Jeffrey M. Zigman, M.D., Ph.D. - US grants

DESCRIPTION (provided by applicant): Obesity is the cause of much morbidity and mortality in the US. Great strides have been made recently in determining the endocrine mechanisms and neuroanatomical pathways that are involved in the development of obesity. One such mechanism involves ghrelin, a hormone that stimulates food intake in animals and whose levels are raised in association with hunger, diet-induced weight loss and certain forms of obesity in humans. The primary purpose of this proposal is to gain a better understanding of ghrelin's role in the regulation of feeding behavior and body weight. The proposed experiments include transgenic and neuroanatomical approaches to determine the central expression patterns of ghrelin's receptor (GHSR), to determine the chemical phenotypes of these ghrelin-responsive neurons and to determine whether these ghrelin-responsive neurons innervate key autonomic, neuroendocrine and behavioral control sites in the brain. Other proposed experiments include disruption of ghrelin's signaling capacity, by deletion of its receptor, to determine if an intact, ghrelin-engaged neuronal circuitry is required for normal body weight homeostasis and the coordinated responses to fasting. Finally, the proposal aims to resolve a debate in the scientific community regarding whether ghrelin is expressed in the brain. We hope that this study will better enable the design of therapeutics to treat and prevent obesity and its co-morbid conditions.

DESCRIPTION (provided by applicant): Obesity is a global public health problem. An increased understanding of the basic physiology and neurobiology of body weight homeostasis is critical in the prevention and treatment of obesity and related co-morbidities such as diabetes mellitus and coronary artery disease. Over the last decade, several critical metabolic signals and candidate central neuronal pathways that mediate their effects have been identified. Ghrelin, the endogenous ligand for the growth hormone secretagogue receptor (GHSR, ghrelin receptor) has emerged as one of these potentially key metabolic signals. Ghrelin is a hormone that stimulates food intake, promotes the accumulation of body fat, and decreases energy expenditure. Previous work has also indicated that ghrelin signaling is required for development of the full phenotype of diet-induced obesity. Ghrelin also affects blood glucose levels, the release of insulin and possibly the sensitivity to insulin. The primary purpose of this current proposal is to gain a better understanding of ghrelin's role in the regulation of body weight homeostasis. The proposed experiments include neuroanatomical approaches to determine the projections of ghrelin-responsive neurons. In addition, a novel mouse model will be used to investigate the role of ghrelin receptor expression in two specific central nervous system sites -- the arcuate nucleus and the dorsal vagal complex -- in ghrelin's effects on food intake, the development of diet-induced obesity, glucose homeostasis, and locomotor activity. The proposed research will be performed under the mentorship of an internationally recognized expert in functional neuroanatomy, and is part of a training and career development plan designed to facilitate the applicant's transition to successful independent clinical scientist. Other elements of the development plan will include laboratory technique training, data presentations and various laboratory, mentor and scientific meetings. The proposed research will take place in the Division of Endocrinology, Diabetes and Metabolism at Beth Israel Deaconess Medical Center, where all the resources (equipment, technical expertise) to carry out the proposed research are readily available.

