The applicant has immediate and long-term goals of maintaining a vigorous molecular biology and biochemistry laboratory with focus on genes whose enzyme and protein products involve intermediary metabolism. Attention will be directed to factors that relate to human disease, specifically including diabetes mellitus, obesity, and genetically determined defects in fatty acid metabolism. Start-up funds and a research grant from the American Diabetes Association have been used to initiate studies that are described in the Preliminary Data section of the present proposal. The NIDDK 15A is being sought in order to provide a sustained period of salary support during a formative period. A well-equipped laboratory has been established within the MGH East Diabetes Unit. Opportunities for interaction with former colleagues in the Molecular Biology and Genetics Departments, other MGH East departments, and new colleagues in the Endocrinology units are abundant. The applicant will devote >95% effort to research during the period of the 15A. The objective of studies described in this proposal is the characterization of determinants and regulators of human hepatic carnitine palmitoyltransferase I (hCPT-I) gene expression. It is hypothesized that genes encoding elements of the mitochondrial fatty acid Beta-oxidation and ketogenic pathways are coordinately regulated; that this regulation is conferred in part by the peroxisome proliferator activated receptor alpha (PPARalpha), a nuclear transcription factor whose activity is regulated by extramitochondrial fatty acyl intermediates; and that this activity provides a mechanism by which insulin and counterregulatory hormones can influence the expression of metabolic enzyme genes through regulation of fatty acid flux to liver from the periphery. RNA will be harvested from primary hepatocytes and rat tissues for blotting studies. Cells will be cultured in medium supplemented with PPARalpha activators including medium- or long-chain fatty acids, pharmacological manipulators of mitochondrial Beta-oxidation, thiazolidinediones, fibrates; or with insulin, glucagon, glucocorticoids, thyroid hormone, or retinoic acid in order to assess regulation of hCPT-I expression by stimuli known to influence expression of other metabolic enzyme genes, or to influence fatty acid metabolism in vivo. Complementary studies will involve characterization of the hCPT-I 5, regulatory region(s) using HepG2 cotransfections with chimeric CPT-I promoter/reporter plasmid constructs, devoting particular attention to the PPARalpha: and to addressing mechanisms for phenomena observed in the RNA blotting studies.
