Jean E. Schaffer - US grants

Although long chain free fatty acids are an important energy substrate utilized by cardiac cells, it is unclear how these hydrophobic molecules enter cells. The purpose of this investigation is to define the mechanism of fatty acid uptake in myocardium. In an attempt to elucidate the molecular mechanism of efficient tissue- specific fatty acid uptake, known genes encoding intracellular proteins which interact with free fatty acids will be over-expressed in cells which do not significantly metabolize long chain free fatty acids. If fatty acid interactions with such a protein are required and sufficient for fatty acid uptake, over-expression will confer on cells the ability to internalize fatty acids. If these candidate proteins do not enable fatty acid uptake, then the method of expression cloning will be used to isolate a gene encoding a postulated plasma membrane fatty acid transport protein from rat ventricular myocytes. The gene will be characterized, the structure and functional domains of the protein will be defined. Subsequently, the expression of the protein involved in fatty acid transport will be examined in animal models of ischemic heart disease and cardiac hypertrophy. Transport of energy substrates into cells is a process through which metabolism of the substrate may be regulated. Furthermore, the ability of cardiac cells to internalize and use long chain free fatty acids as an energy substrate is modified by ischemia and varying mechanical workloads. Elucidation of the mechanism of uptake of fatty acids is an important step toward understanding the heart's ability to use this energy substrate under normal conditions. Moreover, definition of the molecular mechanism of fatty acid uptake into cells may provide further insights into cellular alterations in cardiac ischemia and hypertrophy.

Efficient and regulated import of long chain fatty acids (LCFAs) into specific tissues is critical for normal lipid homeostasis. Mismatch between LCFA import and utilization in the pancreas, skeletal muscle, vascular endothelial cells, and the heart may play an important role in the pathogenesis of diabetes and heart failure. When present at high, pathophysiologic concentrations, un-ionized LCFAs undergo rapid translocation from one leaflet of the bilayer to the other by a non- protein-mediated mechanism. However, evidence is emerging that proteins play an important role in the trafficking of LCFAs across the plasma membrane of cells at low, physiologic LCFA concentrations. In previous work, we identified the long-chain fatty acid transport protein (FATP1) and acyl-CoA synthetase (ACS1) as proteins that facilitate LCFA transport into cells. This project is designed to molecularly characterize FATP1 and its role in vectorial fatty acid transport across the membranes of mammalian cells. We will test the hypothesis that FATP1 functions as a component of an oligomeric complex that transports and esterifies LCFAs. In Specific Aim 1 we will analyze structure-function correlates for FATP1. In Specific Aim 2 we will characterize the FATP1 oligomeric complex. In Specific Aim 3 we will evaluate the role of esterification in FATP1-mediated transport. Together these studies will help to define the molecular mechanisms by which LCFAs are transported across the plasma membrane of mammalian cells. Characterization of these mechanisms has relevance to common human diseases such as diabetes and heart failure in which abnormal lipid homeostasis contributes to pathogenesis.

DESCRIPTION (provided by applicant): Excess lipid accumulation in non-adipose tissues is associated with cellular dysfunction and cell death that may contribute to the pathogenesis of insulin resistance, non-alcoholic steatohepatitis, and cardiomyopathy in diabetes and obesity. Mechanisms involved in these pathophysiological responses have been studied in cultured cells by supplementation of growth media with high concentrations of free fatty acids. The saturated fatty acid, palmitate, leads to apoptosis in cultured cells by a mechanism involving oxidative and endoplasmic reticulum stress. This proposal will test the hypothesis that cells with gene disruptions that prevent lipotoxic cell death may have defects in fatty acid import, in fatty acid channeling, in lipid-induced oxidative stress, or in the response to perturbations of endoplasmic reticulum structure and function. Our first two aims will employ biochemical and genetic approaches in cultured cells to identify molecular targets and signaling pathways in the lipotoxic response. In our third aim, we will translate our findings to mouse models relevant to diabetic cardiovascular disease, in which lipid accumulation in cardiomyocytes is associated with heart failure. The results of these studies will provide new insights into the lipotoxic response to excess lipid accumulation in non-adipose tissues in diabetes and obesity. PUBLIC HEALTH RELEVANCE: Heart failure is a serious medical complication of obesity and diabetes that is linked to alterations in fat metabolism in the heart. The studies proposed in this application will characterize how excess fat leads to dysfunction and death of cells and extend these findings to genetically modified mouse models of diabetes and obesity to understand how these mechanisms affect heart muscle function. Given the prevalence of heart failure in diabetic and obese patients and its associated morbidity and mortality, further understanding of this disease process will facilitate the development of new treatments and preventative strategies.

