Martin Myers - US grants

DESCRIPTION (provided by applicant): This application, entitled, "Molecular Determinants of Leptin Receptor/Jak2 Signaling," is a revised competitive renewal of DK56731. The long (or LRb) isoform of the leptin receptor mediates signaling and the physiologic action of leptin to regulate energy balance (decrease feeding and increase energy expenditure) and neuroendocrine function. It is not clear why leptin fails to adequately protect from obesity and the predisposition to Type 2 diabetes in humans; it is thus critical to understand the molecular details of LRb signaling in order to understand potential mechanisms of leptin resistance and/or to identify potential targets for the therapy of obesity. The long-term outlook of our previous and future studies is to understand the regulation and mechanisms of signaling by LRb. LRb is a type 1 cytokine receptor that, although devoid of enzymatic activity, mediates phosphotyrosine-dependent signaling by means of an associated Jak2 tyrosine kinase. Although we have already learned a great deal about the biology of LRb action, there have arisen a number of important questions regarding mechanisms of signaling that are mediated by the LRb-associated Jak2 independently of LRb phosphorylation (which we refer to as Jak2-autonomous signals); Jak2-autonomous signals likely mediate important leptin actions in vivo. We hypothesize that phosphorylation sites on Jak2 regulate Jak2 activity as well as mediating Jak2-autonomous signals during LRb signaling. The focus of this application is to understand Jak2-autonomous signals and regulatory inputs to Jak2 during LRb activation by defining phosphorylation sites within Jak2 and their roles in LRb action in cultured cells and in vivo. In addition to shedding light upon the biology of LRb specifically, our analysis will illuminate basic mechanisms of cytokine receptor/tyrosine kinase signaling. We propose to: Specific Aim 1: Identify Jak2 phosphorylation sites. Specific Aim 2: Define the function of Jak2 phosphorylation sites in LRb signaling. Specific Aim 3: Determine the phosphorylation and function of Jak2 during leptin signaling in vivo. Each of these aims is crucial to addressing the central hypothesis of this proposal- that Jak2-autonomous signals and Jak2 phosphorylation are critical to the regulation and propagation of physiologic leptin action.

DESCRIPTION (provided by applicant): This proposal, entitled,"Mechanisms of Leptin Receptor Signal Attenuation in vivo, "is an application for renewal of DK57768. The long (or LRb) isoform of the leptin receptor mediates signaling and the physiologic action of leptin to regulate energy balance (decreasing feeding and increasing energy expenditure) and neuroendocrine function. It is not clear why leptin fails to adequately protect from obesity and the predisposition to Type 2 diabetes in humans; it is thus critical to understand the molecular details of LRb signaling and especially mechanisms by which LRb signaling is attenuated in order to understand potential mechanisms of leptin resistance and/or to identify potential targets for the therapy of obesity. The long-term outlook of our previous and future studies is to understand the regulation and mechanisms of signaling by LRb and their role in physiologic leptin action. LRb is a type 1 cytokine receptor that, although devoid of enzymatic activity, mediates phosphotyrosine-dependent signaling by means of an associated Jak2 tyrosine kinase and two phosphorylation sites on the intracellular LRb. Tyr1138 activates STAT3, which is responsible for important positive leptin signals as well as for the induction of SOCS3 by leptin. In addition to its role in SHP- 2/ERK signaling in cultured cells, Tyr985 binds SOCS3 to mediate some elements of LRb signal attenuation in vivo as well as in cultured cells; SOCS3 also binds to the LRb-associated Jak2 to directly inhibit Jak2 signaling. We propose to study the function of SOCS3 binding to Jak2 and Tyr985 in signal attenuation in cultured cells and in vivo. In addition to shedding light upon the biology of LRb specifically, our analysis will illuminate basic mechanisms of cytokine receptor/tyrosine kinase signaling. We propose: Specific Aim 1: Determine the molecular mechanisms of LRb signal attenuation in cultured cells. Specific Aim 2: Examine the role of LRb phosphorylation sites in leptin sensitivity in vivo. Specific Aim 3: Address the mechanism(s) of SOCS3-mediated inhibition of LRb action in vivo. Each of these aims is crucial to addressing the central hypothesis of this proposal- that leptin-induced SOCS3 expression mediates feedback inhibition on LRb action in vi\o by multiple mechanisms, and that each of these mechanisms differentially affects leptin sensitivity in mammals.

