Michael D. Schwartz - US grants

Altered food intake is a fundamental component of the adaptive response to changing energy demands. One of the most striking examples of this phenomenon is the marked increase of food intake (hyperphagia) induced by uncontrolled insulin-dependent diabetes mellitus, both in animal models and in man. Although widely perceived to result from impaired glucose metabolism or its sequelae, the applicant proposes that diabetic hyperphagia results from a deficiency of insulin in the central nervous system (CNS). This hypothesis is supported by studies which suggest that the entry of circulating insulin to the brain plays a major role in body weight regulation, and that an important component of this action is to suppress food intake. Accordingly, insulin-deficient states, such as untreated diabetes and food deprivation, cause a marked increase of food intake. Recent work by the applicant suggests that one consequence of low levels of insulin in the CNS is to increase hypothalamic biosynthesis of neuropeptide Y (NPY), a potent stimulant of food intake. Expression of the NPY gene by neurons of the hypothalamic arcuate nucleus, an area rich in insulin receptors, is increased in both food-deprived and uncontrolled diabetic animals, and in both conditions, increased NPY biosynthesis is inhibited by administration of insulin directly into the brain. The applicant therefore hypothesizes that the induction of CNS insulin deficiency by conditions such as fasting or diabetes stimulates hypothalamic NPY gene expression, which leads to hyperphagic feeding. The aim of this proposal is to use the model of diabetic hyperphagia as a tool to investigate the role of insulin and its interaction with hypothalamic neuropeptides in the control of food intake. By quantifying hypothalamic neuropeptide gene expression using the methods of in situ hybridization and radioimmunoassay (to determine neuropeptide mRNA and peptide levels, respectively) and by altering levels of insulin and NPY within the hypothalamus using both intracerebroventricular and intrahypothalamic infusion techniques, studies in this application propose to l) infuse insulin into the brain of diabetic rats at doses which do not alter peripheral manifestations of diabetes in order to quantify the extent to which CNS insulin deficiency underlies the increased hypothalamic NPY gene expression and hyperphagia characteristic of the diabetic state; 2) administer antibodies specific to NPY into the CNS of diabetic rats to assess whether increased NPY release is responsible for diabetic hyperphagia; 3) administer antibodies specific to insulin into the hypothalamus of normal, nondiabetic rats to determine if CNS insulin deficiency induced by this method causes hyperphagia via an increase in hypothalamic NPY biosynthesis; and 4) determine the effect of diabetes and CNS insulin deficiency to alter hypothalamic expression of other neuropeptides involved in feeding (galanin, corticotrophin releasing hormone, and cholecystokinin). In addition to providing insight into altered feeding behavior in diabetic animals, these studies will help clarify basic mechanisms underlying regulation of feeding behavior and body weight. This information will be useful not only to the study of human diabetes, but to disorders such as obesity as well.

This application investigates the hypothesis that energy balance and body fuel stores in the form of adipose tissue are subject to homeostatic regulation. The key elements of this regulatory system include hormonal signals such as leptid (the adipocyte hormone encoded by the ob gene) and adrenal glucocorticoids, and hypothalamic peptides such as neuropeptide Y (NPY, a peptide that stimulates food intake and promote weight gain), and corticotrophin releasing hormone (CFH, which promotes weight loss by reducing caloric intake and increasing expenditure). Leptin is proposed to be a negative feedback signal secreted in proportion to the level of adipose sores that acts in the brain to promote weight loss by the combined effect of inhibiting NPY and stimulating CRH signaling pathways in the hypothalamus. Glucocorticoids are proposed to modify this leptin effect by opposing its hypothalmic actions. The Specific Aims of this proposal seek to test this model of neuroendocrine control of energy homeostasis by determining 1) the compensatory mechanisms(s) by which mice with genetic NPY deficiency maintain normal energy balance, 2) whether the relative contributions of NYP and CRH as mediators of leptin action can change as a result of weight loss or genetic obesity, 3) if NPY acts directly in the brain to inhibit CRH biosynthesis, and 4) whether CRH signaling can influence food intake and energy balance independently of its effects on the hypothalamic- pituitary-adrenal axis. To accomplish these objectives, studies are proposed using methods established in the applicants' laboratory, including cannulation of the rodent cerebroventricular system for delivery of neuropharaceuticals, detailed measurements of food intake and body composition, determination of plasma hormone levels by radioimmunoassay, and quantitation of neuropeptide mRNA levels by in situ hybridization. Fulfilling the objectives of t his application will make an important contribution to our understanding of the fundamental processes that underlie the regulation of body adiposity. Substantial progression the development of effective therapeutic strategies for disorders of body weight across the spectrum ranging from obesity to wasting illness can be anticipated from this new knowledge.

The three groups of specific aims of this project are intended to determine how metabolic signals, particularly insulin and adrenal glucocorticoid hormones, contribute to the normal regulation of body energy balance and adiposity. The first of these aims proposes to quantify insulin uptake into the CNS and its regulation in the dog. The effects of hormones, food deprivation, and overfeeding on this uptake will be evaluated and compared with the changes in food intake during refeeding. We hypothesize that regulated insulin uptake is a critical contributor to energy balance and is predictive of the response to dietary intervention. The second group of aims will test the hypothesis that insulin and glucocorticoids interact in the brain to regulate energy balance by controlling the biosynthesis and release of neuropeptide Y, a critical neuroregulator of food intake synthesized in the hypothalamic arcuate nucleus and released into the paraventricular nucleus. We also propose to determine how abnormal regulation of this system participates in experimental obesity in the Zucker fa/fa rat. The role of insulin and glucocorticoids in the regulation of NPY mRNA expression will be studied in the rat brain during caloric restriction and refeeding hyperphagia using in situ hybridization. The importance of NPY to food intake will also be assessed by infusion of an NPY antagonist into the cerebral ventricle of normal and genetically obese rats. The third group of aims proposes to assess metabolic control of NPY gene transcription in vivo in phenotypically normal mice heterozygous for a targeted mutation ("knockout") of the NPY gene and in cell culture systems. The goal is to determine whether increased hypothalamic NPY gene expression induced by food deprivation in vivo reflects increased transcription of the NPY gene. This will be accomplished using mice expressing an NPY promoter-reporter gene construct. Additional studies in transgenic mice expressing a full-length mouse NPY gene are proposed to determine the biologic effect of excessive arcuate nucleus NPY gene expression on food intake and body weight. Cell culture systems will then be used to evaluate the site of hormone action on the NPY promoter and/or the effect of hormones on NPY mRNA stability. Ultimately, we wish to understand the insulin/glucocorticoid/NPY body weight regulatory system and determine how this system might be modulated to influence the development of obesity.

