COMPENSATION OF MELANOCORTIN-3 AND MELANOCORTIN-5 RECEPTORS IN THE BRAIN OF MELANOCORTIN-4 RECEPTOR KNOCKOUT RATS. A thesis submitted to the

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2 COMPENSATION OF MELANOCORTIN-3 AND MELANOCORTIN-5 RECEPTORS IN THE BRAIN OF MELANOCORTIN-4 RECEPTOR KNOCKOUT RATS A thesis submitted to the Kent State University Honors College in partial fulfillment of the requirements for General Honors by Addison Spriggs August, 2014

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4 Thesis Written by Addison Spriggs Approved by, Advisor, Chair, Department of Biology Accepted by, Dean, Honors College ii

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6 TABLE OF CONTENTS LIST OF FIGURES...IV LIST OF TABLES....VI ACKNOWLEDGMENTS..... IX CHAPTERS INTRODUCTION....1 ENERGY HOMEOSTASIS. 3 MELANOCORTIN RECEPTORS.. 5 COMPENSATION...6 METHODS SUBJECTS 8 TISSUE COLLECTION...9 SECTIONING OF BRAIN... 9 MICRODISSECTION AND RNA EXTRACTION QUANTIFYING RNA AND CDNA..13 iii

7 QUANTITATIVE PCR...14 STATISTICAL ANALYSIS RESULTS ARC.17 PVN.18 PEFLH 18 DMH 19 VMH.20 DISCUSSION AND IMPLICATIONS REFERENCES APPENDIX iv

8 LIST OF FIGURES Figure 1:.. 10 Brain Regions of Interest Figure 2:...17 Graph of ARC MCR Expression Levels Figure 3: Graph of PVN MCR Expression Levels Figure 4:...18 Graph of PeFLH MCR Expression Levels Figure 5:...19 Graph of DMH MCR Expression Levels Figure 6:...20 Graph of VMH MCR Expression Levels v

9 LIST OF TABLES Table 1:.. 2 Abbreviations utilized vi

10 ACKNOWLEDGMENTS I would like to extend my deepest gratitude to Dr. Colleen Novak for tirelessly guiding me while writing this thesis paper. Her endless corrections, proof-reading and patience, made this paper possible. Thank you for being more than willing and patient to teach me all the techniques and concepts that I needed to master. I would like to thank Dr. Novak s lab members for always being willing to offer their help and guidance. I would also like to thank Dr. Jenny Marcinkiewicz, Dr. Elda Hegmann, and Dr. Sara Newman for taking time to read my paper and be a part of my defense committee. I would like to thank Heaven Burns for being a great friend and for keeping me motivated at the end. I needed that little extra push and couldn t have done it without you. Finally, I would like to extend my deepest thanks to my parents and family, for listening to me complain and giving me critical feedback when necessary. You all helped me finish. vii

11 1 Chapter 1 Introduction Obesity is the most prevalent health problem in the world. In the U.S. alone, 35% of adults are obese [2]. Obesity increases risks for heart disease, stroke, diabetes, and many cancers [2]. With obesity on the rise, it is crucial to understand the mechanistic causes of the disease to devise treatment plans for patients. These treatment plans may then address the changes caused by genetic mutations, such as deficits in production of or response to necessary hormones or neurotransmitters, or interruptions in the endocrine system. Studies have revealed the heritability of adiposity and have shown a genetic contribution of more than 60% of an individual s fat content [3]. Several gene mutations underlie spontaneous syndromes that result in atypical phenotypes characterized by obesity and abnormal body fat distribution in humans. Many of these extreme phenotypes have resulted from the disruptions in the leptin-melanocortin system [4]. In these cases, early onset of severe obesity is the presenting trait of the leptin-melanocortin disruption. Mutations in genes encoding leptin, leptin receptors, prohormone convertase 1 (PC1), pro-opiomelanocortin (POMC), and the melanocortin 4-receptor (MC4R) have all been identified in cases of severe obesity [5]. It is this mutation, the MC4R, which was studied further in this investigation.

