Roger D. Cone, Ph.D. - US grants

DESCRIPTION (adapted from the applicant's abstract): Graves' hyperthyroidism is caused by autoantibodies which stimulate the thyroid by binding to and activating the thyroid stimulating hormone receptor (TSH-R). This phenomenon has been difficult to study due to the extreme scarcity of the receptor protein. Recently, Dr. Cone's laboratory has isolated a full length cDNA encoding a functional human TSH receptor. With this new reagent, he plans to determine the molecular mechanism by which TSH and Graves' autoantibodies bind to and activate the TSH receptor. A rare class of autoantibodies, which act as competitive antagonists of TSH, will also be studied. Normally a negative feedback of thyroid function results from thyroid hormone repression of TSH secretion and TSH beta chain gene expression. In Graves' disease TSH, levels are depressed; however, the levels of stimulatory autoantibodies remain constant resulting in continuous overstimulation of thyroid activity. While most receptors are downregulated by exposure to antagonists, this does not appear to be the case in Graves' disease. In addition to determining the structural mechanisms by which Graves' autoantibodies activate the TSH-R, the investigator would like also to understand the biological mechanism by which the autoantibodies persistently stimulate thyroid activity without apparent desensitization. Towards this end, he will examine the regulation of receptor mRNA and protein by TSH, Graves' autoantibodies, cAMP and thyroid hormone. The internationalization and recycling of the TSH-R protein at the plasma membrane will also be examined. Additional autoimmune phenomena occur in a small percentage of Graves' patients, resulting in an ophthalmopathy or dermopathy. Both of these conditions are characterized by lymphocytic infiltration and metabolic stimulation of resident fibroblasts or adipocytes. The TSH-R is expressed in some adipocytes, and this has led numerous investigators to propose that an autoimmune reaction against the TSH-R, expressed in retro-orbital muscle or the skin, leads to Graves' ophthalmopathy or dermopathy. To address these problems, the investigator proposes the following Specific Aims: 1) to develop a detailed structure/function model for binding and activation of the receptor by TSH and Graves' autoantibodies; 2) to study the expression and regulation of the receptor, and its role in stimulating thyroid growth and metabolism in normal thyroid, Graves' disease, and other abnormal thyroid growth states; and 3) to characterize the extrathyroidal sites of TSH receptor expression and test the hypothesis that extrathyroidal TSH receptor expression, or expression of antigenically related molecules, plays an etiological role in Graves' ophthalmopathy and dermopathy.

The proopiomelanocortin peptide alpha-MSH regulates melanin production and growth of melanocytes by binding to and activating the MSH receptor. Recently we reported the cloning of the genes encoding the human and murine MSH receptors. Three additional related receptors, the receptor for ACTH, and two unique neural melanocortin receptors (MC30R & MC4-R) that recognize a variety of ACTH and MSH peptides were also clone and characterized. The murine MSH receptor (MSH-R) was subsequently found to map to the distal portion of chromosome 8 in the mouse near a gene locus affecting pigmentation, called extension. Variant extension locus alleles in the mouse were found to result from point mutation s which alter MSH recaptor function. In mice, the recessive yellow extension allele (e) results from a frameshift producing a prematurely terminated non-functioning receptor. The sombre (Eso & Eso-31) and tobacco darkening (Eob) alleles, which both have dominant melanizing effects, result from point mutations which produce hyperactive MSH receptors. The Eso-eJ receptor is constitutively activated, while the Eob receptor remains hormone responsive, and produces a greater activation of its effector, adenylyl cyclase, than does the wild type allele. The extension locus is an homologous gene locus for which variant alleles have been described in many mammalian species. We propose to characterize variant MSH-R/extension alleles in man and other mammals to determine the general role of these alleles in man will be designed to identify correlations with skin, hair, and eye color, melanoma susceptibility, and other pigmentation disorders. Study of the MSH receptor will not only further our understanding of pigmentation; this receptor is perhaps one of the best models for the study of G protein coupled peptide receptor structure and activation. The MSH receptor is the only known G protein coupled with naturally occurring functional variants. The cloning of four melanocortin receptor with differing ligand specificities (MSH-R, ACH-R, MC3-R,MC$-R) and the natural occurrence of MSH receptors with variable activity also provide a unique resource for the study of G protein coupled receptors. Data obtained from cloning nd characterization of variant MSH-R alleles, from biochemical and in vitro mutagenesis studies of the MSH receptor, and from characterization of other POMC receptors will also be used to develop a detailed model of the structure and activation of this receptor.

