Henri Brunengraber - US grants

Lipid emulsions used extensively for parenteral nutrition have undesirable side effects which are, in part, caused by the insolubility of fat in water. We would like to explore the potential of 1,3-butanediol and 1,3-pentanediol either as DL mixtures or as L-isomers, as fuels for intravenous nutrition. These diols are water soluble and non-ionized. They are at the same time carbohydrates and lipid precursors. D-1,3-butanediol is metabolized to physiological ketone bodies D-3-hydroxybutyrate and acetoacetate. Ketone bodies are excellent fuels for peripheral tissues. The other diols (L-1,3-butanediol, D and L-1,3-pentanediol) are oxidized to hydroxyacids which are analogs of physiological D-3-hydroxybutyrate. Ultimately the diols are converted to acetyl-CoA (butanediol) or acetyl-CoA + propionyl-CoA (pentanediol). Propionyl-CoA is a gluconeogenic precursor in liver and an anaplerotic substrate in all tissues. There is good reason to hypothesize that the diols, in particulr the L-diols, are a good source of calories for parenteral nutrition. The investigations for which support is sought will study the metabolism of the diols and of the corresponding hydroxyacids in perfused livers and hearts from rats. We shall also conduct intravenous infusion studies on live rats and dogs. We hope that these studies will generate the biochemical and physiological basis for clinical trials on human to be conducted in a subsequent project.

The common theme of the 3 sections of this proposal is the metabolism of ketone bodies. In the first section, we describe our ongoing research on the potential of R- and S-1, 3-butanediol as parenteral nutrients. We emphasize the interest in S-1, 3- butanediol as a nutrient in diabetes. The metabolism of the 2 enantiomers and of their corresponding 3-hydroxyacids will be studied in perfused organs and in vivo. In the second section, we build on our recent finding that acetone is converted to acetate in the liver. We propose investigations aimed at quantitating the metabolism of acetone via two- and three-carbon pathways. In the third section we propose to investigate the artifact of pseudo-ketogenesis in peripheral tissues. Pseudo-ketogenesis is a process which dilutes the specific activities of plasma acetoacetate and R-3-hydroxy-butyrate, by label exchange via the reversal of the reaction catalyzed by 3-oxoacid-CoA transferase. This artifact leads to overestimations of the rate of ketogenesis measured in vivo by isotope dilution techniques.

The long-term goal of the investigations for which a continuation of support is sought are: (i) To develop new nutrienti for parenteral and enteral nutrition, and for nutritional treatment of diabetes. (ii) to improve our understanding of alterations of ketone body metabolism occurring in diabetes. During the next five years, we wish to achieve four specific aims: 1. To study the metabolism of the R- and S- isomers of 1,3-butanediol and 1,3-pentanediol in perfused rat organs, in live rats, dogs and monkeys. 2. To validate the non-invasive chemical biopsy of human liver, under minimal or zero load of xenobiotic. This technique will then be used: 3. To quantitate pseudoketogenesis in extrahepatic tissues, and to develop non-invasive methods for assessing the net metabolic load of ketone bodies. 4. To assess (i) the distribution of acetone metabolism between two-carbon and three-carbon pathways, and (ii) the origin of plasma acetate.

DESCRIPTION: A number of pyruvate esters and thioesters have been synthesized and shown to be potentially useful in preventing and treating reperfusion injury and also for preventing hepatic ethanol toxicity when given in pharmacological doses. In this project, the investigators will test the hypothesis that dipruvyl-acetyl glycerol (DPAG), pyruvyl-acetyl- dihydroxyacetone (PADA), and pyruvate N-acetylcysteine ethyl ester (PNACE) can deliver therapeutic levels of pyruvate without causing lactic acidosis and sodium overload. Preclinical studies will be carried out: 1) to scale up the synthesis of these compounds and prepared new analogs of these compounds; 2) to characterize the metabolism and pharmacodynamics of these compounds in rats, dogs and swines; 3) to determine if the cardioprotective effect of pyruvate esters and thioesters are the result from I) anaplerosis if citric acid cycle intermediates, and ii) inhibition of fatty acid oxidation via increased malonyl-CoA; and 4) to characterize in rats and dogs given ethanol, the effect of pyruvate esters and thioesters on ethanol oxidation, perturbation of intermediary metabolism and free radical production.