12 2 Table 1: Abbreviations used with their corresponding meaning. Abbreviation AGRP ARC CRH dct DMH EE HCR IACUC LCR MCR NPY PA Meaning Agouti-Related Protein Arcuate Nucleus Corticotrophin-Releasing Hormone Change in Receptor Concentration Dorsomedial Hypothalamic Nucleus Energy Expenditure High Capacity Runners Institutional Animal Care and Use Committee Low Capacity Runners Melanocortin Receptor Neuropeptide Y Physical Activity PC1 Prohormone convertase 1 PeFLH POMC PVN qpcr SD SEM SNS TRH Perifornical area of the Posterior Lateral Hypothalamus Pro-opiomelanocortin Paraventricular Nucleus Quantitative Polymerase Chain Reaction Standard Deviation Standard Error of the Mean Sympathic Nervous System Thyrotrophin-Releasing Hormone

13 3 VMH Ventromedial Hypothalamic Nucleus Energy Homeostasis Many signals are integrated to determine feeding habits; long-term signals include those from fat, like leptin, and from pancreatic cells, including insulin. These are integrated with short-term signals from the gut: peptide YY, glucagon-like peptide 1, and cholecystokinin, among others. All of these deter feeding, and ghrelin also from the gut prompts feeding [6]. These signals act together to affect the brain and regulate feeding habits and energy expenditure; other non-homeostatic signals also play a role, but will not be discussed here [7]. Leptin, a well-known signaling molecule, is primarily secreted from white adipose tissue and circulates in proportion to the amount of adipose tissue [8-9]. This hormone also exerts a direct influence on neuronal receptors in the brain including receptors within hypothalamic region. Leptin acts on the arcuate nucleus (ARC) of the hypothalamus, particularly the central projecting neurons of that region. This signal is then relayed to secondary regions of the hypothalamus, some of which can sense leptin directly as well [7].The secondary regions of interest examined here include the dorsomedial hypothalamic nucleus (DMH), the ventromedial hypothalamic nucleus (VMH), the paraventricular nucleus (PVN), and the perifornical area of the posterior lateral hypothalamus (PeFLH). These regions will be described in further detail below.

14 4 The ARC is a primary source of receptors that control appetite and energy homeostasis. Within this region, neurons synthesizing the POMC precursor produce peptide α-msh, an anorexigenic peptide which decreases food intake and increases energy expenditure [10]. Other neurons produce orexigenic peptides such as neuropeptide Y (NPY) and agouti-related protein (AGRP) which serve to increase food intake and decrease energy expenditure. AGRP is an inverse agonist of not only MC4R but also MC3R. These peptide producing neurons project to other hypothalamic brain regions, including the PVN and the PeFLH, which contain second-order neurons that are important for energy homeostasis. The PVN region induces satiety while increasing energy expenditure, physical activity, and sympathetic nervous system (SNS) outflow [11].The neurons in the PVN also release thyrotropin-releasing hormone (TRH) and corticotrophin-releasing hormone (CRH), both of which are critical signals in the endocrine system and also act centrally to increase energy expenditure, EE, and physical activity while simultaneously reducing food intake [12]. These are just two of several signals that act in or are released from the PVN which affect energy balance. The DMH plays a role in energy expenditure and is particularly important in SNS outflow which impacts metabolism, for example lipolysis [13]. Similarly, the VMH has been implicated by functional studies in SNS outflow that impacts metabolism, including skeletal muscle [14]. In the past, the VMH was considered the satiety center because lesions of this region resulted in obesity and hyperphagia. The thought that the VMH,