The H-P-A axis is a key physiological system for detecting and responding to stress. While corticotropin releasing factor (CRF) regulates the response of this axis at the pituitary, hormones and neuropeptides derived from proopiomelanocortin (POMC) regulate the H-P-A axis at the adrenal cortex and in the central nervous system. Several lines of evidence suggest that adrenocortical responsiveness to ACTH is highly regulated and a component of H-P-A axis control. For example, the adrenal cortex demonstrates a circadian responsiveness to ACTH. POMC is produced not only in pituitary, but also in the hypothalamus and brainstem, and numerous effects of POMC peptides on control of neuroendocrine and other autonomic functions have also been reported. For example, evidence for short-loop feedback control of CRF production by centrally produced melanocortins has been described. Recently, this laboratory has cloned the adrenocortical receptor for ACTH and two neural receptors for the melanocortin peptides produced form POMC. These reagents are valuable tools for the analysis of H-P-A axis control both in the adrenal cortex and the central nervous system. The basic pharmacological properties of these receptors will be defined, and functional properties of these receptors will be examined in normal animals, in animals subjected to acute and chronic stress, and in transgenic mouse mouse models which perturb the H-P-A axis. After pharmacological characterization of the cloned ACTH receptor, the regulation of this receptor will be examined both in cell culture and animal models. ACTH binding experiments have suggested that this receptor will be examined both in cell culture and animal models. ACTH binding experiments have suggested that this receptor is actually upregulated by its ligand, ACTH, and as such this receptor displays an unusual type of receptor regulation. The experiments proposed should provide mechanistic explanations for this unique mode of receptor regulation. The role of the ACTH receptor in a human disease, familial ACTH resistance, will be also be examined, and may provide information regarding the etiology of this disease, which is suggested to result from an ACTH receptor defect. The two neural melanocortin receptors, MC3-R and MC4-R, will be characterized pharmacologically, and using histochemical methods to determine their neuroanatomical distribution in the brain. These receptors will also be deleted from the mouse genome using homologous recombination in ES cell in order to define their role in H-P-A axis control. These experiments should contribute to our understanding of the role of the adrenal cortex and central nervous system in H-P-A axis.

DESCRIPTION: (Adapted from the applicant's abstract) The agouti peptide plays a central role in determining the type of melanin (eumelanins or phaeomelanins) produced within the melanocyte. The normal action of agouti is most likely as an antagonist of the MSH receptor, MC1-R. In the mouse, ectopic expression of agouti leads to obesity, potentially mediated through an interaction with (hypothalamic) MC4-R. The aims of the proposed project are: 1) To study the structure the agouti peptide to define the specificity of its effects on pigmentation and obesity; 2) to determine the binding site and mechanism of antagonism of melanocortin receptors; 3) to test the hypothesis that agouti may act independent of melanocortin receptor antagonism; 4) to identify other members of the agouti family that may be more relevant to endogenous antagonism of MC4-R; and, 5) to test the hypothesis that antagonism of MC4-R is responsible for agouti-mediated obesity.

Obesity is a prevalent health problem as an important risk factor for chronic diseases such as diabetes, hypertension, and heart disease. There are essentially no effective pharmaceutical treatments for obesity, and its incidence is on the rise. Obesity, as defined by a body mass index above 25, increased from an incidence of 24% in 1980 to 33% in 1990 in a large representative sample in the United States. One reason why it is difficult for obese individuals to lose weight is the multifactorial neuroendocrine processes that function to maintain energy homeostasis. Decreased caloric intake results in a decrease in basal metabolic rate, and even decreased energy requirements for work, that act to offset the decreased energy intake. Thus, pharmaceuticals that simply decrease food intake for alter metabolic rate are ineffective weight loss agents. A much deeper under of the neuroendocrine control of energy homeostasis is required, and this is the overall goal of this program. This application will focus on two groups of neurons strongly implicated in energy homeostasis, the arcuate nucleus neurons containing the orexigenic neuropeptide Y (NPY) and agouti gene related peptide (AGRP), and the inhibitory arcuate neurons expressing pro-opiomelanocortin (POMC). The first goal of this application will be to determine the role of the POMC and AGRP/NPY neurons in energy homeostasis. Dr. Cone (project 1) will determine the normal physiological inputs to energy homeostasis that operate via release of melanocortin or AGRP peptides in the mouse, and the mechanisms by which melanocortin and AGRP peptides regulate feeding and energy./expenditure. Dr. Cone (project 1) will determine the normal physiological inputs to energy homeostasis that operate via release of melanocortin or AGRP peptides in the mouse, and the mechanisms by which melanocortin and AGRP peptides regulate feeding and energy/ expenditure. Dr. Low (project IV) will examine the role of the opioid POMC peptide, beta-endorphin, collaboration with Dr. Cone, and the function of the POMC neurons exclusive of POMC peptide release. Dr. Simerly (project V), using normal mice and transgenic and knockout strains provided by Drs. Low and Cone, will examine the development of the POMC arcuate projections to better define the neural substrate for energy homeostasis in the rodent. Dr. Cameron (project III) will characterize the unique differences in the POMC and NPY/AGRP systems and the physiological role(s) of NPY and melanocortins in the non-human primate. Finally, Dr. Boston in collaboration with Drs. Low and Cone, will look for mutations in the POMC gene that gained in the first goal into new knowledge of and potential treatments for obesity in humans. Basic studies from the work of Drs. Cone, Low, and Simerly (projects I, IV, V) will serve to better define the fundamental neuroendocrine pathways involved in energy homeostasis, thus pointing to candidate genes to examine for linkage to human obesity and identifying potential targets for drug development. Work from Dr. Cameron's group (project III) will serve to validate these concepts in a non-human primate and also characterize aspects of these pathways that are unique to the primate. Finally, the specific hypothesis that altered levels of POMC gene expression may be a risk factor for obesity will be directly tested by Dr. Boston and his collaborators (project II).

