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<p> The present thesis examined the relative contribution of hypothalamic and nucleus accumbens dopaminergic activity to the control of sucrose sham feeding. Bilateral intracranial infusions of the dopamine antagonist, haloperidol at doses of 1.25, 2.5, 5 and 7.5 ug, into the hypothalamus and nucleus accumbens produced dose-dependent suppressions of sham feeding of a 15% sucrose solution. No differences in temporal pattern of intake were observed between hypothalamic and nucleus accumbens infusions under drug or vehicle conditions. Also, motor deficits were observed with the highest dose of haloperidol (7.5 ug) while the three lower doses inhibited sham feeding without obvious signs of motor impairment. These results suggest that mesolimbic as well as hypothalamic dopaminergic activity mediate the hedonic aspects of feeding. </p> / Thesis / Master of Science (MSc)

To test the hypothesis that hypothalamic β-endorphin levels were related to the body weight changes occurring with ovarian steroid treatments, 80 adult female Sprague-Dawley rats were ovariectomized and allowed to recover for 7 days. Four treatment groups of 20 each were subjected to daily injections for 14 days with A) oil, B) β-estradiol benzoate (2µg), C) β-estradiol benzoate (2µg) plus progesterone (5mg), or D) progesterone (5mg). Weight gain was significantly (p < 0.01) lower in the estrogen group when compared to the control, estrogen/progesterone, or progesterone groups. The estrogen/progesterone-treated group gained significantly less weight than either the control or progesterone group. A significant (p < 0.05) decrease in food intake was also observed in the estrogen and estrogen/progesterone groups when compared to the control and progesterone groups, but not between each other. The progesterone-treated group was not significantly different from the controls in either weight gain or food intake. Hypothalamic β-endorphin (ng/mg protein) concentrations were significantly (p < 0.05) higher in the estrogen- and estrogen/progesterone-treated groups compared to the control and progesterone groups. Again, much like the weight gain, food intake, and hypothalamic β-endorphin (ng/mg protein), the estrogen and estrogen/progesterone groups had significantly (p < 0.05) heavier adrenal weights when compared to the controls and progesterone groups. There was no significant difference in plasma corticosterone levels between any of the groups. In this study, hypothalamic β-endorphin (ng/mg protein) appeared to be effected by ovarian steroid hormone treatment. Whether this is related to the weight gain also observed with the treatments remains to be determined. / Ph. D.

Appetite is a complex behavior which can be influenced by factors within the animal's body as well as the environment around it. Internal factors include hormonal and nutrient concentrations found in the blood stream and subsequent neuropeptide and neurotransmitter signaling in the hypothalamus. External factors, such as high ambient temperature (HAT), can indirectly affect appetite regulation through other neuroendocrine systems such as the hypothalamo-pituitary-adrenal (HPA) axis. Understanding the physiological responses to endogenous factors and HAT exposure in birds will have implications in both the agricultural and biomedical fields. Thus, the purpose of this dissertation research was to explore the hypothalamic molecular mechanisms associated with food intake in broiler type chickens and Japanese quail, and the effect of HAT exposure on food intake in broiler type chickens. Broiler type chickens have undergone intense artificial selection for traits that promote rapid growth, consequently driving them to consume feed incessantly. It is hypothesized that broiler type chickens lack a mechanism that signals satiety, causing them to eat significantly more than layer type chickens. Selection for rapid growth of meat (muscle tissue) has made the broiler more susceptible to the deficits associated with HAT exposure, as animals composed primarily of muscle dissipate less heat while also producing more heat than those composed of fat. The Japanese quail have undergone relatively minor artificial selection compared to the chicken, suggesting that use of this model may provide insight into the mechanisms of appetite regulation in wild-type bird species. This research involved administrating appetite associated factors into the avian brain via an intracerebroventricular (ICV) injection including gastrin releasing peptide, vasoactive intestinal polypeptide, neuropeptide AF, and prostaglandin D2. Additionally, I explored the effects of HAT on food intake, and on the efficacy of several ICV administered appetite associated factors including neuropeptide Y, corticotropin releasing factor and α-melanocyte stimulating hormone. After treatment administration, I measured changes in food