Christopher B. Newgard - US grants

phosphorylases; isozymes; enzyme mechanism; allosteric site; adenosine monophosphate; complementary DNA; muscle metabolism; liver metabolism; gene expression; fusion gene; enzyme structure; chemical structure function; genetic manipulation; site directed mutagenesis; reporter genes; glycogenolysis; glycogen storage disease type VIII; gel electrophoresis; laboratory rat; molecular cloning;

Glucose-stimulated insulin secretion occurs as a result of, and in proportion to, the rate of glucose metabolism in beta-cells of the islets of Langerhans. This proposal seeks to utilize techniques of molecular biology to alter the rate and regulation of glucose metabolism in clonal cells derived from islet and non-islet neuroendocrine tissues. We will evaluate the effects of altering several key metabolic regulatory activities in the AtT-20ins cell line system, and in parallel, in the rat insulinoma cell line RIN 1046-38 or in glucose unresponsive primary islet cells isolated from male Zucker Diabetic Fatty (ZDF) rats. The specific aims of the grant are as follows: 1) to test whether GLUT-2 and GLUT-1 have distinct effects on glucose-stimulated insulin release in islet cells or cell lines, as they clearly do in AtT-20ins cells, 2) to examine the effects of altering glucose phosphorylating capacity and kinetics by overexpressing native and mutant forms of glucokinase, and by reducing the level of expression of hexokinase in insulin secreting clonal cells, and 3) to examine the effects of increasing phosphofructokinase activity in insulin secreting clonal cells by overexpression of native and phosphatase deficient mutants of the fructose-2,6-kinase/fructose-2,6-- bisphosphatase enzyme. Alterations of glucose phosphorylation and phosphofructokinase will be studied in the presence and absence of GLUT-2 or GLUT-1 overexpression. The consequences of all of the molecular manipulations listed above will be examined at two levels, 1) the metabolic fate of glucose, measured by administration of strategically labeled glucose substrates (i.e., 2-, 3-, or 5-3H glucose) and by measurement of accumulation of certain metabolic end products (glycogen, lipids), and 2) the glucose-stimulated insulin secretion response. One long term goal of this research understand the metabolic consequences of manipulation of key proteins involved in regulation of carbohydrate metabolism in eucaryotic cells. A benefit of the systems chosen for this work (AtT-20ins and islet cells and cell lines) is that a second major goal can be pursued simultaneously, namely, the development of a clonal cell that faithfully mimics the glucose-stimulated insulin secretion function of islet beta-cells. Our hope is that such cells can be used in the future in a therapeutic strategy for insulin-dependent diabetes mellitus (IDDM), involving implantation of appropriately encapsulated engineered cells for cell-based insulin delivery in response to variations in circulating glucose.

African American; hyperlipidemia; myocardial ischemia /hypoxia; gene therapy; noninsulin dependent diabetes mellitus; hyperglycemia; oxidation; disease /disorder model; fatty acids;

Despite decades of investigation, the biochemical pathways involved in regulation of insulin secretion by glucose are incompletely understood. The overall goal of this project (DK046492-22), now entering its 23rd year of funding, is to gain a better understanding of metabolic coupling mechanisms in the ?-cell, and how they are altered when islets fail in type 2 diabetes (T2D). Since the last competitive renewal of the program in 2011, we have identified two metabolic pathways that stimulate insulin granule exocytosis, the first involving anaplerotic metabolism of pyruvate, export of mitochondrial substrates and their engagement with the cytosolic, NADP- dependent isoform of isocitrate dehydrogenase (ICDc), and activation of the glutathione/glutaredoxin (GRX) system. This pyruvate/isocitrate pathway connects to glutathione metabolism in two important ways. First, the NADPH produced in the ICDc reaction is used to maintain glutathione (GSH) and GRX in their reduced states, thereby activating GRX-mediated granule exocytosis via SENP-1. Second, the ?-ketoglutarate produced by the ICDc reaction contributes to maintenance of the GSH pool via transamination to glutamate. Using patch- clamped ?-cells from human T2D subjects, we find that NAPDH, isocitrate, and GSH all rescue insulin granule exocytosis in otherwise glucose-unresponsive beta-cells. We also identified the purine/nucleotide pathway intermediate adenosuccinate (S-AMP) as a glucose-regulated metabolite that stimulates exocytosis in normal human ?-cells and rescues secretory function in human T2D ?-cells. Thus, our work has identified two novel pathways of GSIS, and demonstrated that intermediates from both pathways rescue secretion in dysfunctional human ?-cells. Based on these findings, we propose the following new specific aims: 1. To study the pyruvate/isocitrate/GSH and S-AMP pathways via metabolic flux analysis; 2. To investigate manipulation of the NAD/NADP salvage (NAMPT) pathway as a strategy for enhancing flux through the pyruvate/isocitrate/GSH and S-AMP pathways of insulin secretion, and for reversing ?-cell dysfunction in T2D; 3. To test potential additive effects of co-activation of the S-AMP, pyruvate/isocitrate/GSH and NAMPT pathways, and to define targets within these pathways for rescue of ?-cell dysfunction in T2D.