DESCRIPTION: (Adapted from the applicant's abstract) - The known systemic effects of pituitary-derived melanocortin peptides result primarily from ACTH-mediated adrenocortical glucocorticoid production. The identification of a widely-distributed peripheral receptor for ACTH/MSH peptides, called the melanocortin-5 receptor (MC5-R), raises the possibility of non-steroidally mediated systemic effects of these peptides. Targeted disruption of the MC5-R produced a mouse with a severe defect in water repulsion and thermoregulation due to a defect in the production of sebaceous lipids. Further investigation demonstrated high levels of MC5-R in multiple exocrine tissues, including the Harderian, preputial, lacrimal, and sebaceous glands. The MC5-R is also required for the production of porphyrins by the Harderian gland, and regulation of protein secretion by the lacrimal gland via the MC5-R. The data show a requirement for the MC5-R in multiple exocrine glands for the production of a diverse set of products, including lipids, proteins, and porphyrins, and suggest the existence of a coordinated system for the regulation of exocrine gland function by melanocortin peptides. The MC5-R may thus have clinical applications to a number of human diseases involving exocrine gland dysfunction, such as acne, dry eye syndromes, and blepharitis. In this application the investigator proposes to characterize the extent of expression of the MC5-R in exocrine tissues, to begin to identify exocrine gland products whose production and secretion is regulated by the MC5-R, to examine the physiological significance of this regulation, and to determine the potential utility of MC5-R agonists and antagonists for the regulation of exocrine gland function in vivo, using the mouse as a model.

The discovery and characterization of single-gene obesity syndromes in the mouse has led to dramatic progress in our understanding of the neuroendocrine control of energy homeostasis. Cloning of the obesity locus led to the discovery of the adipostatic hormone, leptin, while characterization of the agouti obesity syndrome led to the finding that the arcuate POMC neurons exert a tonic inhibitory effect on feeding and energy storage. Agouti encodes a peptide normally expressed only in skin that regulates pigmentation by acting as an antagonist of the melanocyte- stimulating hormone receptor (MCl-R). Mice containing certain dominant alleles of the agouti peptide appear to become obese because ectopic expression of the peptide in the brain aberrantly antagonizes the related hypothalamic melanocortin-4 receptor. Intracerebroventricular (icv) administration of MC4-R agonists and antagonists in the mouse were used to test this hypothesis; icv administration of melanocortin agonists inhibited feeding, while an antagonist was stimulatory (Fan et al., 1997). This finding was corroborated by deletion of the MC4-R from the mouse, which recapitulated the unique constellation of phenotypes seen in the agouti obesity syndrome (Huszar et al., 1997), including hyperphagia, hyperinsulinemia, obesity, and increased linea4r growth. Recent studies have identified a second agouti brain, is expressed almost exclusively within the arcuate nucleus of the hypothalamus. Like deletion of the MC4-R or ectopic expression of agouti, ubiquitous over-expression of AGRP causes the agouti obesity syndrome. These data argue strongly that POMC peptide agonists and the AGRP antagonists act in concert on the MC4-R to regulate energy homeostasis just as agouti and alpha-MSH act in concert on the melanocyte to determine pigmentation. As this grant was being completed, two independent cases of familial obesity resulting from deleterious mutations in POMC were reported, demonstrating related pathophysiology in humans as well. However, while pathophysiological disruption of MC4-R signaling causes obesity in these models, little is known regarding the normal hormonal, nutritional, or neural inputs to energy homeostasis that are dependent upon the POMC neurons for their transmission. Furthermore, little is known regarding the mechanisms by which the central melanocortin system regulates energy homeostasis and how information derived from the melanocortin system integrates with other pathways known to be involved in regulation of energy homeostasis. The melanocortin system may well be important in common forms of human obesity since a quantitative trait locus for obesity maps near the POMC gene. In this project we will utilize genetic pharmacological, physiological, and neuroanatomical approaches in the mouse to determine the physiological inputs to the melanocortin system relevant to the regulation of feeding and metabolism, and to characterize the mechanisms by which the central melanocortin system regulates energy homeostasis.