15 5 along with the feeding center of the lateral hypothalamus, worked in opposition to regulate feeding behavior has now been discarded in favor of a more nuanced view of many interrelated, overlapping pathways regulating metabolism in concert [15]. Within the PeFLH, orexinergic neurons increase appetite and also decrease PA and EE. Orexin is unique in that it increases food intake while also increasing PA and EE [16]. Orexin appears to be an important signal to coordinate daily rhythms with behavioral outputs like appetite, physical activity, and EE. These five regions the PVN, the PeFLH, the DMH, the VMH, and the ARC are the hypothalamic regions studied in this investigation. As noted above, many of these regions are also important in regulating autonomic nervous system output and metabolism. The MC4R plays an important role in this regulation and will be detailed below [17]. Melanocortin Receptors Five different melanocortin receptors have been found in various locations in the body and with differing functions. All of the receptors are 7-transmembrane G proteincoupled receptors from the rhodopsin family [18]. The first melanocortin receptor (MC1R) is found in melanocytes which typically reside in the skin. MC1R is fundamental in cascades which result in pigmentation of hair and skin [19]. The second melanocortin receptor (MC2R) resides in the adrenal cortex and binds ACTH and thus

16 6 plays an important role in the endocrine system [20]. Both MC4R and MC3R are located in the brain, heavily concentrated in the hypothalamus and the five regions of interest. In addition to central receptors, MC3R can also be found in the placenta and gut, whereas MC4R is restricted to the brain [21]. These two receptors MC3R and MC4R play roles in satiety, feeding efficiency, nutrient partitioning, lean mass accumulation, insulin secretion, energy expenditure, and lipid handling [22]. The final receptor, MC5R is spread throughout the body at low concentrations, including the brain, and may play heretofore unknown roles in regulating metabolism and behavior [23]. MC receptors 3, 4, and 5 are currently understood to be the only 3 MC receptors expressed in the adult mammalian brain [10]. In this study, the expression of the melanocortin receptors 3 and 5 was measured when the fourth receptor was deleted. Compensation Gene deletions, such as knockouts, can result in compensatory effects during development. An individual missing a gene may or may not display normal development due to the linked nature of some genes and compensation effects may make up for any abnormalities from the knocked-out gene. There have been numerous examples of compensatory mechanisms in science. For instance, investigating plants with damaged cell walls resulted in up-regulation of genes producing glycosylphosphatidylinositolattached proteins to protect the plant. Or in the case of yeast, knocking out a gene necessary for the organism is not lethal as other genes are up-regulated [24-25]. I

17 7 examined rats with a loss-of function mutation in the gene encoding MC4R; in this case a single exon which produces MC4R underwent a targeted disruption to produce a premature stop codon. It is not known where and to what extent this missing receptor will effect changes in other systems and receptors. To address this question of compensatory effects, I examined expression of the melanocortin receptors in the brain; all 3 relevant MCRs are expressed differently in hypothalamic nuclei and this will impact function [10]. I predicted I would see increased levels of expression in MCR 3 and 5 in the brains of the rats without function MC4R. Which regions show increased expression will impact the potential change in function.

18 8 Chapter 2 Methods In order to address the question posed above, I measured expression of MC3R and MC5R in the brains of rats lacking functional MC4R and rats with a single functional copy of the gene (MC4R +/- ), compared with their wild-type counterparts (MC4R +/+ ). To execute the experiment, I collected brains from these rats, used micropunches to isolate specific regions of the hypothalamus known to contain MC receptors and impact energy balance. I then isolated RNA from the tissue samples and converted the RNA to cdna which reflects the proportional amount of RNA. The cdna for each receptor subtype was then quantified using qpcr. Subjects For this study, male rats (N=16), were obtained from Taconic Industries, NY, of which three genotypes were used. The first genotype was wild-type (MC4R +/+ ) (n=5), the second genotype was heterozygous for the receptor gene deletion (MC4R +/- ) (n=6), and the last genotype was homozygous (MC4R -/- ) (n=5) for the mutation. The animals were housed individually in polycarbonate cages, and fed ad libitum food and water in an approved animal facility. The animals were housed in a room at a constant temperature of 70ºF, ± 1 or 2 F, on a 12:12 light dark cycle with lights-on at 0700 Eastern Standard Time. Handling of the animals was in agreement with the Kent