intake and behavior, activation of hypothalamic nuclei including the arcuate nucleus, dorsomedial nucleus, lateral hypothalamus, paraventricular nucleus, and the ventromedial nucleus, and the nucleus of the hippocampal commissure. I then measured changes in gene expression in both whole hypothalamic samples and specific hypothalamic nuclei. The data from non-HAT associated studies provided information on the hypothalamic nuclei which respond to the various appetite associated factors and the molecular mechanisms mediating changes in appetite. The data from the HAT study provided information on the hypothalamic nuclei involved in the avian response to HAT exposure, and the molecular mechanisms involved in the effect on food intake. Overall, these data provide insight on the mechanisms associated with short-term regulation of appetite, and pathways associated with stress and food intake. / Doctor of Philosophy / Appetite regulation can be affected by factors both in the body and out in the environment. Understanding how both internal and external factors affect appetite regulation can have positive implications in both the agriculture industry as well as the biomedical field. In agriculture, animals exposed to high ambient temperatures often exhibit several deficits including immunosuppression, decreased body weight, and ultimately an increased risk of mortality. It is hypothesized that the factor linking negative wellbeing to heat exposure is a reduction in food intake. Animals aside, the prevalence of eating disorders has doubled worldwide every 6 years since the year 2000. These numbers have increased even more during the recent COVID-19 pandemic. In order to improve the wellbeing of both humans and animals exposed to stressing stimuli, it is imperative we understand how individual appetite associated factors affect food intake, and how external stressors can impact the normal physiology of the hypothalamus. Thus, the purpose of this dissertation was to elucidate the hypothalamic mechanisms mediating appetite regulation using broiler type chickens and Japanese quail as models. Related pathways and molecular mechanisms were explored for several appetite associated factors including gastrin releasing peptide, vasoactive intestinal polypeptide, neuropeptide AF, and prostaglandin D2. Additionally, the effect of high ambient temperature on food intake, on the efficacy of several appetite associated factors including neuropeptide Y, corticotropin releasing factor and α-melanocyte stimulating hormone, and the hypothalamic pathways and molecular mechanisms mediating heat-induced anorexia were assessed.

Energy balance includes energy intake and energy expenditure. Either excessive food intake or insufficient physical activity will increase the body mass and cause obesity, a worldwide health problem. In the US, more than two-thirds of people are obesity or overweight. Conversely, it is well accepted that reducing energy intake can increase the life span and the resistance to age-related diseases. MicroRNAs are highly conserved non-coding RNA molecules with a length of 21-23 nucleotides. Recent studies show that numerous microRNAs are associated with the regulation of oxidative stress, inflammation, insulin signaling, apoptosis, and angiogenesis that relate to obesity. However, the role of microRNAs in the regulation of energy balance in central nervous system remains unknown, especially within the hypothalamus, a primary site of energy balance control. In this project, microRNA, and mRNA were profiled using microarray technology. Furthermore, quantitative proteomics were used to identify differential protein levels during fasting, and in a genetically obese mouse model, Mice were given either a 24-hour fast, or ad libitum access to food. Hypothalamic RNA and microRNA samples were analyzed by microarray, using both the Affymetrix and Toray 3D mRNA and microRNA platforms. No microRNAs were found to be differentially expressed between two treatments, whereas numerous mRNAs were significantly regulated by fasting, including 7 cell cycle related genes. Hypothalamic protein samples from WT and N2KO mice treated either to ad lib feeding or 24-hour fasting were analyzed by MSE quantitative proteomics. Over 650 proteins were identified with some proteins showing significantly different abundances between or among the four groups. Between ad lib fed WT and N2KO mice, 53 proteins were differentially expressed, with some of these linked to neurodegeneration, NAD synthesis, and the citrate acid cycle (TCA). Overall, the results of this study suggest that while microRNA-mediated mechanisms are not significant modulators of hypothalamic gene expression upon a 24 hour fast, cell cycle gene expression changes represent a major contributor to the fasting response. Moreover, Nlhl2 might play an important role in the neurodegeneration and mitochondrial metabolism. / Ph. D.