DESCRIPTION: (Adapted from the Investigator's Abstract): The hypothalamic melanocortin-4 receptor (MC4-R) has been shown to have an important role in controlling energy balance in both rodents and humans. Neuronal fibers of at least two different types converge on sites of MC4-R expression in the paraventricular nucleus (PVH) of the hypothalamus. These include proopiomelanocortin (POMC) neurons that release both a-MSH and CART, and neurons that release both NPY and AGRP. Signaling via MC4-R appears to reflect a balance between a-MSH and AGRP release at the adipostat cells in the PVH that express this receptor. The detailed mechanism by which a-MSH and AGRP interact at MC4-R is unknown. It is also unclear how the product of the mahogany gene modulates the effect of AGRP on adipostat neurons. Two systems have been developed to allow further characterization of MC4-R signaling in the PVH. These include a hypothalamic slice preparation that permits electrophysiological recordings to be made from individual cells that are responsive (via GABAergic input) to both a-MSH and AGRP analogs. The second system involves a cultured hypothalamic neuronal cell line (GT-1) that expresses mRNA for MC4-R, mahogany, and leptin, and demonstrates specific binding of an AGRP analog. The Specific Aims of the proposal are i) to define the signaling modalities used by MC4-R and verify the GABAergic interneuron hypothesis, ii) to determine the mechanism by which AGRP opposes a-MSH action in the PVH, and iii) to determine the mechanism by which mahogany facilitates the antagonism of a-MSH signaling by AGRP.

DESCRIPTION (provided by applicant): The Fifth International Melanocortin Meeting will be held in Sunriver, Oregon, 25 - 28 August 2002. This meeting is the single most important international gathering for researchers studying melanocortin peptides, secreted peptides in the brain, blood, and periphery involved in the critical functions of weight regulation, cortisol production, secretion from exocrine glands, and pigmentation. Since the Fourth Meeting in 1993 in France, the field has experienced exponential progress. At that time, only melanocortin receptors Mc1r and Mc2r were known; since then, 3 more receptors (Mc3r, Mc4r, and Mc5r) have been identified and physiologic models have been developed for all 5 receptors. In addition, studies have elucidated the role of melanocortin receptor agonists and interactions among components of this system. Mutations in several different parts of the melanocortin system have been linked to a significant percentage of cases of human obesity and additional linkage studies are ongoing. Given these milestones, there is a compelling need for researchers to gather in 2002, and we anticipate 300 - 400 participants. Fifty-five speakers (30 from the United States and 25 from 12 other countries, including 9 women) will address the topics of peptide and small molecule chemistry, receptor structure and function, energy homeostasis, pigmentation, adrenocortical function, and behavior. Two poster sessions, a reception, a banquet, and informal social activities will provide critical opportunities for junior scientists, postdoctoral fellows, and graduate students to interact with leaders in the melanocortin field. To facilitate the gathering of a large international audience, funds are specifically requested to cover travel expenses for 17 speakers.