19 9 State University Institutional Animal Care and Use Committee (IACUC) and in accordance with the guidelines outlined in the Guide to the Care and Use of Laboratory Animals. Tissue Collection Upon completion of calorimetry, physical activity, and calorie restriction experiments, a tissue collection occurred to accumulate samples for further testing. The rats were sacrificed, by rapid decapitation according to the protocols set by the IACUC and the Guide to the Care and Use of Laboratory Animals. The brain was removed and placed into isopentane pre-cooled in a liquid nitrogen bath. Isopentane is excellent for freezing the brain rapidly without shattering it, as commonly occurs with liquid nitrogen. After the tissue was frozen, the brain was removed from the isopentane, wrapped in labeled foils, placed on dry ice, and then stored at -80 C. Sectioning of Brain To access the hypothalamic region for our experiments, the brains were sectioned at 40 µm using a Leica CM1950 cryostat. The chamber and stage temperatures were set at -20 C. The forebrain was attached to cryostat chucks using Tissue-Tek OCT (Sakura Finetek, Torrance, CA). The brains were placed on the rapid freeze mechanism for ten minutes to solidify. The brains were sectioned, utilizing the Paxinos and Watson rat brain

20 10 atlas for reference to determine the correct sections to collect [26]. Brain sections were placed on Fisherbrand Superfrost Plus slides. The slides were placed in a bed of dry ice for transportation and stored at -20 C. A. B.

21 11 C. D. Figure 1: Regions of interest shown (A) the arcuate nucleus (ARC) and paraventricular nucleus (PVN), (B) the perifornical region of the lateral hypothalamus (PeFLH), (C) the dorsomedial hypothalamic nucleus (DMH), and (D) the ventromedial hypothalamic nucleus (VMH) [26].

22 12 Microdissection and RNA Extraction To isolate the ARC, PVN, PeFLH, DMH, and VMH microdissection tools were employed. The slides with tissue sections were placed on dry ice. Landmarks of the brain were utilized to find the region desired, including the fornix, the median eminence, and optic chiasm. Depending on the size of the region of interest, a 1mm punching tool was used for the smaller regions, the ARC, or a 2mm tool was used on larger regions, the PVN, PeFLH, VMH, and DMH. The dissected samples were placed into labeled microcentrifuge tubes. The tissue samples were homogenized using TRI Reagent from Ambion. 1 ml of TRI Reagent was placed in each tube containing tissue sections. They were then placed in a homogenizer (Fisher Scientific) for 30 seconds. The homogenates were incubated at room temperature for 5 minutes. 1mL of the homogenates was transferred to a 1.5mL microcentrifuge tubes. Under the fume hood, 100µL of BCP (Molecular Research Center, Cincinnati, Ohio) was added to each tube. The tubes were vortexed at the maximum speed for fifteen seconds using a Vortex Genie 2 and then incubated again at room temperature for five minutes. The tubes were then centrifuged with an Eppendorf Centrifuge 5417R at 12,000 g for 10 minutes at 4 C to separate the mixture. The clear, upper aqueous portion (the supernatant) contained RNA, while the red, lower phases (the precipitant) contained proteins and DNA. Of the aqueous portion, 400µL was transferred to new microcentrifuge tubes. 200µL of 100% ethanol was added to the RNA containing phases and immediately vortexed to avoid RNA precipitation.

