.

Specifically, AMPK has been shown to be activated by stressors that deplete cellular ATP thus increasing the ADP/ATP ratio (Corton et al., 1994). Once AMPK is activated, it restores energy homeostasis by switching on catabolic pathways that generate ATP (fatty acid oxidation and autophagy), and switching off anabolic pathways that consume ATP such as gluconeogenesis and lipogenesis (Hardie et al., 2012, Hardie et al., 2012). It is found essentially in all eukaryotes and expressed ubiquitously in the brain and virtually every other organ system (Shirwany and Zou

2014).

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Table 3: Evidence for Involvement of Feeding-Associated Neuropeptides in Nicotine-Mediated

Effects on Body Weight

It is worth noting that inhibition of AMPK increases POMC expression in the ARC of nicotine- treated rats and it decreases the expression of orexigenic neuropeptides such as AgRP and NPY

(Martinez de Morentin et al., 2012). Tying together the role of AMPK in energy balance with the melanocortin system, it has been proposed that stimulation of the melanocortin system inhibits

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AMPK activity in PVN as shown by the Mc4R receptor agonist Melanotan-II. This is in contrast to the orexigenic facet of the melanocortin system (with AgRP) whose stimulation increases

AMPK activity (Minokoshi et al., 2004). Thus, it appears that alterations in AMPK activity in the

PVN are mediated through the melanocortin system. Specifically, Minokoshi et al. (2004) state that leptin stimulates POMC neurons in the ARC to inhibit FI by suppressing AMPK activity in the ARC and PVN. The first route of this circuit is that leptin inhibits AMPK activity in

NPY/AGRP neurons by suppressing their activity and subsequent neuropeptide gene expression.

This leads to activation of Mc4R receptor signaling in the second-order PVN neurons. Mc4R activation decreases AMPK activity in the PVN, which probably further enhances the neurotransmission required for suppression of FI and reduction in BW. Furthermore, re-feeding and leptin administration fail to inhibit AMPK activity in the PVN in the Mc4R-/- mouse

(Minokoshi et al., 2004). In summary, AMPK represents a potentially important link in how nicotine is able to increase POMC gene expression that ultimately causes a reduction in FI and

BW.

Although much of the research of the role of AMPK in energy balance has been in the hypothalamus, several studies also indicate that the brainstem nuclei are important for AMPK- melanocortin interactions that effect FI and BW. Studies looking at the role of AMPK in energy balance in the nucleus of the solitary tract (NTS) reveal that energy state alters hindbrain AMPK activity. Specifically, it has been shown that energy status (food deprived vs. ad libitum-fed or re- feeding after deprivation) in freely moving rats alters AMPK activity in NTS-enriched lysates

(Hayes et al., 2009). It has also been shown that leptin induced FI inhibition is mediated by

AMPK activity in the dorsal vagal complex (Hayes et al., 2009). In particular, pharmacological inhibition of hindbrain AMPK activity by intracerebral ventricular infusion of the AMPK inhibitor, compound C, suppresses FI and gain in BW. Additionally, the intake-reducing effects

74 of hindbrain leptin infusion was mediated by AMPK signaling. Evidence for this was obtained by increasing hindbrain AMPK activity with 4th ventricle infusion of AICAR. The presumed increase in AMPK activation reversed the suppression of FI by hindbrain leptin delivery demonstrating a role for the AMPK activity. Thus it was suggested that the dorsal vagal complex

(DVC), in addition to the hypothalamus, is important for energy balance (Hayes et al., 2009).

In summary, the relevant brain circuits and cellular mechanisms (as well as neuropeptide markers) that have been proposed to describe mechanistically how nicotine works to reduce FI and BW include an interaction between AMPK and the central melanocortin circuit.

4: Interactions between Homeostatic and Reward Systems

Lastly, it is worth noting that due to the abundance on nAChRs in the brain, a conceptualization of how nicotine regulates whole-body energy balance is gaining attention. Is nicotine working on two complex and possibly intertwined brain circuits either in series or in parallel? These circuits comprise a hypothalamic homestatic circuit and a hedonic cortico-limbic- striatal circuit. It is well accepted that nicotine powerfully affects brain reward systems (DeBiasi

& Dani 2011). However, it is unknown which nAChR subtype (s) or circuits specifically mediate the nicotine-enhancing effects. It is also possible that nicotine effects on these two systems could have opposing outcomes or in some way alter the balance between the homeostatic and reward systems. Therefore, it may be possible that under conditions whereby the hedonic system is dissociated from the homeostatic system, nicotine treatment may result in a more amplified effect in the homeostatic system, possibly leading to an even greater anti-obesity effect. Such a result would portray nicotine as a more powerful anti-obesity drug.

E: Hypotheses To Be Tested

The abundance of studies documenting an inverse association between BW and nicotine in both smokers and in animal studies compelled me to study the role of nicotine as a

75 pharmacological intervention to treat obesity. Mechanistically, the literature shows that nicotine may be working on multiple subtypes of nAChR to reduce BW. As well, the literature is replete with inconsistencies concerning nicotine administration and the expression of critical neuropeptides involved in energy balance. In addition, only one study has documented a role for nicotine-induced changes in AMPK activity concomitant with an increase in POMC expression.

These reasons, along with my belief that more studies need to be done to elucidate mechanistically how nicotine works to exert its effect on BW led me to propose the following hypotheses:

Hypothesis 1: Nicotine-induced weight loss is mediated through the melanocortin system.

Hypothesis 2: Nicotine-induced slowing in weight gain is mediated through both activation and desensitization of the α4β2* nAChR subtype

Hypothesis 3: Nicotine-induced slowing in weight gain is mediated through a mechanism involving inhibition of hypothlamic AMPK.

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II: MATERIALS AND METHODS

A: Animals

All experimental procedures were performed on male homozygous loxTB Mc4r -/- mice, male

C57BL/6J mice, and male β2 knockout mice (β2-/- mice). The animals used in the 30-day intraperitoneal injection (IP) once daily nicotine study were are as follows: 6 week old male homozygous loxTB Mc4r (-/-) mice (n=5) and age-matched C57BL/6J (n=16) mice; adult 14-20 wk Mc4r (-/-) mice (n=12), and age-matched C57BL/6J (n=16) mice. The animals used in the osmotic pump implantation studies were 14-20 wk homozygous loxTB Mc4r (-/-) mice (n=24) and age-matched C57BL/6J (n=24) mice. The animals used in the 21-day twice-daily subcutaneous injection (SC) of nicotine study were 12 wk old C57BL/6J (n=39). The animals used in the non-drug study (weight monitoring) were adult β2-/- mice and age-matched

C57BL/6J mice. As mentioned in Chapter I, all homozygous mice used in these studies exhibited severe obesity due to a loxP-flanked transcriptional blocking (loxTB) sequence that prevents normal endogenous gene transcription and translation from the endogenous locus. As such, all homozygous mice used in this study are devoid of functional mRNA in all tested regions of the brain that endogenously express Mc4r. The Mc4r (-/-) mice were reared in an in-house breeder colony at Georgetown University, and were born from the same homozygous breeder pair purchased from Jackson Laboratories (Bar Harbor, Maine, United States.) Control C57BL/6J mice were all purchased from Jackson Laboratories. The β2-/- mice were born from homozygous breeder pairs generously provided by researchers at the University of Colorado (Boulder) and were reared in an in-house breeder colony at Georgetown University. All animals used in this study were housed singly under a 12-hr light/ dark cycle under controlled temperature and humidity except for the animals in the 21-day SC injection study that were housed under a reverse 12-hr light/dark cycle (lights on at 7 PM) under controlled temperature and humidity.

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Mice were given at least 7 days to acclimatize to the reverse light cycle and all animals were acclimatized to single housing. All animals had ad libitum access to food and water throughout the duration of the study. The Georgetown University Animal Care and Use Committee,

Washington, DC, approved all animal procedures in accordance with the National Institutes of

Health guidelines for the care and use of animals in research

B: Drugs

Nicotine (bitartrate salt) was purchased from Sigma Chemical Co., USA. Sazetidine-A dihydrochloride (Saz-A) was synthesized by RTI International (Research Triangle, NC) and supplied by the National Institute on Drug Abuse (Rockville, MD). Dihydro-β-erythroidine hydrobromide (DHβE) was purchased from Tocris Bioscience, Ellisville, MO. All drugs were constituted in phosphate buffered saline (pH 7.4) and injected either IP or SC (depending on the study), as well as administered subcutaneously via osmotic mini-pump (Alzet, Durect Corp.). All doses of nicotine bitartrate were calculated as the free base. More than one person weighed out the necessary nicotine (salt) to ensure against user error. In addition, another individual was present to oversee the individual weighing of nicotine and ensure that the proper amount was being weighed on the scale.

