James E. Cox, Ph.D. - US grants

The syndrome of obesity accompanied by dramatically elevated food intake in experimental animals after lesions of the ventromedial hypothalamus (VMH) has occupied a central place in the physiological analysis of feeding and energy balance for several decades. The classic view that the obesity stems from a deficit in the central registration satiety has been largely discredited. Recent years have seen growing interest in peripheral changes, particularly in function of the autonomic nervous system, as a potential basis for the syndrome. Specifically, severing the vagus nerves in the abdomen reverses the hyperphagia and obesity, suggesting a critical role for vagal hyperactivity. Experiments proposed will investigate the basis of this effect of vagotomy. Experiments will examine the role of changes in the vagal-insulin system in VMH obesity in rats. Plasma insulin changes will be determined in response to sham feeding in VMH rats with transections of various branches of the abdominal vagus and related to observed effects of the latter surgery on food intake. One experiment will examine the effectiveness of insulin replacement for restoring hypothalamic hyperphagia. Similar experiments will investigate the role of vagally mediated hyperinsulinemia in the adiposity that develops in VMH rats when food intake is restricted to control amounts. One characteristic of the VMH lesion/vagotomy syndrome is the continued overconsumption of certain diets, such as high fat mash. It is often assumed that this effect reflects continued VMH finickiness after vagotomy. Alternatively, however, it might reflect postingestive actions of the diets. Experiments employing sham feeding and intrasgastric infusion of diets will seek to determine whether the overconsumption results from preingestive or postingestive effects. In light of current interest in metabolic factors in human obesity, study of the VMH syndrome promises to provide clinical insights as to etiology and treatment. At the same time the major changes to insulin release suggest relevance to diabetes.

ABSTRACT NOT PROVIDED

Metabolomics is the quantitative measurement of all metabolites over time. The objective of this Core will be to quantify metabolites in tissues, biological fluids, cells or organelles that are affected by changes in either heme synthesis or metal concentrations. Members of the Utah CEMH have special expertise in the analysis of heme and porphyrin metabolites and of metals (not exactly a metabolite). We have performed determinations of these metabolites and elements for investigators outside of Utah. We propose to extend our analysis to a wide variety of other metabolites, including amino acids, organic acids, monosaccharides, disaccharides, alcohols, and aromatic amines and lipids. Specific attention will be given to mitochondrial metabolites for two reasons. First, malregulation of heme synthesis and many iron disorders affect mitochondrial metabolism. Second, analyzing the relatively restricted number of mitochondrial metabolites will provide proof of principle for metabolomic analysis. Careful analyses of all reactions known to occur in mitochondria have led to the predictions of 164 and 230 metabolites in yeast and human mitochondria, respectively. Separation, identification and quantification of approximately 200 metabolites is realistically achievable using commercially available instruments.

[unreadable] DESCRIPTION (provided by applicant): It has been proposed that food cues, especially from foods high in fat and calories, may trigger overeating in obese individuals. The neural basis of this effect is thought to be exaggerated activity within a distributed circuit referred to as the reward system, which includes such brain regions as the amygdala and orbitofrontal cortex. The proposed studies will use functional magnetic resonance imaging (fMRI) to compare reward-system activation elicited by pictures of high and low calorie foods in obese participants in a 12-week weight loss program vs. normal-weight controls. In the first study, fMRI scans will be performed prior to the program to test the predictions (1) that food images, especially of the high calorie foods, elicit greater reward-system activation in the obese men and women than in controls and (2) that degree of activation will predict subsequent weight loss, such that obese individuals with the greatest activation will lose the least weight. For the second study, scans will be performed after completion of the program. This study will test the prediction that those obese individuals whose elicited activity has been most normalized over the course of the treatment, will be those who show the greatest weight loss, both by the end of the program and at one-year follow-up. Both studies will also investigate the relationship of psychological variables -- appetite stimulated by food images, ratings of the emotional valence of the food images, reward sensitivity, and dietary restraint -- to brain activation patterns and to weight loss. Because obesity is one of the most serious health problems in the United States, there is a pressing need to understand underlying factors and to develop effective long-term treatments. It is hoped that this project will contribute to the development of weight-management techniques which are effective by reducing reactivity to food cues in obese individuals. An important factor underlying obesity may be hypersensitivity to food cues, such as the sight of appetizing foods. This study will use functional brain imaging to investigate brain activation patterns by obese participants in a weight loss program in response to viewing pictures of appetizing foods. We wish to determine whether long-term weight-loss is related to the amount of activation elicited by these images prior to the program and to the degree to which activation is normalized in a scan after completion of the program. [unreadable] [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): The Metabolomics Core at the University of Utah was initiated in 2006 with funding from NIH/NIDDK in the form of a Centers of Excellence in Molecular Hematology. From its inception the Core has performed non-targeted analysis of the metabolome using gas-chromatography based mass spectrometry (GC-MS). The Core initially focused on the relationship of metals in metabolism, searching for the underlying causes of disease states when key metal metabolites were either in excess or limiting. It has broadened its focus to other disease, especially those caused by mutations in mitochondrial proteins. The Core has been highly successful in this endeavor with resulting data has been included in a number of publications. Not all metabolites are detected using this GC-MS based approach. The Core over the past year has used an older PE SCIEX API 365 triple quadrupole LC-MS/MS to perform targeted metabolomics with success. Unfortunately this instrument suffers from major limitations; it is older, unreliable and does not perform non-targeted analysis. This instrument needs to be replaced with a modern, high-end instrument which has increased sensitivity, greater dynamic range and importantly improved reliability. We propose the purchase of an Agilent 6550 Quadrupole-Time-Of-Flight (QTOF) mass spectrometer with an Agilent 1290 Infinity ultrapressure liquid chromatography system. This system will complement our current Waters GCT-Premier GC-MS and will be used immediately at full capacity. We are pursuing a number of medically related metabolomics projects currently, from basic research in understanding the causes of disease to actual patient studies.