Obesity is a leading cause of morbidity and mortality as a risk factor for diabetes, cardiovascular disease, and cancer. The central melanocortin system is a critical circuit in the regulation of body weight and composition, with haploinsufficiency of the melanocortin-4 receptor (MC4-R) in humans the most common monogenic cause of severe obesity, accounting for up to 5% of cases. Consequently, a thorough understanding of the central melanocortin system will increase our understanding of obesity, improve differential diagnosis of obesity syndromes, and ultimately identify potential targets for drug development for the treatment of disorders of energy homeostasis. Much of the research in the field has been focused on the regulation of the arcuate proopiomelanocortin (POMC) neurons by leptin, and their role in mediating the long-term adipostatic leptin signal. Work from the first funding period of this grant also demonstrated an important role of the melanocortin system in satiety, and sensing of nutrient intake. Using an electrophysiological system developed in this laboratory for recording from arcuate POMC and NPY neurons, as well as neuroanatomical methods, we have discovered that these neurons not only respond to leptin, but are also regulated by acute hunger and satiety factors such as PYY3-36, insulin, CCK, and ghrelin. Likewise, in preliminary data provided here we show that MC4-R signaling also has an impact on gastric emptying, identifying a novel efferent pathway for regulation of satiety by this system. Indeed, data just published on MC4-R haploinsufficient humans documents a profound hyperphagia, and perhaps even binge eating in these individuals. These findings raise two intriguing hypotheses. First, most hunger and satiety signals are transmitted to the CNS via vagal afferents and humoral inputs to the brainstem. POMC is also expressed in a poorly characterized set of neurons in the nucleus of the solitary tract (NTS) in the brainstem, and the dorsal motor nucleus of the vagus, a key site of motor neurons regulating GI function, is a dense site of MC4-R expression. Thus, the central melanocortin system may be an important pathway for sensing and responding to hunger and satiety siqnals acting through vaqa! and humoral inputs to the brainstem melanocortin system, and perhaps, in the case of some humoral factors even acting directly on arcuate POMC neurons. Secondly, arcuate and brainstem POMC neurons send projections to a number of MC4-R-containing sites involved in autonomic outflow, like the PVN, DMH, IML, PBL, and DMV, and stereotaxic injection of melanocortJn compounds into the PVN can profoundly impact feeding behavior. Thus, the regulation of autonomic outflow to the gut by the melanocortin system may be an important pathway by which this system participates in the regulation of energy intake. In this next funding period, we will continue to characterize regulatory inputs to the central melanocortin system, and effector output pathways, with a focus on the role of the system in sensing and responding to acute hunger and satiety signals.

[unreadable] DESCRIPTION (provided by applicant): The obesity epidemic, and resulting co-morbidities, are estimated to result in direct medical costs (U.S.) of $75 billion per year, however there are few effective treatments available. Consequently, identification of obesity-related genes may be indispensable for the development of effective therapeutics. Study of monogenic obesity mutants in the mouse and candidate gene approaches have led to the identification of a few dozen genes that play important roles in aspects of energy homeostasis. Given the complexity of the process, there are likely to be hundreds. The recent discovery of the adipostatic hormone leptin (1994), and the putative hunger factor ghrelin (1999) underscore the early stage of the discovery process in this field. Whole genome forward genetic screening for obesity-related mutations in a vertebrate system would be a highly valuable approach since entire collections of genes involved in energy homeostasis could be identified in an unbiased fashion, allowing identification both of previously unknown steps in existing pathways as well as entirely new pathways. Certain elements of the adipostat appear functionally conserved in teleosts, since the zebrafish ortholog of agouti-related protein (AgRP), a component of the hypothalamic melanocortin system, stimulates feeding when administered in the teleost brain, is upregulated by fasting, and causes obesity when overexpressed in fish. Thus, it is possible to use the zebrafish to screen for mutations that effect the central melanocortin system, and many of these mutations are likely to be relevant to mammalian energy homeostasis. In order to identify and characterize these genes using zebrafish the work proposed in this grant will focus on two essential components: 1) characterization of a collection of unique mutations affecting hypothalamic development, identified by screening the Hopkins collection of zebrafish early developmental mutants for genes that alter the expression of zebrafish POMC and AgRP and 2) completion of a large scale forward genetic screen of random retroviral insertional mutations in the zebrafish for genes that alter expression of hypothalamic POMC and AgRP genes. The genes identified in these two screens are likely to include novel genes involved in hypothalamic development, in the expression of POMC and AgRP, and possibly even genes involved in the regulation of these proteins by metabolic state. Certain elements of the adipostat, characterized in mammals, appear functionally conserved in teleosts, such as the laboratory zebrafish, and thus, it is possible to use the zebrafish to screen for mutations that effect the control of energy homeostasis, by screening for defects in conserved elements of the system, such as central melanocortin system. In this application, we propose to screen random retroviral insertional mutants in the zebrafish to identify genes that alter expression of hypothalamic POMC and AgRP. The genes identified in these two screens are likely to include novel genes involved in hypothalamic development, in the expression of POMC and AgRP, and possibly even genes involved in the regulation of these proteins by metabolic state. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Obesity is a significant problem throughout industrialized nations. In particular central/visceral obesity is a major constituent of the metabolic syndrome, a group of cormorbidities including insulin resistance, hypertension, dyslipidemia, and increased prothrombotic and proinflammatory factors, associated with an elevated risk of cardiovascular disease (CVD). Lipotoxicity, the accumulation of excess lipid in non- adipose tissue, is a common problem associated with obesity and the metabolic syndrome. This imbalance in lipid homeostasis is linked to cell dysfunction and apoptosis. A number of rodent models of obesity show lipotoxicity including diet-induced obese (DIO) and leptin deficient Lepob/Lepob mice. The melanocortin system plays a critical role in the regulation of energy homeostasis in rodents and humans. Genetic deletion of the melanocortin receptors MC3-R and MC4-R led to the generation of two distinct models of obesity. Both show an overall increase in percentage body fat, and adipocyte hypertrophy however, in the MC4-R null mouse this increase in percentage body fat is accompanied by hyperinsulinemia, hypercholesterolemia, proinflammatory changes, and lipotoxicity of the liver, or hepatic steatosis, while the MC3-R null appears to be protected from these comorbidities of metabolic syndrome. This suggests that melanocortin signaling may be important in the regulation of glucose homeostasis, lipid homeostasis, and inflammation, and that the MC3-R may represent a good pharmacological target for the treatment of aspects of metabolic syndrome. This research plan proposes a multi-disciplinary approach to examine the effect of modulating melanocortin signaling on hyperinsulinemia, hypercholoesterolemia, steatosis, and inflammation. MC3-R specific agonists and antagonists will be developed to probe the role of the MC3-R in metabolic syndrome, and tissue specific knockouts of the MC3-R will be used to probe the tissues and mechanisms involved in the apparent protection from metabolic syndrome that results from MC3-R blockade. PUBLIC HEALTH RELEVANCE Deletion of the melaocortin-3 receptor causes a novel obesity syndrome lacking the insulin resistance, fatty liver, and pro-inflammatory changes seen in other murine models of obesity. This application seeks to identify the sites and mechanisms of action of the MC3-R in this phenomenon, so as to better understand the etiology, and eventually discover better treatments for metabolic syndrome.