[unreadable] DESCRIPTION (provided by applicant): Ghrelin is a hormone with diverse actions, the most studied of which are its effects on body weight homeostasis. For instance, ghrelin levels rise in association with hunger and fasting. Also, ghrelin stimulates food intake, decreases energy expenditure, and induces obesity when present in high concentrations. Ghrelin's actions are mediated by interaction with its receptor, the growth hormone secretagogue receptor (GHSR; ghrelin receptor), which has a well-defined, discrete pattern of expression within the brain. This includes a high degree of expression in dopamine-containing neurons within a part of the brain known as the ventral tegmental area (VTA). These dopaminergic VTA neurons have been highly studied due to their involvement in brain reward circuits, such as those associated with addiction. The current application provides a series of studies designed to increase our understanding of the involvement of ghrelin in promoting reward- seeking behaviors and the role of the VTA in ghrelin action. In particular, the role ghrelin plays in motivated behaviors aimed at obtaining both food rewards and cocaine will be investigated. To accomplish this, unique mouse models in which either expression of the ghrelin receptor has been deleted or the functioning of the ghrelin receptor has been blocked by the administration of a specific antagonist will be used. These mice will be subjected to a battery of tests that will allow us to determine the effect of genetic and pharmacological blockade of ghrelin signaling pathways on food-reinforced and cocaine-reinforced reward-seeking behaviors. Also, a unique mouse model in which ghrelin receptor expression can be selectively targeted to dopaminergic VTA neurons will be used in order to investigate the sufficiency of ghrelin's engagement of these particular neurons for its actions on reward behaviors and body weight. This selective targeting will involve state-of-the- art neuroanatomical and transgenic techniques. It is hoped that these studies will result in new targeted therapies to treat the unrelenting food-seeking behaviors characteristic of certain forms of obesity, such as Prader-Willi Syndrome, as well as other maladaptive reward behaviors, such as those associated with addiction. PUBLIC HEALTH RELVANCE The experiments proposed in this study have been designed to investigate the role ghrelin plays in motivated behaviors aimed at obtaining both food rewards and addictive drugs such as cocaine. It is hoped that these studies will eventually result in new targeted therapies to treat the unrelenting food-seeking behaviors characteristic of certain forms of obesity, such as Prader-Willi Syndrome, as well as other maladaptive reward behaviors, such as those associated with addiction. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Ghrelin is a hormone with diverse actions, the most studied of which are its effects on body weight. Our recent work also demonstrates antidepressant-like behavioral effects of ghrelin. As such, ghrelin levels rise not only in association with hunger and fasting, thus leading to stimulation of food intake and conservation of energy stores, but also upon chronic stress. These elevations with stress seem to minimize the depression-like behaviors normally associated with stress, as acute peripheral infusions of ghrelin into wild-type mice induce antidepressant-like behaviors, and as mice lacking ghrelin receptors (growth hormone secretagogue receptor; GHSR) demonstrate even more depression-like behaviors than wild-type mice. Ghrelin rises also mediate the antidepressant-like effects of prolonged caloric restriction. These ghrelin effects presumably occur via interaction with its receptors in one or more of the CNS sites comprising GHSR's well-defined, discrete pattern of expression. For instance, GHSRs are expressed within dopaminergic ventral tegmental area (VTA) neurons that are involved in brain reward circuits and that also play an obligatory role in the development of rodent measures of depression. GHSRs also are highly expressed within the hippocampus, another site with well established effects on mood and antidepressant efficacy. In the current application, we provide studies designed to further explore ghrelin's actions in promoting antidepressant-like behaviors. We will investigate the effects of chronic ghrelin administration and pharmacologic blockade of ghrelin action on these behaviors in both male and female mice. We will employ unique genetically-manipulated mouse models in which GHSR expression can be site-selectively targeted to or deleted from dopaminergic VTA neurons or hippocampal neurons as well as CNS site-specific ghrelin microinjection studies to determine the role of the VTA and hippocampus in ghrelin's antidepressant effects. Finally, we will further explore the mechanism of ghrelin's antidepressant actions by examining the role of brain derived nuclear factor (BDNF) signaling cascades within both VTA and hippocampal circuits. We hope that these studies will uncover new pathways involved in the development of mood disorders, including the depression so closely linked to conditions with known perturbations of ghrelin physiology. PUBLIC HEALTH RELEVANCE: The experiments proposed in this study have been designed to investigate the role ghrelin plays in mood and the responses to chronic stress. It is hoped that these studies will uncover new pathways involved in the development of depression and will result in new therapies to treat mood disorders, and particularly the depression so closely linked to chronic stress and conditions with known perturbations of ghrelin physiology.