Recent advances in the physiology of energy homeostasis provide a well-defined and testable model for understanding how uncontrolled insulin-deficient diabetes mellitus affects feeding behavior. This model is based on the hypothesis that negative feedback control of body adiposity involves the hormones, insulin and leptin, that circulate at concentrations proportional to body fat content. These hormones are hypothesized to reduce food intake and body weight by acting upon discrete hypothalamic signaling systems, referred to here as "central effector pathways." The effect of weight loss induced by uncontrolled diabetes to lower circulating levels of insulin and leptin is thus proposed to cause diabetic hyperphagia. This response is hypothesized to result in part via activation of hypothalamic neurons that co-express neuropeptide Y (NPY) and an endogenous melanocortin receptor antagonist, known as "Agouti-related protein" (Agrp) (both of which stimulate food intake), and by the inhibition of neurons that contain melanocortins (which reduce food intake). The objectives of this application are 1) to determine the importance of leptin deficiency as a mediator of the effect of uncontrolled diabetes on these hypothalamic neurons. This will be accomplished by infusing leptin systemically at a dose that precisely replaces the physiological leptin level in diabetic mice that are leptin-deficient, and by measuring hypothalamic expression of these neuropeptide mRNAs by in situ hybridization; 2) To use mice with genetic NPY deficiency to test the hypothesis that NPY is required for the hyperphagic response to diabetes; 3) To determine whether glucocorticoid hormones act in combination with insulin and leptin to regulate hypothalamic neuropeptide gene expression in diabetic rats; and 4) to determine if uncontrolled diabetes activates other hypothalamic neuropeptide systems implicated in the control of food intake, such as pathways containing melanin concentrating hormone and the orexins, and whether changing levels of glucocorticoids, leptin or insulin mediate these responses. These studies will 1) improve our understanding of a behavioral disorder commonly encountered among patients with diabetes, and 2) help to clarify the interactions between hormones involved in energy homeostasis and the targets upon which they act.

DESCRIPTION (adapted from the applicant's abstract): The purpose of this application is to test a new model for the physiology of energy homeostasis. We hypothesize that hormones that circulate at concentrations proportional to body fat content, such as insulin and leptin, reduce food intake and body weight by acting upon discrete hypothalamic signaling systems referred to as central effector pathways. The combined actions of insulin and leptin in the hypothalamus are proposed in turn to potentiate the response of brainstem to afferent, meal-satiety signals such as cholecystokinin (CCK) that lead to the termination of single meals. Though this mechanism, the amount of consumed during individual meals is proposed to decrease when hypothalamic leptin (or insulin) signaling is increased, leading to weight loss. Conversely, the effect of a sustained energy deficit to deplete body fat stores is hypothesized to reduce hypothalamic leptin signaling and thereby increase meal size, since animals become relatively insensitive to signals that terminate the meal. The first major objective of this application is to clarify the interactions between insulin and leptin in the control of food intake and hypothalamic neuropeptide gene expression. This will be accomplished by 1) determining if brain responsiveness to insulin can be restored in leptin-deficient ob/ob mice by infusing leptin systemically at a low dose, and 2) by determining if the effect of leptin to lower circulating insulin levels limits its ability to reduce food intake. The second major objective is to investigate the effect of leptin on the satiety and brainstem response to CCK. Studies will investigate 1) if reduced CCK responsiveness induced by fasting or genetic leptin deficiency is reversed by site or both; and 3) if central effector peptides that are regulated by leptin act in the hypothalamus to modulate the brainstem and feeding responses to CCK. By improving our understanding of the integration of long and short-term regulators of energy intake, these studies will help to identify sites for therapeutic intervention in the treatment of obesity and other weight disorders.

DESCRIPTION (provided by applicant): Although the cellular and molecular workings of the circadian pacemaker located in the suprachiasmatic nucleus (SCN) are being elucidated in striking detail, the neural mechanisms underlying diurnality versus nocturnality remain poorly understood. Current vertebrate research relies overwhelmingly on nocturnal rodent models; yet, diurnal species, including humans, present drastically different circadian rhythms. Recent work has revealed an endogenous rhythm in Fos-immunoreactivity in the lower subparaventricular, zone (LSPV), a principal target of the SCN that is reversed in a diurnal rodent species, the unstriped Nile grass rat, relative to that seen in lab rats. The research proposed here will test the hypothesis that the LSPV acts to 'switch' the coupling between the pacemaker and circadian rhythms in physiology and behavior in grass rats.

The Animal Physiology Core will play an essential role in many studies in each of the projects. Procedures supported by this core include cannulation of brain ventricles and specific hypothalamic nuclei of rats and mice, jugular vein catheterization for blood sampling, collection of brain and other tissues at that time of study termination and preparation of those materials for storage or for further analysis, metabolic studies including oxygen consumption and respiratory quotient by calorimetry, physical activity measurements, meal pattern analysis, and body composition analysis by magnetic resonance spectroscopy. Finally, this core will support the performance of specific biochemical or molecular assays that are common to each project including real time PCR ofhypothalamic rnRNA species in rats and mice and western blot analysis for detection of specific signaling molecules (e.g., phospho-ACC and phospho-PKB). Animal Physiology Core personnel already have the expertise and access to resources necessary to perform all of the experiments included in this proposal, and existing protocols will ensure that the goals of this core are efficiently met.

DESCRIPTION (provided by applicant): For more than 25 years, the research program supported by this grant has focused on the hypothesis that insulin functions as an "adiposity negative feedback signal" that circulates at concentrations proportionate to body fat mass, enters the brain, and acts upon discrete neuronal systems that control food intake and autonomic function. The net effect of this central action is 'catabolic' in nature, promoting reductions of food intake and body weight via actions in the hypothalamic arcuate nucleus (ARC). Following the cloning of the ob gene, this model was expanded to include the adipocyte hormone, leptin. It has subsequently become evident that many of leptin's CNS effects overlap with those induced by insulin, and our data indicate that the anorexic effects of both hormones requires signaling via a single intracellular pathway -- the insulin receptor substrate-phosphotidylinositol 3-OH kinase (IRS-PI3K) pathway -- in hypothalamic neurons. One way in which insulin and leptin may reduce food intake is by hastening the onset of satiety, a biological state that is elicited by neurohumoral stimuli including cholecystokinin (CCK) that act in hindbrain areas such as the nucleus tractus solitarius (NTS). To explain this interaction, we propose that a descending projection from the hypothalamus links the response to adiposity-related signals to hindbrain circuits that respond to satiety signals. The first Specific Aim of this proposal is to determine if actions of insulin and leptin in the ARC regulate the hindbrain response to CCK via a mechanism that requires PI3K signaling. An exciting new area of study revolves around the hypothesis that acute neuronal effects of insulin and leptin converge upon those induced by intracellular long-chain fatty-acyl CoA (LCFAcoA) molecules. Specific Aims 2-4 will determine if hypothalamic LCFAcoA content is regulated by acute changes of energy balance, and whether insulin and leptin exert effects on hypothalamic neurons that are dependent on FACoA signaling. Specific: Aim 5 investigates whether hypothalamic FACoA signaling, like that of insulin or leptin, potentiates the hindbrain response to satiety signals. Together, these aims constitute a body of work with the potential to revise our understanding of energy homeostasis and obesity pathogenesis, and will provide important new direction to ongoing efforts to develop more successful approaches to obesity treatment.

