Title: Hypothalamic Melanocortin Regulation of Energy Balance and Metabolism
Description: Genetic and environmental factors both contribute to obesity, however studies in twins and adoptees demonstrate that genetic predisposition and susceptibility are driving factors in the development of this disease. Although numerous human mutations are associated with an increase in obesity prevalence, melanocortin-4 receptor (MC4-R) mutations are the most common monogenic form of severe obesity and genetic deletion of this receptor in rodents also leads to an obese phenotype. The G-protein coupled MC4-R is a target for the peptide products of Proopiomelanocortin (POMC) and Agouti-related peptide (AgRP) neurons residing in the arcuate nucleus of the hypothalamus. POMC-derived alpha-melanocyte-stimulating hormone (MSH) is an agonist for the MC4-R and promotes negative energy balance, while the melanocortin-receptor antagonist AgRP promotes positive energy balance. Given the strong influence of the hypothalamic melanocortin system on energy balance, this thesis sought to investigate unexplored aspects of this system in relation to obesity.

POMC is post-translationally processed to biologically active peptides with opposing actions. Alpha-MSH is well established to decrease food intake and increase energy expenditure, however POMC-derived beta-endorphin (beta-EP) has been shown in limited studies to increase food intake. Our experiments in intracerebroventricular (icv) cannulated rats demonstrate that the effects of beta-EP alone on feeding are complex. Beta-EP acutely stimulated food intake during both the light and dark cycle, however orexigenic effects were not sustained in a chronic model; in fact, higher doses of chronic beta-EP decreased food intake. Subthreshold doses of beta-EP also reversed alpha-MSH-induced suppression in feeding and weight gain in an acute fasting and refeeding model as well as a chronic infusion model. Beta-EP 1-27, a product of C-terminal beta-EP cleavage reported to have reduced opioid activity, did not stimulate food intake alone, nor could it reverse alpha-MSH-induced suppression in feeding.

These studies show that POMC-derived peptides alpha-MSH and beta-EP can interact to regulate food intake and body weight and highlight the importance of understanding how the balance between these peptides is maintained, as well as the potential role of differential POMC processing in regulating energy balance. AgRP is also a critical component of the melanocortin system; however, studies in which the AgRP peptide was deleted show only a mild phenotype suggesting that developmental compensation exists in this model. Consequently the role of the AgRP peptide was investigated using a novel AgRP inhibitor developed by TransTech Pharma, Inc. Results show that this inhibitor was extremely effective in reversing exogenous icv AgRP-induced metabolic and neuroendocrine parameters in rats, and these parameters were unaffected in saline infused rats receiving this drug. This AgRP inhibitor also reduced food intake, weight gain and adiposity in diet-induced obese (DIO) and ob/ob mice and increased thyroxine (T4) levels in DIO mice, consistent with AgRP's reported effects; however this drug did not affect food intake or weight gain in lean chow fed mice.

The AgRP inhibitor also suppressed rebound feeding and potently reduced food intake in mice immediately upon initiation of a high fat diet (HFD). As some of these effects were also observed in AgRP knockout (KO) mice, this indicates that there are clear off-target effects that are not due to AgRP antagonism. Although there are many potential reasons why a drug may yield anorexia and weight loss, the fact that these effects were only observed in obese models or in the presence of increased dietary fat, suggests the possibility that another molecule that promotes positive energy balance and fat intake is also being targeted. As the melanocortin system can also regulate pituitary function, this thesis investigated circulating and pituitary prolactin levels in models with genetic manipulation of this system. Previous studies have shown that acute stimulation of the melanocortin system by alpha-MSH or antagonism by AgRP decreases and increases prolactin levels, respectively. However, the effects of chronic melanocortin manipulation were unknown.

Male mice with selective MSH overexpression and AgRP deletion were found to have decreased blood prolactin levels under both stressed and unstressed conditions, and mice with AgRP deletion also had lower prolactin content in the pituitary. As prolactin is tonically inhibited by dopamine and acute melanocortin regulation of prolactin release has been shown to be dopamine-mediated, we sought to show this mechanism in these genetic models. Measurements of mediobasal hypothalamic (MBH) tyrosine hydroxylase mRNA levels as well as dopamine and 3,4-dihydroxyphenylacetic acid (DOPAC) content did not differ between groups, nor did prolactin measurements after functional dopamine receptor antagonism. However, these results do not preclude changes in dopamine activity as dopamine turnover was not directly investigated. This is the first report of reduced baseline and stress-induced prolactin release and pituitary prolactin content in mice with genetic alterations of the melanocortin system and suggests that changes in hypothalamic melanocortin activity may be reflected in measurements of serum prolactin levels.

The melanocortin system has been shown to mediate some of the adaptive responses after introduction to the HFD including attenuation of hyperphagia and weight gain; thus, preliminary studies sought to investigate the effects of the HFD on hypothalamic melanocortin gene and peptide expression, as well as expression of enzymes responsible for POMC post-translational processing. Mice administered a 60% HFD were found to have increased levels of POMC precursor peptide after both acute (3 and 7 days) and chronic (8 week) HFD administration. After 3 days of HFD feeding, increased POMC peptide levels were accompanied by increased Pomc mRNA and alpha-MSH and beta-EP, however after chronic HFD feeding POMC peptide levels were elevated without an increase in these parameters. Additionally, chronic HFD feeding suppressed AgRP mRNA and peptide levels and tended to increase the alpha-MSH/beta-EP ratio and suppress mRNA levels of prolylcarboxypeptidase (Prcp), an enzyme responsible for inactivating alpha-MSH.

These data suggest that the HFD can modulate melanocortin gene and peptide expression with distinct time-related changes and are consistent with the hypothesis that POMC-derived peptides act in concert to regulate energy balance and metabolism. However, these results are preliminary and further investigation of the effects of HFD feeding on peptide and processing enzyme expression in more anatomically discrete hypothalamic nuclei as well as dynamic studies of peptide release are required. These data provide a contribution to the field of melanocortin regulation of energy balance by further elucidating the effects of POMC processed peptides both alone and in interaction, by providing insight into the effects of a novel AgRP inhibitor on energy balance and metabolism, by investigating the effects of genetic models of chronic melanocortin manipulation on the pituitary hormone prolactin, as well as by examining the effects of HFD feeding on melanocortin gene and peptide expression.