Over the past decade, our group has been actively engaged in development of engineered cell lines as potential alternatives to insulin injection or islet transplantation for insulin replacement in insulin dependent diabetes mellitus (IDDM). A major hurdle faced in bringing such an approach to fruition is to provide protection against immune destruction of the transplanted cell lines. This application seeks to develop a combinatorial approach for overcoming this obstacle. The first part of our strategy is to use genetic engineering approaches to provide protection against the cytotoxic effects of inflammatory cytokines such as IL-1beta and gamma-IFN that are known to be involved in beta-cell destruction in IDDM. We have recently shown that overexpression of MnSOD in INS-1 insulinoma cells provides complete protection from IL-1beta-mediated cytotoxicity and also blocks iNOS upregulation by the cytokine. The first specific aim seeks to understand the mechanism of this protective effect of MnSOD by a combination of biochemical and molecular approaches. MnSOD overexpressing cells are not protected from the cytotoxic effects of gamma-IFN. Thus, the second specific aim seeks to define and discriminate the pathways by which gamma-IFN and IL-1beta exert their cytotoxic effects. We will also engineer insulinoma cell lines for expression of the anti-apoptotic gene bcl-2, and test whether this maneuver provides protection against one or both of the cytokines. The third and final specific aim will involve transplantation of the new cell lines engineered for resistance to cytokine damage into rodents. These experiments will include the second part of our strategy, which is to encapsulate the engineered cell lines in a cell-impermeable device provided by industrial collaborators that will prevent contact between cellular elements of the immune system and the transplanted cells. The broad goal of the application is to determine whether this combinatorial approach will allow long-term survival of transplanted, insulin producing cells in rodent allograft and IDDM models. If we are successful, these studies could lead to development of viable strategies for cell-based hormone replacement in IDDM and other diseases.

Measurement of hepatic glucose output from plasma glucose 13C-isotopomers has been successfully accomplished in 24-hour fasted anesthetized rats that underwent adenovirus loading with b-galactosidase, glucokinase, and glucose-6-phosphatase (G6Pase) genes. The animal was given a 0.5 mg/kg min infusion of [1,6-13C]glucose and 2.5 mg/kg/min of [U-13C] propionate over 2 hours. 13C NMR spectra were obtained with a 1.5 ml blood sample from a putative G6Pase overexpressing animal. The spectrum collection time was approximately 1 hour, demonstrating adequate sensitivity. In the 13C spectrum, the contribution of [1,6-13C]glucose can be differentiated from all other 13C glucose molecules, including the background natural abundance signal since it generates an unique doublet as a result of long-range 13C-13C-coupling between carbons 1 and 6. From the 13C and 1H NMR spectra, the fraction of [1,6-13C]glucose in the plasma, hence its dilution, can be measured. Hepatic glucose output is given by the product of the dilution and infusion rate. We are confident that the amount of [1,6-13C]glucose can be cut from 0.50 to 0.15 mg/kg/min without compromising its measurement by 13C NMR. Analysis of the C2b glucose multiplet components provides metabolic flux information at the level of PEP and the citric acid cycle. Assuming that glucose is derived quantitatively from PEP after 24 hours fasting, absolute flux through anaplerosis, pyruvate kinase and citrate synthase can be estimated by indexing the gluconeogenic PEP flux to hepatic glucose output. (Service 1) REPORT PERIOD: (09/01/97-08/31/98)