Prolactin-releasing peptide (PrRP) is an endogenous hypothalamic neuropeptide that when exogenously injected increases food intake in chickens, but decreases it in rodents and goldfish. We designed three sets of experiments to elucidate the mechanisms of PrRP's orexigenic effect in chicks. In experiment one, food and water intake were evaluated in chicks after receiving intracerebroventricular (ICV) injection of the vehicle, 0.75, 3, 12, 47 or 188 pmol PrRP. The administration of 12 and 47 pmol PrRP increased food intake for up to 120 min after injection, and 188 pmol increased it for up to 180 min. The lowest effective dose was 3 pmol, which increased food intake for up to 60 min after injection. Water intake was not affected. To investigate the molecular mechanisms, c-Fos immunohistochemistry was performed and mRNA expression of some appetite-associated neurotransmitters was measured in chicks that received either vehicle or 188 pmol of PrRP. The rostral paraventricular nucleus (PVN) was activated which coincided with increased neuropeptide Y (NPY) mRNA expression in the whole hypothalamus. In experiment two, food and water intake were evaluated in chicks fed a high carbohydrate (HC), high fat (HF) or high protein (HP) diet after ICV injection of vehicle, 3 or 188 pmol PrRP. Chicks fed the HP diet increased food intake at a lower dose than chicks fed HF and HP diets after ICV PrRP injection. In addition, ICV injection of vehicle, 3 and 188 pmol PrRP were performed in chicks fed all three diets, and ICV PrRP injection induced preferential intake of the HP diet over HC and HF diets. The expression of some appetite-associated neuropeptides in the hypothalamus was also measured in chicks fed the HC, HF or HP diet after ICV injection of vehicle or 188 pmol PrRP. There was a diet effect on mRNA abundance of all appetite-associated genes measured (P < 0.05), with greater expression in chicks fed the HF or HP than HC diet. While neuropeptide Y (NPY) mRNA abundance was similar between vehicle and PrRP-injected chicks that consumed HP or HF diets, expression was greater (P < 0.05) in PrRP- than vehicle-injected chicks that consumed the HC. In experiment three, the orexigenic effect of PrRP was tested in chicks selected for low (LWS) and high (HWS) body weight after central administration of vehicle, 24, 94 and 375 pmol PrRP. The LWS chicks had a lower threshold and higher magnitude of food intake increase in response to PrRP injection. Results demonstrate that PrRP is a potent orexigenic factor in chickens and that effects are likely mediated through the hypothalamus. The orexigenic effect of PrRP was influenced by dietary macronutrient composition, and diet in turn influenced the food intake response to PrRP. These results may contribute to a novel understanding of appetite regulation. / Master of Science

Feeding behavior is a complex trait that is regulated by various hypothalamic neuropeptides and neuronal populations (nuclei). Understanding the physiological regulation of food intake is important for improving nutrient utilization efficiency in agricultural species and for understanding and treating eating disorders. Knowledge about appetite in birds has agricultural and biomedical relevance and provides evolutionary perspective. I thus investigated hypothalamic molecular mechanisms associated with appetite in broilers, layers, chicken lines selected for low (LWS) or high (HWS) body weight, and Japanese quail, which provide a unique perspective to understanding appetite. Broiler-type chicks have been genetically selected for rapid growth and consume much more feed than do layer-type chicks which have been selected for egg production. Long-term selection has caused the LWS chicks to have different severities of anorexia while the HWS chicks become obese, thus making these lines a valuable model for metabolic disorders. Lastly, the Japanese quail have not undergone as extensive artificial selection as the chicken, thus this model may provide insights on how human intervention has changed the mechanisms that regulate feeding behavior in birds. This research involved applying a variety of different treatments including fasting and refeeding, diets differing in macronutrient composition, and/or central administration of neuropeptide Y, xenopsin, neuropeptide K, oxytocin, mesotocin, gonadotropin-inhibitory hormone, and prolactin-releasing peptide, after which I measured feeding behavior and various aspects of hypothalamic physiology. I measured nuclei activation in hypothalamic appetite-associated regions including the lateral hypothalamus, paraventricular nucleus, ventromedial hypothalamus, dorsomedial nucleus, and arcuate nucleus and I measured gene expression of various appetite-associated factors in the whole hypothalamus and individual nuclei. These data provided information about the regions of the brain involved in mediating effects on appetite and the molecular pathways involved in the effect on appetite. There were differences in dose threshold sensitivity to various injected factors in the different stocks, differential responses to fasting and refeeding, and differences in nuclei and genes that were activated in response to the various treatments. These data provide valuable insights on the molecular mechanisms that are associated with the short-term regulation of feeding behavior and pathways that may be genetically stock-dependent. / PHD