DESCRIPTION (provided by applicant): The melanocortin circuitry of the CNS is a critical component of the adipostat. Activation of these circuits inhibits food intake and stimulates energy expenditure and thus the melanocortin-4 receptor has been a target of the major pharmaceutical companies for the development of drugs for the treatment of common obesity. Two clinical trials of potent MC4-R agonists exhibited unwanted pressor activity in some individuals. Recently, allosteric modulators of GPCRs have been recognized as a method of restoring normal spatio-temporal activity of physiological systems without the toxicity resulting from potent orthosteric agonists. Such allosteric modulators of the MC4R might have application to both severe syndromic obesity, as well as common obesity. Severe early onset obesity due to defective melanocortin signaling is linked, in up to 5% of cases, with non- synonymous coding mutations causing haploinsufficiency of the MC4R. It would not be unusual to expect that 10-30% of severe childhood obesity may thus result from defective melanocortin signaling, assuming MC4R promoter mutations, and mutations in other genes in the pathway may ultimately be discovered. The majority of MC4R mutations disrupt trafficking of receptors to the cell surface, rather than affinity for ligand. In contrast to common obesity, where excessive MC4R stimulation may cause unwanted side effects, successful treatment of severe obesity due to melanocortin receptor haploinsufficiency may involve increasing MC4R protein levels to physiological levels, thus potentially avoiding side effcets. Indeed, relative hypotension has been demonstrated in MC4R haploinsufficient obese patients. In this application, we propose to identify allosteric modulators of the MC4R, beginning with a high throughput screen of 162,000 compounds with the Vanderbilt High Throughput Screening Core. Initial hits will be validated, and a subset will be extensively characterized in cell culture models. Compound optimization will then be performed with the Vanderbilt Synthetic Chemistry Core, to develop preclinical lead compounds. We also propose to utilize two assays we present here, an electrophysiological slice preparation for MC4R function and a mouse model of MC4R haploinsufficiency, and to characterize antibodies against the MC4R to fully characterize the mechanism of action of allosteric modulators identified in the screen. Finally, we will attempt to identify serum biomarkers for melanocortin signaling as a less invasive tool for analysis of allosteric modulators of melanocortin signaling. PUBLIC HEALTH RELEVANCE: The melanocortin-4 receptor has been a target of the pharmaceutical industry for the development for therapeutics for the treatment of obesity because the receptor regulates body weight homeostasis, and haploinsufficiency of the receptor is the most common cause of severe early onset obesity. Clinical trials of potent orthosteric MC4-R agonists have not been successful, however, and pressor activity has been reported in at least some individuals in one trial. In this application we will conduct a high throughput screen with the Vanderbilt High Throughput Screening Facility, perform mechanism-of-action studies, and begin the process of optimization of hits with the Vanderbilt Synthetic Chemistry Core with the goal of identifying allosteric modulators of the MC4R that may maximize the normal activity of the receptor without tachyphylaxis or unwanted side effects.