? DESCRIPTION (provided by applicant): This proposal is a request for funding for a Q-ToF Maxis HD mass spectrometer (Bruker Daltonics) for the University of Utah Mass Spectrometry and Proteomics Core facility. The instrument will serve two primary purposes: 1) replacement of two old, low-performance ESI/MS, and non-functional mass spectrometers with a high-performance instrument that provides advanced research capabilities, and 2) increase sample analysis efficiency and capacity for the Mass Spectrometry and Proteomics Core for approximately 80 to 90 investigators at the University of Utah, annually. Ten major users from the University of Utah, with 19 active NIH-funded grants, have been identified with current and future research projects, which ensures immediate and long-term continued use of the Maxis HD instrument. There are about 70 to 75 additional minor users, all from the University of Utah. The group of major users will utilize the high mass accuracy capabilities for MS and MS/MS data of the Maxis HD. The research projects of the major users vary widely, but applications include: Proteomics (protein ID and protein PTM discovery), protein sequence confirmations and identification of proteolytic cut sites from intact protein molecules, structural characterizaton of modified peptides (natural and synthetic), structural characterization of drugs and other synthetic small molecules, RNA analysis and other nucleic acid investigations, and synthetic peptide/drug conjugate structure work. The Q-ToF Maxis HD is a high-performance mass spectrometer that provides high sensitivity, high mass accuracy, high resolving power, and versatility for both MS and MS/MS applications, but at the same time, it's relatively easy to operate and is a dependable, workhorse mass spectrometer that is well-suited for the environment of a mass spectrometry core facility. The Q-ToF Maxis HD instrument will be housed in the MS and Proteomics Core facility, a central mass spectrometry shared resource. The MS Core is administered by the Institution, within a well-structured Core Research Facilities framework, including Oversight and Advisory committees. There is very strong Institutional financial support to ensure long-term utilization of the instrument, including service agreements and full-time Ph.D. mass spectrometrists to operate the instrument. The long-term impact to the University of Utah and regional institutions will be addition of new mass spectrometry resources for the University scientific and medical research community. This state-of-the-art mass spectrometry will stimulate new research projects and innovations for many years. The analytical power of the Maxis HD instrument will play a major role in accelerating research in many areas, particularly, proteomics, natural-product research, and many drug-delivery systems under investigation at the University.

PROJECT SUMMARY   The Metabolomics Core will provide analytic measurements of biologic building blocks, amino acids, nucleotides, lipids and carbohydrates to investigators of the Center for Iron and Heme Disorders (CIHD). Metabolomics is the unbiased survey of metabolites found within a tissue, biological fluid, organism, culture or other biological source. Currently metabolomics is a comparative science; typically an analysis is performed to identify the differences found between biological samples that have been subjected to a treatment or condition. This can be a genetic mutation, drug treatment or environmental constraint. No one method is fully capable of completely profiling the metabolome of a cell or tissue. To maximize the number of metabolites observed, the Core is equipped with three chemical analysis platforms, Gas Chromatography-Mass Spectrometry, Liquid Chromatography-Mass Spectrometry, and Nuclear Magnetic Resonance spectroscopy. The research base of the CIHD focuses on questions that can easily be addressed with these services. The staff of the proposed Metabolomics Core will assist investigators in training sessions for sample preparation, equipment use and analysis. Program staff will also assist users in preparation of materials for publications and presentations within the CIHD and externally to national and international meetings.