Mammalian and avian species differ in some appetite-related aspects including how and which neurotransmitters and hormones regulate appetite. The objective of this research was to determine how two satiety-inducing neuropeptides regulate feeding behavior in avian models. Neuropeptide FF (NPFF) was intracerebroventricularly (ICV) injected into Japanese quail and decreased food intake at a dose of 32 nmol. NPFF-injected quail had increased expression levels of hypothalamic melanocortin subtype 3 receptor and decreased expression levels of neuropeptide Y receptor subtype 1 mRNAs compared to vehicle-injected controls. In a second study, gastrin was ICV injected into broiler chicks and decreased food intake at a dose of 500 ng (0.12 nmol). There was increased c-Fos immunoreactivity in the lateral hypothalamus (LH), paraventricular nucleus (PVN), arcuate nucleus, nucleus of the solitary tract, and area postrema at 1 h post-injection. Although a variety of genes were measured in those activated nuclei, there were only differences in melanin-concentrating hormone mRNA in the LH and corticotropin-releasing factor (CRF) mRNA in the PVN, suggesting that CRF signaling was involved in the hypothalamic response to gastrin. However, co-injection of gastrin and astressin, a CRF receptor antagonist did not affect gastrin-induced suppression of food intake, implying that the CRF receptors may not be directly associated with gastrin-induced satiety. Identifying the molecular pathways that mediate the effects of anorexigenic neuropeptides in birds will lead to the development of novel treatment options for appetite-related diseases and increased understanding of factors that affect production efficiency in commercial poultry and survival/resource allocation in wild birds. / Master of Science

Obesity is a major health condition in which little is known about the molecular mechanisms that drive it. The hypothalamus is the primary control center for controlling both food intake and energy expenditure in order to maintain the body's energy balance and dysregulation of molecular processes in this region have been implicated in the development and progression of obesity. Recently, several studies have shown altered DNA methylation of critical appetite genes, including the satiety gene Pomc, in the hypothalamus of rodents fed a high fat obesogenic diet. However, it has not previously been studied whether diet-induced changes in DNA methylation of critical appetite genes in the hypothalamus contributes to the development and persistence of the obesity phenotype. Further, DNA 5-hydroxymethylation (5-hmC) is one type of DNA methylation that is 10 times more abundant in the brain than peripheral tissues. However, to date, no study has been conducted examining whether DNA 5-hmC becomes altered in the brain following weight gain and/or contributes to the obesity phenotype. Additionally, there is also evidence to support that exposure to a high fat diet dysregulates the activity of the ubiquitin-proteasome system, the master regulator of protein degradation in cells, in the hypothalamus of male rodents. Despite this, whether this can occur in both sexes and directly contributes to abnormal weight gain has not been investigated. Here, we used a rodent diet-induced obesity model in combination with quantitative molecular assays and CRISPR-dCas9 manipulations to test the role of hypothalamic 1) DNA 5-hmC levels, 2) Pomc methylation, and 3) dysregulated ubiquitin-proteasome signaling in abnormal weight gain following exposure to obesogenic diets. We found that males, but not females, have decreased levels of DNA 5-hmC in the hypothalamus following exposure to a high fat diet, which tracked body weight. Short-term exposure to a high fat diet, which does not result in significant weight gain, resulted in decreased hypothalamic DNA 5-hmC levels, suggesting these changes occur prior to obesity development. Moreover, decreases in DNA 5-hmC persist even after the high fat diet is removed. Importantly, CRISPR-dCas9 mediated upregulation of DNA 5-hmC enzymes in the male, but not female, hypothalamus significantly reduced the percentage of weight gained on