23 13 The samples were transferred to a filter collection tube from the Ambion Ribopure kit. The filter tubes were centrifuged at 12,000g for 30 seconds at room temperature to push the liquid through the filter. The eluate was discarded and the filters were returned to the same collection tubes. The RNA was then bound to the filter cartridges. 500µL of a wash solution, from the Ambion Ribopure kit, was added to the filter tubes to remove excess organic material such as DNA and protein and they were centrifuged for 30 seconds at 12,000g. The eluate was discarded and a second wash was repeated. The tubes were centrifuged for 1 minute at room temperature and 12,000g. The filter cartridges were placed in new filter columns, and 100µL of an elution buffer was added to the tubes and incubated at room temperature for two minutes. The tubes were centrifuged for 30 seconds at 12,000g at room temperature to elute the RNA from the filter. The RNA could be found in the eluate of the collection tubes; filters were discarded. Quantifying RNA and cdna Estimation of RNA concentration is crucial to determine purity levels of the samples and prepare cdna. Using a NANOdrop ND-100 spectrophotometer, a blank of the elution solution (a solution containing no organic material) from the Ambion Ribopure kit was used to calibrate the machine. 1µL of each sample was used to determine the RNA concentration (ng/µl) of each sample. The ratio of A260nm/A280nm was read as well. Values of this ratio that are between are indicated as very pure

24 14 samples (Appendix). To prepare the cdna, a kit from Applied Biosystems was utilized that contained TaqMan reverse transcription reagents. For each reaction, the nonenzymatic components were combined first into autoclaved microtubes: 2µL of 10X RT buffer, 4.4µL of MgCl 2, 4 µl of dntp mix, 1 µl of random hexamers, and 5.7µL of nuclease-free water. This solution was vortexed briefly before the addition of the enzymatic components: 0.4µL of RNase Inhibitor, 0.5µL of MultiScribe RTase, and 2µL of the RNA sample. More RNA sample was added if the concentration found in the previous step was below 10ng/ µl. The microtube was inverted, and the contents transferred to a 96-well reaction plate. The plate was briefly centrifuged in a Sorvall RT7 Plus centrifuge before being placed in an Agilent Technologies Stratagene Mx3005P thermoblock. The thermoblock program consisted of 10 minutes at 25 C, followed by 30 minutes at 48 C, and ending with 5 minutes at 95 C. The concentrations of cdna, which reflect the same concentration of mrna, could then be measured using quantitative polymerase chain reaction, qpcr. Quantitative PCR Microcentrifuge tubes were labeled, with the probes for MC3R, MC5R, and one with the control, Glyceraldehyde 3-phosphate dehydrogenase GAPDH. GAPDH is one of the most common housekeeping genes is often used as a control with gene expression data [27]. Into each tube, 450µL of nuclease-free water was pipetted along with 540µL of 2x Brilliant III Ultra-Fast QPCR Master Mix and 54µL of the target/control probe. The

25 15 tubes were vortexed briefly before aliquoting 62µL the target probe and control probe into each of 16 labeled sample tubes, with randomized sample numbers. 2µL of each sample was added to the labeled tubes before vortexing briefly. The samples were aliquoted, 20µL from each tube, into a 96-well plate; each reaction was run in triplicate. The plate was sealed and placed inside the Agilent Technologies Stratagene Mx3005P thermoblock. The settings of the Mx program included the detector: FAM and the reference dye: ROX. The data was saved and analyzed as described below. Statistical Analysis The mrna levels of the MC3R and MC5R were compared after completion of qpcr. GAPDH, a housekeeping gene, was used as a standard curve upon which the expression levels of the target genes were compared to; this calculation resulted in number of cycles necessary to reach the threshold value (dct) relative to GAPDH. By calculating 2 -dct, high 2 -dct (dct) values indicate high levels of expression of MC3R and MC5R [28]. The samples, which were placed in triplicate in the plate, were averaged (for each target gene and brain region), yielding a single number. The means were then analyzed using a one-way ANOVA to compare the expression levels of each target gene dct, the dependent variable, between the 3 groups, the independent variable. A linear model was utilized for multiple comparisons for observed means and equal variance was assumed. Expression levels of each target gene, MC3R and MC5R, were analyzed

26 16 separately for each brain region. Post-hoc comparisons between groups were made using the LSD test and significance was indicated by p<0.05.