DESCRIPTION (provided by applicant): This proposal, entitled, "Molecular Mechanisms of Leptin Receptor/Jak2 Action," is an application for competitive renewal of DK56731. The long-term outlook of our previous and future studies is to understand mechanisms of LepRb signaling and to determine how LepRb signals contribute to the regulation of neural function and thence to the control of energy balance, glucose homeostasis, and neuroendocrine function. LepRb mediates tyrosine phosphorylation (Tyr(P))-dependent signaling by means of an associated Jak2 tyrosine kinase. Leptin binding stimulates the Tyr(P) of Jak2 and tyrosine residues on LepRb;each Tyr(P) site mediates a unique complement of intracellular signals. To this point, we have defined the mechanisms of LRb/Jak2 interaction, defined the function of Jak2 Tyr(P) sites, and examined the biology of LepRb Tyr1138AESTAT3 signaling and LepRb Tyr985AESHP2/SOCS3 signaling. Recently, we defined a third Tyr(P) site on LepRb (Tyr1077), which regulates STAT5 signaling and potentially other LepRb signals, and have generated novel mouse models to probe the function of Jak2 and Tyr1077 in LepRb action in vivo. Preliminary data suggest that contributions from LepRb phosphorylation sites are required for most known leptin effects, including some actions that are independent of Tyr1138 and Tyr985. In contrast, Tyr1077 is crucial to the regulation of glycemic control and potentially other physiologic leptin effects. In the context of a variety of data that suggest the importance of the acute (non-transcriptional) effects of leptin in the short-term regulation of energy balance and glycemic control, these data suggest the hypothesis that LepRb Tyr1077 mediates cellular signals required for the acute effects of leptin. In addition to testing this core hypothesis, the proposed research will define the signals and mechanisms by which leptin mediates a variety of physiologic effects and by which leptin modulates blood glucose levels. We propose to: (1) Understand the roles for LepRb signals in the regulation of physiology, focusing on Jak2 and Tyr1077. (2) Define the leptin-mediated regulation of neural function in mouse models of altered LepRb signaling. (3) Determine the signaling mechanisms by which LepRb Tyr1077 and/or other Jak2 and LepRb Tyr(P) sites control physiology and neural function. This approach will reveal the molecular underpinnings of leptin action and delineate the role of each leptin signal in the regulation of neural and organismal physiology by leptin. The mechanistic insights derived from these studies will suggest potential molecular targets for therapeutic intervention in metabolic disease. PUBLIC HEALTH RELEVANCE: Leptin is a key regulator of body energy homeostasis and metabolism, and impaired leptin action may contribute to a variety of metabolic diseases. Understanding the molecular mechanisms of leptin action is thus crucial for our understanding of processes that may be dysregulated in metabolic diseases, as well as for defining potential targets for therapeutic intervention. We have thus been working to define the mechanisms by which the leptin receptor, LepRb, mediates cellular signaling and to understand how each of these signals contributes to the physiologic actions of leptin in vivo. While LepRb Tyr1138/STAT3 signaling is crucial for long-term energy balance, this signaling pathway fails to explain many important aspects of leptin action. LepRb Tyr985, which mediates feedback inhibition on LepRb to attenuate leptin action in vivo, cannot account for residual leptin action. Recent data from our ongoing analysis suggests important roles for LepRb Tyr1077 in leptin action in vivo;signals emanating directly from Jak2 may contribute, as well. We will thus analyze the role of Jak2 and LepRb Tyr1077 in the neural and physiologic actions of leptin by utilizing mouse models in which we have altered LepRb to specifically affect those signals. We will furthermore utilize a set of in vivo systems to define the molecular mediators that lie downstream of Jak2 and/or LepRb Tyr1077 in the regulation of these processes. Overall, these studies will define crucial mediators of leptin action and reveal specific mechanisms by which leptin controls particular physiologic endpoints.