The long term goal is to devise new strategies for the treatment of nutritional diseases such as diabetes and specific inborn errors of energy metabolism. The specific aims: 1) to characterize the pharmacokinetics, metabolism, and safety of 1,3-butanediol diacetoacetate (ketone esters) in humans and its usefulness in patients suffering from pathologies which could be alleviated by therapeutic ketosis. 2) to devise a non-invasive clinical test to assess the activity ratio of pyruvate carboxylase; pyruvate dehydrogenase in the liver of normal subjects and of children with inborn errors of pyruvate metabolism or diabetes.

ketone body; fatty acid metabolism; pharmacokinetics; diol; human therapy evaluation; steroid biosynthesis; pyruvate dehydrogenase; diet therapy; inborn metabolism disorder; insulin dependent diabetes mellitus; epilepsy; pyruvate carboxylase; noninvasive diagnosis; fatty acid biosynthesis; gluconeogenesis; parenteral feedings; oral administration; young adult human (21-34); dogs; human subject; adolescence (12-20); laboratory rat; Primates; child (0-11); nutrition related tag;

gluconeogenesis; biomedical resource; clinical research;

DESCRIPTION (provided by applicant): This application is for the acquisition of a continuous flow gas chromatograph combustion/ paralysis-isotope ratio mass spectrometer (GC-CF-IRMS) with modules for the on-line assay of low enrichments of compounds labeled with 13C, 15N, 18O, and 2H. The applicants are five established and two junior faculties of Case Western Reserve University and two colleagues from Youngstown State University (Ohio) and the University of Oklahoma, all with expertise in gas chromatography-mass spectrometry. The instrument will be used for basic science and clinical investigations on enzymatic reaction mechanisms, gluconeogenesis, lipogenesis, ketone body metabolism, protein metabolism, fatty acid oxidation, and inborn metabolic disorders. No such instrument is presently available at CWRU or in Greater Cleveland. The instrument will be set up in the Center for Metabolic Mass Spectrometry of the Department of Nutrition of CWRU. It will be made available to all scientists of the Cleveland area. The costs of operating and maintaining the instrument will be shared by the users. The availability of this instrument will considerably increase the scope of the basic science and clinical investigations conducted by the applicants.

DESCRIPTION (provided by applicant): The general aim of metabolomics is to identify, measure and interpret the complex time-related concentration, activity and flux of endogenous metabolites in cells, tissues, and other biosamples. We propose a three-pronged approach to metabolomics: (i) upgrading metabolite measurements by focusing on instrumentation to increase sensitivity, (ii) employing C-labeled substrates and 2H2O to generate data enabling estimates of fluxes in metabolic networks via mass isotopomer analysis, and (iii) utilizing state of the art models and multivariate statistics to test the hypothesis that specific tracers add information to metabolomics. The three aims of the study are: 1. To expand analytical procedures for the concentration and mass isotopomer distribution of metabolites (acyl-CoAs, acylcarnitines, aminoacids, carboxylic acids, as well as intermediates of glycolysis, the pentosephosphate pathway, and the citric acid cycle) extracted from perfused rat livers, as well as from the plasma and urine of control, and insulin resistant rats. A matrix isotopomer balance method will be used to fit isotopomer labeling patterns to fluxes. 2. To study the temporal patterns of concentration and mass isotopomer distribution of both known and unknown metabolites extracted from perfused liver, plasma, urine and organs of mice. Changes in the profiles following an intervention will identify discriminating intermediates and isotopomers, which may play a role in metabolic regulation. We will apply multivariate statistical methods to reduce the dimensions of the data set of unknown peaks, detecting those that discriminate between treatments. 3. To investigate the temporal pattern of concentration and mass isotopomer distribution of metabolites labeled from 2H-enriched water in perfused organs and in vivo. 2H2O, which is non-toxic at low enrichment, distributes evenly in all body compartments, and is an ideal as a metabolomics probe for human studies. Multivariate statistical tests will determine if 2H2O enhances concentration-based metabolomics. The significance of our study is two-fold. First, we provide a bridge in developing metabolomics, moving from a solid foundation in hypothesis-based research to a new data-driven "omics" approach. Second, we provide a rigorous test of the hypothesis that the mathematical analysis of mass isotopomer data enhances concentration-based metabolomics to provide new avenues for understanding metabolic diseases.