Measurement of hepatic glucose output from plasma glucose 13C-isotopomers has been successfully accomplished in 24-hour fasted anesthetized rats that underwent adenovirus loading with b-galactosidase, glucokinase, and glucose-6-phosphatase (G6Pase) genes. The animal was given a 0.5 mg/kg min infusion of [1,6-13C]glucose and 2.5 mg/kg/min of [U-13C] propionate over 2 hours. 13C NMR spectra were obtained with a 1.5 ml blood sample from a putative G6Pase overexpressing animal. The spectrum collection time was approximately 1 hour, demonstrating adequate sensitivity. In the 13C spectrum, the contribution of [1,6-13C]glucose can be differentiated from all other 13C glucose molecules, including the background natural abundance signal since it generates an unique doublet as a result of long-range 13C-13C-coupling between carbons 1 and 6. From the 13C and 1H NMR spectra, the fraction of [1,6-13C]glucose in the plasma, hence its dilution, can be measured. Hepatic glucose output is given by the product of the dilution and infusion rate. We are confident that the amount of [1,6-13C]glucose can be cut from 0.50 to 0.15 mg/kg/min without compromising its measurement by 13C NMR. Analysis of the C2b glucose multiplet components provides metabolic flux information at the level of PEP and the citric acid cycle. Assuming that glucose is derived quantitatively from PEP after 24 hours fasting, absolute flux through anaplerosis, pyruvate kinase and citrate synthase can be estimated by indexing the gluconeogenic PEP flux to hepatic glucose output. (Service 1) REPORT PERIOD: (09/01/97-08/31/98)

model

DESCRIPTION (Provided by applicant): Over the past decade, our group has focused on application of the tools of molecular biology to the development of insulin secreting cell lines that might serve as islet surrogates for transplantation therapy of diabetes. The focus of this proposal is on the design and implementation of methods for preventing impairment of function and/or islet cell damage by inflammatory cytokines and other small molecular weight mediators of the immune response. This renewal application was first funded in 1998 as part of an RFA entitled "Cellular and Molecular Approaches to Achieving Euglycemia." In the ensuing three years, we have made several discoveries pertaining to methods for protecting insulin secreting cells from damage by the immune system. Most important among these has been the development of a method for selecting cells that are resistant to the combined cytotoxic effects of the inflammatory cytokines IL-1 beta + IFN-gamma, involving culture of INS-1 insulinoma cells in iteratively increasing concentrations of these agents and collection of surviving cells. We have also demonstrated that resistance to IL-1 beta is related to impairment of NF-kB translocation and inducible nitric oxide synthase (iNOS) induction, while the resistance to IFN-gamma is due to a large induction in expression of signal transducer and activator of transcription (STAT)-1 alpha. Moreover, we have demonstrated that adenovirus-mediated overexpression of STAT-1 alpha confers resistance to IFN-gamma and IFN-gamma-IL-1 beta-induced cytotoxicity. Finally, we have recently learned that our selection strategy also confers resistance to cytokine-induced impairment of insulin secretion. In our view, these studies provide the platform from which novel and robust genetic engineering strategies can be developed for protecting insulin secreting cells from the immune system. To this end, the specific aims of this grant are: 1) To investigate the mechanisms by which resistance to IL-1 beta-induced cytotoxicity is conferred in cells subjected to our selection protocol; 2) To investigate the mechanism by which STAT-lalpha overexpression in insulinoma cells blocks cytokine-induced cytotoxicity; 3) To investigate the mechanism by which cytokines cause impairment of glucose-stimulated insulin secretion, and to determine the mechanism by which our newly developed selection and engineering methods confer resistance to this impairment. These studies have the potential to foster new methods for preserving beta-cell mass in both major forms of diabetes.