C: Chronic Nicotine via Daily Intraperitoneal Injection (IP)

For the first nicotine study (30-day), C57BL/6J mice or Mc4r (-/-) mice were randomized into the various treatment groups (n=5-10/group) and injected with saline or nicotine (0.5-3 mg/kg;

IP). A 3 mg/kg was administered for the first 3 days of the study. However, due to the severe adverse affects (convulsions, tremors, ataxia, sedation, straub-tail), the dose of nicotine was lowered to 1 mg/kg but ataxia and sedation was still observed. Hence, the nicotine dose was further reduced to 0.5 mg/kg. Because there was tolerance observed with this dose, nicotine was increased back to 1 mg/kg. This latter dose was the dose used for the remainder of the study.

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Mice were pre-handled for at least three days upon arrival and injected daily for 30 days between

4 and 5 PM.

D: Osmotic Pump Priming for Chronic Delivery of Nicotine and Sazetidine A (SazA)

The Alzet osmotic mini-pumps used in these studies were purchased from (Durect Corp.,

Cupertino, CA). The model used (#2002) had delivery rate of 0.5 µl/hr over a 14 day at a drug delivery period. The osmotic pump is powered by the osmotic difference between the pump and the body fluid of in an animal. When implanted into an animal, the interstitial fluid enters the pump via a semi-permeable membrane that encloses the impermeable reservoir of the pump. The osmotic difference developed between the interstitial fluid and the salt concentration of the pump will cause an expansion of the salt layer in the pump which compresses the pump reservoir where the drug is contained, thus ejecting the drug under pressure out of the delivery portal at a controlled rate. Generally, the osmotic pump has a start-up gradient where the pumps absorb fluid and fluid increases to the in vivo temperature of the animal. During this equilibration period, the pumps are slowly building up to their actual drug release rate. This process should automatically occur in vivo but to ensure steady-state delivery after implantation, each pump was

“primed” overnight prior to implantation. This involved filling each pump with its respective drug, then allowing for it to stand overnight in a petri dish of sterile 0.9% saline at 37°C.

E: Osmotic Pump Implantation for Chronic Delivery of Nicotine and Sazetidine A (Saz-A)

All animals were anesthetized with isoflurane and a dorsal skin incision (~1 cm) was made over the cervo-thoracic spine and the skin bluntly separated from the underlying tissues to form a pocket above the right scapular area. An Alzet osmotic mini-pump was inserted into the pocket and the skin closed with Vicryl (5-0). At the time of pump replacement, the skin was incised at the same site, and the previously implanted pump was removed. A new pocket was then made over the left scapular area in which and a new pump (same model as before) was inserted. The

79 skin was then sutured closed as described before. Each pump was filled with either sterile saline

(0.9% NaCl; SAL) or nicotine bitartrate (NIC) dissolved in sterile saline. Animals were implanted with either the nicotine-containing mini-pump or the saline vehicle-containing mini- pump. Nicotine was administered via the osmotic mini-pumps at three different consecutively increasing doses of: 18 mg/kg/day, 36 mg/kg/day and 72 mg/kg/day. My starting dose of nicotine at18 mg/kg/day is considered high for other species, but is justified by the fact that the elimination rate of nicotine is very rapid in mice (Marks et al, 1983). Furthermore, the low dose of nicotine (18 mg/kg/day) was chosen to facilitate comparison with results from Turner et al.

(2010), who reported anorexigenic effects using the novelty-induced hypophagia (NIH) test were observed. All mice were subdivided into 4 groups: (1) MC4R- SAL, (2) MC4R-NIC, (3) WT-

SAL, and (4) WT-NIC groups. For experiments involving Saz-A (a partial agonist at α4β2 nAChRs;), mice were implanted with osmotic pumps that contained given Saz-A (6 mg/kg/day expressed as the salt). This dose was chosen based on in vivo studies, which report that that Saz-

A does not upregulate nAChRs in rat CNS (Hussmann et al., 2012).

F: Chronic Nicotine via Twice Daily Subcutaneous Injection (SC)

For the second nicotine injection study (21-day), C57BL/6J mice were randomized into the various treatment groups (n = 7-10/group) and injected twice daily SC with either: saline (0.9%

NaCl); nicotine (0.75 mg/kg), SazA (2-3 mg/kg) or DHβE (2 mg/kg) between 9-11 AM in the morning and again between 3-5 PM in the afternoon. To minimize stress, mice were injected in their home cages. Initially, nicotine at 1.5 mg/kg was administered twice a day based on the data of Hur et al. (2010). Due to toxicity of this dose (clonic convulsions, straub tail, etc.), I reduced the dose of nicotine to 0.75 mg/kg. This dose did not produce any untoward side effects such as tremors, ataxia and sedation.

G: Feeding and Weight Measurements

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Mice were acclimated to individual housing for at least 3 days before measurement. Mouse chow was weighed and provided ad libitum. Low fat Purina Diet 5001 (4.5% fat)) was used for all groups of animals except for those in the 21-day SC study that were fed a high fat diet (HF,

D12451: 44.9% Fat)which was obtained from Research Diets Inc. (New Brunswick, NJ). Mice were singly housed for 5-15 days of baseline food intake measurements and body weight monitoring before surgery (i.e., osmotic pump implantation), and injections. Food intake measurements were made at the same time of day (between 3-5 pm) by weighing each pellet manually, taking into account food spillage.

H: Data Analysis

To adjust for individual differences in pretreatment weight and food intake values, the mean pretreatment weight and food intake were subtracted from post-treatment weight and food intake data. For all experiments, excluding the ß2 subunit of the nAChR knockout (ß2-/-) weight study, a 5-15-week pretreatment baseline was acquired. Baseline-corrected data were analyzed using a repeated-measures mixed-model ANOVAs. Post hoc comparisons were performed in MATLAB

(MathWorks, Natick, MA). In all cases, p < 0.05 was the criterion used to determine statistical significance. Data are presented as mean ± SEM. In addition, body weight data of both the 8 wk and 15 wk WT mice compared with the ß2-/- mice for each time point was analyzed via two-way

ANOVA followed by uncorrected Fisher’s LSD post hoc comparisons revealed a significant effect of genotype (p < .05).

I: Micro-dissection of Hypothalamic and Brainstem Tissue

After cessation of the 21-day nicotine study (within 24 hours), all mice were killed by an overdose of pentobarbital followed by decapitation. Brains were rapidly removed and samples were then flash frozen in liquid nitrogen and stored at −80 C until processing. Briefly, to collect

NTS-enriched tissue, brains were positioned with the dorsal surface up, and the cerebellum was

81 carefully separated from the medulla. The calamus scriptorius (most caudal portion of the fourth ventricle) provides a landmark for isolating NTS-enriched dorsomedial medullary tissue. Coronal cuts were made ~2 mm anterior and ~1 mm posterior to the calamus scriptorius under a dissecting microscope. Bilateral sagittal cuts were made ~1mm lateral to the midline. Similarly, a single horizontal cut was made at the dorsal tip of the central canal. Similar technique was employed to obtain hypothalamic tissue, except the tissue was trimmed horizontally just above the dorsal tip of the ventral third ventricle (~1mm dorsal to the ventral surface). Sagittal cuts were ~ 1mm both sides of the midline, whereas coronal cuts were at the level of the caudal most part of the medien eminence and the ventromedial hypothalamus.

J: Preparation of Membrane Homogenates for Western Blot

Each hypothalamic and NTS-enriched brainstem was separately suspended in ice-cold TEE buffer [10mM Tris, 5mM EDTA, 5mM EGTA, pH 7.5] with protease and phosphatase inhibitors

(Halt Protease and Phosphatase Inhibitor Cocktail, Thermo Scientific), and homogenized with a

Polytron homogenizer. The homogenates were centrifuged at 35, 000 g for 10 minutes at 4°C.