Seeinstructions): The goal of the Metabolism Training Program is to provide our graduate students and postdoctoral scholars with in-depth training in modern metabolic research. Our training environment integrates traditional areas of metabolism with the latest advances in molecular biology, cellular biology, genetics, proteomics, mass spectrometry, and nuclear magnetic resonance. Our training faculty, drawn from basic and clinical science departments of the School of Medicine, have active, grant-supported research programs and an outstanding record of training. Each trainee is admitted to the program uncommitted to an individual scientist, and selects an advisor from the training faculty after extensive interviews. The Steering Committee, chaired by the PI, manages all academic and research aspects of the Program. In particular, the Steering Committee closely monitors the matching of trainees with advisors, as well as the research and academic progress of the trainees. An extensive set of courses in metabolism has been established between the Departments of Nutrition, Biochemistry, Genetics, Molecular Biology, Pharmacology, Physiology and Biomedical Engineering. Trainees participate in a Journal Club and in monthly seminars and discussion groups in metabolic regulation. Research training ranges from the use of isotopic tracers to study whole body metabolism in humans to the construction of gene knock-outs to test the function of regulatory proteins on metabolic processes. Postdoctoral trainees include PhD and MD scholars with backgrounds in clinical medicine. Graduate students, drawn from a number of departments of the School of Medicine, are enrolled in the PhD or MD/PhD programs of CWRU. A high percentage of our trainees hold academic positions at US universities and medical schools. The Program is dedicated to increase the number of minority scientists. It has helped establish a Minority Scholars Program for college graduates interested in a career in the biomedical sciences. RELEVANCE (Seeinstructions): About one-half of the US population dies of metabolic and nutritional diseases. At present, few basic science and clinical investigators have expertise in modern techniques for investigating metabolism in health and disease. The goal of our Metabolism Training Program is to train young investigators to conduct research on metabolic diseases.

DESCRIPTION (provided by applicant): The general aim of metabolomics is to identify, measure and interpret the complex time-related concentration, activity and flux of endogenous metabolites in cells, tissues, and other biosamples. We propose a three-pronged approach to metabolomics: (i) upgrading metabolite measurements by focusing on instrumentation to increase sensitivity, (ii) employing C-labeled substrates and 2H2O to generate data enabling estimates of fluxes in metabolic networks via mass isotopomer analysis, and (iii) utilizing state of the art models and multivariate statistics to test the hypothesis that specific tracers add information to metabolomics. The three aims of the study are: 1. To expand analytical procedures for the concentration and mass isotopomer distribution of metabolites (acyl-CoAs, acylcarnitines, aminoacids, carboxylic acids, as well as intermediates of glycolysis, the pentosephosphate pathway, and the citric acid cycle) extracted from perfused rat livers, as well as from the plasma and urine of control, and insulin resistant rats. A matrix isotopomer balance method will be used to fit isotopomer labeling patterns to fluxes. 2. To study the temporal patterns of concentration and mass isotopomer distribution of both known and unknown metabolites extracted from perfused liver, plasma, urine and organs of mice. Changes in the profiles following an intervention will identify discriminating intermediates and isotopomers, which may play a role in metabolic regulation. We will apply multivariate statistical methods to reduce the dimensions of the data set of unknown peaks, detecting those that discriminate between treatments. 3. To investigate the temporal pattern of concentration and mass isotopomer distribution of metabolites labeled from 2H-enriched water in perfused organs and in vivo. 2H2O, which is non-toxic at low enrichment, distributes evenly in all body compartments, and is an ideal as a metabolomics probe for human studies. Multivariate statistical tests will determine if 2H2O enhances concentration-based metabolomics. The significance of our study is two-fold. First, we provide a bridge in developing metabolomics, moving from a solid foundation in hypothesis-based research to a new data-driven "omics" approach. Second, we provide a rigorous test of the hypothesis that the mathematical analysis of mass isotopomer data enhances concentration-based metabolomics to provide new avenues for understanding metabolic diseases.