Responsibility for administration of this program will reside in the Administrative core (Core C), directed by the overall PI of the program, Dr. Christopher B. Newgard, who will devote 5% of his time to this effort. He will be assisted in this role by James R. McCurdy, Business Manager of the Sarah W. Stedman Nutrition and Metabolism Center at Duke, and Ms. Brenda Aston, Chief Administrator of the Advanced Imaging Center at UTSW. This team will work closely with the project and core Pis in preparation of yearly progress reports, monitoring of personnel changes that occur during the course of the program, and oversight of the program budget, including yearly summaries of expenditures. Core C will also be responsible for assuring that the program is in compliance with institutional standards for human and animal research, including the timely submission of protocols to the relevant institutional review committees. A travel budget has been requested in Core C to allow four face-to-face meetings of the UTSW and Duke Pis per year, and for yearly meetings of the Scientific Advisory Board. Dr. Newgard and his staff will plan and coordinate those meetings. In the unlikely event that Dr. Newgard is rendered unable to fulfill his duties as Principal Investigator of this Program Project for any reason. Dr. Sherry will take full responsibility for completion of the program, and will work closely with the other investigators and senior administrative personnel at Duke and UTSWMC to ensure continued coordination of effort and delivery of the program milestones. RELEVANCE (See instructions): The administrative core of this progam will provide oversight that ensures timely achievement of the core goals of the program, and will optimize communication between team members. It will also monitor all budgetary allocations, time commitment of key personnel, and progress reporting requirements.

Over the first five years of funding of this program project grant (PPG), our laboratory has been applying an interdisciplinary approach for defining metabolic abnormalities of liver, pancreatic islet [unreadable]-cells, and skeletal muscle in diabetes and obesity. Over the same time period, other members of this PPG team have developed technologies for functional imaging, targeted delivery of genes and other molecular cargo, and customized gene activation switches. The most compelling advances made by the PPG team have occurred in the area of pancreatic islet biology and related technologies. We have therefore chosen to focus the competitive renewal of this application on development of new strategies for understanding and reversing [unreadable]-cell dysfunction of type 2 diabetes. The goal of this project (Project 1) is to investigate and validate novel pathways for control of [unreadable]-cell function and growth that have emerged from our work in the prior funding period. The project will make extensive use of extraordinary technologies resident in Core B for [unreadable]-cell specific gene delivery in adult animals, and in Core C for comprehensive MS- and NMR-based metabolic analysis of islets and [unreadable]-cell lines. The specific aims of the project are: 1) To investigate mechanisms by which manipulation of the homeodomain transcription factor Nkx6.1 and its target genes affect glucose-stimulated insulin secretion (GSIS) in pancreatic islets;2) To investigate mechanisms by which manipulation of the homeodomain transcription factor Nkx6.1 and its target genes affect pancreatic islet growth;3) To test the potential protective or restorative effect of Nkx6.1 and its target genes in preservation of [unreadable]-cell mass and function in cellular and animal models of type 2 diabetes.

Over the first five years of funding of this program project grant (PPG), our laboratory has been[unreadable] applying an interdisciplinary approach for defining metabolic abnormalities of liver, pancreatic islet[unreadable] beta-cells, and skeletal muscle in diabetes and obesity. Over the same time period, other members of[unreadable] this PPG team have developed technologies for functional imaging, targeted delivery of genes and[unreadable] other molecular cargo, and customized gene activation switches. The most compelling advances[unreadable] made by the PPG team have occurred in the area of pancreatic islet biology and related technologies.[unreadable] We have therefore chosen to focus the competitive renewal of this application on development of new[unreadable] strategies for understanding and reversing beta-cell dysfunction of type 2 diabetes. The goal of this[unreadable] project (Project 1) is to investigate and validate novel pathways for control of beta-cell function and growth[unreadable] that have emerged from our work in the prior funding period. The project will make extensive use of[unreadable] extraordinary technologies resident in Core B for beta-cell specific gene delivery in adult animals, and in[unreadable] Core C for comprehensive MS- and NMR-based metabolic analysis of islets and beta-cell lines. The[unreadable] specific aims of the project are: 1) To investigate mechanisms by which manipulation of the[unreadable] homeodomain transcription factor Nkx6.1 and its target genes affect glucose-stimulated insulin[unreadable] secretion (GSIS) in pancreatic islets; 2) To investigate mechanisms by which manipulation of the[unreadable] homeodomain transcription factor Nkx6.1 and its target genes affect pancreatic islet growth; 3) To test[unreadable] the potential protective or restorative effect of Nkx6.1 and its target genes in preservation of beta-cell[unreadable] mass and function in cellular and animal models of type 2 diabetes.

