The objective of this proposal is to identify the molecular basis for the glucocorticoid dependence of the genetically inherited obesity of the Zucker fa/fa rat and to explore its relationship to the fa gene which causes the obesity. The hypothesis to be investigated is that certain genes that are normally either positively or negatively regulated by glucocorticoids are overexpressed in obese fa/fa rats because of the absence of, or abnormality in, an inhibitory transcription factor or other protein modulator of transcription. We shall investigate this hypothesis by comparing glucocorticoid control of a positively regulated gene (hepatic tyrosine aminotransferase [TAT]) and a negatively regulated gene (the glucocorticoid type II receptor) in lean and obese fa/fa rats. Western blots will be used to examine the relationship of receptor protein levels to corticosterone binding. Northern blots and Nuclear run on assays will be used to investigate the regulation of transcription of the receptor type II gene and the tyrosine aminotransferase gene in response to adrenalectomy and glucocorticoid replacement in lean and obese rats. The possible roles of enhanced receptor occupancy and increased nuclear localization of receptors to increased TAT gene transcription will be investigated. Three strategies will be used to identify differences in transcription factors or other nuclear proteins in lean and obese rats: 1. Subtractive hybridization of hepatic c-DNA from lean rats with hepatic mRNA from obese rats and use of the subtracted probe to identify the differentially expressed genes in a cDNA library of lean rats. 2. Two dimensional gel electrophoresis of nuclear proteins followed by microsequencing of any proteins that are absent or abnormal in fa/fa rats; 3. DNAase I footprinting and gel retardation analysis of the 5' upstream regulatory component of the tyrosine aminotransferase gene incubated in the presence and absence of nuclear fractions from lean and obese rats. The effectiveness of any nuclear factors identified to alter transcription of a construct of the 5' upstream elements of the TAT gene linked to the chloramphenicol acetyltransferase reporter gene will be investigated. cDNA probes or probes synthesized on the basis of protein sequence information will be used to isolate genes from genomic libraries and to study the regulation of these genes in lean and obese rats in response to diet and adrenal steroids. The relationship of any message difference identified to the fatty gene will be investigated.
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