27 17 Chapter 3 Results ARC The ANOVAs revealed no significant main effects of group on MC3R or MC5R expression levels. The expression of MC3R and MC5R in the ARC of the homozygotes (MC4R -/- ) was not statistically significant compared to the wild-type (MC4R +/+ ) or the heterozygotes (MC4R +/- ), as shown in Figure 2 below. Both values failed to reach the cutoff of p<0.05 but instead p-values were calculated at p=0.20 for MC3R and p=0.80 for MC5R. A. B. Figure 2: In the arcuate nucleus (ARC), rats lacking functional MC4R (MC4R -/- ) did not have significantly higher expression of MC3R (p=0.20) or MC5R (p=0.80) compared to rats heterozygous for the mutated gene or wild-type rats.

28 18 PVN The ANOVA revealed a significant main effect of group. Specifically, the expression of MC3R in the PVN of the rats homozygotic for the gene deletion (MC4R -/- ) was significantly greater than either the wild-type (MC4R +/+ ) or the heterozygotes (MC4R +/- ) as shown in Figure 3A below. As for the MC5R, there was a trend toward overexpression in the homozygotes (p=0.051) but the trend did not make the significance cutoff. A. B. Figure 3: In the paraventricular nucleus (PVN), rats lacking functional MC4R (MC4R -/- ) had significantly higher expression of MC3R. Though MC5R expression C. showed a similar trend, this did not reach significance D. (p=0.051). *p<0.05 PeFLH The ANOVA revealed no significant main effects of group. The expression of MC3R and MC5R in the PeFLH of the homozygotes (MC4R -/- ) was not statistically significant compared to either the wild-type (MC4R +/+ ) or the heterozygotes (MC4R +/- ),

29 19 as shown in Figure 4 below. Both values failed to reach the cutoff of p<0.05 but instead p-values were calculated at p=0.33 for MC3R and p=0.26 for MC5R. A. B. Figure 4: In the perifornical area of the lateral hypothalamus (PeFLH), rats lacking functional MC4R (MC4R -/- ) did not have significantly higher expression of MC3R (p=0.33) or MC5R (p=0.26). DMH The ANOVAs revealed no significant main effects of group. The expression of MC3R and MC5R in the DMH of the homozygotes (MC4R -/- ) was not statistically significant compared to the wild-type (MC4R +/+ ) or the heterozygotes (MC4R +/- ), as shown in Figure 5 below. Both values failed to reach the cutoff of p<0.05 for significance but instead displayed strong trends; p-values were calculated at p=0.06 for MC3R and p=0.10 for MC5R.

30 20 A. B. Figure 5: In the dorsomedial hypothalamic nucleus (DMH), rats lacking functional MC4R (MC4R -/- ) did not have significantly higher expression of MC3R (p=0.06) or MC5R (p=0.10). VMH The ANOVA revealed no significant main effects of group. The expression of MC3R and MC5R in the VMH of the homozygotes (MC4R -/- ) was not statistically significant compared to the wild-type (MC4R +/+ ) or the heterozygotes (MC4R +/- ), as shown in Figure 6 below. Both values failed to reach the cutoff of p<0.05 for significance, but instead MC3R expression showed a strong trend toward significance (p=0.073); for MC5R, p=0.84. A. B. Figure 6: In the ventromedial hypothalamic nucleus (VMH), rats lacking functional MC4R (MC4R -/- ) did not have significantly higher expression of MC3R (p=0.073) or MC5R (p=0.84).

31 21 Chapter 4 Discussion and Implications The MC4R is critical for homeostatic regulation of energy expenditure, physical activity, metabolism, and autonomic nervous system outflow [18, 22]. MC receptors 3, 4, and 5 are expressed throughout nuclei in the hypothalamic region of the brain, as well as elsewhere in the brain, in varying amounts. When one of these receptors is removed or mutated, abnormalities such as obesity can result; the extreme adiposity of the MC4R -/- rats demonstrates this. Because the MC4R -/- rats lack the receptor throughout development, compensations in the leptin-melanocortin system may have occurred. By quantifying the amount of RNA in specific regions of the hypothalamus known to contain MCRs, I was able to determine if over-expression of MC3R and MC5R occurred. My data indicate that, indeed, while MC4R -/- showed significant increase in only MC3R expression in the PVN, all regions showed examined trends toward the same pattern in both MC3R and MC5R expression. Altogether, these data implicates global compensation of the leptin-melanocortin system in the hypothalamus. After analyzing the concentrations of MC3R and MC5R among five different brain regions, only one region, the PVN, showed significant over-expression of the remaining brain MCRs in the MC4R -/- rats, specifically in MC3R expression. The other nuclei displayed trends toward this same pattern of over expression of MC3R and MC5R (Figure 2, Figures 4-6); further studies are necessary to determine if the trends seen in other regions represent true effects that were not detectable due to the low statistical