Excess lipid accumulation in non-adipose tissues is associated with cellular dysfunction and cell death that may contribute to the pathogenesis of insulin resistance, non-alcoholic steatohepatitis, and cardiomyopathy in diabetes and obesity. Mechanisms involved in these pathophysiologic responses have been studied in cultured cells by supplementation of growth media with high concentrations of free fatty acids. The saturated fatty acid palmitate leads to apoptosis in cultured cells by a mechanism involving oxidative stress and initiation of ER stress. This proposal will test the hypothesis that fatty acid perturbation of normal endoplasmic reticulum function is an early event in this lipotoxic cell death. Moreover, cells with gene disruptions that prevent lipotoxic cell death may have defects in fatty acid import and channeling or in the response to lipid-induced perturbations of endoplasmic reticulum function or oxidative stress. Our first three aims will employ biochemical and genetic approaches to identify molecular targets and signaling pathways in the lipotoxic response, In our fourth aim, we will translate our findings to mouse models relevant to diabetic cardiovascular disease in which lipid accumulation in cardiomyocytes is associated with heart failure. The results of these studies will characterize fundamental aspects of cellular lipid homeostasis. Moreover, these studies will provide new insights into the lipotoxic response to excess lipid accumulation in non-adipose tissues in diabetes and obesity.

DESCRIPTION, OVERALL (provided by applicant): This proposal is in response to NIDDK RFA-DK-06-014 and is entitled "Washington University Diabetes Research and Training Center (WU DRTC)". The overall goal is to support centralized resources, facilities, and expertise shared by diabetes investigators at Washington University Medical School (WUMS), to promote the discovery of fundamental mechanisms leading to the disease and its complications, and to translate this information into prevention, treatments, and cures. Currently in its 30th year of funding, the WU DRTC has a proud history of major contributions to diabetes research. Positive momentum has continued during the funding period of 2002-present with a 65% increase in new members, a similar increase in NIH funding for diabetes-related research, a vibrant Pilot and Feasibility Program (PFP), significant productivity in terms of total number of publications attributable to the DRTC Cores (872), and substantial changes in the Core facilities to make them more responsive to the needs of the users. In the next funding cycle we plan to continue to evolve so that the WU DRTC is responsive to the changing expectations of the diabetes research community. We are particularly sensitive to the need to demonstrate the relevance of our research advances to the needs of society and to translate basic laboratory advances to improvements in the care of diabetics. Most notable is the development of a major new collaboration with St. Louis University School of Public Health (SLU-SPH) initiated in the fall of 2005. The resources of this School of Public Health, which is the only such school in Missouri, when coupled with the strengths at WUMS in basic and clinical diabetes research, provides a potent partnership that promises to be able to continue to make significant advances in diagnosis, treatment, and prevention of diabetes. In summary, the WU DRTC has now positioned itself to continue to meet the challenges of the current RFA by 1) increasing the DRTC user base along with a commensurate increase in diabetes-related research funding, 2) forming an alliance with SLU-SPH for expanding translational research and community outreach, 3) developing closer interaction with the WU CNRU for sharing of core resources and enrichment activities, 4) enhancing our presence in the important area of diabetic cardiovascular disease by improved interaction with the Center for Cardiovascular Research, 5) forming a partnership with the Institute of Clinical and Translational Sciences that will markedly enhance our members'opportunities to conduct human research, and 6) developing the WUMS-Barnes Jewish Diabetes Center, that provides state-of-the-art care of diabetic patients in our community. We believe that these new directions will result in 1) greater efficiency in utilizing enabling technologies by individual investigators;2) greater collaboration aimed at preclinical development of potential therapeutic strategies;and, 3) faster translation of initial discoveries into useful treatments for diabetes and related metabolic and endocrine disorders. The specific aims proposed here are designed to accomplish these goals.