DESCRIPTION (provided by applicant): The current treatment of patients with inherited fatty acid oxidation defects (FOD) involves providing most of the dietary fat as medium even-chain triglycerides (mostly trioctanoin) for long-chain disorders or simply dietary fat restriction for medium- and short chain disorders. This treatment does not prevent, in many cases, the progressive deterioration of cardiac, muscular, and/or retinal function. An initial clinical trial has shown that replacing trioctanoin in the diet by triheptanoin, a medium odd-chain triglyceride, leads to a rapid improvement of the patients'clinical condition and quality of life. We hypothesize that heptanoate, and the C5-ketone bodies derived from its initial hepatic metabolism, exert their beneficial effects by (i) providing propionyI-CoA, an anaplerotic substrate for the citric acid cycle (CAC), and (ii) compensating for partial CAC blockade when the flux through one or more CAC enzymes is restricted. We also hypothesize that odd-chain compounds such as tripentanoin or esters of C5-ketone bodies could be useful in the treatment of medium-chain FOD. Lastly, we hypothesize that a trimer of the C5-ketone body beta-hydroxypentanoate could be used as a slow enteral release form of beta-hydroxypentanoate. We propose a research program to be conducted in mice deficient in very long-chain acyI-CoA dehydrogenase (VLCAD, Aims 1 -4) and in mitochondrial trifunctional protein (MTP, Aim 5). Our specific aims are: To characterize the metabolism and metabolic effects of the odd-chain compounds in vivo and in perfused organs (heart, muscle and liver). This will be achieved using compounds labeled with 13C in their propionyl or acetyl moiety, to evaluate anaplerosis and oxidation, respectively, by mass isotopomer analysis. 2. To test whether the odd-chain compounds (i) improve the survival of knock-out mice stressed by fasting or/and cold exposure, and (ii) improve energy metabolism and mechanical performance of the heart and muscle under stress-induced conditions (high workload or adrenergic stimulation). 3. To test new avenues for the acute treatment of FOD decompensation, i.e., parenteral preparations of odd-chain anaplerotic substrates (triheptanoin, tripentanoin, glycerol beta-ketopentanoate). To test the practicality of the cyclical trimer of beta-hydroxypentanoate (triolide) as a slow enteric release form of the propionyl-CoA precursor, suitable for nocturnal coverage of patients. To improve the survival of newborn MTP -/- mice.

DESCRIPTION (provided by applicant) The goal is to use metabolomics coupled to mass isotopomer analysis to study the metabolic and oxidative stress exerted by two related compounds, i.e.,1,4butanediol (14BD) and gamma-butyrolactone. These industrial solvents are used extensively in the chemical industry in building materials and in consumer products. Lastly, they are precursors of the dangerous drug of abuse gamma-hydroxybutyrate (GHB) which is very popular among young people. The investigators have outlined a strategy to accelerate the disposal of gamma-hydroxybutyrate in the liver. The coupling of metabolomics to mass isotopomer analysis will provide new avenues for understanding xenobiotic stress. The aims are: 1. To determine if compounds identified by the metabolomics approach provide new insight into the metabolism of 14BD and GHB. The investigators will generate a database of metabolic information obtained by mass spectrometric analyses of plasma, urine and liver of control and 14BD- or GHB- exposed rats. Multivariate statistical methods will reduce the dimensions of the data set of unknown peaks, detecting those that discriminate between treatments. A number of techniques will be evaluated, including Principal Component Analysis, Fisher Discriminant Analysis and Partial Least Squares. 2. To study the temporal patterns of concentration and mass isotopomer distribution of metabolites extracted from (i) isolated rat livers perfused with unlabeled and uniformly 13C-labeled 14BD and GHB and (ii) the plasma, urine, liver and kidney of rats infused with these compounds. The rats will be normal or pre-treated with compounds that interfere with the metabolism of 14BD and/or GHB (ethanol, methylpyrazole). The patterns of change in the profiles following an intervention will be analyzed with a new software tool, Metran, which produces flux estimates with statistical confidence from a pathway model and isotopomer data. The data will also be analyzed by multivariate statistics described in aim 1 to identify discriminating isotopomers, which may provide clues to regulatory mechanisms. 3. To test the hypotheses that the metabolism of GHB in liver and kidney can be accelerated by glucuronolactone, precursors of alpha-ketoglutarate or by taurine. This will involve an investigation of the mechanisms of the enzymes that catalyze the conversion of GHB to succinic semialdehyde. 4. To characterize the response of the liver to oxidative stress by a noninvasive technique to measure the turnover of glutathione in liver. This will be achieved by administering low doses of 2H20 and acetaminophen, followed by measuring the 2H-labeling of urinary acetaminophen-glutathione adduct.