DESCRIPTION (provided by applicant): Ghrelin is a peptide hormone secreted primarily by endocrine cells that line the stomach and intestine. The most important actions of ghrelin include stimulation of food intake, inhibition of energy expenditure and promotion of adiposity. We have recently shown that ghrelin mediates pleasurable aspects of eating, and in particular, enhances the rewarding value of foods high in fat and increases the motivation to obtain palatable, fatty foods. Despite a substantial literature characterizing ghrelin action in the regulation of the food intake, relatively little is known about the mechanisms responsible for ghrelin's action on hedonic aspects of eating. In the current grant, we propose to better characterize the neuronal circuits mediating ghrelin's action on reward- based eating behaviors. In particular, we will test the hypothesis that corticotrophin releasing factor (CRF)- producing neurons of the central amygdala (CeA) contribute to ghrelin's actions on reward-based eating. We will test this hypothesis as part of three independent, yet related aims. We will determine if rises in ghrelin that occur upon ghrelin injection or naturally upon caloric restriction activate CA CRF neurons. We will determine if ghrelin's ability to enhance the rewarding value of fatty food requires CRF signaling. Also, we will determine if neurons that express the neuropetide orexin mediate ghrelin's effects on CeA CRF neurons. For these studies, we will use a newly developed transgenic mouse line in which green fluorescent protein marks the location of CRF neurons within the brain. These mice will be used together with other mouse models, including those in which CRF or orexin are missing, to elucidate the ghrelin-engaged neuronal circuits of interest. We will also use pharmacologically administered agents, including antagonists to receptors for ghrelin, CRF and orexin, in order to further characterize these pathways. Food reward behaviors will be assessed using a conditioned place preference behavioral task that we have adapted and developed for the specific study of food reward behavior in mouse models. We believe these experiments will yield important insights about behaviors related to eating, and eventually may lead to new therapies for obesity. This research will be performed in collaboration with Mario Perello, as an extension of NIH grant 1R01DA024680-01, 6-1-08 to 4-30- 13. They will be performed both at UTSW Medical Center and in the laboratory of Neurophysiology of the Multidisciplinary Institute of Cell Biology (IMBICE-CONICET/PBA), located in the city of La Plata, Argentina. They are designed to extend and enhance the research interests of both Drs. Zigman and Perello, and will help build the research and research capabilities of Dr. Perello and the IMBICE.

? DESCRIPTION (provided by applicant): Ghrelin is an orexigenic, glucoregulatory and antidepressant peptide hormone derived mainly from a distinct group of ghrelin cells dispersed throughout the gastric mucosa. Its plasma levels rise upon mild caloric restriction to stimulate food intake and fat storage and upon severe caloric restriction to prevent life-threatening falls i blood glucose. Ghrelin levels also rise following psychosocial stress, minimizing stress-induced depression and inducing food reward behavior. These key physiologic and behavioral effects of ghrelin action are emphasized by the hypoglycemic and depression phenotypes observed in mouse models of ghrelin, ghrelin receptor, and/or ghrelin O-acyltransferase deficiency upon exposure to prolonged caloric restriction or chronic psychosocial stress. Despite critical roles fo ghrelin in glucose homeostasis and the coordinated response to psychosocial stress, relatively little is known about the mechanisms regulating ghrelin secretion. The central theme of this proposal is to identify mechanisms mediating ghrelin secretion at the level of the ghrelin cell, with a focus on ß1-adrenergic receptor (ß1-AR) signaling. The experiments have been designed to determine if ß1-AR signaling within ghrelin cells is required for the ghrelin response to calorc restriction, if ghrelin cell ß1-ARs are required for appropriate ghrelin secretory and mood responses to chronic psychosocial stress, and if the pentose phosphate pathway and ChREBP (carbohydrate response element-binding protein) mediate the effects of ß1-ARs and glucose on ghrelin cell secretory responses, as hypothesized. The proposal will utilize a one-of-a-kind collection of techniques and tools developed to study and manipulate the ghrelin cell. This includes a system for primary culture of dispersed gastric mucosal cells with which to assess ghrelin secretion ex vivo, ghrelin-Cre transgenic mice which permit selective deletion of ß1-ARs and functional ChREBP from ghrelin cells when crossed with novel conditional ß1-AR and functional ChREBP knockout lines, and ghrelin- hrGFP transgenic mice that report on the location and allow for fluorescent-activated cell sorting of ghrelin cells. This unique collection f mouse lines and techniques and the accompanying in vivo metabolic and behavioral studies, ex vivo secretion experiments, and other assessments will allow our proposed hypotheses regarding ghrelin secretion and the ghrelin response to different extremes of caloric restriction, food intake and psychosocial stress to be rigorously tested. Ultimately, it is hoped that the planned studies advance our understanding of the coordinated physiologic and behavioral responses to caloric restriction and chronic psychosocial stress so that new treatment modalities for extremes of body weight, chronic stress, depression, and eating disorders such as anorexia nervosa may be uncovered.