The pellets were resuspended in fresh ice-cold TEE buffer and sonicated with a Tekmar

Sonicator. This procedure was repeated and the final pellet was frozen at −80°C until used for western blotting.

K: Western Blot Analysis

Western blotting was performed on tissue lysates (whole hypothalamic and NTS-enriched brainstem) obtained as described above. Twelve micrograms of lysate were added to denaturing sample buffer [62.5 mM Tris, pH 6.8, 10% (v/v) glycerol, 2% (w/v) SDS, 5% (v/v) β- mercaptoethanol, and 1% (w/v) bromophenol blue], boiled for 2 minutes, and subjected to polyacrylamide gradient (4-20%) gel electrophoresis under denaturing conditions. This procedure was followed by transfer to polyvinylidene difluoride (PVDF) membranes (PerkinElmer,

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Waltham, MA, USA). Transfer efficiency was assessed using Ponceau S (Amresco, Solon, OH,

USA). Membranes were blocked in 5% (w/v) nonfat dry milk in TBST [20 mM Tris-HCl pH 7.5,

150 mM NaCl, 0.1% (v/v) Tween-20] and then subsequently incubated with mouse anti- phosphorylated AMP kinase-α and mouse anti-AMP kinase- α primary antibody (Cell Signaling

Technology, Danvers, MA, USA, 1:1,000). Antibody-antigen complexes were visualized on film following incubation with HRP-conjugated goat anti-rabbit IgG secondary antibody (EMD

Millipore, Temecula, CA, USA, 1:5,000) and Amersham ECL Prime Western Blotting Detection

Reagent (GE Healthcare Life Sciences, Pittsburgh, PA, USA) as per manufacturer's instructions.

L: Quantification of Western Blot

Quantification was performed using ImageJ (National Institutes of Health, Bethesda, MD, USA).

Phosphorylated AMP-kinase-α (p-AMPKα) and unphosphorylated (total) AMP-kinase-α

(AMPKα) were normalized to β-actin and expressed as a percentage of values obtained with saline vehicle treatment.

M: Statistical Analyses (for Western Blot)

Data are expressed as mean ± SEM. Statistical differences between the levels of p-AMPK expression in nicotine versus saline (n=6 per treatment group) were evaluated by the non- parametric Mann–Whitney U test. A non-parametric statistical test was chosen here due to the fact that the microdissections (of the hypothalamus and brainstem) were not identical to each other; hence, no assumption of normality for the population could be made. Significance was based on a criterion level of p < 0.05.

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III: RESULTS

A: Effects of nicotine administered intraperitoneally (IP) daily to juvenile and adult C57BL6/J and Mc4r (-/-) mice on body weight

My studies with nicotine as mentioned in the INTRODUCTION were initiated by the exciting report of Mineur and colleagues (2011) demonstrating the robust effect of daily administered 3 mg/kg (free base) nicotine on BW of juvenile 6 week old C57BL6/J mice. This dose administered over 30 days prevented C57BL6/J mice from adding approximately 5 g of weight over a month’s time. They also provided data suggesting that the CNS melanocortin circuit mediated the effect of nicotine.

In my study, animals were assigned to the following treatments: Group 1, Adult Mc4R (-/-)- saline (KO-SAL, n=6); Group 2, Adult Mc4R (-/-) - nicotine (KO-NIC, n=6); Group 3, Adult

WT-saline (WT-SAL, n=8); Group 4, Adult WT-nicotine (WT-NIC, n=8); Group 5, juvenile

Mc4R (-/-)-nicotine (J-KO-NIC, n=5), Group 6, juvenile WT- saline (J-WT-SAL, n=8); Group 7, juvenile WT-nicotine (J-WT-NIC, n=8). Except for the vehicle groups, all treatment groups received nicotine (3 mg/kg as the base) once daily via IP injection. The dose of nicotine used was derived from the study of Mineur et al. (2011), who examined whether activation of POMC neurons by nicotine was necessary for the resultant nicotine-induced hypophagia. A similar dose was also tested in juvenile C57BL/6J mice and was found to robustly decrease in body weight without reporting any adverse effects (Mineur et al., 2011). Mineur et al. (2011) also tested the effect of nicotine in animals in which the Mc4R was knockdown in the PVN (by direct administration of AAV-shRNA into the PVN where POMC efferents terminate). While nicotine- induced hypophagia (dose unreported) was blunted in these animals, any corresponding BW data was not reported. The Mc4R-/- mouse model used in my study has the Mc4R knocked down anywhere the receptor is endogenously expressed, which is in contrast to the model used by

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Mineur et al. (2011); in their model it is only deleted in the PVN. Since, Mineur et al. (2011) did not show any BW data in their Mc4R knockdown study, I was interested to know if chronically administered nicotine could reduce BW and FI in a conventional Mc4R knockout mouse model.

In addition, I was interested to know if there were any age-related differences in nicotine- mediated effects. Hence, both juvenile (6-week) and adult (8-week) mice were used in my study.

In theall 7 groups treated, nicotine did not produceshow any significant change in BW. More importantly, the dose of nicotine used (3 mg/kg free base; IP) proved to be toxic, as both adverse side effects (such as ataxia and convulsions that are consistent with nicotine overdose) and sedation were observed in all control WT mice . Because of the severe toxicity associated with the 3-mg/kg dose of nicotine, I lowered the dose to 1 mg/kg. The tremors were lessened at this dose; however, ataxia, sedation, and the occasional straub-tail could still be observed. This necessitated a further reduction in the nicotine dose to 0.5 mg/kg for 4 days. No adverse reactions were apparent at this dose; however, I suspected that the mice had developed tolerance since the change in BW remained unaffected. This suspicion proved to be correct when the dose was increased back to 1 mg/kg and no adverse effects were seen. Because the 1mg/kg nicotine dose did not induce any discernible side-effects, it was used for the remainder of the study. At the end of the 30-day period, the survival rate of all the mice receiving nicotine is displayed in Figure 1: Graph showing the effect of chronically

Figure 1. Due the severe side effects such as administered nicotine (0.5, 1.0 and 3.0 mg/kg) on % survival rate at the end of a 30-day period. [Note: the tremors, ataxia, increased heart rate, and adult Mc4R KO mice given nicotine were protected from sedation etc. withinthe adult WT group (WT- the toxicity of this drug.]

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NIC group), survival rate was 12.5% by the end of the 30-day period, and within the juvenile WT group receiving nicotine treatment (J-WT-NIC), the survival rate was 50% (see Figure 1). As can be noted from Figure 1, adult Mc4R (-/-) treated with the same dose of nicotine as all the control WT mice appeared to be protected against nicotine-induced toxicity. There was a 100% survival rate at the end of the 30-day period indicating a role for genotypic differences in the either the threshold for nicotine-induced toxicity or protection against nicotine-induced toxicity.

In summary, due to the administration of a toxic dose of nicotine, no conclusions could be drawn on the effect of nicotine on BW or FI in this study. The only conclusion that I was able to draw was that adult animals with Mc4R deficiency appear to be protected against nicotine-induced toxicity.

B: Effect of nicotine infusion by osmotic mini-pump on body weight and food intake of obese

Mc4r (-/-) mice

Since the daily dose of 3mg/kg nicotine (previously demonstrated by Mineur et al., 2011, to cause robust weight loss in 6 week old C57BL6/J) proved to be toxic in my previous experiment

(see Figure 1 above), no conclusions on its effect on BW could be drawn. Instead, the finding from the previous experiment highlighted the need to establish proper dosing and administration routes for testing nicotine-mediated effects on energy balance.