DESCRIPTION (provided by applicant): This application seeks support from the National Heart, Lung &Blood Institute (NHLBI) through the NIH P30 mechanism (OD-09-005) for resources to recruit a newly independent assistant professor to the Diabetic Cardiovascular Disease Center (DCDC) at Washington University. DCDC is a recently established interdisciplinary research center that brings together scientists from diverse fields in work to advance our understanding of the mechanisms that contribute to the excess burden of myocardial and vascular disease in diabetics, and to rapidly translate new findings into development of diagnostic tools and therapeutic approaches. Under the auspices of DCDC, interdisciplinary teams of scientists are exploring the interactions between derangements in glucose and lipid metabolism and diabetic cardiac disease. One of the goals of ongoing projects is the development of new blood-based and imaging biomarkers. The outstanding individual we propose to recruit, Jeffrey Henderson, MD, PhD, will bring a new focus to the Center at the intersection between inflammation, infection and atherosclerosis. Dr. Henderson also has unique expertise in the use of sensitive mass spectrometry techniques for identification of urinary biomarkers. The resources of the P30, combined with the resources being provided by the Department of Medicine and DCDC and the thorough mentoring and career development plan described herein, will set the stage for the future success of this well-trained and highly promising individual in an area of high relevance to NHLBI. Diabetes is associated with serious cardiovascular complications including aggressive atherosclerosis and heart failure that is unrelated to atherosclerosis. The goal of the Diabetic Cardiovascular Disease Center is to develop new biomarkers for more effective diagnosis and treatment of this cardiovascular disease. This P30 program will support the recruitment to the Center of a well trained, promising young scientist.

DESCRIPTION (provided by applicant): Diabetes is associated with serious cardiovascular complications that include heart failure. Data from studies conducted in animal models and human subjects suggest that alterations in fatty acid metabolism, independent of atherosclerosis, are involved in the pathogenesis of heart failure in diabetic cardiomyopathy. This study will test the overall hypothesis that excessive myocardial fatty acid delivery and utilization is associated with cardiomyopathy in patients who have type 2 diabetes mellitus. Moreover, we hypothesize that alterations in lipid metabolism can be exploited to develop novel biomarkers for early detection of myocardial lipid exposure and diabetic cardiomyopathy. To test these hypotheses, we will study the relationship between fatty acid metabolism, lipid biomarkers and cardiac function in carefully characterized men and women who span physiological and pathophysiological ranges in insulin sensitivity, plasma lipid biomarkers and cardiac function. The goals of this study are to 1) identify novel plasma biomarkers for early detection and management of diabetic cardiomyopathy;and 2) determine whether manipulations of fatty acid delivery to the heart affect the functional manifestations of diabetic cardiomyopathy. This study will provide a better understanding of the relationship between systemic and myocardial fatty acid metabolism and cardiac function, which could also lead to new strategies for diagnosis, prevention, and treatment of diabetic cardiomyopathy. PUBLIC HEALTH RELEVANCE: Diabetes is associated with serious cardiovascular complications including heart failure that is unrelated to coronary artery disease. Scientific evidence suggests that blood fat levels may play a major role in this complication. Our study will investigate the link between blood fat levels and heart function in adults with type 2 diabetes. Our goal is to develop new blood-based biomarkers of heart disease in diabetics and to provide insight into new therapeutic strategies.