PROJECT SUMMARY/ABSTRACT Defense against hypoglycemia is critical for survival, and is of particular importance in the clinically-significant setting of insulin-induced hypoglycemia. Insulin-induced hypoglycemia is prevalent in Type 1 and advanced Type 2 diabetes mellitus, and is associated with a far-ranging negative impact, including reduced work productivity and quality of life, decreased adherence to or recommendations for intensive insulin regimens (resulting in poorer glycemic control), increased incidence of hypoglycemia-related morbidity and unfortunately, occasionally death. Several facets of ghrelin biology suggest that the ghrelin system may participate in the counter-regulatory response to insulin-induced hypoglycemia: Ghrelin secretion is directly stimulated by low glucose. The ensuing raised ghrelin has at its disposal many potential downstream targets with which to influence glucose handling, including stimulation of food intake, reduction of insulin secretion and insulin sensitivity, and enhancement of hepatic gluconeogenesis and hepatic autophagy. Also, the ghrelin system directly interacts with several arms of the traditional counter-regulatory response to hypoglycemia, including stimulation by ghrelin of glucagon and GH secretion, elevation by ghrelin of circulating glucocorticoids and induction of ghrelin release by increased sympathetic nervous system activity. Thus, ghrelin is well-positioned to play a central protective glucoregulatory role during insulin-induced hypoglycemia. However, while ghrelin regulation of blood glucose and by blood glucose have been evaluated in some contexts (e.g. fasting, postprandial, etc.), the overall role of the ghrelin system in the counter-regulatory response to insulin-induced hypoglycemia has not been fully assessed experimentally, for instance by re-creating insulin-induced hypoglycemia in mice without an intact ghrelin system. Here, we will test the concept that ghrelin plays a key, protective, counter-regulatory role in the body's response to insulin-induced hypoglycemia. We will accomplish this by studying effects of insulin-induced hypoglycemia on both ghrelin action and ghrelin secretion using a unique collection of recombinant mouse models. These models include ghrelin-knockout mice and three novel lines: mice lacking insulin receptors exclusively from ghrelin cells, mice lacking GHSRs exclusively from preproglucagon cells, and mice with preproglucagon cell-selective expression of GHSRs on an otherwise GHSR-null background. We will emulate insulin-induced hypoglycemia in these mouse models of altered ghrelin action and secretion using 2 ?gold-standard? paradigms for assessing the counter-regulatory response: insulin-induced (hyperinsulinemic)-hypoglycemic clamp and an insulin bolus-induced hypoglycemia protocol, neither of which have been reported before in mice with altered ghrelin systems. Together with these novel mouse models, our team's experience in studying both ghrelin action and secretion and our expertise with clamp technology and glucose metabolism in mice uniquely position us to address important questions regarding the impact of the ghrelin system on the counterregulatory response to insulin-induced hypoglycemia.