In consultation with Dr. Ken Kellar (Pharmacology Department, Georgetown University), I administered nicotine via osmotic pumps to mice over a 2 wk period. I conducted this study for 3 reasons: (1) to avoid the nicotine-induced toxicity observed in my first study; (2) to reduce the stress that mice undergo with daily IP injections; and (3) to mimic the way that nicotine is administered by a patch, a method that had recently been shown to reduce BW in human volunteers (Newhouse et al., 2012). The osmotic mini-pump delivery system was considered to be most appropriate as it provided a method by which a continuous steady rate of nicotine could

86 be administered by the SC route without the stress caused by IP injections. Mc4R (-/-) and

C57BL/6 mice were assigned (5-6 per group) to the following 4 treatments: Group 1, Mc4R (-/-) vehicle saline (KO-SAL, n=6); Group 2, Mc4R (-/-) nicotine (KO-NIC, n=6); Group 3, age- matched control WT saline (WT-SAL, n=6); and Group 4, age-matched control WT nicotine (WT-NIC, n=5). For nicotine treated animals, the initial dose of nicotine was 18 mg/kg/day (expressed as free base). Although, this dose is high, it is justified by the fact that elimination rate of nicotine is very rapid in mice (Marks et al, 1983). Furthermore, the

18mg/kg/day dose has been used previously to assess nicotine's anorexigenic effects on the novelty-induced hypophagia test (Turner et al., 2010).

At conclusion of the 14-day treatment interval, no significant effect of nicotine (18 Figure 2: Effect of chronic nicotine treatment on body weight in

male Mc4R-/- and C57BL/6J wildtype mice. (A) Graph illustrating mg/kg/day) was observed on BW in either the the effect of chronic nicotine for 3 consecutive 14-day intervals on

Mc4r (-/-) animals or in the WT mice (Figure body weight. [Note: For nicotine treatment groups, dosage

increased across the 3 treatment intervals (18 mg/kg/day, interval 1; 2), which is contrary to its positive 36 mg/kg/day, interval 2; and, 72 mg/kg/day, interval 3). Data are pharmacologic effect on the novelty-induced expressed as mean +/- SEM of the body weight. (B) Graph showing

the average change in cumulative FI during each nicotine treatment hypophagia test (Turner et al, 2010). interval as in A. = p<0.05 using a repeated measures mixed-model

Despite the fact that an anorexigenic effect ANOVA. Abbrev.: KO-SAL, Mc4R (-/-) vehicle saline; KO-NIC,

Mc4R (-/-) nicotine; WT-SAL, age-matched control WT saline; WT- was not observed for 18 mg/kg/day nicotine in NIC, age-matched control WT nicotine. 87

Mc4R (-/-) and WT mice, it is important to note that this dose did not produce any adverse behaviorial effects or stereotypies in any of the treated animals. Since no anorexigenic effect was seen with the 18 mg/kg/day nicotine, the dose was increased to 36 mg/kg/day, and the study was extended for another 14-day treatment interval. While no statistically significant effect was observed for BW in Mc4R (-/-) mice, nicotine at this dose significantly reduced BW of the WT mice [F(1,9)=7.30, p<0.05]. In addition, the 36mg/kg/day nicotine dose did not produce any adverse behavioral effects or stereotypies in both the WT mice and Mc4R (-/-) transgenic animals. To further assess effects on BW, and because no side-effects were seen at either of the two previous doses, a third 14-day treatment interval was added during which nicotine via osmotic pumps was administered at dose of 72 mg/kg/day. At this dose, a treatment effect in weight gain for MC4R(-/-) mice was observed [F(1,10)= 6.10, p<0.05]. Similarly, a treatment effect was also observed for WT mice [F(1,9)=17.89, p<0.05] (see Figure 2A). Again, no evidence of adverse behavioral effects or stereotypies was observed at the 72 mg/kg/day dose.

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To investigate the mechanism of nicotine induced changes in BW, concurrently recorded solid FI data was also analyzed. Consistent with the lack of any nicotine-induced weight change at 18 mg/kg/day, FI was unaffected by nicotine treatment in either Mc4R (-/-) mice or WT mice (see Figure

2B). Neither was FI affected at the 36 or

72mg/kg/day nicotine dosed, suggesting that the observed weight change in WT animals may have been due to an increase in energy expenditure (EE).

While there seemed to be an apparent decrease in

FI of Mc4R (-/-) mice at the 72 mg/kg/day nicotine dose compared to vehicle controls, it was not significant. The FI difference between the two groups was driven by increased consumption in the vehicle treated animals. In summary, although I observed nicotine-induced changes in weight gain, the effect is not attributable to a reduction in FI but rather may be due to mechanisms that increase in Figure 3: Effect of chronic SazetidineA (SazA) treatment on

EE. body weight in male Mc4R-/- and C57BL/6J wildtype mice.

C: Effect of Saz-A infusion by osmotic mini-pump (A) Graph showing the effect of chronic Saz-A on body weight. (B) Graph illustrating the average change in cumulative on body weight and food intake of MC4R(-/-) FI during each nicotine treatment interval as in A. Data are mice expressed as mean +/- SEM of the body weight. = p<0.05 using a repeated measures mixed-model ANOVA. Abbrev.: Mc4R (-

Nicotine can activate multiple nAChR subtypes /-) Saline, Mc4R (-/-) vehicle saline; Mc4R (-/-) Saz-A, Mc4R (-/-) precluding its use in determining whether a specific Saz-A(6mg/kg); WT SAL, age-matched control WT saline; WT Saz-A, age-matched control WT Saz-A (6mg/kg). nAChR is responsible for producing decreases in

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FI and BW. Additionally, by administering nicotine by the osmotic pump method, I assumed that effects obtained were due to desensitization of the nAChRs rather than activation of the nAChRs.

Two recent studies have concluded that the a3β4 subtype of nicotine receptor is responsible for decreases in FI and BW (Mineur et al, 2011; Martinez de Morentin et al, 2012). Lacking selective agonists or antagonists of the α3β4 nAChR subtype, I chose to study the effect of the nicotinic partial agonist, Saz-A. The rationale for using this drug is that it has a high affinity for the α4β2 nAChR and very low affinity for all other nAChR subtypes (Xiao et al., 2006). Most importantly,

Saz-A has a high affinity for the desensitized state of the receptor (Xiao et al., 2006). Therefore,

Saz-A was used to assess whether desensitization of the α4β2 nAChR will affect FI and BW in

Mc4R (-/-) and WT mice.

Parallel to my nicotine study, Saz-A effect on BW and FI was examined in Mc4R (-/-) (n=6) and

WT (n=5) mice. A dose of 6 mg/kg/day Saz A (expressed as the salt) was delivered at constant

rate for a 14-day treatment interval via osmotic mini-pump. The dose chosen was based on the

studies of Hussmann et al. (2012) who looked at the ability of Saz-A to upregulate nAChRs in

the rat brain. No effect of Saz-A treatment, relative to saline, was observed in Mc4R (-/-) mice

(Figure 3A). This was in contrast to the Saz-A treated WT mice, who showed a significant

attenuation in weight gain [F(1,9)=23.03, p<0.05] (Figure 3A). Consistent with an anorexigenic

mechanism of action, specific to WT animals, analysis of FI data found no difference in intake

for Saz-A treated Mc4R(-/-) mice [F(1,10)=0.35, p > 0.05], but a reduced FI in Saz-A treated

WT mice [F(1,9)=16.33, p<0.05], relative its saline treated cohorts (Figure 4).

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D: Effect of nicotine administered SC on body weight, food intake, and AMPK in diet-induced obese mice

I further assessed the ability of nicotine and drugs that act at the a4β2 nAChR on BW and FI in a diet-induced obesity (DIO) model. In addition to nicotine (0.75 mg/kg; SC; BID), drugs that were studied include Saz-A (6 mg/kg; SC; BID) and the competitive α4β2 nAChR antagonist DHβE (3 mg/kg;

SC; BID). These drugs further addressed the issue of receptor desensitization (i.e., Saz-A and DHβE should elicit similar results assuming that receptor antagonism is analogous to chronic receptor Figure 4: Effect of chronic Saz-A treatment on FI desensitization). In my previous study, nicotine, 72 in male Mc4R-/- and C57BL/6J wildtype mice. Data are expressed as mean +/- SEM of the body mg/kg/day, administered via osmotic mini pump was weight.*= p<0.05 using a repeated measures found to lower the rate of weight gain in the Mc4R (-/- mixed-model ANOVA. Abbrev.: Mc4R (-/-) Saline, Mc4R (-/-) vehicle saline; Mc4R (-/-) SazA, ) mouse obesity model without any adverse behavioral Mc4R (-/-) Saz-A(6mg/kg); WT SAL, age-matched effects or stereotypies. However, this effect was not control WT saline; WT SazA, age-matched control WT Saz-A (6mg/kg). very robust. Hence, I decided to move to the DIO model of obesity and investigate whether nicotine administered via SC injection over a 21-day period could elicit a robust change in BW. As mentioned before (see Introduction), nicotine exerts a greater effect on BW in rodents when they are on a high fat diet (Bellinger et al., 2005;