DESCRIPTION (provided by applicant): Diabetes is associated with serious cardiovascular complications that include atherosclerotic coronary artery disease and myocardial dysfunction even in the absence of underlying coronary artery disease, a disorder termed diabetic cardiomyopathy (DCM). Data from studies of animal models and human subjects provide evidence that alterations in myocardial lipid metabolism is central to the pathogenesis of DCM, which early on can be asymptomatic, but which can progress to symptomatic heart failure. The ability to identify new disease markers to facilitate early detection and intervention is limited b inadequacies of existing measures of systemic and myocardial lipid metabolism in humans. In our Preliminary Studies, we have addressed this problem by using sensitive mass spectrometry-based metabolomics to identify two plasma very long-chain ceramides, Cer(22:0) and Cer(24:0), that are highly correlated with asymptomatic systolic dysfunction in obese and type 2 diabetic humans. Cell biological and mouse model studies suggest these species arise from the unique intersection of ectopic lipid accumulation and activation of innate immune signaling pathways. We hypothesize that plasma Cer(22:0) and Cer(24:0) reflect systemic alterations in lipid metabolism that can be exploited as novel biomarkers for DCM. While the diagnosis of cardiac dysfunction can be readily made noninvasively by echocardiogram, Cer(22:0) and Cer(24:0) track with pathophysiological consequences of ectopic lipid accumulation and thus have potential to predict individuals at risk, to further our understanding of disease mechanism, and to identify new treatment targets. We have assembled a multidisciplinary team to extend these findings by 1) Developing a robust high-throughput clinical assay for Cer(22:0) and Cer(24:0); 2) Validating and extending these findings in two existing cohorts of human subjects; 3) Exploring the mechanistic links between very long-chain ceramides and cardiac dysfunction in relevant mouse models of DCM; and 4) Defining the direction of causality in the relationships among lipid exposure, plasma ceramides, and cardiac function in humans with type 2 diabetes. Our approach has the potential to define an integrated measure of pathophysiologically relevant lipid exposure that can be used to track intervention success, data linking phenotype to a modifiable risk factor that is currently undertreated in the target population (dyslipidemia), and marker for future disease risk that can be acted upon to prevent clinically apparent morbidity and mortality.

DESCRIPTION, OVERALL (provided by applicant): This proposal is in response to NIDDK RFA-DK-06-014 and is entitled Washington University Diabetes Research and Training Center (WU DRTC). The overall goal is to support centralized resources, facilities, and expertise shared by diabetes investigators at Washington University Medical School (WUMS), to promote the discovery of fundamental mechanisms leading to the disease and its complications, and to translate this information into prevention, treatments, and cures. Currently in its 30th year of funding, the WU DRTC has a proud history of major contributions to diabetes research. Positive momentum has continued during the funding period of 2002-present with a 65% increase in new members, a similar increase in NIH funding for diabetes-related research, a vibrant Pilot and Feasibility Program (PFP), significant productivity in terms of total number of publications attributable to the DRTC Cores (872), and substantial changes in the Core facilities to make them more responsive to the needs of the users. In the next funding cycle we plan to continue to evolve so that the WU DRTC is responsive to the changing expectations of the diabetes research community. We are particularly sensitive to the need to demonstrate the relevance of our research advances to the needs of society and to translate basic laboratory advances to improvements in the care of diabetics. Most notable is the development of a major new collaboration with St. Louis University School of Public Health (SLU-SPH) initiated in the fall of 2005. The resources of this School of Public Health, which is the only such school in Missouri, when coupled with the strengths at WUMS in basic and clinical diabetes research, provides a potent partnership that promises to be able to continue to make significant advances in diagnosis, treatment, and prevention of diabetes. In summary, the WU DRTC has now positioned itself to continue to meet the challenges of the current RFA by 1) increasing the DRTC user base along with a commensurate increase in diabetes-related research funding, 2) forming an alliance with SLU-SPH for expanding translational research and community outreach, 3) developing closer interaction with the WU CNRU for sharing of core resources and enrichment activities, 4) enhancing our presence in the important area of diabetic cardiovascular disease by improved interaction with the Center for Cardiovascular Research, 5) forming a partnership with the Institute of Clinical and Translational Sciences that will markedly enhance our members' opportunities to conduct human research, and 6) developing the WUMS-Barnes Jewish Diabetes Center, that provides state-of-the-art care of diabetic patients in our community. We believe that these new directions will result in 1) greater efficiency in utilizing enabling technologies by individual investigators; 2) greater collaboration aimed at preclinical development of potential therapeutic strategies; and, 3) faster translation of initial discoveries into useful treatments for diabetes and related metabolic and endocrine disorders. The specific aims proposed here are designed to accomplish these goals.