DESCRIPTION (provided by applicant): The ongoing epidemic of obesity in the United States represents a public health emergency that remains unchecked and without specific therapy. To design specific treatments to prevent and treat obesity, we must first understand the mechanisms that regulate feeding and energy expenditure in order to identify potential therapeutic targets. In this proposal, entitled, Role of the lateral hypothalamic area in leptin action, we will analyze novel leptin-regulated neural pathways in the lateral hypothalamic area (LHA) that likely contribute to body energy homeostasis. We have defined the existence of a novel population of LRb-expressing, leptin-responsive LHA neurons that project locally as well as densely innervating the ventral tegmental area (VTA). We hypothesize that LRb-mediated signaling in the LHA is crucial to the regulation of the VTA and activity of the mesolimbic dopamine system, and thereby for the regulation of feeding and energy balance. We will thus study the regulation of LHA LRb neurons and their role in the regulation of physiology by leptin. We propose the following specific aims: 1. Understand the regulation of LHA LRb neurons. Analyze the regulation of gene expression and activity of LHA LRb neurons, and define the mechanisms by which leptin and other factors mediate this regulation. 2. Define the action of LHA LRb neurons on downstream neurons. Define the downstream target neurons of LHA LRb neurons and the role of LHA LRb neurons in the regulation of these downstream neurons, including the mesolimbic dopamine system. 3. Determine the function of LHA LRb neurons in physiologic leptin action. Examine the physiologic function of LHA LRb neurons by examining energy balance and behavior in animals following a variety of genetic and pharmacologic manipulations directed at these neurons. These studies will enable us to understand the mechanisms by which leptin regulates LHA function, and the mechanisms by which LHA LRb neurons interact with other areas of the CNS in order to contribute to the regulation of feeding and energy balance. This information will in turn lay the groundwork for understanding mechanisms by which LHA-driven feeding may be regulated, which is crucial as we seek to determine the pathogenesis of, and potential therapeutic targets for the ongoing epidemic of obesity. PUBLIC HEALTH RELEVANCE: The ongoing epidemic of obesity in the United States represents a public health emergency that remains unchecked and without specific therapy. To design specific treatments to prevent and treat obesity, we must first understand the mechanisms that regulate feeding and energy expenditure in order to identify potential therapeutic targets. In this proposal, we will analyze novel neural pathways in the brain that likely contribute to body energy homeostasis in order to define these mechanisms and the potential therapeutic targets that they represent.

DESCRIPTION (provided by applicant): This proposal, entitled, "Developmental mechanisms of leptin resistance," is an application for competitive renewal of DK57768-09. The long-term outlook of our previous and proposed studies under this award is to understand molecular and neural mechanisms that impair leptin action and promote obesity and metabolic dysfunction. During the current funding period, we focused upon the mechanisms by which LepRb regulates hypothalamic physiology and may mediate feedback inhibition. Major conclusions from this work include the roles for Tyr985 (along with other LepRb signals) in the attenuation of LepRb action. Multiple lines of evidence now support the notion that cellular processes that attenuate LepRb signaling contribute to obesity and leptin resistance in vivo. Additional neural/developmental mechanisms contribute to the inception of obesity, however. Importantly, altered perinatal nutrition initiates a program that promotes obesity and metabolic syndrome in adulthood. Lack of leptin action or alterations in nutrient availability during this crucial perinatal window disrupts the development of projections from ARC neurons to their target nuclei, suggesting a potential role for leptin and the development of this ARC circuit in early metabolic programming. Many issues regarding these alterations in ARC projections remain to be addressed, however. As these issues are difficult to address with standard tools, we have generated a number of novel transgenic systems that will enable us to probe these issues in this application. The requirement for diverse intellectual and technical expertise in this proposal dictates an intimate collaboration between the Myers and Simerly labs. Together, we will: (1) Define the functional consequences of leptin deficiency on the development of leptin-regulated neural circuitry. (2) Examine the programming of leptin-regulated neural circuitry by perinatal undernutrition. (3) Determine the mechanisms that underlie the leptin-mediated developmental programming of the ARC neural circuitry. These studies will define the neural consequences of altered leptin and perinatal nutritional status within a core component of the neural circuitry that regulates energy homeostasis, as well as defining the mechanisms underlying these processes. The mechanistic insights derived from these studies will define processes that likely contribute to the inception of metabolic disease. PUBLIC HEALTH RELEVANCE: Leptin is a key regulator of body energy homeostasis and metabolism, and impaired leptin action may contribute to a variety of metabolic diseases. Understanding the mechanisms that may interfere with leptin action to mediate obesity and metabolic dysfunction is thus crucial. We have thus been working to define the mechanisms by which the leptin receptor, LepRb, mediates feedback inhibition of leptin action and to define the contribution of these processes to obesity in vivo. While these processes can contribute to the regulation of adiposity, they explain the propagation rather than the onset of obesity. In contrast, perinatal metabolic programming clearly underlies important aspects of the inception of obesity. Developmental alteration of hypothalamic leptin-responsive circuits represents a likely mediator of this perinatal programming. We have thus generated a number of novel mouse genetic models with which to analyze the mechanisms and consequences of perinatal hypothalamic programming by leptin and altered nutrition.