32 22 power of this study. A larger population size would be necessary to determine this; only 16 animals were tested in our study, only 5 were homozygous MC4R -/- (n=5), and that may not have been a large enough population to determine significance. The lack of any evidence of compensation in the heterozygotes is interesting and implies that a total deletion of function may be necessary to induce compensatory effects. These finding suggest that humans with monogenic obesity involving total suppression of MC4R function may show compensatory effects through other MCRs, while people with partially functioning (heterozygous) MC4R and those with polygenic obesity impacting MC4R may not. These increased expression of PVN MC3R seen in rats lacking MC4R (Figure 3) provide support for compensation in the brains of animals missing both copies of MC4R gene. Compensation is seen in this study as the targeted disruption of the MC4R gene affects other related genes and systems in our rat model, causing modifications to other MCR expression levels. This change in the expression of MCRs may alter satiety, metabolism, physical activity, and EE, all of which are impacted by the PVN. If a conditional knockout occurred, where the rat would undergo development with its normal complement of MCRs, the compensatory effects would be bypassed and the animal would not develop compensation mechanisms during development. This could result in even more extreme cases of adiposity. This would further solidify the role that MC4R is the most crucial melanocortin receptor for maintaining correct adiposity, metabolism, EE, and satiety.

33 23 It is known that different hypothalamic nuclei contribute in regulating energy balance, but all of the nuclei in the hypothalamus play overlapping roles in the regulation of appetite, EE, metabolism, and SNS outflow. The roles differ slightly and show substantial overlap. The PVN is crucial for increasing SNS outflow and EE as well as producing signals to control feeding behavior, particularly inducing satiety. Increased expression of MC3R and MC5R will differentially regulate these signals and behaviors in knockout animals. The potential impact of altered MCR expression and activity is illustrated by differential receptor expression seen in high capacity running (HCR) and low capacity running (LCR) rats. HCR have more lean mass, higher metabolism, and increased energy expenditure while LCR have lower metabolism and decreased EE as well as high adiposity[29]. The MCRs are expressed differently in the brain regions of HCR-LCR [10]. Specifically, obesity-prone rats (LCR) have lower PVN MC4R expression than their lean (HCR) counterparts. MC agonists in the PVN increase physical activity and EE, implying that compensatory increases in MC3R in the PVN could potentially lead to increases in PA and EE relative to a rat with lower levels of MC3R in the PVN [10]. The leptin-melanocortin system is crucial for maintaining appropriate homeostatic regulation of adiposity, feeding behaviors, metabolism, and energy expenditure. Disruptions in this system result in phenotypic abnormalities, like obesity. Targeted disruption of a single gene in this system, MC4R, resulted in compensation of other MCRs in the hypothalamic region of the brain. This compensation will alter the feeding behaviors, EE, and metabolism of the animals with the disrupted gene.