Hur et al., 2010). To examine the effect of the above drugs on BW and FI, twelve week old WT mice were assigned (6-10 per treatment group) to the following 4 treatments: Group 1, WT vehicle-saline (WT-SAL, n=10); Group 2, WT nicotine (WT-NIC, n=7); Group 3, WT Saz A

(WT-SAZA, n=9); and Group 4, WT mice DHβE (WT- DHβE, n=6). All animals were fed a

91 high fat diet consisting of 45% kcal from fat for 9 weeks prior to treatment. Analysis of the final time point at the end of the 21-day period reveals a significant effect of treatment on BW

[F(3,28)=16.47, p<.05]. There was a significant reduction in BW in WT-NIC compared to WT-

SAL animals at the end of the 21-day period

(Figure 5A; p<0.05). The WT-NIC animals lost greater than 5 grams of BW at the end of the 21- day period. In addition, analysis of the final time point reveals a robust effect of drug treatment on reducing BW in the WT-SAZA (n=9) in comparison to WT-SAL animals (Figure 5A; Figure 5. Effect of nicotine (0.75mg/kg), Saz-A (6mg/kg) and DH E (3mg/kg) on net body weight p<0.05). No significant effect of treatment on BW β in diet-induced obese male mice (A) Graph showing was observed in the WT- DHβE (n=6) group in comparative loss of body weight in wildtype mice

at the end of a 21 day treatment period. (B) Graph comparison to WT-SAL animals (Figure 5A). In of the average change in cumulative food intake addition, there was a significant difference between during entire duration of drug treatment. *=p<0.05

using repeated-measures mixed-model ANOVA. the WT-NIC group and the WT-SAZA group

(Figure 5A; p <0.05) indicating that nicotine is more efficacious at reducing BW than SazA.

To investigate the mechanisms of nicotine’s ability to robustly reduce BW in the DIO model of obesity, cumulative FI collected throughout the 21-day treatment period was also analyzed.

Analysis of the entire treatment period revealed, a significant effect of treatment on FI

[F(3,28)=5.13, p<0.05], which can also be seen in the final time point alone [F(3,28)=2.99, p<0.05]. Consistent with the weight reducing effect of nicotine and Saz-A in treated animals

92 compared to vehicle controls, the WT-NIC group and the WT-SAZA group both had a significant

Figure 6. Western-blot images showing the effect of 0.75 mg/kg nicotine (NIC) and saline (VEH) administration

twice daily on p-AMPKα in NTS-enriched brainstem (A) and whole hypothalamus (B). (C) Graph showing the levels phosphorylated AMPK and total AMPK (D). Data expressed as mean +/- SEM. reduction in cumulative food intake during the 21-day period relative to the WT-SAL group

(Figure 5B; p <0.05).

Because of the interesting and highlighted findings of the Lopez's group (Martinez de Morentin et al., 2012; Seoane-Collazo et al., 2014) on the role of hypothalamic AMPK in mediating nicotine’s effect on BW in rats, I assessed the effect of the 21-day nicotine treatment on AMPK activity in my DIO mice. I evaluated the effect of chronic nicotine treatment on phosphorylated

AMPKα (p-AMPKα) expression levels in both enriched hypothalamus tissue and enriched

NTS/brainstem tissue. Enriched NTS/brainstem tissue was examined because of data published by Hayes et al. (2009) indicating that AMPK activity plays a role in this part of the brain to 93 control BW. In the NTS/brainstem, nicotine treatment (0.75 mg/kg; twice daily) for 21 days produced no change in the levels of p-AMPKα in comparison to vehicle treated controls (n=6 per group). The lack of change in p-AMPKα between nicotine treated and vehicle treated animals was also associated with unchanged protein concentrations of the nonphosphorylated form of the

AMPKa isoform (i.e., total AMPKα levels in the brainstem were equivalent between nicotine and vehicle groups; Figure 6A). In contrast to the results from NTS/brainstem tissue, nicotine treatment (0.75 mg/kg; twice daily) for 21 days showed significant upregulation (p <0.05) of p-

AMPKα levels in enriched hypothalamic tissue in comparison to vehicle treated controls (n=6).

The increase in p-AMPK in the nicotine treated animals in the enriched hypothalamic tissue was associated with unchanged protein levels of the nonphosphorylated forms of the AMPKa isoforms (Figure 6B). These data are in contrast to the findings of the Lopez group (Martinez de

Morentin et al., 2012; Seoane-Collazo et al., 2014) who report that nicotine inhibits phosphorylation of hypothalamic AMPK. My data suggest a previously unknown link between nicotine-induced BW loss and activation of hypothalamic AMPK.

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E: Preliminary Studies of Mice with Knockout of the β2 nAChR subunit

My data showing Saz-A induced effect on BW and FI implicates the α4β2* nAChR subtype in mediating nicotine's anorexigenic effect. To determine this, I generated a colony of β2 knockout nAChR subunit mice. My goal in studying these mice is to determine whether nicotine (and Saz-

A) will exert a weight-reducing effect. This study will be conducted in the same way that my DIO study was conducted where nicotine was shown to produce a robust reduction in

BW (see Figure 4A). In preparation for my study, I have found that 8 wk old β2-/- mice in comparison to age-matched WT controls showed no differences in BW at any time point over an 18-day period. In contrast 15-wk old β2-/- mice weighed significantly weighed less than age-matched WT controls at all time points in the 18-day period (Figure7; p

<0.05). Measurement of FI revealed no significant differences between β2-/- mice and

WT mice.

Figure 7: Graphs comparing differences in weight gain over an

18-day period in a 8-wk and 15-wk nicotinic receptor β2 knockout mouse and age-matched WT controls (n = 6-8 per

group). Data are expressed as the mean +/- SEM.

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IV: DISCUSSION

A: Magnitude of reduction in body weight produced by chronic administration of nicotine using 3 different study designs

A comparison of the effectiveness of nicotine in slowing the gain of weight in mice using

3 different study designs is shown in Table 4. As can be noted, the greatest effect was seen when nicotine was administered as a constant SC infusion by osmotic mini-pump in the Mc4R-/- mice.

This occurred with the highest dose of nicotine (72 mg/kg/day). Surprisingly, no statistically significant effect was observed with the lower doses of infused nicotine due to the high variability in the BW responses (see Chapter II: results, Figure 2A). This robust effect was of greater magnitude (i.e., greater weight reducing magnitude) than the nicotine response in WT mice (13% vs. 4.7 %). Indeed the 13% magnitude response was greater than the effect on BW observed in the studies reviewed in the INTRODUCTION of this thesis. Reasons for the robust effect of nicotine may be related to the fact that the Mc4R -/- mice are obese, and it is known that the obese rodent exhibits a greater weight reducing response with nicotine administration (e.g.,

Hur et al., 2010; Mangubat et al., 2012). Alternatively, it is also possible that the robust nicotine response was a reflection of the extraordinary high dose of nicotine that was given to the mice. It should be noted that no adverse side effects were noted in these mice.

The only other study that shows such a robust effect of nicotine on BW is that of Mineur et al. (2011) where 3 mg/kg nicotine given IP exhibited a magnitude of effect of approximately

27 %. I tried this does of nicotine in my initial study, and observed that all mice exhibited severe adverse side effects including convulsions. It was clear that mice could not tolerate this dose and method of administration; therefore, I had to lower the dose in order to finish the study. Adverse effects to this dose were also noted by Mineur, et al. but not reported. Inquiry into the adverse

96 effects of this dose from the senior investigator of the study corroborated my observations.

“Although not quite as severe as you describe, we definitely saw adverse side effects at the highest (3 mg/kg) dose of nicotine. We included the highest dose for completeness, but I think that the flat weight curve at that high dose was aberrant and was due in part to the aversive effects of the highest dose (e-mail correspondence).” A most interesting finding did emerge from my initial study that was unrelated to BW effects. What I observed was that nicotine treated adult Mc4R-/- had a 100 % survival rate. In contrast, survival was less than 10 % in age-matched wild type mice that received nicotine. In reviewing the literature, I found no indication that

Mc4R -/- rodents are protected from nicotine toxicity or from other drugs known to cause CNS excitation. Additional studies need to be conducted to pursue this observation.