PROJECT SUMMARY/ABSTRACT Defense against hypoglycemia is critical for survival, and is of particular importance in the clinically-significant setting of insulin-induced hypoglycemia. Insulin-induced hypoglycemia is prevalent in Type 1 and advanced Type 2 diabetes mellitus, and is associated with a far-ranging negative impact, including reduced work productivity and quality of life, decreased adherence to or recommendations for intensive insulin regimens, increased incidence of accidents and other morbidities, and occasionally death. While the body has developed a highly integrated defense system with which to prevent or correct hypoglycemia, in diabetes patients experiencing recurrent hypoglycemia due to over-insulinization, these counter-regulatory defenses are compromised, contributing to what can become a vicious cycle of hypoglycemia and often hypoglycemia unawareness (hypoglycemia-associated autonomic failure; HAAF) stemming from an abrogated sympathoadrenal arm of the counter-regulatory response. Several facets of ghrelin biology suggest that ghrelin may participate in the counter-regulatory response to insulin-induced hypoglycemia: 1) ghrelin secretion is directly stimulated by low glucose and sympathoadrenal activation; 2) the ensuing raised ghrelin has at its disposal many potential downstream targets with which to influence glucose handling, including interactions with several traditional counter-regulatory response hormones. However, while ghrelin regulation of blood glucose and by blood glucose have been evaluated in contexts such as caloric restriction, the overall role of the ghrelin system in the counter-regulatory response to insulin-induced hypoglycemia has not been fully assessed experimentally, for instance by re-creating insulin-induced hypoglycemia or HAAF in mice without an intact ghrelin system. Here, we will test the concept that ghrelin plays a key, protective, counter-regulatory role in the body?s response to insulin-induced hypoglycemia. In particular, we will dissociate the direct effects of insulin versus hypoglycemia on ghrelin secretion by performing insulin bolus-induced hypoglycemia tests, hyperinsulinemic-hypoglycemic clamps, and ex vivo ghrelin secretion studies in mice lacking insulin receptors selectively in ghrelin cells and phloridzin-hypoglycemic clamps in ghrelin-KO mice. We will determine requirement and sufficiency for GHSRs in AgRP neurons or SF1 neurons on the counter-regulatory response using Cre-lox transgenic mice and chemogenetic technology. Also, we will determine if ghrelin deletion increases susceptibility to HAAF by assessing the ghrelin response to hypoglycemia in wild-type mice with HAAF and the overall counter-regulatory response following recurrent hypoglycemia in mice lacking ghrelin. Our one-of-a-kind toolbox of recombinant mouse models targeting the ghrelin system, our team?s expertise in studying both ghrelin action and secretion, and our expertise in glucose metabolism including performing glucose clamps in mice will allow us to gain important and novel insights into not only hypoglycemia counter- regulation and the development of HAAF, but also ghrelin cell physiology and ghrelin action.

PROJECT SUMMARY Exercise is often highly effective for prevention of weight gain, for prevention of weight regain after weight loss, and for weight loss, however the hormonal and neuronal mediators that influence the complicated and highly variable exercise-associated changes in appetite and food intake and the exercise-associated effects on energy expenditure and related physiology such as exercise endurance, are poorly understood. In the current proposal, we aim to investigate the role of the hormone ghrelin and its receptor, the growth hormone secretagogue receptor (GHSR), in mediating the effects of exercise on food intake as well as in mediating exercise endurance. The proposal follows up on recently published findings in mice demonstrating that exercise transiently raises circulating levels of the hormone ghrelin and that without the action of this increased ghrelin, post-exercise food intake and exercise endurance are both markedly diminished. The proposal uses a unique collection of genetically-engineered mouse models to investigate the mechanisms regulating exercise- induced ghrelin release and the neuronal sites and other mechanisms through and by which ghrelin and its receptor mediate metabolic responses to exercise and exercise endurance. In particular, we will determine if ghrelin-dependent or ghrelin-independent (constitutive) signaling via GHSRs is required for the metabolic responses to exercise by evaluating the effects of ghrelin deletion and a GHSR mutation (which eliminates GHSR constitutive activity) on food intake, energy expenditure, neuronal excitability, and exercise endurance. We will use a mouse model lacking ?1-adrenergic receptors exclusively in ghrelin cells to determine if activation of these ghrelin cell ?1-adrenergic receptors, as induced by the sympathoadrenal system, is required for the ghrelin response to exercise. We also will use mice lacking GHSRs selectively from arcuate hypothalamic AgRP neurons or ventromedial hypothalamic SF1 neurons and mice with chemogenetically- inhibited mediobasal hypothalamic GHSR-expressing neurons to determine if those populations of hypothalamic neurons mediate the exercise-associated effects of the ghrelin system on metabolism and exercise endurance. Our studies will provide fundamental insight into the metabolic effects of exercise, exercise endurance, arcuate hypothalamic AgRP and POMC neuronal excitability, ghrelin cell physiology, and ghrelin action. Ultimately, we hope that studying the role of the ghrelin system in the metabolic responses to exercise may allow us to determine and therapeutically harness the mechanisms by which exercise promotes whole-body health.