This Program Project proposes to facilitate an improved understanding of biological systems governing food intake, body fat mass and obesity pathogenesis. While many key molecules and neuronal cell types have been identified, it is the interactions between them, and the impact of environmental factors on these interactions, that ultimately determine the level of body fat that is defended. Identifying these interactions, and distinguishing those that are critical from those that are not, provides the overarching focus of this proposal. Because of the complexity inherent in this undertaking, this goal is best met by uniting investigators with complementary expertise and resources in an effective and productive collaboration using a multidisciplinary approach and state-of-the-art technology. Our proposal brings Dr. Greg Barsh from Stanford University School of Medicine together with established, NIH-funded investigators at the University of Washington--Drs. Michael W. Schwartz, David E. Cummings and Denis G. Baskin--in three highly-interrelated Projects that will clarify how insulin, leptin and ghrelin interact with nutrient-related signals to regulate both feeding behavior and the function of key neuronal subsets in the hypothalamic arcuate nucleus. The success of each project will depend upon interactions between them and on support provided by a Histochemistry Core and an Animal Physiology Core. Day-to-day oversight of the entire Program Project will be provided by an Administrative Core. In the event of its funding, Dr. Paul Ramsey, Dean of the University of Washington School of Medicine, has made a major commitment of new laboratory space to support the success of this endeavor. Progress in understanding signaling networks in energy homeostasis requires an interactive, multidisciplinary research program and is critical for ongoing efforts to develop more effective strategies for obesity treatment.

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[unreadable] DESCRIPTION (provided by applicant): Neuroendocrine Control of Energy Balance and Insulin Sensitivity In response to input from hormones such as insulin and leptin, which circulate in proportion to body fat mass, hypothalamic neuronal systems adjust feeding behavior and autonomic outflow in ways that promote homeostasis of both energy stores and fuel metabolism. Recent work from our program and elsewhere has established the hypothalamic arcuate nucleus (ARC) as a key brain area involved in both processes and identified neuronal signal transduction via the insulin receptor substrate - phosphatidylinositol-3-OH kinase (IRS-PI3K) pathway as a pivotal mediator of the actions of both insulin and leptin in this brain area. A key, unanswered question is whether signaling downstream of PI3K involving mammalian target of rapamycin (mTOR), atypical protein kinase C (aPKC) isoforms, or both pathways mediate the effects of adiposity- related hormones on energy balance and glucose metabolism; and whether signaling via these pathways is disrupted in common forms of obesity. The overarching aims of this proposal are therefore 1) to clarify the role of the ARC in comparison to other leptin-sensitive brain areas as a mediator of leptin action in the control of glucose homeostasis; 2) to determine the roles played in the ARC by mTOR and aPKCs as mediators of neuronal signal transduction downstream of PI3K in the action of leptin on glucose metabolism; and 3) to determine whether dysfunction within either or both of these signal transduction pathways contribute to the effect of diet-induced obesity (DIO) on glucose metabolism. To accomplish these goals, we will employ state-of-the-art histochemical, biochemical, physiological and gene therapy techniques in both normal rats and in genetically obese, insulin-resistant Koletsky (fak/fak) rats that lack all leptin receptor to investigate whether disorders of this control system cause both weight gain and insulin resistance, cardinal features that link obesity with type 2 diabetes. [unreadable] [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): [unreadable] The broad, long-term objective of this training grant renewal application is to provide a group of M.D. and Ph.D. postdoctoral fellows with the research training to permit them to become independent investigators in the fields of metabolic and reproductive disorders. The training program will utilize investigators at the University of Washington who are performing funded research, both basic and clinical, in these related areas as preceptors. Use will also be made of many basic scientists with whom these core faculty collaborate. The program will provide the trainees with research experience in both basic and clinical investigation in preparation for independent research careers. In keeping with trends in research, there will be a strong focus on molecular and cell biology. A series of lectures and seminars related to the topics of diabetes, obesity and dyslipidemia as well as in scientific methods and ethics, will complement the research training. The area of metabolism has obvious health-relatedness. Complications of atherosclerosis remain a major cause of morbidity and mortality in both males and females of all ethnic groups in the United States. A better understanding of the way that metabolic disorders alter the endocrine/metabolic process has important health implications. [unreadable] Trainees entering this program will have attained the M.D. an/or Ph.D. degree, and M.D.s will usually have completed residency. Some candidates will have had some prior research experience. Ph.D. trainees will have demontrated ability in a basic science discipline and will have shown capability for research related to the focus of this program. Selection of 8-10 new trainees/year supported through various funding sources will be made from among the 20-30 qualified applicants who have continued to seek research training in metabolism and endocrinology at the University of Washingto [unreadable] [unreadable]