the high fat diet relative to controls. Next, we used the CRISPR-dCas9-TET1 and dCas9-DNMT3a systems to test the role of Pomc DNA methylation in the hypothalamus in abnormal weight gain following acute exposure to a high fat diet in male rats. We found that exposure to a high fat diet increases Pomc DNA methylation and reduces gene expression in the hypothalamus. Despite this, we found that CRISPR-dCas9-TET1-mediated demethylation of Pomc was not sufficient to prevent abnormal weight gain following exposure to a high fat diet. Moreover, CRISPR-dCas9-DNMT3a-mediated methylation of Pomc did not alter weight gain following exposure to standard or high fat diets. Finally, we found that both males and females showed dynamic downregulation of proteasome activity, decreases in proteasome subunit expression and an accumulation of degradation-specific K48 polyubiquitinated proteins in the hypothalamus. However, while the CRISPR-dCas9 system was able to selectively increase some forms of proteasome activity, it was unable to prevent diet-induced proteasome downregulation or abnormal weight gain. Collectively, this data reveals novel, sex-specific differences in the engagement of the ubiquitin proteasome system and role of DNA 5-hydroxymethylation in the hypothalamus during the development of the obesity phenotype. / Doctor of Philosophy / Obesity affects 34% of the American population at an annual cost of more than $340 billion in healthcare and is a risk factor for the development of diabetes and certain cancers. Genetic and environmental factors have also been shown to influence the expression of genes that play a role in the development of obesity. The hypothalamus coordinates many integral activities such as hormone regulation and feed intake and numerous studies have observed altered hypothalamic gene regulation in obesity models. Recently, several studies have shown altered DNA methylation of critical appetite genes, including the satiety gene Pomc, in the hypothalamus of rodents fed a high fat obesogenic diet. However, it has not previously been studied whether diet-induced changes in DNA methylation of critical appetite genes in the hypothalamus contributes to the development and persistence of the obesity phenotype. Further, DNA 5-hydroxymethylation (5-hmC) is one type of DNA methylation that is 10 times more abundant in the brain than peripheral tissues. However, to date, no study has been conducted examining whether DNA 5-hmC becomes altered in the brain following weight gain and/or contributes to the obesity phenotype. Additionally, there is also evidence to support that exposure to a high fat diet dysregulates the activity of the ubiquitin-proteasome system, the master regulator of protein degradation in cells, in the hypothalamus of male rodents. Despite this, whether this can occur in both sexes and directly contributes to abnormal weight gain has not been investigated. In this document, I outline a series of experiments designed to elucidate novel, sex-specific differences in the role of the ubiquitin proteasome system and DNA 5-hydroxymethylation in the hypothalamus during the development of the obesity phenotype.

Appetite regulation is primarily mediated by the hypothalamus, within which many neurotransmitters that regulate feeding are shared by the stress response circuitry. Stressors, especially those occur during critical periods of life, influence epigenetic programming and gene expression in the long-term. Therefore, the aim of this dissertation was to elucidate how hypothalamic mechanisms of appetite regulation correlate with the stress response and epigenetic modifications, using avian models and intracerebroventricular administration of various appetite-regulating factors. We first administered two methylation modifiers, S-adenosylmethionine (SAM), a methyl donor, and 5-azacytidine (AZA), a methylation inhibitor, to determine their effects on appetite. When measuring food intake immediately post-injection, SAM didn't affect fed or fasted chickens from a line selected for low bodyweight (LWS, individuals with anorexia), but suppressed feeding in fed and fasted broilers. In Japanese quail, SAM transiently induced satiety in fed but not fasted chicks. Intriguingly, AZA increased feeding in fasted LWS but decreased it in fed chicks. While it didn't affect either fed or fasted broilers, AZA induced satiety in both fed and fasted quail. These results suggests that SAM/AZA can directly affect appetite depending on genetics and nutritional state. The LWS chickens, when injected with SAM or AZA on day of hatch, didn't show increased feeding to the orexigenic stimulation of neuropeptide Y central injection on day 5 post-hatch. This suggests that epigenetic modifications occurred following SAM/AZA injection and affect