Academic Medical Centers; Animals; Area; Arts; B9 endocrine pancreas; Beta Cell; Biochemical; Biology; Biotechnology, Genetic Engineering; CRISP; Cell Function; Cell Process; Cell model; Cell physiology; Cells; Cellular Function; Cellular Physiology; Cellular Process; Cellular model; Collaborations; Computer Retrieval of Information on Scientific Projects Database; D-Glucose; Development; Dextrose; Diabetes Mellitus; Diabetes Mellitus, Adult-Onset; Diabetes Mellitus, Ketosis-Resistant; Diabetes Mellitus, Non-Insulin-Dependent; Diabetes Mellitus, Noninsulin Dependent; Diabetes Mellitus, Slow-Onset; Diabetes Mellitus, Stable; Diabetes Mellitus, Type 2; Diabetes Mellitus, Type II; Disease; Disorder; Dysfunction; Functional disorder; Funding; Generalized Growth; Genes; Genetic Engineering; Glucose; Goals; Grant; Growth; Homeo Domain; Institution; Insulin Cell; Insulin Secreting Cell; Intermediary Metabolism; Investigators; Islands of Langerhans; Islet Cell; Islet Cells; Islets of Langerhans; Laboratories; Life; METBL; MODY; Mass Spectrum; Mass Spectrum Analysis; Maturity-Onset Diabetes Mellitus; Measurement; Medical Imaging, Positron Emission Tomography; Medical center; Metabolic; Metabolic Processes; Metabolism; Molecular; Molecular Biology, Genetic Engineering; NIDDM; NIH; National Institutes of Health; National Institutes of Health (U.S.); Nesidioblasts; Non-Insulin Dependent Diabetes; Non-Insulin-Dependent Diabetes Mellitus; Nuclear Magnetic Resonance; P01 Mechanism; P01 Program; PET; PET Scan; PET imaging; PETSCAN; PETT; Pancreas, Endocrine; Pancreatic Islets; Pars endocrina pancreatis; Pathway interactions; Performance; Photometry/Spectrum Analysis, Mass; Physiopathology; Positron Emission Tomography Scan; Positron-Emission Tomography; Program Project Grant; Program Research Project Grants; Programs (PT); Programs [Publication Type]; Proton Magnetic Resonance Spectroscopic Imaging; Rad.-PET; Recombinant DNA Technology; Research; Research Personnel; Research Program Projects; Research Resources; Researchers; Resources; Role; Source; Spectrometry, Mass; Spectroscopy, Mass; Spectrum Analyses, Mass; Spectrum Analysis, Mass; Subcellular Process; T2D; T2DM; Technology; Texas; Tissue Growth; Type 2 diabetes; Type II diabetes; United States National Institutes of Health; Universities; University Medical Centers; adult onset diabetes; base; beta cell development; diabetes; disease/disorder; endocrine pancreas; endocrine pancreas development; gene discovery; homeodomain; in vivo; insulin secretion; interdisciplinary approach; islet; islet development; islet progenitor; ketosis resistant diabetes; maturity onset diabetes; measurement of metabolism; metabolomics; molecular imaging; new technology; novel; ontogeny; pathophysiology; pathway; programs; social role; transcription factor