ANIMAL STUDIES CORE I. OVERVIEW OF CHANGES TO THE CORE During the current funding cycle, the Animal Studies Core has been revised with respect both to its organizational structure and to the scope and sophistication of services offered to Affiliate Investigators. These changes have been made possible, in part, through a major increase of institutional commitment in support of the Core's various functions and due to the changing needs of Affiliate Investigators. The following summary highlights these changes: [unreadable] Organizational changes. Prior to this competing renewal, the CNRU supported basic animal research through two distinct Core laboratories - the Rodent Energy Metabolism and Body Composition Core (Dr. Michael Schwartz, Director) and the Nutrient Gene Core (Dr. Renee LeBoeuf, Director). To better meet the needs of Affiliate Investigators (and in response to input from our External Advisory Board), these two Core laboratories are now merged into a single entity termed the "Animal Studies Core", with Dr. Schwartz serving as Director, Dr. LeBoeuf as Co-Director, and Dr. Gregory Morton as Associate Director. [unreadable] Institutional commitment. To meet expanding demand for Core services, and in conjunction with the implementation of the new NIH-funded Mouse Metabolic Phenotyping Center (MMPC), the University of Washington School of Medicine has committed new, state-of-the-art research space and animal housing space within its newest biomedical research facility located at the South Lake Union (SLU) campus in downtown Seattle. Institutional funds have also been committed by UW in support of this program, as summarized below: [unreadable] Research space. Included is an approximate doubling of space for metabolic cage studies, expanding this resource from its current size (8 mouse and 4 rat cages;total of 12 cages) to accommodate 16 cages for rats and 16 for mice (total of 32 cages). New wet lab space will also be made available both for surgical procedures (e.g., implantation of body temperature monitor and chronic indwelling venous and arterial line insertion) and in vivo study procedures (e.g., glucose clamp studies), reflecting an overall marked expansion of research space committed to the Animal Studies Core. (Equipment funds are requested to support this expansion;see Animal Studies Core Budget.) [unreadable] Adjacent animal housing space. In addition to wet lab space, new animal housing quarters will be installed adjacent to the Animal Studies Core laboratory, allowing study animals received from Affiliate Investigators to be boarded until such time as studies are performed, averting the potential risk to animals located in the regular animal housing area, one floor below the Core laboratory. [unreadable] Creation of a new UW Obesity/Diabetes Center of Excellence, based at the SLU campus. A key priority of this Center will be to meet current and future animal research needs of investigators involved in obesity research. Additional priorities include support for junior investigators, additional pilot and feasibility funding, and promotion of interdisciplinary research across programs, departments and schools both within and outside of UW. [unreadable] Transfer of some services to the Analytic Core. During previous funding cycles, the Nutrient Gene Core participated in limited numbers of microassays of proteins, lipids, and carbohydrates, which now has largely been taken over by the Analytic Core. However, staff of the Genetics Component of the Animal Studies Core will continue to teach and advise with respect to quantification of mRNA species, thereby enabling Affiliate Investigators to set up appropriate tests in their own laboratory. Techniques include mRNA isolation from adipose and other tissues. Services offered directly by the Animal Studies Core are: Physiology Component (Dr. Morton, Associate Director): [unreadable] indirect calorimetry (e.g., metabolic rate, heat production) [unreadable] locomotor activity/total physical activity [unreadable] meal pattern analysis/continuous measures of total daily food and water intake [unreadable] continuous body temperature monitoring [unreadable] noninvasive body composition analysis [unreadable] biopsy sample triglyceride content [unreadable] euglycemic glucose clamp Genetics Component (Dr. LeBoeuf, Director): [unreadable] genotyping of mutant models [unreadable] breeding strategy for mouse colonies [unreadable] quantitative PCR of mRNA in tissue samples [unreadable] Meal preparation for nutritional interventions [unreadable] Bone marrow transplantation procedures Services offered indirectly via affiliated cores, with the Animal Studies Core serving as a conduit are: [unreadable] Diabetes complications (microvascular, macrovascular, cardiac) [unreadable] Molecular biology (gene sequencing, riboprobe preparation) [unreadable] Insulin secretion/islet morphology [unreadable] Creation of transgenic mice These services will be performed on a fee-for-service basis by the Diabetes Endocrinology Research Center (DERC), the new Mouse Metabolic Phenotyping Center (MMPC), and the Center for Ecogenetics and Environmental Health (CEEH). Arrangements have been made with these Centers (see letters of collaboration) for CNRU Affiliate Investigators to have access to the services noted above.

This training grant provides research training that will allow M.D., Ph.D., and M.D./Ph.D postdoctoral fellows to become independent investigators in the fields of diabetes, obesity, and metabolism. Research training will be provided by a large group of highly skilled and experienced investigators at the University of Washington and affiliated institutions who will serve as preceptors. Based on 1) the continued outstanding success of this program and large numbers of applicants, 2) an expanded emphasis on basic science aspects of obesity and diabetes, 3) a major new commitment from the University of Washington to create and support a Diabetes and Obesity Center of Excellence in a new research campus, and 4) a substantial increase in the size of our program faculty, we request an increase in the number of training slots from four to six. M.D. candidates will have completed residency training as well as a first, clinical year of fellowship prior to funding via this program. This arrangement allows both M.D. and Ph.D. fellows to spend at least 80% of their time on research activities while they are supported by this training grant. Selection of fellow applicants will be done by the Fellowship Training Executive Committee, and will benefit from a large pool of qualified candidates who apply to the Division of Metabolism, Endocrinology and Nutrition at the University of Washington for their research training. Among the criteria for selection are 1) a strong interest in the fields of diabetes, obesity and glucose or energy metabolism (frequently accompanied by previous research experience) and 2) demonstrated potential for a successful research career. This program provides trainees with research experience in both basic and clinical aspects of diabetes and obesity, with special emphasis on molecular and cell biology, phsyiology, and translational research. Research training will be complemented by didactic lectures and seminars that cover diabetes, obesity, glucose homeostasis, energy metabolism and related aspects of endocrinology, with additional seminars on scientific methods, manuscript preparation, grantsmanship, and biomedical ethics. Diabetes and obesity are among the most pressing public health concerns. Despite impressive advances at the basic science level, major unanswered questions regarding the pathogenesis of these disorders contintue to limit our capacity to treat them. Effective training for scientists in obesity and diabetes is therefore a fundamental priority.