DESCRIPTION (provided by applicant): Bariatric surgery is currently the only effective treatment for severe obesity, and the only effective cure for type II diabetes. Research on the mechanism of action of the different bariatric surgical procedures in humans and model systems including pigs, dogs, rats, and mice supports the hypothesis that the beneficial effects result from more than the restrictive or malabsorptive effects of the procedures on food intake. Indeed, data argue that neuroendocrine changes in gut-brain signaling resulting from the Roux-en-Y and gastric sleeve procedures alter satiety, hunger, food preferences, and glucose homeostasis prior to the achievement of significant weight loss. Understanding the cellular and molecular basis of these changes induced by bariatric surgery might lead to the development of pharmaceutical interventions, or improved surgical procedures for the treatment of obesity and diabetes. While several animal models can be used for research on the physiology of bariatric surgery, the mouse provides the best model for studies of cellular and molecular mechanisms because transgenesis can be used to alter individual genes, and to label specific cell types. We show results here demonstrating successful creation of murine bariatric surgery models at Vanderbilt, and the use of the models to identify the first gene that plays an essential role in th efficacy of RYGB for long term maintenance of significant weight loss. The unique hypothesis to be tested is that the efficacy of bariatric surgery results not solely from a collection of changesto Gl signaling, but rather that essential changes in both Gl signaling AND in the plasticity and responsiveness of CNS homeostatic and hedonic circuits act synergistically to restore glucose homeostasis, and create a new weight set point. In this interdisciplinary team grant application, we bring together leading experts in human and murine bariatric surgery, murine pathology, Gl anatomy and function, obesity and diabetes, and quantitative human genetics to jointly study surgical preparations from humans and mice in order to identify the genes and cell types mediating the efficacy of bariatric surgery.

DESCRIPTION (provided by applicant): The melanocortin-4 receptor (MC4R) is a well-validated drug target for the development of therapeutics for the treatment of obesity and disease cachexia. More recent studies suggest potential applications for MC4R compounds in diabetes and aspects of metabolic syndrome, depression related anorexia and anhedonia, and obsessive compulsive disorder. Clinical trials for treatment of common obesity using potent orthosteric agonists of the MC4R have failed, however, due to unacceptable target-mediated pressor activity. Two independent studies, however, have identified peptide MSH analogues that produce significant weight loss without a pressor response. Therefore, we hypothesize that the weight loss and pressor actions of MC4R can be discriminated pharmacologically, given a more thorough understanding of the mode(s) and site(s) of action of MC4R signaling in weight loss and cardiovascular regulation. During the previous funding period, we conducted a high throughput screen for positive allosteric modulators of the MC4R that identified a collection of 165 receptor-specific compounds in multiple mechanistic classes, and have demonstrated in vivo activity for several of these. Allosteric modulators of the MC4R should be applicable to treatment of syndromic obesity through restoration of normal levels of receptor activity in melanocortin receptor haploinsufficiency, a syndrome responsible for up to 5% of early onset obesity, and indeed a subset of our compounds are currently in the drug development pipeline at GSK. However, allosteric modulators of GPCRs, known to often exhibit excellent receptor subtype, ligand, and signaling mode specificity, are also outstanding tools for probing receptor function. We have also made significant progress in the identification of differentiated modes of MC4R signaling in vivo. During the previous funding period, we identified two novel signaling modalities of the receptor, melanocortin receptor associated protein 2 (MRAP2) mediated receptor-sensitization, and coupling of the receptor to an inwardly-rectifying K channel, Kir7.1 that is essential for depolarization of hypothalamic MC4R neurons by ?-MSH. In this application, we propose to use the unique pharmacological tools described above, and a set of tissue-specific knockout mice that delete G?s, Kir7.1, MRAP2, and ?-arrestin1 signaling in MC4R neurons to test the hypothesis that MC4R PAMS can correct melanocortin haploinsufficiency, and to identify the mode(s) and site(s) of action of MC4R in mediating its well-characterized weight loss, pressor, and cardioacceleratory effects. The results of this research program should 1) advance our understanding of the unique pharmacological properties of the MC4R, 2) enhance our understanding of the central control of energy homeostasis, 3) provide a unique set of pharmacological and genetic tools for the research community, and 4) provide the basic knowledge necessary to effectively utilize the MC4R as a drug target.