DESCRIPTION (provided by applicant): Genetic studies of complex diseases like obesity and diabetes have so far not succeeded in explaining the large genetic contribution of heritability to these disorders. In humans, the studies are compromised by limited phenotyping and environmental variation. In mice, intercross studies are limited by poor mapping resolution and the lack of biological replication. The Collaborative Cross is a large community resource under development that will generate ~300 recombinant inbred strains derived from eight founder strains. The founder strains together account for ~90% of all the genetic diversity of all the inbred mouse strains, which it is widely believed, carry about as much genetic variability as the human population. The strains will be fully genotyped and will carry enough recombinations to afford high-resolution genetic mapping. We plan to carry out detailed phenotyping of these strains on high-fat diets, bringing together physiological phenotyping (e.g. glucose tolerance, lipid biochemistry) with phenotypes derived from the application of various "-omics" technologies (e.g. transcriptomics, metabolomics). For the first time, we plan to genetically map flux measurements;i.e. the true rates of synthesis of proteins, fatty acids, cholesterol, and glucose. This study is a pilot project on the eight parental strains ofthe Collaborative Cross. Our aim is to ascertain the range of variability of the phenotypes will measure so that we can justify a larger study of the recombinant inbred strains. We have assembled an experienced team with complementary expertise;1) Alan Attie (University of Wisconsin;mouse genetics &genomics, lipid metabolism);2) Chris Newgard (Duke University;metabolomics);3) Gary Churchill (The Jackson Laboratory;conceived and developed the Collaborative Cross, statistical methods for the investigation of complex disease-related traits in the mouse);and 4) Henri Brunengraber (Case Western Reserve University;stable isotope technology (GC-MS and NMR) to measure changes in metabolic flux associated with chronic diseases).

In the past funding period, we have identified strong associations between circulafing levels of branched-chain amino acids (BCAA-Leu, Val, He) and chronic metabolic diseases in mulfiple human cohorts. In each case, principal component analysis identified a correlating group of metabolites comprised of all three BCAA, aromafic amino acids (Phe, Tyr), and C3 and C5 acylcarnitines, with disease associafions much stronger than for any lipid-related principal component. Feeding studies involving BCAA supplementation of high fat diets demonstrated a contribufion of BCAA to development of insulin resistance independent of body weight. However, the increases in BCAA, aromafic amino acids, and related metabolites that we observe in the blood of humans with metabolic diseases is not necessarily driven only by protein consumption, and could also be influenced by rates of amino acid catabolism and protein turnover, or changes in hormones, such as the substanfial decrease in IGF-1 levels that we observe in obese humans. Herein, and in close collaboration with the other projects and cores, we will test an evolving model for BCAA-mediated impairment of insulin action involving decreased disposal of BCAA in adipose tissue, and consequent accumulafion of BCAA metabolites in skeletal muscle. The role of decreased lGF-1 levels in control of amino acid homeostasis will also be studied. The overarching goal of Project 1 is to fully understand the metabolic and molecular changes that lead to perturbed BCAA homeostasis and loss of insulin sensitivity in animal models, thereby leading to better understanding of possible cause/effect relafionships between BCAA and metabolic disease in human subjects. Specific aims are: 1. To study the time course of changes in pathways of BCAA metabolism during development of insulin resistance in Zucker-obese rats; 2. To expand upon our prior studies of dietary supplementafion of BCAA in normal rats to include Leu alone and aromatic amino acids; 3. To investigate the impact of three maneuvers designed to reverse defects in BCAA metabolism and insulin resistance in Zucker-obese rats; 4. To investigate the effects of HF or HF + BCAA feeding on insulin sensitivity, BCAA levels, and BCAA metabolism in mice with reduced circulating IGF-1.