The Administrative Component (Core) of the Washington University School of Medicine (WUMS) Diabetes Research Center (DRC) provides the structure for effective and efficient operation and maintenance of the DRC in compliance with NIH and University guidelines. The Administrative Core encompasses the roles of the Director, co-Director, Associate Directors, Internal and External Advisory committees, and the Program Manager. Its aims are to: 1) Provide leadership and organization for and allocate funds for the DRC programs; 2) Provide scientific direction and vision for the DRC program; 3) Foster the environment for diabetes and metabolism research at WUMS; and 4) Represent the interests and needs of WUMS diabetes researchers to internal and external authorities. To accomplish this we have assembled leaders in diabetes research at WUMS who are committed to the mission of the NIDDK and the success of the WUMS DRC. Each of these individuals has been involved in leading the DRC during the past funding period. The accomplishments of the WUMS DRC outlined in this renewal application attest to the dedication and skill of this leadership group and the successful design of the administrative plan.

DESCRIPTION (provided by applicant): The mission of the Washington University School of Medicine (WUMS) Diabetes Research Center (DRC) is to support and enhance research in diabetes and related metabolic diseases through Biomedical Research Core services reflecting the evolving needs of diabetes investigators, a vibrant Pilot & Feasibility Program and a dynamic Enrichment Program. Now in Its 35th year of continuous NIDDK funding, this DRC is located at an outstanding research institution with a longstanding tradition of excellence in diabetes investigation. The WUMS DRC Research Base is organized in three Focus Groups: Metabolic Regulation, Complications, and Islet Biology & Immunology. Investigators from each of these groups participate in DRC programs that address two central, interacting scientific themes-a) Approaches Across the Translational Spectrum, and b) Prevention of Diabetes Complications. Evidence that the WUMS DRC continues to successfully pursue the mission outlined in this renewal application Includes a record of outstanding productivity reflected by publications and peer-reviewed funding in diabetes and related research. Our research strategy will build on these accomplishments by: 1. Creating an environment that supports important as well as innovative research In diabetes and related metabolic disorders; 2. Supporting cutting edge basic and clinical research related to etiology, pathogenesis, prevention and cure of diabetes; 3. Raising awareness and interest in fundamental and clinical diabetes research in addition to enhancing multldisciplinary approaches to diabetes and its complications; and 4. Translating new knowledge in diabetes to improved treatment of patients with diabetes.

DESCRIPTION (provided by applicant): Niemann-Pick type C1 (NPC1) disease is a rare, progressive neurodegenerative disorder characterized by accumulation of cholesterol and other lipids in the viscera and central nervous system. A barrier to the development of treatments for NPC1 disease is the lack of readily quantifiable outcome measures to evaluate efficacy of therapy in clinical trials. Through broad-based metabolomic efforts, we have discovered in NPC1 subjects cholesterol oxidation product (oxysterol) biomarkers that reflect the unique intersection of oxidative stress and unesterified cholesterol storage - the biochemical hallmark of NPC1 disease. This proposal tests the highly innovative hypothesis that oxysterol biomarkers, together with other cholesterol homeostatic markers, can serve as outcome measures to assess the effect of disease-modifying therapies (e.g., 2-hydroxypropyl-¿-cyclodextrin, HP-¿-CD) on cholesterol metabolism in the CNS and to monitor disease progression. 24(S)-HC, which is synthesized almost exclusively in large neurons in the CNS, and CSF cholesteryl esters (CE) offer potential quantitative, non-invasive metrics to guide dosing and to monitor drug response. Likewise, cholestane-3¿, 5¿, 6¿-triol (triol), which we have previously shown to be elevated in the CSF of NPC1 subjects, will inform with respect to the effect of HP-¿-CD on intraneuronal cholesterol storage. This hypothesis will be tested in NPC1 animal models administered intracerebroventricular (ICV) HP-¿-CD (Aim 1), and in NPC1 human subjects enrolled in a natural history study and in a Phase 1 trial for ICV HP-¿-CD at the NIH Clinical Center (Aim 2). The proposal will also explore the possibility that the exceptional receiver operating characteristics (ROC) of the triol assay can be harnessed to develop a newborn screen to identify NPC1 patients earlier and thus intervene in pre-symptomatic patients (Aim 3). The proposed newborn screen for NPC1 disease is innovative and would be the first for a non-enzymatic lysosomal disorder, as well as the first for an inborn error of sterol metabolism. While the goal of this project is to develop a prototype newborn screen for NPC1 disease suitable for implementation at the statewide or regional level, the tandem mass spectrometry methods developed for extraction and detection of the oxysterols could be readily extended to metabolites that accumulate in other sterol disorders (e.g., Smith-Lemli-Opitz Syndrome), thereby permitting multiplexed screening for several inherited disorders within the context of a single screen. The studies in this proposal are highly significant because we address the critical unmet therapeutic and diagnostic needs of NPC1 disease.