DESCRIPTION (provided by applicant): Rodent models of disease, especially genetically-modified mouse models, have provided powerful tools to investigate the processes underlying metabolic diseases, including obesity and Type 2 diabetes. In order to understand the physiology of such models and the alterations in metabolism that underlie their phenotypes, equipment capable of specialized and precise measurements must be employed to examine energy balance and metabolic variables. The Comprehensive Laboratory Animal Monitoring System (CLAMS) continuously and simultaneously monitors activity, feeding, and drinking, as well as VO2 and VCO2 for the determination of metabolic rate and RQ. Our Animal Phenotyping Core facility has a single 8-cage CLAMS that cannot keep pace with the high and accelerating demand for this service;this demand results from the increased number of investigators and mouse models that require this analysis. The current system is also inadequate for the analysis of animal models that employ multiple control groups. The purchase of a second, 12-cage system will permit the analysis of these animal models in a more timely fashion and the two systems together will enable the analysis of up to 20 animals at one time.

Animal Phenotyping Core A thereugh understanding ef the precesses centrelling the response te nutrients and ef the mechanisms that centribute te obesity and related metabolic diseases is required if we are te effectively combat these condifions. The detailed analysis of animals with altered metabolism (e.g. due te dietary, molecular, genefic, or pharmacological manipulation) at a level that reveals basic underiying mechanisms of control requires specialized expertise and technology not normally available to individual investigators. Established in 2006, the goals efthe MNORC Animal Phenotyping Cere was are te provide state-of-the art equipment, services and censultafive advice regarding the detailed metabolic phenotyping of rodent models of metabolic diseases. The Animal Phenotyping Core makes the metabolic analysis of rodent models of disease available, expedifious, affordable, effective, and convenient for individual investigators. In addition to providing educafion, consultation and advice regarding the analysis ef rat and mouse models with altered metabolism, the Core provides phenotyping services on specialized equipment that it operates. Specifically, the cere determines body eempesitien and utilizes the CLAMS apparatus and other systems to examine metabolic rate, respiratory quotient, food consumption, and acfivity in rodent models ef metabolic disease. The Cere also examines the response te exercise and examines cardiovascular and ether parameters by telemetry in rodents. The Core performs hyperinsulinemic/euglycemic clamp studies including specialized analysis ef metabolite storage and release in rats and mice, as well as providing catheterizafion/cannulafien services and fissue harvesting in rodents. Thus, overall, the Animal Phenotyping Core will consultatively aid individual investigators in designing an appropriate experimental plan for the metabolic analysis of animal models relevant to obesity and then provide the tools and services necessary to effect this analysis. This research is relevant to public health because it will increase our understanding ef the events that underiie the development ef obesity and its complications, and hence will facilitate the develepment ef improved diagnostic, prevenfion and treatment strategies.

DESCRIPTION, OVERALL (provided by applicant): The Michigan Diabetes Research and Training Center (MDRTC) is a multidisciplinary unit of The University of Michigan Health System. The MDRTC is now in its thirtieth year, having been funded by the NIH/NIDDK since 1977. This application is for a seventh five-year continuation of funding for the period February 1, 2008 through January 31, 2013. The Center exists to meet the needs of investigators and thereby support and strengthen the University's interdepartmental activities in research, training and outreach in the field of diabetes, its complications and related endocrine and metabolic disorders. The resources provided by the MDRTC have expanded and enriched the base of investigators involved in diabetes research, the Center's most important resource, and have facilitated innovative multidisciplinary biomedical and translational research programs. The overall goals of the MDRTC are to: 1. Facilitate and focus basic molecular and cellular research in the areas of diabetes, its complications and related endocrine and metabolic disorders. 2. Promote the application of relevant new knowledge to innovative, relevant, feasible, and cost-effective approaches to the prevention and control of diabetes and its complications through behavioral, clinical, epidemiologic, and health services research. 3. Evaluate, refine and disseminate new knowledge regarding diabetes, its complications, and related disorders into sustainable, widespread community practice, especially in communities at increased risk. 4. Recruit, train, motivate and retain an effective workforce of basic, clinical, epidemiologic, and health services investigators and health care providers in the areas of diabetes, endocrinology and metabolism.