Project Summary Ion channels are membrane proteins that selectively regulate the movement of ions across cellular membranes. Ion channels play a role in many pathophysiological states, such as pain, epilepsy, metabolic disorders, obesity, diabetes, cardiac arrhythmias and renal disease. Studying the role of ion channels in disease is often slow because traditional patch clamp electrophysiology, although precise, is also a low throughput technique that can only be done by researchers who have specialized training and equipment. This bottleneck restricts the number of ion channel research problems that can be addressed by any lab or institution, compared to other drug targets, such as the G-protein-coupled receptors (GPCRs). Recent advances in ion channel technologies have demonstrated that studying ion channels can be done in high- throughput (HT). The first instrument that was specifically designed to study ion channels in HT was IonWorks HT. IonWorks HT was breakthrough technology because it enabled the study of ion channels in a 384-well ion channel instrument for the first time. The current market leader in HT electrophysiology (HT ephys) is Nanion. Nanion's SyncroPatch 384PE can record 384 cells in parallel, which can generate 20,000 data points per day. This gigaseal HT ephys instrument can measure the physiology and pharmacology of all classes of ion channels, including rapidly sensitizing ones such as alpha 7 nicotinic receptors. The Center for Chemical Genomics (CCG) is the well established home of HT biology and pharmacology at the University of Michigan (U-M), and has conducted over 200 high throughput screens over the last decade. The CCG's research team collectively has eight decades of pharmaceutical experience which provides them extensive knowledge of best practices in HTS assay development, validation, execution and informatics, and has the expertise to implement HT ion channel screening. However, there is currently no HT ephys instrument at U-M. Adding this highly scalable technology to the CCG, will allow the full exploration and exploitation of ion channel research and ion channel drug discovery at U-M and our collaborating partners.

Obesity and overweight affect more than one third of the world population, and are significant risk factors for a number of comorbidities including cardiovascular disease, cancer and diabetes. Common obesity is usually accompanied by elevated circulating levels of leptin, the primary adipostatic factor in mammals. Methods augmenting leptin sensitivity may represent safe and effective means of treating obesity. This proposal presents compelling new preliminary data showing that peripheral administration of a specific histone deacetylase 6 (HDAC6) inhibitor (Tubastatin A) to diet-induced obese mice suppresses food intake and reduces obesity, in an HDAC6-dependent manner, with up to 50 percent decrease in fat mass. These improvements are accompanied by significantly reduced hepatic steatosis, and improved systemic glucose homeostasis. Tubastatin does not induce weight loss in leptin receptor mutant db/db mice, or lean wild type mice, but increases the sensitivity of animals to exogenous leptin administration. Tubastatin-induced metabolic improvements are independent of central HDAC6 activity, and in large part depends on adipose tissue HDAC6 expression. The current application is centered on the hypothesis that peripheral HDAC6 inhibition confers central leptin sensitization, and proposes to identify the anatomical (Aim 1) and molecular (Aim 2) mechanisms of HDAC6 inhibition-mediated amelioration of obesity and diabetes using a combination of genetic, pharmacological and biochemical approaches. Aim 3 will explore the central mediators of leptin sensitization, and how blood brain barrier permeability is potentially altered by HDAC6 inhibitors. It will further study the role of the non-receptor tyrosine kinase Pyk2 as a potentially novel regulator or leptin receptor signaling. This proposal presents HDAC6 as a novel regulator of energy homeostasis and as a potential target for development of novel therapeutic approaches against obesity. More generally, this work will establish a fundamental platform for basic and clinical research for the treatment of obesity and eating disorders.

Neural systems regulating feeding behavior and reproduction communicate bidirectionally to gate reproductive events and to regulate energy stores for changes in reproductive states, such as pregnancy. The melanocortin-3 receptor (MC3R) is expressed in multiple nuclei that regulate reproduction and energy homeostasis such as the AgRP neurons of the arcuate nucleus, and exhibits a large sexual dimorphism in its level of expression in both humans and mice. Loss of the MC3R has particularly profound consequences for female animals. First, we demonstrate that MC3R is essential for fasting-induced suppression of the HPG axis in females. Second, we have identified fascinating sexual dimorphisms in feeding behavior in the absence of MC3R. For example, In the novelty suppressed feeding (NSF) assay in which fasted animals must enter an open field to retrieve food, MC3RKO males eat equivalent amounts to WT mice, while female MC3RKO fail to explore, and cease feeding entirely. In contrast, male but not female mice exhibit enhanced anorexia in response to restraint stress. AgRP neurons, the majority of which express MC3R, send information regarding nutritional state directly to reproductive and metabolic control centers, like the PVH and AVPV, and to behavioral control centers like the ventral tegmental area (VTA), paraventricular nucleus of the thalamus (PVT), and lateral parabrachial nucleus (PBl), and project indirectly to the insular cortex, involved in sensory interpretation of food cues and implicated in the pathophysiology of anorexia nervosa. This proposal will conduct the first systematic mapping and characterization of the MC3R neurons involved in the coordination of energy homeostasis, reproduction and feeding behavior, determine the mechanism(s) underlying the requirement of MC3R for fasting-induced suppression of the HPG axis, and identify the MC3R neurons and circuits mediating sexually dimorphic feeding behaviors.