To serve a wider scientific community, we propose to leverage the successes and experience of the Washington University Diabetes Research Center (WUMS DRC) in a Shared National Resource Component with two distinct aims: 1, To extend our Pilot & Feasibility Program to diabetes investigators at institutions without NIDDK Diabetes Centers, we propose to establish subcontracts with University of Kentucky and University of Utah. These two universities have outstanding research bases in diabetes with a shared strong focus on diabetic cardiovascular complications. Philip Kern and Dale Abel, internationally recognized diabetes researchers with strong records of leadership, will serve as the subcontract Program Directors at Kentucky and Utah respectively. Each subcontract will fund two meritorious Pilot & Feasibility grants per year at each institution, and the program will be well-integrated with the successful Pilot & Feasibility Program at WUMS. 2. To make available the services of the WUMS DRC Immunoassay and Mass Spectrometry Cores to diabetes investigators on a national level, we propose to dedicate resources specifically for work performed in these Cores for diabetes investigators at other institutions. This effort will build on the substantial expertise of these two long-standing, highly functioning cores with track records of service to outside users.

? DESCRIPTION (provided by applicant): In non-alcoholic fatty liver disease (NAFLD), excess lipid accumulation can damage hepatic tissues leading to steatohepatitis or cirrhosis. Given the high prevalence of NAFLD and the serious consequences of liver dysfunction, understanding the pathogenesis of lipotoxicity in the liver is of high importance to human health. Our recent studies uncovered an unanticipated role for small nucleolar RNAs (snoRNAs) encoded in the rpL13a locus in the response of non-adipose tissues to lipid overload. These non-coding RNAs likely function by targeting 2??-O-methylations to specific cellular RNAs, although the precise targets of rpL13a snoRNAs in lipotoxic responses are not known. The first aim of this study will probe the mechanism of action of the rpL13a snoRNAs by using two complementary systems biology approaches to identify the targets of the snoRNAs in hepatocytes under lipotoxic conditions. The second aim of this study will leverage snoRNA loss-of-function murine models to analyze the physiological contributions of the rpL13a snoRNAs to diet-induced steatohepatitis.

ABSTRACT: Expanded Pilot and Feasibility Program The purpose of the Expanded Pilot and Feasibility Program (ePFP) of the Washington University Diabetes Research Center (DRC) is to extend our Pilot and Feasibility Program to diabetes investigators at institutions without NIDDK-supported Diabetes Research Centers. In this application, we propose to establish subcontracts with the University of Kentucky, the University of Utah, and the University of Wisconsin-Madison, three universities with outstanding research bases in diabetes. Philip Kern, Simon Fisher, and Alan Attie are internationally recognized diabetes researchers with strong records of leadership, who will serve as subcontract Program Directors at Kentucky, Utah, and Madison, respectively. Each subcontract will fund two meritorious pilot grants per year at each institution, and the program will be well integrated with the successful Pilot and Feasibility Program at Washington University. The ePFP leverages the success and experience of the Washington University DRC to serve a wider scientific community and enhances scientific interchange between investigators at the four institutions.