DESCRIPTION (provided by applicant): The general aim of metabolomics is to identify, measure and interpret the complex time-related concentration, activity and flux of endogenous metabolites in cells, tissues, and other biosamples. We propose a three-pronged approach to metabolomics: (i) upgrading metabolite measurements by focusing on instrumentation to increase sensitivity, (ii) employing C-labeled substrates and 2H2O to generate data enabling estimates of fluxes in metabolic networks via mass isotopomer analysis, and (iii) utilizing state of the art models and multivariate statistics to test the hypothesis that specific tracers add information to metabolomics. The three aims of the study are: 1. To expand analytical procedures for the concentration and mass isotopomer distribution of metabolites (acyl-CoAs, acylcarnitines, aminoacids, carboxylic acids, as well as intermediates of glycolysis, the pentosephosphate pathway, and the citric acid cycle) extracted from perfused rat livers, as well as from the plasma and urine of control, and insulin resistant rats. A matrix isotopomer balance method will be used to fit isotopomer labeling patterns to fluxes. 2. To study the temporal patterns of concentration and mass isotopomer distribution of both known and unknown metabolites extracted from perfused liver, plasma, urine and organs of mice. Changes in the profiles following an intervention will identify discriminating intermediates and isotopomers, which may play a role in metabolic regulation. We will apply multivariate statistical methods to reduce the dimensions of the data set of unknown peaks, detecting those that discriminate between treatments. 3. To investigate the temporal pattern of concentration and mass isotopomer distribution of metabolites labeled from 2H-enriched water in perfused organs and in vivo. 2H2O, which is non-toxic at low enrichment, distributes evenly in all body compartments, and is an ideal as a metabolomics probe for human studies. Multivariate statistical tests will determine if 2H2O enhances concentration-based metabolomics. The significance of our study is two-fold. First, we provide a bridge in developing metabolomics, moving from a solid foundation in hypothesis-based research to a new data-driven "omics" approach. Second, we provide a rigorous test of the hypothesis that the mathematical analysis of mass isotopomer data enhances concentration-based metabolomics to provide new avenues for understanding metabolic diseases.

DESCRIPTION (provided by applicant): This application is presented on behalf of the Mouse Metabolic and Phenotyping Center (MMPC) network () and its Advisory Committee. The MMPC network groups five Centers (located at Case Western Reserve University, University of Cincinnati, University of Texas SouthWestern, University of Washington, Vanderbilt University, Yale University) and a Coordinating and Bioinformatics Unit (located at the Medical College of Georgia). The goal of this five-year project is to expand the educational and training activities of the MMPC network by organizing a series of courses and hand-on workshops on the use of isotopic techniques for metabolic investigations. Although the MMPC network was created to provide phenotyping testing in mice, it is integrated in the wider research and training activities of NIDDK, NHLBI and the NIH. Therefore, the scope of the planned courses and workshops on isotope technology will not be limited to investigations of mouse metabolism. The faculty who will run the courses and workshops have wide expertise in the use of isotopes for metabolic studies in animals and humans. They are also involved in the development of new isotopic, metabolomic and proteomic techniques which will allow major expansion of metabolic investigations conducted from the mouse to the bedside. This project builds on the teaching and training expertise of Dr Robert R. Wolfe, a member of the MMPC External Advisory Committee. Dr Wolfe has created in 1992, and taught eight times with colleagues, a course on the use of isotopes for metabolic studies. This course drew trainees from all over the United States. In October 2007, Dr Wolfe and MMPC faculty joined forces to teach a new version of the isotope course. This new course, which was very well received by about 80 trainees, is the template for the courses to be taught under the present application. In addition, we plan to conduct each year one specialized hand-on workshop in the laboratory of a member of the course faculty.

"This award is funded under the American Recovery and Reinvestment Act of 2009 (Public Law 111-5)."

Case Western Reserve University has been awarded a grant to build computational tools to facilitate research in metabolic profiles. Metabolites are molecules that are intermediates and products of metabolism. Current blood-labs in most hospitals and health care centers in the country can only measure in a blood sample small numbers of (less than 15) metabolites. However, recent advances in high-energy physics technologies has now made it possible (for researchers for the time being) to measure much larger numbers of metabolites in a blood sample, up to about 300 of them. When these measurements differ from those in a healthy individual (i.e., the control subject), the basic question is: what do they mean?

Currently, the above question can only be answered manually by experts in metabolic biochemistry. This project will test the validity of computationally and algorithmically deduced first-cut interpretations of such large sets of metabolite measurements. These interpretations are made available through web-based, database-enabled, easy-to-use, online and reliably correct software systems that are
- Scalable to the complete metabolic network, not parts of it and
- Grounded by the well-known and accepted metabolic biochemistry principles,
instead of being done manually as there are not enough metabolic biochemistry experts around to manually interpret each such lab result.

This project develops a web-based resource and its tools to enable the research community to help answer the above-listed question. The tools of the resource include
- Observed Metabolite Analysis tool that eliminates those metabolic paths that cannot have caused the observed measurements (i.e., inactive metabolic paths in the metabolic network), and provides a small list of activated metabolic paths that may have caused the observed measurements. Note that no effort is made to return actual flux estimations in the network, simplifying the task at hand,

- Metabolomics-Oriented Querying tool, that allows researchers/users to further focus on activated metabolic paths within a subnetwork (by allowing them to formulate queries about activated paths, futile cycles, etc.), and provides users with explanations of its query answers,

- Metabolomics-Oriented Visualization tool, which provides visualizations of activated paths within the complete metabolic network or zoomed-in and focused parts of the network, allowing users to visually interpret and analyze the results.