DESCRIPTION (provided by applicant): Both behavioral and metabolic consequences of uncontrolled, insulin-deficient diabetes mellitus (uDM) arise in part from the response of key brain areas such as the hypothalamic arcuate nucleus (ARC) and ventromedial hypothalamic nucleus (VMN) to changes in the humoral milieu, including marked decreases in the circulating levels of both insulin and leptin, and elevated levels of ghrelin. Rodent models of uDM therefore constitute a unique and valuable tool with which to study these neuroendocrine control systems. Among ARC neuronal subsets activated in uDM are those that express orexigenic (or 'food intake-stimulatory') peptides such as neuropeptide Y (NPY) and agouti-related peptide (AgRP), whereas adjacent anorexigenic, melanocortin-producing (or 'POMC') neurons are inhibited, a combination of responses implicated in the pronounced increase of food intake characteristic of uDM (termed "diabetic hyperphagia"). At the cellular level, signaling via the insulin receptor substrate-phosphotidylinositol-3 kinase (IRS-PI3K) pathway plays a critical role in insulin action in peripheral tissues and while this pathway is also critical for both leptin and insulin action in the CNS, the specific neuronal subsets involved remain to be determined. Signaling molecules downstream of PI3K include protein kinase B (PKB) and mammalian target of rapamycin (mTOR), both of which are also implicated in hypothalamic control of food intake. Growing evidence also suggests that hypothalamic neurocircuits that sense input from insulin, leptin and ghrelin participate in the control of insulin sensitivity in peripheral tissues, and our recent work implicates dysfunction of these neurocircuits, triggered by reduced PI3K signaling, in the progressive insulin resistance seen in rats with uDM induced by the b-cell toxin, streptozotocin (STZ). Based on these observations, we propose in Specific Aim 1 to employ mouse models of STZ-induced uDM that enable us to identify the specific brain areas and neuronal subsets in which signal transduction via the IRS-PI3K-PKB pathway regulates food intake and glucose metabolism. Specifically, we will use a combination of Cre-loxP genetic and adenoviral gene therapy techniques to increase PKB specifically in NPY/Agrp neurons, POMC neurons, neurons that express leptin receptors, or VMN neurons (that express the transcription factor SF- 1) in mice with STZ-induced uDM. In this way, we will identify neuronal subsets in the ARC and VMN in which reduced PKB signaling contributes to feeding and metabolic consequences of uDM. Similarly, Aim 2 seeks to delineate the role of reduced hypothalamic mTOR signaling in behavioral and metabolic responses to uDM in rats. In Aim 3, we investigate mechanisms underlying increased plasma ghrelin levels in uDM and determine the contribution made by this increase to hyperphagia and insulin resistance in this setting. Together, this information will shed new light on neuroendocrine mechanisms controlling food intake and insulin sensitivity and help to clarify how dysfunction within these systems contributes to the pathogenesis of disordered feeding behavior and glucose metabolism in obesity and diabetes. PUBLIC HEALTH RELEVANCE Obesity and diabetes mellitus are closely related metabolic disorders that take an increasing toll on human health. Recent findings suggest that dysfunction of key hypothalamic neurons that regulate food intake, autonomic function and glucose metabolism contributes to the link between these metabolic disorders. The identification of neuronal circuits upon which hormones such as insulin, leptin and ghrelin act to control both food intake and glucose metabolism is therefore an important scientific priority. Towards this end, this proposal employs rat and mouse models of uncontrolled diabetes mellitus to further define neuroendocrine mechanisms controlling food intake, body fat storage and insulin sensitivity in peripheral tissues. By improving our understanding of how mechanisms driving obesity and diabetes are linked to one another within the brain, our ultimate goal is to find new strategies for their prevention and treatment.

DESCRIPTION (provided by applicant): Obesity and diabetes are complex disorders that have emerged as major health concerns worldwide. The complexity inherent in these disorders with respect both to their pathogenesis and metabolic consequences has heightened the need for interactive groups of investigators that unite multidisciplinary approaches with overlapping research interests. The goal of this proposal is to provide a group of five closely interacting investigators with a comprehensive, state-of-the-art resource for energy balance/metabolic phenotyping of rodent models relevant to nutrition, obesity, diabetes or related disorders. Each investigator is NIH-funded and employs mouse and/or rat models to investigate the pathogenesis of obesity, inflammatory anorexia, insulin resistance and metabolic dysfunction, and each has a well-defined requirement for improved metabolic analysis capability. Meeting this need will be accomplished through the purchase of the TSE LabMaster / PhenoMaster (TSE Systems, Midland, MI) apparatus, which offers numerous advantages over alternative systems for rodent metabolic phenotyping. This system provides a series of interrelated measures pertinent to rodent energy metabolism in up to 16 rats or mice simultaneously. Indirect calorimetry is used to monitor rates of oxygen consumption and carbon dioxide production by monitoring gas concentrations entering and exiting the chamber and computing the difference between them. This information in turn yields measures of the rates of oxygen consumption (VO2, a measure of energy expenditure), CO2 production (VCO2), heat production, and respiratory exchange ratio (RER, also known as respiratory quotient, which describes the relative oxidation of fat versus carbohydrate as a source of fuel by the animal). Combined with simultaneous measures of locomotor activity, wheel running activity, and body temperature (by telemetry), this system provides not only measures of total energy expenditure, but clarifies whether differences in energy expenditure are due to changes of body temperature, physical activity, metabolic rate, or some combination of these. Moreover, the optimal parameter for normalizing calorimetry data is lean body mass, and this will be determined for each animal using one of our two state-of-the-art magnetic resonance spectroscopy (MRS) units (one for rats, the other for mice) purchased from Echo Medical Systems and housed in space adjacent to where the TSE calorimetry apparatus will be located. Lastly, this system also offers real-time measures of food and liquid intake, allowing quantitative analysis of energy intake (including meal patterning), as well as energy expenditure. This system, therefore, provides comprehensive, quantitative information that is critical for metabolic phenotyping of rodent models of obesity and diabetes. PUBLIC HEALTH RELEVANCE: The prevalence of obesity has reached epidemic proportions in the US and other developed countries, and is causally linked to a dramatic, global increase in the prevalence of type 2 diabetes. Despite major technological advances, including the application of genetic, physiological and pharmacological tools in rodent models, the pathogenesis of these common metabolic disorders remains poorly understood, and treatment options are limited. The current proposal is intended to support quantitative metabolic phenotyping of rodent models of obesity and related disorders undertaken by a group of interactive, NIH-funded investigators who will soon relocate to a new research facility. Obtaining the requested equipment will critically advance the ability of this group to conduct their studies and will support synergistic interactions between investigators with distinct but overlapping interests.