Cholesterol is an essential component of cellular membranes, a regulator of membrane protein function, and an obligate metabolic precursor. Exogenous cholesterol is acquired principally through receptor-mediated endocytosis of cholesteryl ester-laden low-density lipoproteins and trafficked to the lysosome. Lysosomal acid lipase liberates unesterified cholesterol, which is transferred by the Niemann-Pick C1 and C2 (NPC1, NPC2) proteins to the limiting lysosomal membrane and subsequently to other cellular organelles. Cholesterol exiting the lysosome is distributed to multiple compartments ? including the plasma membrane, ER, Golgi, mitochondria and peroxisomes ? and serves to regulate key cholesterol homeostatic responses. While several proteins have been implicated in post-lysosomal cholesterol trafficking, the components and relative contribution of each pathway and specific trafficking itinerary of the lipoprotein-derived cholesterol are poorly understood. This represents an important gap in our knowledge and an area in which advances have the potential to identify new therapeutic targets for treating both atherosclerotic and neurodegenerative diseases. We have generated novel cholesterol probes that can substitute for cholesterol and allow for the selective capture of cholesterol interacting proteins for unbiased identification by mass spectrometry. These probes provide a powerful new tool to study previously elusive post-lysosomal cholesterol trafficking pathways. We hypothesize the specific itinerary and metabolic fate of lipoprotein cholesterol involves both NPC1-dependent and -independent distribution pathways that can be identified based on unique cholesterol cargo interactomes. Elucidation of these protein networks through quantitative proteomics will identify major nodes through which lipoprotein cholesterol traffics and novel therapeutic targets..

? DESCRIPTION (provided by applicant): This application is the competitive renewal of the Principles in Cardiovascular Research Training Program (T32-HL007081) at Washington University School of Medicine (WUSM). The program, which began in 1975, has had a strong record of training postdoctoral fellows who are committed to careers in cardiovascular research. The current application, which seeks support for 6 MD, MD/PhD or PhD postdoctoral trainees per year, incorporates a number of new programmatic features developed to enhance the training mission, as will be detailed herein. The goal of this training program is to develop independent cardiovascular scientists who are trained to utilize basic and/or applied principles of clinical cardiovascular research to improve outcomes of patients with cardiovascular disease. The centerpiece of the program remains the commitment to a sustained period of research training (2 years) that focuses on a research project supervised by a faculty mentor who has (1) a proven track record of mentoring and training postdoctoral fellows and (2) the resources to support the work conducted by postdoctoral fellows for the duration of the appointment in this training program. A hallmark of the program is the recognition that true mentoring and skills development need to be individualized. There are presently 24 faculty preceptors from seven different Departments in the Medical School and the Danforth campuses, and from six different divisions within the Department of Medicine. All of the faculty participants in this program are committed to excellence in cardiovascular research and to the training, mentoring and professional development of postdoctoral trainees to prepare these individuals for independent, productive careers at the forefront of cardiovascular disease-focused research. New to this application, the training faculty has been organized into four thematic research areas: genetics; cardiac and electrical remodeling of the heart; vascular pathobiology; and cardiac imaging. The program training faculty bring expertise in biochemistry; biostatistics; bioengineering; epidemiology; cardiovascular medicine; cell, molecular, and vascular biology; computer sciences; pathology; pharmacology; physics; physiology; and radiology. The training faculty are well-funded, interactive, collaborative, and productive investigators committed to providing the training, experience, resources, intellectual enthusiasm, and mentoring needed to facilitate the scientific and professional development of the trainees. The didactic aspect of this training program provides lectures and hands on workshops in the Responsible Conduct for Research and Ethics, as well as a Core Curriculum in Cardiovascular Research that complements the four research themes of the training program. In addition to the research training provided, the trainees participate in the weekly Trainee Research Seminar series and the Cardiovascular Research Seminar series, as well as our annual Cardiovascular Research Day Symposium. (End of Abstract)

PROJECT SUMMARY/ABSTRACT The current epidemic of obesity and metabolic syndrome has been linked to alterations in diet and lifestyle. Although human tissues demonstrate remarkable metabolic plasticity in response to physical activity and dietary exposures, our understanding of the mechanisms that underlie the beneficial or deleterious effects of exercise or nutrient exposures on skeletal muscle and hepatic metabolism are incomplete. The goal of this study is to uncover new mechanisms that underlie enhanced skeletal muscle insulin sensitivity following an acute bout of exercise and changes in hepatic glucose homeostasis and insulin sensitivity in refeeding responses and time-restricted feeding paradigms. I will test the radically novel hypothesis that remodeling of ribosomes in these tissues rapidly generates changes in the proteome and serves as a mechanism for metabolic plasticity. This work will advance our understanding of how environmental cues shape metabolic physiology and has the potential to identify new targets for preventive therapies aimed at improving human metabolic fitness.