The resulting online tools will be an excellent web-based educational tool for educating students in metabolic biochemistry, biology, bioinformatics, and metabolomics--to train them on the use of bioinformatics tools in terms of metabolic network models. Additional information about the project may be found at http://nashua.case.edu/.

Description (as provided by applicant): The Administrative Core is directed by the PI, Dr Henri Brunengraber, who will oversee the whole management of the Case MMPC with the help of faculty and administrative staff. The Administrative Core will meet twice monthly with the Committee of Core Directors, and with the external Steering Committee (which will come to Cleveland once a year). The Administrative Core will interact with the other MMPCs, the NIDDK and NIHLB Program Directors in charge of the MMPCs, and with users. The Administrative Core will coordinate the operations of the cores and will manage all financial aspects of the MMPC administration. As an extension of the Administrative Core, the Training and Education Core (Drs Brunengraber, Croniger, Puchowicz) mentors users in the use of isotopic techniques, protocol design, analytical procedures, trouble shooting, and data interpretation. Also, the PI co-directs a one-week annual MMPC course on the use of isotopes for metabolic research.

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).

Abstract of Core A: Administrative Core The Administrative Core is directed by the PI, Dr Henri Brunengraber, who will oversee the whole management ofthe Case MMPC with the help of faculty and administrative staff. The Administrative Core will meet twice monthly with the Committee of Core Directors, and with the external Steering Committee (which will come to Cleveland once a year). The Administrative Core will interact with the other MMPCs, the NIDDK and NIHLB Program Directors in charge ofthe MMPCs, and with users. The Administrative Core will coordinate the operations ofthe cores and will manage all financial aspects ofthe MMPC administration. As an extension of the Administrative Core, the Training and Education Core (Drs Brunengraber, Croniger, Puchowicz) mentors users in the use of isotopic techniques, protocol design, analytical procedures, trouble shooting, and data interpretation. Also, the PI co-directs a one-week annual MMPC course on the use of isotopes for metabolic research.

ABSTRACT: Core C: Analytical & Metabolomic The Case MMPC specializes in the use of stable isotopes, mass spectrometry and metabolomics to investigate the regulation of pathways of intermediary metabolism in vivo and ex vivo. Emphasis is on quantitative measurements of inter-organ and intra-organ carbon fluxes. Our strategy links clinical-type studies (limited to body fluids) and pure biochemical studies (such as the kinetics of purified enzymes). The association of metabolomics and mass isotopomer analysis provides a wealth of information on the regulation of known pathways. It also allows to identify new pathways. Metabolomics developed initially as a non-targeted strategy aimed at gathering sufficient data on a (patho)physiological process (biomarkers), so that one could formulate hypotheses to be tested. A variant of this strategy is to target metabolomic studies to classes of compounds, the concentrations and labeling patterns of which can provide much new information: citric acid cycle intermediates, (hydroxy)acids, acyl-CoA esters, acylcarnitine esters. The Case MMPC occupies a specialized niche in the MMPC consortium, complementing the expertise of the other centers, but with a healthy degree of overlap with the Vanderbiit, Yale, Dallas and Cincinnati centers. The specific aims of the Analytical & Metabolomic Core are: 1. To help users in the design, implementation, analytical procedures and interpretation of metabolic studies conducted either, (i) entirely in the user's lab, (ii) entirely in the Case MMPC, or (iii) partly in the user's lab (animal experiments) and partly in the MMPC (analyses of tissues and body fluids shipped from the user's lab). 2. To set up, at users' request, tests and analytical procedures not currently listed on the web page of the Case MMPC. 3. To develop analytical techniques and isotopic strategies in anticipation of users' needs. 4. To train US and foreign scientists in the proper use of isotopes for metabolic studies. This training takes to form of (i) mentoring individual users, and (ii) contribution to an annual MMPC course.

ABSTRACT : Core B: Animal The AAALAC-accredited Case Animal Research Center (ARC) provides outstanding and state of the art housing and care of animals (including fragile transgenic and knockout mice). To insure that the Animal Care Core is also managed under the general standards of the Case ARC, Dr John Durfee, Director of the ARC, is also the Director of the Animal Care Core. The mice investigated by the MMPC and housed within the ARC during quarantine and conditioning prior to testing. The MMPC Animal Care Core Director ensures that animals are cared for under the uniform standards of the MMPC network. This includes (i) the quarantine conditions upon arrival of the mice at the ARC, and re-quarantine of some mice after imaging or metabolic procedures outside of the main ARC, (ii) the testing for parasites and infectious agents (see below), and (iii) the feeding of mice the special Teklad diet or semi-synthetic diets. The management of multiple groups of mice, each under its own IACUC protocol, but all under the MMPC umbrella, requires special record keeping procedures to make sure that each user is correctly billed for, but only for, the housing and care of her/his mice.

