The major objective is to explore the roles of the SREBPs in the metabolic derangements that accompany insulin resistance, diabetes, lipodystrophy, and fatty liver. The central approach is to use the techniques of homologous recombination to generate new lines of knockout mice with blocks in the SREBP pathway. We will utilize the Cre-loxP system to obtain conditional tissue-specific gene deletions of SCAP, Site-1 protease, and Site-2 protease. Tissue-specific knockouts for SREBP-1c and SREBP-2 will also be generated to determine the tissue-specific requirements for these SREBP isoforms. Novel SREBP-regulated genes will be identified by microarray technology using RNA from livers and adipose tissue from our previously characterized SREBP-1a, -1c, and -2 transgenic mice. The results from these analyses will provide a comprehensive framework of gene clusters coordinately regulated by each SREBP isoform and will identify novel genes central to the development of fatty livers, lipodystrophy and the insulin-resistant phenotype. Livers in rodent models of Type-II diabetes (ob/ob mice and lipodystrophy mice) show a mixed pattern of insulin resistance (increased gluconeogenesis) and insulin sensitivity (increased lipogenesis). We have recently proposed that the insulin resistance is mediated by down-regulation of IRS-2, a key protein in the insulin signaling cascade. The amount of IRS-2 mRNA and protein is reduced in the livers of ob/ob and lipodystrophic mice, and we hypothesize that this leads to increased gluconeogenesis. We have also proposed that the insulin sensitivity as manifested by increased lipogenesis is mediated by SREBP-1c knockout mice and transgenic IRS-2 mice to each of the two mouse models with insulin-resistant diabetes, creating four different double mutant lines. Disruption of the SREBP-1c gene in the diabetic overexpression of the IRS-2 transgene in livers of the diabetic mice should restore the elevated gluconeogenesis to normal, but it may or may not affect the elevated lipogenesis. To understand the mechanism for insulin regulation, we will characterize the promoter/enhancer regions of the SREBP-1c and IRS-2 genes to identify the DNA elements that transduce the insulin signal for activation (SREBP-1c) and repression (IRS-2). Fatty livers can also result from ethanol ingestion. To determine wheter SREBPs play a role, we will investigate SREBP regulation in ethanol-fed rodents. We have recently cloned acetyl CoA synthetase (ACS), the enzyme that activates acetate derived from ethanol. ACS was cloned based on its marked regulation by SREBPs.
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