appetite regulation that persisted. In other studies, we injected broilers with prostaglandin E2 (PGE2) or β-melanocyte-stimulating hormone (β-MSH) since their effects on appetite are unknown in meat-type chicks. We found that they both potently induced satiety, but the effective duration was longer in β-MSH-injected birds (up to 9 hours) than in PGE2-injected chicks (lasted for 1.5 hours). They both activated the paraventricular nucleus of the hypothalamus. The satiety induced by β-MSH mainly involved corticotropin-releasing factor and mesotocin, while the effect of PGE2 included ghrelin and brain-derived neurotropic factor. Nevertheless, all affected appetite-related factors have connections with the stress response. Thus, our results demonstrate that the hypothalamic mechanisms underlying anorexia induced by different neuroactive molecules involve the stress response and epigenetic modifications. / Doctor of Philosophy / Eating disorders (EDs) all involve abnormal eating behaviors and altered body weight. These aberrant conditions are associated with a change in metabolism and pose great risk to human health and animal production, and are generally characterized by two opposite outcomes, anorexia and obesity. Although affected by multiple systems within the body, appetite regulation is mainly controlled by the brain, especially the hypothalamus. Thus, it is important to understand the hypothalamic mechanisms underlying the regulation of eating behavior. In the hypothalamus, many neurotransmitters affect multiple pathways, including the stress response and those that regulate appetite. Additionally, stress, especially when occurring during early life, can influence behaviors later in life through inducing epigenetic modifications (changes to the packaging of the DNA nucleotide sequence) that alter gene expression. Therefore, the aim of this dissertation was to elucidate how hypothalamic mechanisms of appetite regulation correlate with the stress response and epigenetic modifications, using avian models. To focus on the effects within the brain, we directly injected various appetite regulating factors into the brain in each of the experiments. Previously, our group demonstrated that early-life cold exposure and delayed food supply changed DNA methylation and affected expression of appetite-related genes and food intake in a chicken line predisposed to anorexia. We herein injected chicks with one of two methylation modifiers, S-adenosylmethionine (SAM), a methyl donor, and 5-azacytidine (AZA), a methylation inhibitor, to evaluate their effects on feeding behavior. When food intake was measured immediately after injection, SAM did not affect food intake in either fed or fasted line chickens from a genetic line selected for low body weight (LWS, individuals with anorexia), but suppressed food intake in both fed and fasted broiler (meat-type chickens) chicks. In Japanese quail, however, SAM only transiently induced satiety in fed chicks but not in fasted ones. Intriguingly, AZA increased food intake in fasted LWS chicks but decreased it in fed chicks, but AZA had no effects on food consumption in either fed or fasted broilers. Additionally, AZA suppressed food intake in both fed and fasted quail. These results suggest that SAM and AZA affect appetite differently depending on genetic background and nutritional states. LWS chickens, when injected with SAM or AZA on day of hatch, did not eat more after being injected with the potent hunger factor, neuropeptide Y, at 5 days of age. This indicates that epigenetic modifications occurred following SAM/AZA injection and had persisting effects on appetite regulation. In the other two studies, we injected broiler chicks with prostaglandin E2 (PGE2), a fatty acid-based molecule, or β-melanocyte-stimulating hormone (β-MSH), a peptide. These two molecules have been reported to regulate feeding behavior in rodents and layer-type chickens, but effects are unknown in broilers. They both potently decreased food intake in broilers, but the effective duration was much longer in β-MSH-injected birds (up to 9 hours) than in PGE2-injected chicks (lasted for 1.5 hours). They both activated the paraventricular nucleus of the hypothalamus, while β-MSH also activated the arcuate nucleus and ventromedial nucleus. We further found that the anorexia induced by β-MSH involved corticotropin-releasing factor, mesotocin, and their receptors, while the effect of PGE2 was associated with a change in ghrelin and brain-derived neurotropic factor gene expression. Nevertheless, all of these affected factors have connections with the stress response. Thus, results indicate that the hypothalamic mechanisms underlying anorexia induced by different neuroactive molecules involve the stress response and epigenetic modifications.