MCDTR Center Overview Project Summary/Abstract The mission of the MDRC is to promote new discoveries and enhance scientific progress through the support of cutting-edge basic and clinical research by its highly interactive research base. The MDRC research base comprises 145 members: 118 members with $56.8 million of annual direct cost diabetes-related funding at the University of Michigan (UM) along with 27 regional members with $8.6 million of annual funding at three nearby Regional Partner Institutions: Michigan State University (MSU), Wayne State University (WSU) and the University of Toledo (UT). The investigators that make up the MDRC research base perform ground-breaking research in five broad areas relevant to diabetes: Cellular Aspects of Diabetes and Metabolism; Integrative Aspects of Diabetes and Metabolism; Islet Biology; Diabetic Complications; and Clinical Research in Diabetes and Metabolism. To support and empower research by its members, the MDRC will: 1. Coordinate activities that raise awareness of, interest in, and support for basic and translational research in diabetes, its complications, and related endocrine and metabolic disorders at the University of Michigan and beyond. 2. Advance learning and promote scientific exchange related to diabetes, endocrinology and metabolism. 3. Provide research cores that provide shared, specialized technical resources and expertise that enhance the efficiency, productivity, and multidisciplinary nature of research performed by MDRC investigators. 4. Support a Pilot and Feasibility studies grant program. 5. Provide support for research in diabetes, its complications, and related endocrine and metabolic disorders at Regional Partner Institutions. The MDRC provides membership, enrichment activities, an expanded Pilot and Feasibility Grant Program, and access to the MDRC Molecular Genetics Core for researchers at Regional Partner Institutions who study diabetes, its complications, and related endocrine and metabolic disorders.

Administration Core The Administration Core of the Michigan Diabetes Research Center (MDRC) provides leadership, infrastructure, and resources to: ¿ Raise awareness of, interest in, and support for research in diabetes, its complications and related endocrine and metabolic disorders and create an environment that facilitates such research. ¿ Coordinate the activities of the MDRC and provide guidance and coordination to the support for diabetes research at the UM and its regional partners ¿ Recruit new investigators to diabetes research and the MDRC, and support new and established investigators in diabetes research. ¿ Administer Cores that provide MDRC members with expertise and services to support diabetes related research. ¿ Administer the MDRC Pilot and Feasibility Study (P/FS) Grants Programs. ¿ Provide enrichment, education, and training for investigators from diverse schools, departments, and institutes to foster interdisciplinary collaborations. ¿ Maintain the Center's website.

DESCRIPTION (provided by applicant): The epidemics of obesity and diabetes represent a public health emergency. We must understand the mechanisms that link energy balance and glucose homeostasis, as these may represent potential therapeutic targets. In this proposal, entitled, A leptin-regulated neural pathway that modulates the counter-regulatory response, we will analyze novel leptin receptor (LepRb)-expressing neural pathways in the brainstem lateral parabrachial (lPBN) and periaqueductal grey (PAG) nuclei. Together, these regions contain the majority of brainstem LepRb neurons; each contains numbers of LepRb neurons similar to those found in major hypothalamic nuclei. The projections from lPBN and PAG LepRb cells (along with their activation by physiologic stressors such as hypoglycemia and discomfort) suggest the importance of these neurons in modulating the counter-regulatory response. Indeed, ablation of LepRb in a subpopulation of lPBN and PAG LepRb neurons enhances the counter-regulatory response to glucoprivic and noxious stimuli, suggesting that leptin acts on these brainstem LepRb cells to restrain the counter-regulatory response. In this application, we will employ a variety of cre-dependent viral and genetic systems to understand the function, importance, and mechanisms of action of these brainstem LepRb neurons. These studies reveal the mechanisms by which leptin acts in the brainstem to contribute to overall leptin action and aspects of neural function that are crucial for glucose homeostasis. This information will in turn lay the groundwork for understanding mechanisms that modulate glycemic control, which is crucial as we seek to determine the pathogenesis of, and potential therapeutic targets for the twin epidemics of obesity and diabetes.