Project Summary/Abstract Exercise is an effective intervention for both the prevention and treatment of cardiometabolic diseases, but the mechanistic underpinnings of the health benefits of exercise remain incompletely defined. Recent work highlights the importance of inter-organ circuits in mediating healthful exercise responses. We identified ?- aminoisobutyric acid (BAIBA) as a novel small molecule ?myokine? that increases the expression of brown adipocyte-specific genes in vitro, and improves glucose disposal and decreases weight gain in mice. In humans, plasma BAIBA concentrations are increased with chronic exercise and demonstrate a strong inverse association with metabolic risk factors. Our team has also been a leader in characterizing within-tissue responses to exercise and dietary interventions. These experiences, coupled with the high translational relevance of the research problem, motivate us to take a systems wide approach to studying the health benefits of exercise in humans and animal models. To this end, we have formed a multi-institutional, multi-omics center to engage in collaborative studies under the aegis of the NIH MoTrPAC initiative. Our core builds upon ongoing collaborations between teams at Duke, Harvard and the Broad Institute with complementary strengths in metabolomics and proteomics technologies and decades of experience in cardiometabolic research. The core will provide a deep menu of analytical tools for targeted and non-targeted metabolomics, protein profiling, and the analysis of key protein post-translational modifications. Each of the core components has a track record for handling large sample sets, and is well- poised to analyze the expected tens of thousands of tissue and blood samples generated by a national consortium of investigators studying exercise interventions in animal and human cohorts. An additional distinction of our team is the ability to integrate new findings from MoTrPAC with previously collected genomic, proteomic and metabolomic data from large human cohorts. We hypothesize that integrating the metabolomic and proteomic profiles of human tissues and blood during exercise with genetics and detailed human phenotyping will provide novel insights into the inter-organ circuits and within-organ responses that mediate the salutary effects of exercise. All of the primary data generated by this multi- disciplinary proposal will be made rapidly available to the scientific community via a novel information portal at the Broad Institute. Importantly, all four leaders of this proposed core (Carr, Clish, Gerszten and Newgard) have strong track records in the use of metabolomics and proteomics tools for the identification of novel cardiometabolic regulatory and disease mechanisms. These experiences position this proposed core as one that can have maximal impact on the generation, analysis, and interpretation of molecular profiling data.

Project Summary/Abstract Exercise is an effective intervention for both the prevention and treatment of cardiometabolic diseases, but the mechanistic underpinnings of the health benefits of exercise remain incompletely defined. Recent work highlights the importance of inter-organ circuits in mediating healthful exercise responses. We identified ?- aminoisobutyric acid (BAIBA) as a novel small molecule ?myokine? that increases the expression of brown adipocyte-specific genes in vitro, and improves glucose disposal and decreases weight gain in mice. In humans, plasma BAIBA concentrations are increased with chronic exercise and demonstrate a strong inverse association with metabolic risk factors. Our team has also been a leader in characterizing within-tissue responses to exercise and dietary interventions. These experiences, coupled with the high translational relevance of the research problem, motivate us to take a systems wide approach to studying the health benefits of exercise in humans and animal models. To this end, we have formed a multi-institutional, multi-omics center to engage in collaborative studies under the aegis of the NIH MoTrPAC initiative. Our core builds upon ongoing collaborations between teams at Duke, Harvard and the Broad Institute with complementary strengths in metabolomics and proteomics technologies and decades of experience in cardiometabolic research. The core will provide a deep menu of analytical tools for targeted and non-targeted metabolomics, protein profiling, and the analysis of key protein post-translational modifications. Each of the core components has a track record for handling large sample sets, and is well- poised to analyze the expected tens of thousands of tissue and blood samples generated by a national consortium of investigators studying exercise interventions in animal and human cohorts. An additional distinction of our team is the ability to integrate new findings from MoTrPAC with previously collected genomic, proteomic and metabolomic data from large human cohorts. We hypothesize that integrating the metabolomic and proteomic profiles of human tissues and blood during exercise with genetics and detailed human phenotyping will provide novel insights into the inter-organ circuits and within-organ responses that mediate the salutary effects of exercise. All of the primary data generated by this multi- disciplinary proposal will be made rapidly available to the scientific community via a novel information portal at the Broad Institute. Importantly, all four leaders of this proposed core (Carr, Clish, Gerszten and Newgard) have strong track records in the use of metabolomics and proteomics tools for the identification of novel cardiometabolic regulatory and disease mechanisms. These experiences position this proposed core as one that can have maximal impact on the generation, analysis, and interpretation of molecular profiling data.