34 24 References 1. Phinney, S.D., et al., Capacity for moderate exercise in obese subjects after adaptation to a hypocaloric, ketogenic diet. J Clin Invest, (5): p Adult Obesity Facts. [web] 2014 March 28, 2014 [cited 2014 July 22]; Available from: 3. Maes, H.H., M.C. Neale, and L.J. Eaves, Genetic and environmental factors in relative body weight and human adiposity. Behav Genet, (4): p Farooqi, I.S. and S. O'Rahilly, Monogenic obesity in humans. Annu Rev Med, : p Chen, D.L. and A. Garg, Monogenic disorders of obesity and body fat distribution. Journal of Lipid Research, (10): p Cummings, D.E. and M.W. Schwartz, Genetics and pathophysiology of human obesity. Annu Rev Med, : p Konner, A.C., T. Klockener, and J.C. Bruning, Control of energy homeostasis by insulin and leptin: targeting the arcuate nucleus and beyond. Physiology & Behavior, (5): p Guyton, A.C. and J.E. Hall, Textbook of medical physiology. 11th ed. 2006, Philadelphia: Elsevier Saunders. xxxv, 1116 p. 9. Tanaka, T., et al., Central melanocortin signaling restores skeletal muscle AMP-activated protein kinase phosphorylation in mice fed a high-fat diet. Cell Metab, (5): p Shukla, C., et al., Region-specific differences in brain melanocortin receptors in rats of the lean phenotype. Neuroreport, (10): p Kannan, H., Y. Hayashida, and H. Yamashita, Increase in sympathetic outflow by paraventricular nucleus stimulation in awake rats. Am J Physiol, (6 Pt 2): p. R Lechan, R.M. and C. Fekete, The TRH neuron: a hypothalamic integrator of energy metabolism. Prog Brain Res, : p

35 Clapham, J.C., Central control of thermogenesis. Neuropharmacology, (1): p Gold, R.M., Hypothalamic obesity: the myth of the ventromedial nucleus. Science, (4111): p King, B.A., The rise, fall, and resurrection of the ventromedial hypothalamus in the regulation of feeding behavior and body weight. Physiology & Behavior, (2): p Novak, C.M., et al., Associations between behavior, hormones, and Fos responses to novelty differ in pre- and post-pubertal grass rats. Physiology & Behavior, (1): p Sohn, J.W., et al., Melanocortin 4 Receptors Reciprocally Regulate Sympathetic and Parasympathetic Preganglionic Neurons. Cell, (3): p Biebermann, H., et al., The neuroendocrine circuitry controlled by POMC, MSH, and AGRP. Handb Exp Pharmacol, 2012(209): p Huszar, D., et al., Targeted disruption of the melanocortin-4 receptor results in obesity in mice. Cell, (1): p Mountjoy, K.G., et al., The Cloning of a Family of Genes That Encode the Melanocortin Receptors. Science, (5074): p Low, M.J., R.B. Simerly, and R.D. Cone, Receptors for the melanocortin peptides in the central nervous system. Current Opinion in Endocrinology, Diabetes and Obesity, (1): p Evidence of CNS plays an important role to control metabolism. [web] [cited 2014 July 22]; Available from: &title=Compound&sum=Function. 23. Gantz, I., et al., Molecular cloning, expression, and characterization of a fifth melanocortin receptor. Biochem Biophys Res Commun, (3): p

36 Terashima, H., et al., Up-regulation of genes encoding glycosylphosphatidylinositol (GPI)-attached proteins in response to cell wall damage caused by disruption of FKS1 in Saccharomyces cerevisiae. Mol Gen Genet, (1-2): p Tong, A.H., et al., Systematic genetic analysis with ordered arrays of yeast deletion mutants. Science, (5550): p Watson, G.P.a.C., The Rat Brain in Stereotaxic Coordinates. 5th ed. 2005, Burlington: Elsevier Academic Press. 27. Barber, R.D., et al., GAPDH as a housekeeping gene: analysis of GAPDH mrna expression in a panel of 72 human tissues. Physiol Genomics, (3): p Schmittgen, T.D. and K.J. Livak, Analyzing real-time PCR data by the comparative C(T) method. Nat Protoc, (6): p Koch, L.G., S.L. Britton, and U. Wisloff, A rat model system to study complex disease risks, fitness, aging, and longevity. Trends Cardiovasc Med, (2): p

37 27 Appendix Sampl e Numb er RNA Concentrati on A260nm/A280 nm

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