A robust effect of nicotine was also observed in my study where nicotine was given SC twice a day to DIO mice. When I initiated my study, I was guided by the experimental design used by Hur et al. (2010). In their design, they indicated that a SC single dose of 1.5mg/kg nicotine was ineffective, but that dose given twice a day was effective in DIO mice (using the rate of weight gain over 14 days of treatment). They also treated their mice during the light cycle. Since mice are active during the dark cycle, I injected the animals during this cycle (as recommended by Bellinger et al., 2003a). Again, as in the case of trying to use the experimental design of Mineur et al. (2011), I observed toxicity in the mice when the dose regimen of

1.5mg/kg nicotine was administered twice a day. It wasn’t until reducing the nicotine dose to

0.75 mg/kg given twice a day (SC), was I able to avoid toxicity. In contrast to the data reported by Mineur et al. (2011) and Hur et al. (2010), the BW data obtained with nicotine and listed in

Table 4, were from non-toxic doses of nicotine.

B: Mechanism for nicotine-induced reduction in the role of body weight gain

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How does nicotine reduce BW in my studies? To explain this, I will focus on components of the energy balance system (i.e., the tightly regulated processes of FI and EE) and how my findings compare to that of previous investigators. Nicotine treatment at 0.75 mg/kg given twice a day during the dark cycle in the DIO mouse model produced a robust weight- reducing effect, which was mediated by a significant cumulative reduction in FI during the 21-

Table 4: Effectiveness expressed as a percentage and determined by how much drug treatment decreased the weight gain observed in saline treated controls, i.e., difference between % weight gain of mice on saline and % weight gain of mice on drug.

day period of drug administration. In contrast, the ability of nicotine to lower the rate of weight gain in the Mc4R-/- mouse was not mediated by a reduction in FI, but rather by mechanisms that

I presume involved increases in EE (although this was not evaluated). My results from the DIO

98 study fully agree with those of Bellinger and colleagues (2003a, 2003b, 2005), who found that the BW of rats was initially reduced during the dark cycle because of nicotine-induced decreases in FI; even though it normalized eventually, BW remained suppressed compared to vehicle treated controls. Although, it is worth noting that in my results, normalization of FI was not observed. Martinez de Morentin and colleagues (2012) also report reduced FI after nicotine treatment in rats receiving 2 mg/kg (salt) twice daily for 48 hours, which is also consistent with my results.

Three rat studies that show strong similarities to my results with the DIO study are those of Wellman et al. (2005) Bellinger et al. (2010) and Seoane-Collazo et al. (2014). The study of

Wellman et al. (2005) found that suppression of FI by nicotine was larger in rats fed a high fat diet (HFD) compared to rats on the normal diet. Nicotine reduced meal size in rats on both diets, but the usual increase in meal number noted in rats 5 days after nicotine treatment (observed in the study by Bellinger et al., 2003a) was not observed in rats on the HFD (Wellman et al., 2005).

The ability of nicotine to reduce BW was greater in the HFD group than the normal diet group.

Bellinger et al. (2010) used pair-feeding to study nicotine treated and vehicle treated rats. The pair-fed group received a similar amount of food as the nicotine treated group. At the end of the study, both the pair-fed and nicotine treated groups had similar BW gain values, which were less than the vehicle treated groups. This latter experiment shows that the BW loss of nicotine treated rats was due to lowered FI. The authors make the interesting point that during FI suppression, animals react by breaking down energy stores, which would contribute to their weight loss.

Consistent with this point was the finding in the authors’ study that the daily dark and light phase respiratory quotient (RQ) of the nicotine treated rats was significantly reduced. Seoane-Collazo et al., (2014) also showed reduced FI with 2 mg/kg nicotine dose twice daily in DIO rats.

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In terms of the mice studies, with the exception of the Fornari et al. (2007), the results with nicotine experiments are consistent with those of Mineur et al. (2011), Mangubat et al.

(2012) and Hur et al. (2010) who also showed reduced FI as the mechanism by which nicotine lowers BW. My data is especially similar to those of Mangubat et al. (2012) and Hur et al. (2010) who show suppression of FI on mice fed a HFD.

Lastly, there are studies that report nicotine is able to lower BW without any concomitant decrease in FI or report only transient effects on FI despite claims of lowering BW (Schechter and Cook, 1976; Grunberg et al., 1984; Levin et al., 1987; Arai et al., 2001; Bellinger et al.,

2003a; Guan et al., 2004; Bellinger et al., 2005; Kramer et al., 2007a). Therefore, mechanisms underlying EE would be expected to come into play in these studies. This is consistent with my data showing that chronic nicotine lowers BW in Mc4R-/- without any change in FI.

C: Role of the melanocortin brain circuit in nicotine-induced reduction in the rate of body weight gain

The aforementioned study of Mineur et al. (2011) study specifically states that when nicotine reaches the arcuate nucleus of the hypothalamus (ARC), activity of POMC neurons increases through activation of nAChRs located on POMC neurons. These in turn activate of

Mc4Rs located on a second order neurons in the PVN resulting in inhibition of FI. Specifically,

Mineur et al. found that nicotine-induced hypophagia was blunted in mice with knockdown of

Mc4R by AAV-shRNA specifically in the paraventricular nucleus of the hypothalamus (PVN).

My results in the Mc4R-/- study where I show that chronically administered nicotine at a dose of

72 mg/kg/day delivered by an osmotic mini-pump is still able to reduce the rate of weight gain despite the absence of a functional melanocortin system. Therefore, according to my results, in contrast with those of Mineur et al. (2011) indicate that Mc4R is not needed for nicotine to exert a weight reducing effect.

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Several explanations can be offered to account for the discrepancy between my findings and those of Mineur et al. (2011). First, the animal models are different. Mineur et al used the knockdown approach where Mc4R is just absent in the PVN, whereas in my study, the Mc4R is absent wherever it is endogenously expressed. Secondly, Mineur et al. do not report BW data in the study where nicotine-mediated hypophagia was blunted in mice with knockdown of Mc4R by

AAV-shRNA. Since it has been established that BW is the product of tightly regulated homeostatic processes of FI and EE, it is conceivable that FI could decrease without a change in

BW because of engagement of homeostatic mechanisms. It would have been important to know whether in the presence of blunted hypophagia, nicotine could have reduced the BW in the animals with Mc4R knockdown in the PVN. Third, the route of nicotine administration is different in the 2 studies as Mineur et al. used the IP injection route and I used the osmotic pump route.

In my view, the most important finding of my studies on the role of the melanocortin brain circuit in nicotine (and Saz-A) induced reduction in BW is that it was still effective in these mice. In contrast, Saz-A was ineffective in these mice. This finding suggests that nicotine may be acting on two separate brain circuits to reduce the rate of BW gain, namely, the same circuit acted upon by Saz-A (one that is dependent on an intact melanocortin circuit) and a circuit which controls BW independent of the melanocortin circuit.

D: Subtype of nAChR involved in nicotine-induced reduction in the rate of body weight gain

Several types of experiments were conducted to address this issue. To elucidate the subtype of nAChR involved in the ability of nicotine to lower BW, I exploited the unique pharmacological properties of the nicotinic-like drug Sazetidine A (Saz-A). Saz-A is a partial agonist at the α4β2 nAChR and was synthesized from its parent compound A-85380 (it is550 fold selective for binding to the α4β2* than the α3β4 nAChR). Like A-85380, Saz-A displays

101 high affinity binding to α4β2 and is also a very potent desensitizer at these receptors (Xiao et al.,

2006). However, it is worth noting that SazA can have differential agonist properties based on the stoichiometry of the receptor. SazA has been recently shown to be a full agonist at the α4β2 nAChR when 3 (α4) subunits and 2 (β2) subunits are expressed, whereas it had only a 6% efficacy of when the stoichiometry of the receptor was 2 (α4) and 3 (β2) subunits (Zwart et al.,

2008). However, the exact stoichiometry of the α4β2* subtype of nAChR where Saz-A could work to exert behavioral effects in vivo is not known. When Saz-A was chronically administered using osmotic mini-pumps to non-obese (C57Bl6/J) mice, they showed a significant reduction in

BW compared to vehicle treated animals. In addition, SazA administered twice daily by SC injection caused significant weight loss in DIO mice. Furthermore, the data suggest (statistically) that the efficacy of Saz-A to reduce BW in DIO mice was less than that of nicotine. However, a proper dose response curve using nicotine and Saz-A was not conducted; the caveat being that I was unable to go higher with the Saz-A dose as increasing doses result in sedation (Hussman thesis).