DESCRIPTION (provided by applicant): Ethylene glycol (EG;CAS No. 107-21-1) is the toxic chemical found in automotive antifreeze. Ethylene glycol is a frequently encountered etiology of human poisoning and causes significant morbidity and mortality. In 2002, 5816 calls to US Poison Control Centers involved human exposure to ethylene glycol. The medical management of presumed ethylene glycol poisoning involves a significant delay of several hours while waiting for confirmatory testing for EG in a serum sample;this testing is available only at reference laboratories. As the same time, an expensive antidote (fomepizole;4-methylpirazole;AntazolTM, Paladin Labs, Delaware US) and/or invasive empirical treatment (hemodialysis) must be started to prevent renal failure, worsening metabolic acidosis and death. While a reliable and validated qualitative ethylene glycol test kit (EGT, PRN Phramacal, Pensacola, FL) exists for use in veterinary medicine, the clinical management of EG poisoning in humans is not as advanced. Thus, EG poisoning diagnosis is hampered by the absence of a readily available screening test in nearly all hospital laboratories. The veterinary EGT has demonstrated efficacy in a small convenience sample of known EG positive serum samples. In that study (n=24), the EGT had 100% sensitivity and high specificity over a clinically relevant range of EG serum concentrations (27-281 mg/dl). No interference was observed in samples positive for methanol or ethanol. We propose an un-blinded, prospective, case-control study of the clinical utility of the veterinary EG test kit to detect ethylene glycol in human serum samples from known poisoning cases compared to unmatched controls. EGT kit results will be compared against criterion standard testing with gas chromatography/mass spectroscopy performed in duplicate at two independent reference laboratories. We will assess the EGT detection limit in human serum and the sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV) of the platform, as well as any evidence of interfering substances (ethanol, acetone, lactate, methanol, salicylate) leading to false positive or false negative results. If successful, this pilot study will provide preliminary data which will enable pursuit of a much larger, multi-centered, prospective, case controlled clinical trial, of the utility of the EGT kit. PUBLIC HEALTH RELEVANCE: Ethylene glycol is a fairly common occurrence and delay in diagnosis and treatment is a source of significant morbidity and mortality. Gas chromatography/mass spectroscopy (GC/MS), the current gold standard for the diagnosis of EG poisoning, is unfortunately not widely clinically available and confirmation of poisoning takes anywhere between 12 and 24 hours to be completed. Veterinarians have been using a qualitative EG assay in animals that ingest EG which is rapid, inexpensive, and easy to perform;our study will assess the utility of this bioassay in humans with clinical and laboratory suspicion of EG poisoning.

DESCRIPTION (provided by applicant): Inflammation in peripheral tissues is implicated as a key mediator of insulin resistance and other metabolic consequences of obesity. Recent studies show that a similar inflammatory process also occurs in the hypothalamus, and that this process, unlike inflammation in peripheral tissues, is a potential cause (and not just a consequence) of obesity and associated metabolic impairment. These effects of hypothalamic inflammation are mediated in part via impaired neuronal responses to the hormones insulin and leptin, key signals in the central control of both energy homeostasis and insulin sensitivity. Our novel findings that hypothalamic proinflammatory cytokine expression occurs within just 24 h of the onset of high-fat (HF) feeding, an effect that coincides with a marked increase of caloric intake, and that both parameters are gradually return to normal over the subsequent week suggest a link between neuronal inflammatory responses and the hyperphagic response to a HF diet. Coincident with these early responses, microglia (the macrophage of the brain) accumulate in the arcuate nucleus (ARC, a key hypothalamic area for sensing input from insulin and leptin) - but not other brain areas. These and other observations strongly suggest that interactions between hypothalamic neurons and microglia are determinants of weight gain during HF feeding. Further, acute reversal of hypothalamic inflammation fully reverses systemic insulin resistance induced by 3 wk of HF feeding. Here, we propose studies to determine the time course over which hypothalamic microglia and neurons respond to HF feeding, and whether the response of microglia, neurons, or both cell types is required for this inflammation. We will also identify mechanisms underlying microglial accumulation in the ARC during HF feeding, and determine whether disruption of this microglial response predisposes to obesity. Lastly, we will investigate the mechanism whereby hypothalamic inflammatory signaling induced by HF feeding causes insulin resistance. These studies will clarify how interactions between hypothalamic neurons and microglia influence weight gain and metabolic impairment induced by HF feeding, which will inform our understanding of the pathogenesis of both obesity and insulin resistance and facilitate the discovery of new approaches to the treatment and prevention of these disorders. PUBLIC HEALTH RELEVANCE: This proposal focuses on mechanisms whereby high-fat feeding causes obesity and insulin resistance, both of which are major public health problems worldwide. Growing evidence implicates inflammation in peripheral tissues as a major cause of insulin resistance and other metabolic consequences of obesity. During high-fat feeding, inflammation also occurs in the hypothalamus, a key brain area for the control of both body weight and glucose metabolism. Unlike inflammation in peripheral tissues, hypothalamic inflammation is implicated as a cause (and not just a consequence) of both obesity and insulin resistance. By clarifying the mechanisms underlying these effects of hypothalamic inflammation, our studies will advance our understanding of obesity pathogenesis and its links to metabolic dysfunction, and identify new potential targets for the treatment of these conditions.

The expanded Core moved in 2008 from Harborview Medical Center to the SLU campus. Services include: Energy Balance: Advantages of our new Sable System include that it can be used for either mice or rats, generates 4x more data per unit time, and reduces animal stress by employing cages that contain bedding (unlike other calorimeter systems) and thus more closely resemble the animal's home cage. This equipment has also overcome previous obstacles related to food intake measurement and has several additional advantages over other systems (e.g., pair-feeding capability, measurement of food preferences and the ability to conduct metabolic studies in a wide range of temperature environments). Metabolic Imaging Subcore: The overall goal is to assist NORC Als in harnessing advanced technologies that allow for in vivo assessment of metabolic phenotypes and endpoints in both animal and human models by capitalizing on existing state-of-the-art facilities, equipment, and expertise. Although some of these imaging services can be obtained directly via the UW Department of Radiology, our Subcore markedly increases accessibility to while reducing costs to NORC Als for these services. Moreover, many imaging protocols described below were developed and validated by Subcore faculty specifically to meet the needs of NORC Als, and would not otherwise be available. Services components include consultative services for optimization of existing or new protocols, training, and ongoing technological support, effectively facilitating access for NORC Als to contact experts and supporting their ability to acquire and/or analyze imaging data on a fee-for-service basis. These goals are met by taking advantage of the substantial investment in sophisticated equipment and maintenance that exists within UW imaging facilities, under the direction of the UW Department of Radiology. Routine access to these services is limited by the need to independently generate and provide complete image acquisition protocols (which requires significant technological expertise, especially for MRI) and by the fact that once images are acquired, users are also responsible for image processing to measure their qualitative or quantitative radiologic outcomes. Moreover, existing facilities do not provide for animal handling, human subject safety screening, or administration of stimuli during fMRI. Lastly, we are pleased to offer funds to enable new imaging projects to be piloted at no charge to the investigator. By providing these services as well as consultation to Als, the Metabolic Imaging Subcore facilitates access to and use of available advanced imaging technologies.

