This is a competing renewal application that will attempt to identify differentially expressed genes in fat and muscle that are sensitive to regulation by thiazolidenedione (TZD) and in diabetes. The PI will employ cDNA gene array technology with chips purchased from Affymetrix, and will incorporate both mice and humans in the experimental approach.
In specific aim 1, the PI will prepare transgene mice with targeted hyperexpression in fat (aP2 promoter) or muscle (MCK promoter) using constructs containing: i) wild type PPARgamma; ii) pro115gln mutant PPARgamma. This native mutation in a serine phosphorylation site was detected in 4 obese heterozygotes. The mutation impairs serine phosphorylation leading to enhanced expressed and an increase in triglyceride accumulation in 3T3 fibroblasts. iii) a synthetic ser114ala mutation. These mice will be fully phenotyped. Furthermore, in specific aim 2, the mice will be treated with and without troglitazone for 4 weeks, and RNA extracted from muscle, liver, and fat to assess differential gene expression. The Affymetrix M19k chip will be utilized which includes 19,000 mouse genes and ESTs. Specific gene alterations identified by cDNA chip approach will be confirmed using quantitative real-time PCR detection.
In specific aim 3, PPARgamma knockout mice will be generated using the Cre lox-P approach to selectively reduce PPARgamma in fat and muscle. The experiments will involve PPARgamma floxed/floxed, floxed/-, and wild type mice bred with other mice containing Cre transgenes. The knockout mice will be treated with and without TZD, and differential expression assessed by chip technology. These mice will address whether TZD enhanced insulin sensitivity involves primary PPARgamma effects in fat versus muscle.
In specific aim 4, 6 type 2 DM, 6 type 1 DM, and 6 controls will be metabolically characterized and undergo fat and muscle biopsies. Differential expression in these tissues will be assessed by cDNA gene array technology using the Affymetrix 6800 expression chip containing 6,800 genes and the 35,000 chip containing 35,000 ESTs. For genes altered 2-fold compared with controls in at least 3 of the 6 subject pairs, differential expression will be confirmed by real-time quantitative PCR. In addition, 6 type 2 DM having low S1 and SG on fs-IVGTT will be compared with 6 offspring with high S1 and high SG, and 6 normoglycemic obese will be compared with 6 lean subjects, for differential expression in muscle and fat.
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