PROJECT SUMMARY The hormone acyl-ghrelin serves as a key regulator of eating, body weight, blood glucose, and survival upon binding to its receptor, GHSR. In the past project period, we studied regulation of ghrelin secretion. A major finding was that under a severe caloric restriction protocol modeling starvation, ghrelin cell-expressed ?1- adrenergic receptors (?1ARs) mediate ghrelin secretion, which in turn defends against marked hypoglycemia and mortality. Also, following the recent identification of liver-enriched antimicrobial peptide 2 (LEAP2) as an endogenous GHSR antagonist, we extended our studies to characterize LEAP2 function at the cellular level and to determine plasma LEAP2 changes due to various metabolic perturbations in human subjects and mice. We found that LEAP2 both hyperpolarizes and prevents acyl-ghrelin from activating arcuate NPY neurons, suggesting that LEAP2 serves as both a GHSR inverse agonist and antagonist. Also, we found that plasma LEAP2 is regulated by metabolic status: its levels increase with obesity and rising blood glucose and decrease with fasting and weight loss. These changes were mostly opposite of those of acyl-ghrelin. Collectively, this led us to propose the following model of LEAP2 function: 1) In obese states, LEAP2 rises and acyl-ghrelin falls, shifting the plasma LEAP2/acyl-ghrelin molar ratio higher and thus limiting acyl-ghrelin?s capacity to worsen obesity and glucose intolerance by raising food intake, body weight and blood glucose. 2) In nutritionally deficient states, such as that induced by severe caloric restriction, a fall in LEAP2 creates an environment in which elevated acyl-ghrelin can most effectively act to prevent life-threatening hypoglycemia. In the current R01 proposal, we test this model in 3 aims by using a combination of mostly novel tools to delete, knockdown, and/or neutralize LEAP2 in settings of obesity and severe caloric restriction. In Aim 1, we test whether deleting or blocking LEAP2 will exacerbate obesity and glucose intolerance in obesogenic settings, and we determine the extent to which LEAP2 and ghrelin gain access to different CNS regions. In Aim 2, we test whether under a severe caloric restriction protocol modeling starvation, deleting LEAP2 will further enhance the capacity of activated GHSRs to boost blood glucose and survival. In Aim 3, we delete LEAP2 selectively from liver or intestine ? the two predominant sources of LEAP2 ? and assess changes in plasma LEAP2 and metabolism in response to long-term high fat diet exposure and severe caloric restriction. We will use a collection of four new, unpublished recombinant mouse lines that allow us to delete or site-selectively delete LEAP2 and/or ghrelin, together with a novel viral vector that induces knockdown of LEAP2 expression, and a LEAP2 neutralizing monoclonal antibody. Our studies will provide fundamental insight into the functional significance of the recently characterized GHSR antagonist and inverse agonist LEAP2 and the related GHSR agonist acyl- ghrelin in the development of obesity and glucose intolerance under settings of nutritional overabundance and in the development of life-threatening hypoglycemia under settings of severe caloric restriction.