DESCRIPTION (provided by applicant): There presently are 67 Affiliate Investigators (Als) of the NORC (Table 1) representing a diversity of divisions, departments, and schools throughout the University of Washington (UW) system (see Table 2). Although most of the investigators are in clinical departments, the UW is unique in that many of its basic scientists actually have their primary appointments in clinical departments. There has been a longstanding interest in several aspects of nutrition and obesity at the UW. However, over the past several years, Als have tended to focus their research in the following major areas: 1. Body Weight Regulation and Obesity 2. Adipose Tissue Biology and Inflammation 3. Lipids and Atherosclerosis 4. Diabetes

DESCRIPTION (provided by applicant): This training grant provides research training that will allow MD, PhD, and MD/PhD postdoctoral fellows to become independent investigators in the fields of diabetes, obesity, and metabolism. Research training will be provided by a large group of highly-skilled and experienced investigators, from the University of Washington and affiliated institutions, who will serve as preceptors. Based on 1) the continued outstanding success of this program and large numbers of applicants, 2) a major commitment from the University of Washington to create and support a Diabetes and Obesity Center of Excellence in a new research campus, 3) revamped didactic training and oversight and 4) an infusion of new program faculty, including a number of new clinical and translational researchers, we request continued funding of six postdoctoral training positions. In addition, we are requesting six predoctoral positions to support students enrolled in the NIDDK Medical Student Research Program in Diabetes which provides three months of training to eligible medical students. MD candidates will have completed residency training as well as a first, clinical year of fellowship prior to funding by this program. This arrangement allows both MD and PhD fellows to spend a minimum of 80% of their time on research activities while they are supported by this training grant. Fellow applicants will be chosen by the Fellowship Training Executive Committee from a large pool of qualified candidates who apply to the Division of Metabolism, Endocrinology and Nutrition at the University of Washington for their research training. Among the criteria for selection are 1) a strong interest in the fields of diabetes, obesity and glucose or energy metabolism (frequently accompanied by previous research experience), and 2) a demonstrated potential for a successful research career. This program provides trainees with research experience in both basic and clinical aspects of diabetes and obesity, with special emphasis on molecular and cell biology, physiology, and translational research. Research training will be complemented by didactic lectures and seminars that cover diabetes, obesity, glucose homeostasis, energy metabolism and related aspects of endocrinology, with additional coursework in scientific methods, manuscript preparation, grantsmanship, and biomedical ethics. Diabetes and obesity are among our most pressing public health concerns. Despite impressive advances at the basic science level, major unanswered questions regarding the pathogenesis of these disorders continue to limit our capacity to treat them. Effective training fo scientists in diabetes and obesity are therefore fundamental priorities.

DESCRIPTION (provided by applicant): This project is based upon our recently published evidence that the brain can rapidly, potently and selectively increase insulin-independent glucose lowering (referred to as glucose effectiveness, or GE). The overarching goals are to employ state-of-the-art methods in defined mouse models that allow us to identify neurocircuitry underlying this effect, investigate their physiological relevance, and determine if defects in this CNS control system contribute to reduced GE and glucose intolerance in diet-induced obesity (DIO). We propose two specific aims: Specific Aim 1. To identify neurons regulating GE and determine their physiological role in glucose homeostasis. We will use Minimal Model analysis of glucose and insulin data from a frequently sampled intravenous glucose tolerance test (FSIGT) to measure insulin secretion, insulin sensitivity and GE. Proposed studies will 1) test a model of discrete neuronal subsets in hypothalamus and hindbrain proposed to mediate the actions of leptin and FGF19 to increase GE, and 2) determine if glucose intolerance results when the function of these neurocircuits is impaired. Specific Aim 2. To determine if neuronal control of GE is impaired in diet-induced obesity (DIO). Studies will be conducted in mice with DIO to determine 1) if reduced GE and glucose intolerance can be ameliorated by manipulating the activity of specified neurons that comprise the neurociruitry controlling GE, and 2) whether these neurons are targets of obesity-associated gliosis and neuron injury. By expanding our understanding of the role of the brain in the control of GE in normal and obese mice, these studies will shed new light on the pathogenesis of glucose intolerance and diabetes and inform future approaches to the treatment of these disorders.

DESCRIPTION (provided by applicant): Following a glucose challenge, both insulin-dependent and -independent mechanisms contribute to the return to baseline blood glucose concentrations. Referred to as glucose effectiveness (GE), the insulin- independent component contributes as much to overall glucose homeostasis as insulin, but it has been viewed as a fixed and largely unregulated process and hence has not been a research focus for investigators. Several recent observations, however, offer evidence of the brain's capacity to potently induce glucose lowering via insulin-independent mechanisms. Adding to this work is our preliminary data showing that in leptin-deficient ob/ob mice, intracerebroventricular (icv) injection of the ani-diabetic hormone fibroblast growth factor-19 (FGF19) rapidly normalizes glucose tolerance despite having no effect on either insulin secretion or insulin sensitivity. Instead, this effect i mediated entirely by a selective, 3-fold increase of GE. Although the peripheral mechanism underlying this effect is unknown, our data strongly implicate a process whereby glucose is taken up into peripheral tissues via an insulin-independent mechanism, followed by its metabolism to lactate that is subsequently released back into circulation. With this background, we propose Specific Aim 1: To determine how FGF19 increases insulin-independent glucose disposal. Studies in this aim will 1) quantify this brain- mediated increase of glucose uptake and metabolism to lactate in response to FGF19, 2) determine the extent to which it explains the associated increase of GE, and 3) identify the tissues in which it occurs. These goals will be accomplished in mice using a combination of methods ranging from hyperglycemic clamp to metabolomics and biochemical analyses. Could a similar process contribute to the anti-diabetic effects of bariatric surgery? Rodent data implicate the brain in the glucose-lowering effects of bariatric procedures, and in some cases, glucose lowering involves insulin-independent as well as insulin-dependent mechanisms. Moreover, bariatric procedures increase FGF19 secretion from the GI tract. Based on these considerations, we propose Specific Aim 2: To determine if increased GE contributes to the anti-diabetic effect of bariatric surgery. Studies in this aim will determine if bariatric surgery activates CNS mechanisms analogous to those engaged by FGF19, including stimulation of insulin-independent glucose uptake, followed by conversion to lactate, which is then released into circulation. Our finding that FGF19 action in the brain rapidly, potently and selectively increases insulin-independent glucose disposal identifies a novel, CNS-driven mechanism with translational implications for both the pathogenesis and treatment of human diabetes. Studies in this proposal seek to clarify how this occurs and the extent to which it explains the anti-diabetic effect of bariatric surgical procedures.