? DESCRIPTION (provided by applicant): Metabolic reprogramming is one of the hallmarks of cancer. The Warburg effect and glutamine dependency are two well-known metabolic reprogramming events that occur in cancer cells. It has long been known that most cancer cells are dependent on glutamine to grow. However, the mechanisms by which cancer cells become dependent on glutamine are not well understood. PIK3CA, which encodes the p110? catalytic subunit of phosphatidylinositol 3-kinase, is frequently mutated in a variety of human cancers including 20 to 30% of colorectal cancers. Our preliminary studies demonstrate that colorectal cancer cells harboring oncogenic PIK3CA mutations are more dependent on glutamine, suggesting that mutant PIK3CA may be a driving force that reprograms glutamine metabolism in cancer cells. Moreover, our gene expression analyses show that expression levels of GPT2, an enzyme that catalyzes conversion of glutamate to ?-keto-glutarate, are up- regulated in colorectal cancer cells harboring PIK3CA mutations. Knockdown of GPT2 makes PIK3CA mutant cell growth less dependent on glutamine, whereas overexpression of GPT2 renders PIK3CA wild-type (WT) cell more sensitive to glutamine deprivation. Remarkably, we found that aminooxyacetate (AOA), a small compound which inhibits GPT2 enzymatic activity, suppresses xenograft tumor growth of colorectal cancers harboring oncogenic PIK3CA mutations, but not WT PIK3CA. These results lead us to hypothesize that the oncogenic PIK3CA/p110? mutant-GPT2 axis reprograms colorectal cancer metabolism and thus renders cancer cells dependent on glutamine. We propose that targeting glutamine metabolism will be an effective treatment for colorectal cancer patients harboring PIK3CA mutations. To test our hypotheses and to elucidate the molecular mechanisms by which mutant p110? reprograms cancer metabolism, we propose the following aims: (1) delineate the signaling pathway by which mutant p110? up-regulates GPT2 expression; (2) determine how the p110?-GPT2 axis reprograms glutamine metabolism in colorectal cancer; and (3) determine in preclinical models if targeting glutamine metabolism is an effective treatment for colorectal cancers harboring PIK3CA mutations. Our proposed studies investigate an innovative concept that oncogenic PIK3CA mutations reprogram colorectal cancer metabolism and render cancers dependent on glutamine. Moreover, our studies may provide a novel precision therapy that targets glutamine metabolism in colorectal cancer patients harboring PIK3CA mutations. Given that PIK3CA is frequently mutated in a variety of human cancers, we expect that our proposed studies will have broader conceptual and therapeutic impacts that extend beyond colorectal cancer.

ABSTRACT The Administrative Core of the Case-MMPC is headed by Dr. Henri Brunengraber, who is also responsible for the overall leadership and management of the Case-MMPC. In order to ensure success of the Case-MMPC, the Administrative Core will manage all service, development, training and educational activities of the Center. Specific administrative responsibilities include: optimization of fee-for-service and educational activities, uploading all data from the MMPC Cores to the data banks of the Coordinating Bioinformatics Unit, management of all financial aspects of the MMPC, integration of the service and educational activities of the cores, and maintenance of the Case-MMPC website. In support of the training activities of the MMPC the Administrative Core will promote the development of a new Biomarkers of Inflammation division within the Analytical & Metabolomic Core, provide mentorship to the user base including designing protocols, using isotopes for metabolic studies, analytical procedures, troubleshooting the data, interpreting the data, and planning follow-up studies, and organize and lead an annual national MMPC course on the use of isotopes for metabolic studies.