Abstract Obesity represents a burgeoning health threat that predispose millions of people to reduced life expectancy; additionally, obese people incur $2741 higher annual healthcare costs than normal-weight individuals. Unfortunately, few effective medical options exist for the prevention or treatment of obesity. To design effective treatments, we must understand the mechanisms that mediate energy homeostasis. Leptin and its receptor (LepRb) play crucial roles in the control of energy balance. Understanding the molecular and neural mechanisms of leptin action represents the long-term goal of our previous and proposed studies under this project (DK056731). Our findings to date have revealed the importance of STAT3 and a second, pY-independent, LepRb signal for leptin action; have determined the transcriptional targets (potential downstream mediators) of leptin; and have identified crucial neuronal mediators of leptin action. We propose to understand the molecular and neural mechanisms of leptin action by: 1: Testing the hypothesis that specific genes regulated by leptin in LepRb neurons play crucial roles in leptin action and the control of energy balance. 2: Testing the hypothesis that the deletion of specific pY-independent regions of LepRb will alter the leptin-mediated control of neuronal function and energy balance, despite intact STAT3 signaling. 3: Testing the hypothesis that distinct subpopulations of hypothalamic LepRb neurons control POMC and AgRP neurons to modulate energy homeostasis. Overall, these studies will define the molecular and neural mechanisms by which leptin controls energy balance. These pathways may be dysregulated in disease and may represent targets for therapeutic intervention.

Project Summary/Abstract - Administration Core Now entering its 40th year, the Michigan Diabetes Research Center (MDRC) continues to establish, promote, and enhance multidisciplinary and collaborative basic biomedical and clinical research among investigators addressing diabetes, its complications, and related endocrine and metabolic disorders. The Administration Core of the MDRC provides leadership, infrastructure, and resources to support and enhance diabetes research, including the translation of scientific discoveries from bench to bedside. To accomplish this, the Administration Core will: ? Organize, coordinate, integrate, and administer all MDRC components and their activities. ? Identify and recruit new investigators to diabetes research and the MDRC, maintain and curate the MDRC membership roster, and support new and established investigators in diabetes research. ? Raise awareness of, interest in, and support for research in diabetes, its complications and related endocrine and metabolic disorders and create an environment that facilitates such research. ? Assess the productivity, effectiveness, and appropriateness of MDRC activities ? Assess scientific opportunities, and areas for collaboration among MDRC members ? Recruit, organize and utilize Internal and External Advisory Committees. ? Keep records, including regarding the use of MDRC facilities and services; publications; pilot and feasibility awards; and new grant applications resulting from preliminary data enabled by the MDRC. ? Effect interactions with and reporting to other Diabetes Research Centers, the NIDDK, and other appropriate individuals, groups, or organizations. ? Maintain the MDRC website, including curating and overseeing the site and ensuring the proper and seamless integration of the MDRC website with the NIDDK Diabetes Research Center program website.

Abstract Project 1- Project 1: Defining the structure and function of NTS satiety circuits While much recent energy balance-directed research has focused upon hypothalamic circuits, these hypothalamic systems largely mediate their effects on feeding via hindbrain circuits. Furthermore, many of the most effective pharmacological obesity treatments act on brainstem satiety systems. The inadvertent production of aversive symptoms such as nausea, which is mediated by circuits that are intermingled with brainstem satiety systems, represents one of the most important limitations to therapies that target these systems, however. Thus, it will be crucial to distinguish the brainstem circuits that encode satiety from those that promote nausea and other aversive symptoms, since the non-aversive circuits represent ideal targets for therapy. This project will define the neural circuits by which the NTS controls feeding and by which it signals aversive or non-aversive responses, as appropriate for the stimulus. In addition to enhancing our understanding of neural systems important for the control of ingestive behavior, distinguishing non-aversive from aversive satiety systems may permit the design of improved therapies to decrease food intake and combat obesity.