.PROJECT SUMMARY ? R01 Newgard/Herman . Studies performed by our group have helped establish that branched-chain amino acids (BCAA) and related metabolites are associated with insulin resistance and T2D, predictive of diabetes development and intervention outcomes, and highly responsive to therapeutic interventions. The goal of this proposal is to fully understand the metabolic and molecular mechanisms linking concerted dysregulation of BCAA, glucose and lipid metabolism. Key recent findings leading to this proposal emerged when we altered branched-chain ketoacid dehydrogenase (BCKDH) complex activity via pharmacologic and molecular manipulation of its regulatory kinase (BDK) and phosphatase (PPM1K) in rodent models of obesity and metabolic disease. Treatment of Zucker-obese rats with BT2, a small molecule inhibitor of BDK, or a recombinant adenovirus expressing PPM1K lowered circulating BCAA and branched chain ketoacid levels, improved glucose tolerance and insulin sensitivity, and increased fatty acid oxidation while markedly decreasing liver triglycerides. Phosphoproteomics analysis revealed that in addition to their function to modify BCKDH activity, BDK and PPM1K also regulate the phosphorylation of the key lipogenic enzyme, ATP-citrate lyase (ACL). Whereas phosphorylation of BCKDH inhibits its activity, phosphorylation of ACL is an activating post-translational modification that leads to increased de novo lipogenesis. We also demonstrated that overnutrition or fructose feeding activates the carbohydrate sensing transcription factor, ChREBP, which upregulates both BDK and ACL expression while suppressing PPM1K. Altogether, these studies define a novel regulatory node integrating glucose, lipid, and BCAA metabolism that participates in the progression of metabolic disease. The current study seeks to understand the impact of chronic manipulation of the ChREBP/BDK/PPM1K regulatory node in multiple dietary contexts, and to expand our human studies to include evaluation of genetic and dietary variables, via the following specific aims: 1) To test the hypothesis that chronic hepatic BDK overexpression will exacerbate metabolic disease phenotypes; 2) To test the hypotheses that chronic hepatic PPM1K overexpression or ChREBP suppression will attenuate or prevent development of metabolic disease phenotypes; 3) To determine whether consumption of sugar-sweetened beverages (SSB) associates with circulating BCAA levels, and whether genetic variants in the ChREBP/BDK/PPM1K regulatory node interact with sugar consumption to regulate BCAA levels and other metabolic traits in human populations.

Project Abstract/Summary Diabetes currently affects 9.3% of the U.S. population totaling $245 billion annually in U.S. healthcare costs. Current therapies for diabetes are limited in their ability to control blood glucose and/or enhance insulin sensitivity. Given the magnitude of diabetes costs and complications, new therapies with reduced side effects are urgently needed. The common defect in both type 1 and type 2 diabetes is the loss of functional pancreatic islet ?-cell mass. A therapeutic intervention that replenishes the insulin-producing pancreatic beta cells represents a cure for diabetes We propose to develop two distinct molecular scaffolds with demonstrated in vivo proof of concept for stimulating pancreatic beta-cell proliferation. A series of phenylbenzamides, discovered in an unrelated hepatic steatosis screen, were found to stimulate selective beta cell proliferation in rodents, isolated rat and human islets, and will be evaluated and optimized for efficacy and translational potential. The second series - chromenones, discovered in a Nkx6.1 promoter screen, have been shown to robustly stimulate beta-cell proliferation in rodent and human islets. We will perform medicinal chemistry optimization on both scaffolds to produce in vivo chemical probes with translational potential for human therapeutic use.

PROJECT SUMMARY/ABSTRACT ? METABOLOMICS CORE Comprehensive metabolic analysis, or ?metabolomics?, is a technology that defines the chemical phenotype of living systems. Given that metabolic fluxes and metabolite levels are downstream of genomic, transcriptomic, and proteomic variability, metabolomics provides a highly integrated profile of biological status. As such, it has unique potential for discovery of biomarkers that predict disease incidence, severity, and progression, and for casting new light on underlying mechanistic abnormalities. Metabolomic analyses are challenging, however, due to the complexity inherent in measuring large numbers of intermediary metabolites with diverse chemical properties in a quantitatively rigorous and reproducible fashion. The DMPI Metabolomics Core Lab has a long history of collaborative research and has established a strong and reliable infrastructure for conducting measurements for investigators at Duke and at outside institutions. Thus, it is well poised to become the NCDRC Metabolomics Core. While Duke has world-renowned facilities for metabolomics, its use by diabetes investigators outside of Duke (such as WF and UNC researchers) has been limited by bottlenecks, particularly in the analysis and interpretation of data, which the NCDRC seeks to address by establishing the NCDRC Metabolomics Core with support from Research Navigators.