ABSTRACT The mission of the Case-MMPC is to facilitate phenotypic characterization of mice for investigators studying metabolic diseases, with an emphasis and prioritization on diabetes and obesity. We work within the MMPC Consortium to complement and partially overlap the activities of the other six Centers. Our niche is the measurement and interpretation of metabolic fluxes and metabolomic patterns with the help of stable isotope tracer techniques and mass spectrometry, with emphasis on the avoidance of artifacts that often plague such protocols. The combination of metabolomics and stable isotope technology allows the discovery of new pathways and new regulatory mechanisms. This approach was pioneered at the Case-MMPC and has since gained favor with investigators around the world. Our Center was also instrumental in the development of the NIDDK-sponsored annual course on ?Use of Isotopes for Metabolic Studies?, which will be taught for the 8th time in November 2015 by 11 faculty from MMPCs and US universities, and draws an international attendance. The Case-MMPC is currently in its 9th year of operation. This application requests support for increasing the capacity of the Center, expanding the repertoire of tracers provided and metabolites measured to include oxidative stress and inflammation-related pathways. Specific Aim 1 will provide core facilities for investigators to carry out metabolic flux experiments, utilizing our expertise in stable isotope tracers and mass spectrometry. Aim 2 will conduct analyses of murine tissues and body fluids provided by investigators carrying out metabolic studies. This includes investigators working at CWRU and in other institutions, domestic and globally. In Aim 3 we will develop additional innovative tracer and methodological approaches for assessing oxidative stress and inflammation. This involves a new ?sub-core? that will also provide custom synthesis of stable isotopic tracers and mass spectrometric analysis of lipid peroxidation products. In Aim 4 we will provide training and education to investigators and their staff for, (i) proficiency in the use of isotopes for metabolic studies, (ii) understanding the capabilities of the MMPC and designing appropriate experiments for their questions, (iii) carrying out procedures in their laboratory for preparing samples to be analyzed by the MMPC, and/or (iv) learning to carrying out the complete procedure in their own laboratories. The implementation of these aims is expected to significantly advance the field of metabolism and assist investigators in Cleveland, nationally and globally, to discover new pathways and mechanisms that cause diabetes and obesity- related chronic diseases.

? DESCRIPTION (provided by applicant): Diabetes is a public health problem, with an estimated prevalence of 29 M people in the U.S. Over 26 M people in the U.S. suffer from chronic kidney diseases (CKD) (3,4), and diabetic nephropathy (DN) is the leading cause of CKD, accounting for 45% of incident end stage renal disease (ESRD). Among subjects with diabetes and/or CKD, morbidity and mortality is significant, with the most common cause of death due to cardiovascular disease (CVD). The ability to predict which diabetic patients will develop DN or CVD is poor. Current biomarker standards for DN progression, serum creatinine and albuminuria, are suboptimal. Serum creatinine is insensitive; microalbuminuria frequently regresses spontaneously, and diabetics often develop renal dysfunction without albuminuria. In DN diseased glomeruli leak non-esterified fatty acids, which are then exposed to the proximal tubule luminal surface. Tubular atrophy, a strong predictor of DN progression, is linked to proximal tubule reabsorption of palmitate, which leads to lipotoxicity and apoptosis. Data from a longitudinal diabetic cohort revealed that Upalm:creatinine ratio better predicts eGFR decline compared to Ualb:creatinine. The causes and risk predictors for CVD in CKD differ from those in the non-CKD population. The serum concentrations of 5 solutes (trimethylamine oxide (TMAO), symmetric and asymmetric dimethylarginine (SDMA and ADMA), p-cresol sulfate and indoxyl sulfate) rise with CKD and have been proposed as CVD predictors in the general and CKD populations. We developed targeted LC/MS assays for all 5 solutes, and found 2-40 fold greater serum levels in ESRD subjects compared to normal controls. Hypotheses: (1) Urine palmitate is an accurate predictor of eGFR decline in DN, and is a superior biomarker compared to albuminuria. (2) A panel of uremic solutes (TMAO, SDMA, ADMA, p-cresol sulfate and indoxyl sulfate) will have an additive effect to traditional Framingham risk factors for predicting cardiovascular events in a diabetic population. The hypotheses will be tested with the following specific aims: Aim 1: Test urine palmitate:creatinine ratio as biomarker for DN progression. The correlation between baseline Upalm:creatinine vs. Ualb:creatinine and rate of eGFR decline, will be tested in a diabetic cohort with baseline eGFR >60 using a mixed effects model and AUC analysis of ROC curves. A secondary composite outcome (serum creatinine doubling, eGFR decrease by >50%, incident ESRD), will be analyzed using a multivariate Cox proportional hazards model. Analyses will be validated in matched ACCORD trial subjects. Aim 2: Test a panel of 5 retained solutes as a predictor of CVD in CKD. The discrimination ability of the 5 markers (in addition to the traditional Framingham factors) will be evaluated in an ACCORD cohort with stages 1-3 CKD by computing the increase in the estimated AUC from ROC curve analysis. Computation of the integrated discrimination index will be used to assess improvement due to inclusion of the 5 markers.