DESCRIPTION (provided by applicant): Cancer cachexia/anorexia is a common, devastating manifestation of many malignancies and contributes significantly to patient morbidity and mortality. The etiology of cancer cachexia/anorexia is multifactorial and existing treatment strategies are disappointing. Recent work has identified calcitonin/calcitonin-gene relate peptide (CT/CGRP) neurons as the specific neuronal group in the hindbrain that causes anorexia and weight loss due to infection, inflammation and pain - and this same area of the hindbrain, the parabrachial nucleus, is known to be activated in pre-clinical cancer models. The novel genetic mouse models that elucidated the role of CT/CGRP neurons in 'illness' models of weight loss can also be used to definitively test the mechanistic role of these neurons in cancer cachexia/anorexia. Importantly, our proposed studies evaluate mouse cancer models causing gradual, progressive cachexia/anorexia - more similar to clinical situations. The novel genetic mouse models of inducible inhibition of CT/CGRP neurons allow us to investigate treatment strategies for cancer-induced weight loss rather than focusing merely on cachexia prevention. A second genetic strategy will focus on a key Arcuate nucleus neuronal population - NPY/Agrp neurons - and determine whether tumor- related inhibition of these neurons contributes to the mechanism of cancer cachexia/anorexia and whether inducible activation of these neurons can either prevent or treat established cancer cachexia/anorexia. Critically, our studies will also determine the impact of interventions to treat cancer anorexia on lean body mass and survival. Our proposal has the potential to discover novel therapies for treatment of cancer anorexia and thereby potentially improve the quality of life of patients with cancer.

Project Summary Type 2 diabetes (T2D) is among the most common and costly challenges confronting modern society. Although current treatment regimens can transiently normalize glycemia, lasting diabetes remission has yet to be achieved through nonsurgical means. This changed with our recent finding that central administration of fibroblast growth factor 1 (FGF1) can restore normal blood glucose levels to both rat and mouse models of T2D in a manner that is sustained for weeks or months. Our interrogation of underlying mechanisms supports the hypothesis tested in this proposal that FGF1 action in the brain of diabetic animals re-sets the defended level of glycemia to the normal range, and our preliminary data point strongly to the mediobasal hypothalamus (MBH) as a key target for this effect. The overarching goal of the proposed studies is to employ FGF1 as a tool with which to understand how glucoregulatory neurocircuits can be remodeled so as to lower the defended blood glucose level. In this context, FGF1 is used as a tool how this brain effect is achieved. Proposed studies seek to identify both the discrete MBH neurocircuits involved in this FGF1 effect and the underlying cellular and molecular mechanisms, based in part on our preliminary evidence that cross-talk between FGF receptors and integrin receptors is required for FGF1-induced diabetes remission. To delineate the specific cell types and molecular mediators involved, we will employ single cell transcriptomics, histochemistry, biochemistry, pharmacology and electrophysiology, along with established energy homeostasis and glucose metabolic phenotyping methods, in rodent models of T2D. A comprehensive understanding of how FGF1 action in the MBH induces diabetes remission will shed new light on how glucose homeostasis is controlled by the brain and on the potential of interventions that target the brain to improve treatment outcomes for patients with T2D.

The primary function of the Administrative and Enrichment Core of the University of Washington (UW) Nutrition and Obesity Research Center (NORC) is to ensure that the UW NORC effectively achieves its three overarching aims: 1) to offer a balanced array of services in support of research in nutrition and obesity in a manner that evolves to meet the specific needs of our investigators; 2) to advance science by offering, optimizing, and individualizing services that otherwise would be either unavailable to or not cost-effective for the local nutrition and obesity research community; and 3) to proactively support collaboration within, and the educational enrichment of, the local nutrition and obesity research community. Success in achieving these goals (which are discussed in greater detail in the Overall Research Strategy section) hinges on support provided by the Administrative and Enrichment Core. To this end, the Core incorporates programs and personnel that support the entire NORC. This includes programmatic components such as the Enrichment and Pilot and Feasibility Programs. It also includes administrative support for all Cores. Finally, the Biostatistics Subcore, directed by Dr. Sarah Holte, is an additional vital element of the support that the Administrative and Enrichment Core provides, by addressing the biostatistical needs of our 3 biomedical research cores and contributing expertise to our Enrichment and P/F Programs. The specific functions of the Administrative and Enrichment Core include being responsible for 1) organization, coordination, and integration of all NORC components and activities; 2) convening various advisory and executive committees; 3) assessing the appropriateness and effectiveness of NORC services and activities; 4) overseeing the review and approval of applications to become a NORC Affiliate Investigator (AI); 5) assessing the rationale for and feasibility of new services proposed by Core Directors and taking final responsibility for decisions regarding whether to implement them; 6) recordkeeping of meeting minutes, 7) oversight of quality improvement activities; 8) organization and oversight of both the P/F program and the Enrichment activities of the UW NORC; 9) interacting with other programs and institutions and their leadership; 10) addressing the biostatistical needs of the UW NORC and its AIs; and 11) fiscal management and budget oversight.

Project Summary Walking promotes independence, participation, fitness, and exploration in daily life. Like other activities, walking requires metabolic energy from the food we eat, which is ultimately used by our muscles to power movement. Experimentally, we can measure this energy using indirect calorimetry, which monitors oxygen and carbon dioxide as the body converts stored energy into the form used by muscles during activities of daily living. Decades of energetics research has demonstrated that human walking is incredibly efficient. However, for people with cerebral palsy the energetic cost of walking is significantly increased, on average over two times higher than typically-developing individuals. This means that for people with cerebral palsy, walking is as tiring as jogging or climbing stairs. An energetic cost of this magnitude restricts activities of daily living and causes exhaustion. While our team and many others have sought to reduce these costs through surgical interventions, rehabilitation, orthotics, or other assistive devices, these strategies have failed to result in meaningful reductions in energy. To design strategies that successfully reduce walking costs, we must first understand the underlying mechanisms contributing to elevated cost in people with cerebral palsy. The proposed research seeks to fill this knowledge gap by examining biomechanical factors that contribute to elevated energy for people with cerebral palsy. Specifically, we will evaluate the energetic cost of supporting the body (Aim-1) and stabilizing the body (Aim-2) during walking for children with cerebral palsy, and compare these costs to typically-developing peers. These tasks require very little energy during unimpaired walking (e.g., <10% of total walking cost), but their costs remain unknown for people with cerebral palsy and have direct implications for treatment decisions and assistive device design. Building upon decades of energetics research of unimpaired walking, this research will use a mechatronic device to precisely provide support and stabilization assistance during walking and quantify the impact of this assistance on walking cost. In unimpaired adults, similar methods have led to the design of exoskeletons and training programs that effectively reduce walking and running energy. This research will provide the foundation to create evidence-based strategies to decrease energy costs, minimize fatigue, and increase quality of life for people with cerebral palsy and other neurologic injuries.