Results from the SazA experiments suggest a role for a novel α4β2* nAChR-Mc4R pathway in nicotine-induced BW regulation. The location in the brain of this proposed α4β2* nAChR-Mc4R pathway and the exact stoichiometry of α4β2* nAChR involved remains to be determined. Furthermore, the fact that nicotine proved to be more efficacious, than Saz-A in lowering BW in DIO mice lends further evidence to the notion that a drug (Saz-A) working at the

α4β2* nAChR is not the same (in terms of efficacy) as nicotine. The presumed greater efficacy of nicotine in my studies could mean that it is working on an alternative pathway in addition to the proposed α4β2* nAChR-Mc4R pathway to exert greater anorexigenic effects, but this is speculative.

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In comparing the findings from the experiments with Saz-A to those of other investigators, my data is consistent with the weight-reducing effect of Saz-A shown by Hussman et al. (2014) using osmotic mini-pumps in rats. Hussman et al. (2014) showed rats that were previously on nicotine during the 1st phase of treatment gained significantly less weight than animals on saline. When nicotine treated animals were switched over to Saz-A during phase 2 of the treatment, they gained weight at a slower rate than controls and ended up weighing 11% less than the controls at the end of the 4-week treatment period (Hussman et al., 2014). Both my findings with Saz-A and those of Hussman et al. (2014) are consistent with a role for α4β2* nAChR in BW regulation. My data are also consistent with data from Huang et al., (2011) showing that the depolarizing effect of nicotine on the membrane of POMC neurons is significantly decreased with dihydro-β-erythroidine hydrobromide (DHβE), an antagonist at the

α4β2 nAChR, suggesting that α4β2 receptors exist on these neurons and are potentially mediating the nicotine induced depolarization.

Results from my studies with Saz-A should also be considered in the context of data presented by Mineur et al. (2011). Mineur et al. report that the mechanism responsible for the ability of nicotine to reduce FI in a mouse model is through central activation of the α3β4 nAChR located on POMC neurons of the ARC (Mineur et al., 2011). The strongest evidence presented by Mineur et al. is that they tested the nicotine related drug cytisine, a full agonist at the α3β4 nAChR that has weaker effects at other nAChRs (Piccioto et al., 1995). They report that cytisine decreased weight gain over time, and that cytisine mediated hypophagia was blunted with pretreatment with the noncompetitive nicotinic antagonist mecamylamine and β4 subunit knockdown. However, it is important to note that the nicotinic antagonist, mecamylamine, is not selective for α3β4 nAChR and the β4 knockdown approach (that blunted cytisine-induced hypophagia) used by Mineur et al. does not offer any additional information as to how nicotine

103 works; instead it only confirms that cytisine works through α3β4 nAChR (an already known fact). My results from the Saz-A experiments do not necessarily conflict with those of Mineur et al. (2011) but rather suggest the existence of another central pathway where nicotine could be working to reduce BW, one that involves α4β2* nAChR mediating the weight reducing effects of nicotine.

E: Role of desensitization of the nAChR in nicotine-induced reduction in the rate of body weight gain

The benefit of delivering nicotine by osmotic mini-pump is that it allows for precise chronic and controlled drug delivery with a pre-determined amount of drug being delivered every hour. With this route, nicotine build up is slow and steady and as mentioned previously, does not provide the initial excitatory effect seen with IP or SC administration where a brief high concentration of drug is achieved quickly in the blood and brain (HussmannHussman et al.,

2012). More importantly, the reason I am focusing on this method of drug delivery is that receptor desensitization as opposed to activation becomes an issue. It is widely accepted that in the desensitized state, nAChRsthe nAChR have a much higher affinity for agonists than they do in their resting/activatable state (daCosta and Baenziger, 2009). Moreover, in the prolonged presence of agonist such as with chronic nicotine exposure via the osmotic pump method, receptors are recruited into the desensitized state. Lu and colleagues (1999) have shown that nicotine agonists will stimulate the release of GABA from synaptosomes at concentrations that are less than those required to activate the nAChR (subactivating concentrations of agonist). A subactivating concentration of agonist is one that falls below the activation dose but is still enough to desensitize the receptor. Due to slow build up of nicotine occuringsuch as that with the osmotic pump, it is possible that sub-activating concentrations of nicotine may be responsible for chronically keeping the receptor chronicallyin a desensitized state. Since, the α4β2* nAChR is

104 more likely to be affectedeffected by desensitization than the α3β4 nAChR subtype (Meyer et al.,

2002) the question becomes to what extent does desensitization of the α4β2* nAChR play a role in nicotine-induced reductions in BW that were observed with nicotine and Saz-A? To answer this question, in addition to administering Saz-A in the DIO mouse model, I administered DHβE

(3 mg/kg; given twice daily) an antagonist at the α4β2 nAChR to another group of DIO mice to determine if it would mimic the effect of Saz-A on BW. The rationale behind the use of DHβE was that if Saz-A was working primarily through a mechanism involving desensitization (which is functionally equivalent to receptor antagonism in that the desensitized receptor is unable to respond to an agonist), then DHβE per se should also produce weight loss similar to Saz-A.

However, the results with administration of DHβE to the DIO mice showed no statistically significant weight loss. Therefore, I questioned whether or not the use of a competitive antagonist such as DHβE was the proper way to study chronic desensitization of the α4β2* nAChR. Since

DHβE did not act in the same manner that Saz-A (i.e., it did not reduce BW), I turned to a different model with which to study the effect of chronic desensitization of the α4β2* nAChR.

Still reasoning that chronic receptor desensitization is functionally equivalent to loss of function of the α4β2* nAChR, I turned to the knockout mouse model of the β2 subunit of the nAChR.

Results from studies using β2-/- mice reveal that starting at 15 weeks, the weights of the β2-/- are significantly lower than age-matched controls, and remain lowered for more than 2 weeks (entire duration of monitoring). These data suggest an important role for α4β2* in BW regulation. The results from the BW data in the β2-/- mice fed ad libitum are consistent with those of the desensitization of the α4β2* nAChR in mediating nicotine's reducing effects on BW. In other words, the desensitization of the α4β2* nAChR that would be expected to be produced with delivery of nicotine and Saz-A via osmotic pump should be analogous to loss of function of the

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α4β2* nAChR for prolonged periods of time, a scenario comparable to loss of function mutation

(i.e. β2-/- mouse).

In summary, results from experiments with Saz-A and the β2-/- reveal a role for the desensitized α4β2* nAChR in the ability of nicotine to reduce BW. Depending on where this desensitized α4β2* nAChR is in the CNS, and how endogenous neurochemical transmission is disrupted/or altered from having the receptor kept in a chronically desensitized state, could have serious implications on nicotine mediated effects on feeding and BW regulation. More studies need to be done to further elucidate this.

F: Role of AMPK in nicotine-induced decrease in body weight

So far, I have discussed relevant nicotinic receptor subtypes that may be involved in the ability of nicotine to reduce BW, the relevant brain circuits, as well as the role of receptor desensitization. The last mechanism that will be focused on in this discussion is cellular components that regulate energy balance that could also be mediating the ability of nicotine to lower BW. As mentioned earlier in the INTRODUCTION, one potential cellular link between the ability of nicotine to upregulate feeding related neuropeptides (POMC) could be the ubiquitously expressed cellular energy sensor AMPK (Martinez de Morentin et al., 2012;

Seoane-Collazo et al., 2014). Prior to conducting the AMPK experiment, the data from 2 previous studies (by the same group of investigators) indicated that nicotine decreased BW through a mechanism involving inhibition of hypothalamic AMPK (Martinez de Morentin et al.,

2012; Seoane-Collazo et al., 2014).

