This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Coexistence of hyperglycemia and hyperinsulinemia indicates profound disruption of the glucose homeostasis functions of liver and skeletal muscle. This Project (Project 4 of a Program Project headed by Dr. Newgard) is motivated by the desire to understand the interaction of branched chain amino acids (BCAAs) with intermediary metabolism in both organs in human subjects. As outlined in the Overview section and Project 1 of this application, BCAAs may compete with other macro-nutrients for oxidation in skeletal muscle, and anaplerotic fluxes in both muscle and liver may be abnormal. In the presence of a high fat diet, anticipated secondary effects include accumulation of acylcarnitines and intra-cellular lipids in muscle, both markers of skeletal insulin resistance. Of course, specific amino acids also have complex effects on insulin secretion, carnitine metabolism and other aspects of biochemistry, and at this point it is not clear if abnormal BCAA metabolism significantly impacts mitochondrial function in either organ. Our colleagues in Projects 1, 2 and 3 of the Program Project will test these ideas in isolated cells and in animal models, but there will still be a need to measure fluxes in relevant pathways and measure specific metabolites in both the liver and skeletal muscle in relevant human subjects. In spite of the potential power of 13C tracer methods for studying skeletal muscle metabolism, this technology is little used, outside of a handful of labs, for one simple reason: suitable 13C enriched substrates that compete effectively for oxidation in the citric acid cycle have not been identified. A focus is to develop better methods to deliver high 13C enrichment of citric acid cycle intermediates in skeletal muscle and liver simultaneously. Although the immediate target is to assess biochemistry in the setting of elevated plasma BCAAs, these new methods will be widely applicable to studies of human metabolism and are in keeping with the historical theme of this program project of linking technology development with hypothesis-driven diabetes investigation.
The specific aims of the project are as follows: 1) To develop a protocol that labels citric acid cycle intermediates with 13C to a high level (>20%) in both skeletal muscle and liver in conscious rats. In separate experiments, 13C-enriched acetate, ? hydroxybutyrate plus propionate, octanoate, and lactate or pyruvate will be infused intravenously, and 13C enrichment in skeletal muscle glutamate, hepatic glutamate and blood glucose will be determined at steady-state. The combination of tracer(s) that yield the best combination of high 13C enrichment in skeletal muscle and plasma glucose will be selected for further studies in humans. 2) To translate the 13C infusion protocol developed in animals to healthy human subjects and refine 31P magnetization transfer methods. The infusion conditions necessary to achieve steady-state isotope labeling in skeletal muscle will be determined by serial 1H decoupled 13C NMR spectra at 7T. The 13C labeling pattern in plasma glucose will be monitored to assure steady-state in glucose isotopomers and to determine whether 13C enriched glucose, generated by the liver, contributes significantly to plasma glucose. In this aim, we will also compare two 31P NMR methods, inversion transfer and saturation transfer, for measurement of ATP synthesis rates in resting skeletal muscle. 3) To test the primary hypothesis that overweight subjects with elevated BCAAs have altered mitochondrial function in skeletal muscle plus altered liver gluconeogenesis. Selection of patients will be achieved by analysis of BCAA and related metabolites by Core B of this program. Specifically, we anticipate that skeletal muscle mitochondrial function (ATP turnover by 31P NMR) may be normal in these individuals but that intermediary skeletal muscle metabolism in these individuals will be altered as reflected by excess IMCL, excess acylcarnitines and an increase in anaplerosis. We will also test whether the network of glucose production pathways ?glycogenolysis, gluconeogenesis from glycerol and gluconeogenesis from the citric acid cycle ?in liver is abnormal among these subjects. In summary, our goal is to integrate new high field NMR technologies with those already largely in hand for understanding mitochondrial function in liver and skeletal muscle of humans, thereby serving as a project that translates the biological / mechanistic findings of Projects 1, 2, and 3 to human studies. A high priority will be placed on developing a protocol that will be well-accepted by patients.
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