? DESCRIPTION (provided by applicant): This proposal is a request for funding for an Agilent 7200 GC/Q-TOF mass spectrometer for the University of Utah Metabolomics Core Facility. The instrument will serve two primary purposes: 1) replace an aging, lower performance and unreliable GC-MS with a high performance instrument that provides advanced research capabilities, and 2) increase sample analysis efficiency and capacity for the Metabolomics Core for the approximately 40 different users of GC-MS, annually. Based upon recent use of the GCT Premier 10 major users from the University of Utah, with 11 active NIH- funded grants, have been identified with current and future research projects, which ensure immediate and long-term continued use of the Agilent 7200. All of these users have a long term history with the Metabolomics Core, leveraging the Core's GC-MS capabilities to further their NIH funded research. The current GC-MS instrumentation has contributed to over 32 publications, many in high impact journals including Science, PNAS, Cell Metabolism, and Nature Cell Biology. The Agilent 7200 GC/Q-TOF is a high-performance mass spectrometer that provides the high sensitivity, high isotope accuracy, wide dynamic range, and the versatility needed for metabolomics and fluxomics analysis. In addition, this instrument is easy to operate and will require minimal training. The Metabolomics Core currently owns and operates four other Agilent instruments, the operating software as well as the data analysis software are the same across three out of four of these instruments. The 7200 GC/Q-TOF will be housed in the Metabolomics Core which is centrally located to the major users. The Metabolomics Core is administered by the Institution, within a well-structured Core Research Facilities framework, including Oversight and Advisory committees. There is very strong Institutional financial support to ensure long-term utilization of the instrument, including service agreements and full-time Ph.D. mass spectrometrists to operate the instrument. The long-term impact to the University of Utah and the regional institutions will be a state of the art work horse mass spectrometer to further the goals of the NIH funded researchers. The Core has a long history of maximizing the current GC-MS instrumentation to aid in important discoveries in the field of metabolism, cancer research, and the microbiome.

PROJECT SUMMARY/ABSTRACT ? Metabolomics Core The goals of the Metabolomics Core (MC) remain to advance the understanding of metabolism and metabolic flux in relation to iron, heme, hematopoeisis and red cell biology through the use of cutting edge technology, newly developed methodologies and Bioinformatic analyses. The need for the MC continues to expand as the applicability of this technology to model systems is further realized. Our MC core, which has been in existence for over 15 years, has rapidly evolved to meet user demand including the development of extensive education (workshops), which are made available to all users through the CIHD Enrichment activities, high throughput sample processing, improve flux analysis and utilization of bioinformatic tools to process data and interpret results. We have increased personnel focused on metabolomics, lipidomics and bioinformatics as well as adding cutting edge instrumentation (e.g. high resolution orbitrap instrument) to satisfy the demand for high end isotope tracer and flux analysis. The Core will continue to refine its methodologies, standardize reporting using developed custom data analysis reporting software and provide educational opportunities for successful investigator-initiated research and advancement.

Project Summary: The proposed University of Utah Cooperative Centers of Excellence in Hematology (CCEH) brings together 38 investigators whose research activities are focused on various aspects of iron and heme metabolism and non- malignant hematology. Iron plays an essential role in many biological processes including heme synthesis, oxygen transport, cellular respiration and DNA synthesis. Malregulation of iron homeostasis, either from deficiency or excess, results in disease. Heme is a key component of hemoglobin and other hemoproteins, but heme also plays a regulatory role in a number of metabolic pathways. Disorders of heme biosynthesis are responsible for an important group of human diseases, namely the porphyrias. The Utah Center for Iron and Heme Disorders (CIHD) will support the activities of a Research Base of 38 investigators whose research projects focus on the roles of iron, porphyrins and heme in eukaryotic metabolism. The activities of center members encompass both basic and clinical studies designed to identify disease mechanisms. To accomplish our goals, we propose an Administrative Core and four Biomedical Research Cores. The majority of the cores are already present and have been adjusted to meet the needs of the CIHD. These include: an Iron and Heme Core, which can assay and quantitate metals, porphyrins, heme biosynthetic enzymes and iron-binding and other proteins; a Metabolomics Core, which provides metabolomic phenotyping and molecular identification; a Mutation Generation and Detection Core, which provides cutting edge genome editing through CRISPR and TALEN reagents; and a new core in this renewal, the Protein-Metabolite Interactomics Core that provides proteomics services but adds the unique technology of identification of metabolites that interact with a protein and may post-translationally modify the protein in novel ways. The services provided by these cores will enable individual investigators to: 1) identify the role of genes in hematopoiesis or iron overload; 2) determine the effects of gene modification or mutations on metabolism in cultured cells or biological fluids; and 3) identify at the biochemical level the effect of mutations or conditions that affect iron and heme homeostasis on all levels. The Administrative Core will provide budgetary and scientific guidance to CIHD activities. Core recharge fees will be used to enhance and expand core operations. An Enrichment Program is designed for trainees and young investigators in the fields of nonmalignant hematology and for senior investigators who wish to enter this field. The Research Base is drawn from both the University of Utah and other institutions, with half of the members belonging to institutions outside of Utah. We have increased the Research base from 22 to 38 with the addition of ~4 investigators each year. The goals of the CIHD are to be a national resource for studies involving iron and heme and to inspire the next generation of investigators focused on iron, heme and nonmalignant hematology.