My data, looking at the relevance of hypothalamic and brainstem AMPK in the weight- reducing effect of nicotine, does not demonstrate that the well-established anorexigenic effects of nicotine are mediated through inhibition of hypothalamic AMPK. In contrast, the results of my data indicate that chronic nicotine treatment causes activation of AMPK in the hypothalamus as

106 demonstrated by a significant upregulation of levels of hypothalamic p-AMPKα. Furthermore, in the brainstem, there is no change in the levels of p-AMPKα after chronic nicotine treatment. The issue of withdrawal is not relevant here as brains from nicotine-treated animals were harvested within 24 hours of cessation of drug treatment. Martinez de Morentin et al. (2012) saw upregulation of AMPK only when the animals were in withdrawal (during the last 72 hours of cessation of nicotine treatment).

Why is there is a discrepancy between my findings and those of the previously aforementioned studies? One reason is species difference. My results are obtained in DIO mice and the results obtained in the previous studies have all been performed in rats. Significant physiological differences exist between mice and rats at least with respect to nicotine metabolism. The half-life of nicotine in a mouse is significantly shorter (Petersen et al, 1984) than that of the rat (45 minutes in the rat and 6-7 min in the mouse). In addition, mice are less sensitive to the acute effects of nicotine than are rats (de Fibre et al., 2002; Miner and Collins,

1989; Tripathi et al., 1982). Thus mice require a higher dose of nicotine to achieve a similar response (Matta et al., 2006). With this in mind, it is important to note that the dosing regimen of

0.75 mg/kg (free base) twice daily chosen for my studies is only slighter higher (~0.13 mg/kg higher) than the dose of 2 mg/kg (expressed as the salt) used by Martinez de Morentin et al.

(2012). Again, to reiterate, my dose was safe and non-toxic for a mouse (as no abnormal behavioral phenotype was observed) and since mice metabolize nicotine much more rapidly than rats, it is likely that the dose chosen by Martinez de Morentin et al. (2012) was too high. An indication that the dose from the Martinez de Morentin et al. study may have been a toxic one is that the 2 mg/kg dose given twice daily induced several minutes of 100% respiratory depression, which was not a phenomenon, I observed in my study. In checking out possible toxic effects of the 2 mg/kg SC dose used in rats, I administered this dose to 3 rats and observed the classic

107 stereotypic behavior confirming toxicity. Therefore, the dose of 2 mg/kg twice daily in a rat is most likely not a physiologically relevant dose.

In addition, it is worth noting that an animal in a state of respiratory depression could alter AMPK levels and subsequent cellular energetics as it creates a state of hypoxia that AMPK has been shown to be responsive to (Mungai et al., 2011). As mentioned earlier, one of the aims of my studies was to find a non-toxic dose of nicotine that would produce robust weight loss without any adverse effects (i.e., respiratory depression, abnormal locomotor, stereotypies).

Therefore, having used a safe dose of nicotine in DIO mice, my data do not support a role for inhibition of hypothalamic AMPK in the ability of nicotine to reduce BW. In contrast, my data show a potential role for activation of hypothalamic AMPK.

The role of activation of hypothalamic AMPK in the weight-reducing effect of nicotine should also be addressed here. As mentioned previously, activated AMPK optimizes energy utilization by increasing energy-producing pathways to conserve energy and turns off energy- wasting processes. Therefore, it seems counter-intuitive that nicotine reduces BW would turn on this cellular sensor that encourages anabolic processes to be conserved. In thinking of the interplay between nicotine causing weight loss and activation of hypothalamic AMPK, I speculate that one possible explanation for the activation of hypothalamic AMPK after chronic nicotine treatment could be that it represents a compensatory mechanism. In other words, the activation of hypothalamic AMPK could be a homeostatic response to the reduced BW and FI evident after chronic nicotine treatment. However, more studies need to be done to determine whether or not activation of hypothalamic AMPK is involved in the ability of nicotine to reduce

BW or that it is a consequence of nicotine-induced weight loss.

G: The role of endogenously active nAChR in controlling BW

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In further attempting to elucidate signals that inform the brain of the body’s energy status, results from the weight monitoring study of mice with a knockout of the β2 subunit of the nAChR (β2 -/- mice) reveal that nAChRs containing the β2 subunit appears to be part of a system that is endogenously active in controlling BW regulation. Results from the weight monitoring study reveal that knockout of the β2 subunit, anywhere where it is endogenously expressed, lowers BW. Specifically, I have shown that starting at 15 weeks, β2 -/- mice for the entire duration of monitoring (18 days) had significantly lower BWs than age-matched controls. This shows that β2 containing nAChR are involved in an endogenously active system controlling BW.

In much the same way that leptin deficient animals are unable to control their FI due to the fact that leptin is always operating to reduce it in response to changes in energy stores, it appears that as though signaling at the β2 containing nAChR is also always operating to keep the receptor in a desensitized state to maintain BW at a certain set point. The question of how this is accomplished remains to be determined, as well as where it takes place in the brain. Thus in the absence of an endogenously active control state, I would expect that an episodic event such as nicotine-induced hypophagia working through desensitization of the β2- subunit of the nAChR would not work in these animals. The other interesting finding from my studies of β2- subunit knockout mice is there is a "window of time" when the gain of weight of these mice is reduced. Thusfar my data indicate that this "window" is not operating between 0 and 8 weeks and is clearly present at 15 weeks and beyond. According to the published literature, mecamylamine treatment of rats

(Schecter and Cook 1976) and mice (Mineur et al., 2011; Mangubat et al., 2012 has no effect on

BW. Hence, my findings from the β2-/-mice provide the first evidence of an endogenously active nAChR receptor that influences BW.

H: Outcome of Hypotheses Testing, Conclusions, and Future Studies

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My first hypothesis was that the effect of nicotine to cause weight loss is mediated through the melanocortin system. This hypothesis was tested by administering nicotine to mice with genetically deleted Mc4R. When Mc4Rs were missing, nicotine was found to exert its most efficacious effect to reduce the increase in BW overtime. Clearly, the melanocortin system is not required for nicotine to affect BW. On the other hand, in the case of a related drug and one that has greater selectivity for nAChRs, namely, SazA, the effect of SazA to influence body weight is dependent on an intact melanocortin system. My second hypothesis was that the effect of nicotine to cause a slowing in weight gain is through both activation and desensitization of the α4β2 nAChR subtype. Data obtained are consistent with a role for desensitization of nAChR. Data in support of this are the finding with osmotic pump infusion of nicotine and the finding that slower gain in weight occurs in β2-/- mice. My data do not allow me to draw any conclusion about the role of activation of nAChRs. My third hypothesis was that the effect of nicotine to slow the rate of weight gain is mediated through a mechanism involving inhibition of hypothalamic AMPK.

My data indicate that inhibition of hypothalamic AMPK plays no role in the effect of nicotine to influence BW. Indeed, my data show that nicotine has the opposite effect on AMPK, i.e. nicotine activates AMPK.

A frustrating but important experience in my studies with nicotine was that each time I sought information on dose of nicotine to use in my studies of BW, the doses were all toxic. This was true when I attempted to sue the Mineur et al., 2011 dose of nicotine IP in mice, when I attempted to use the Hur et al., dose of nicotine in mice, and when I gave the Morentin et al., dose of nicotine to rats. This experience compels me to question the scientific value of some of the published data obtained with nicotine on BW. Effects obtained and mechanisms revealed with toxic doses may not contribute to current knowledge on nicotine and BW.

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My experience working with nicotine convinces me that a useful therapeutic agent for treating BW problems may come from this line of study. The efficacy of nicotine appears to be similar to other therapeutic agents on the market, and perhaps in combination with the newer drug, lorcaserin, addition or synergism might be observed.

My enthusiasm for future studies is derived from my recent observation of BW changes in β2-/- mice. It would be important to start a new parallel study in α4-/- mice, and assess whether the same effect obtained in β2-/- mice is observed. Another critical experiment is to determine whether knockdown of β2- subunit of the nAChR in normal mice will mimic the effect

I seeiam seeing in the β2-/- mice. Additionally, it would be interesting to see if chronic treatement with mecamylamine during the first 8 weeks of life of the β2-/- mouse has any effect per se, and compare data obtained from studying mecamylamine in β2-/- after 15 weeks of age.

Most important, a future experiement will be to use my DOI mouse model and to determine whether nicotine influences BW in β2-/- mice.

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