The focus of this grant is to identify the molecular basis for the glucocorticoid dependence of the genetically inherited obesity of the Zucker fatty (fafa) rat. In the current grant period, we have shown that mRNA levels of several genes are induced excessively by glucocorticoids in liver of obese rats in comparison to learn littermates. Our data suggests that this hyperresponsiveness to glucocorticoids may result from overexpression of the glucocorticoid receptor gene and increased localization of GR protein in the nucleus. From gel retardation studies with upstream DNA from tyrosine aminotransferase (TAT) and Malic enzyme (ME) we have obtained evidence that three proteins bind in higher quantities in the nuclei of obese rats and that this binding change may be related to excess phosphorylation. The hypothesis we shall investigate is twofold, firstly, that athe increase in GR nuclear localization causes the abnormal transcriptional responsiveness to glucocorticoids and secondly, that alterations in protein phosphorylation may underlie the change in GR nuclear localization and protein binding to DNA. We shall use primary cultures of hepatocytes from lean and obese rats to investigate GR activation and cycling from cytosolic to nuclear compartments in response to dexamethasone stimulation or removal. Northern blots and nuclear run-on assays will be used to investigate if increased rates of transcription underlie the elevated levels of TAT, ME and GR mRNAs that are present in liver cells of obese rats after treatment with glucocorticoids. We shall determine if altered transcriptional activity requires or is independent of the increased localization of GRs in hepatocytes of obese rats. Oligonucleotide probes based upon N-terminal protein sequence information will be used to identify and sequence the genes for the three DNA binding proteins from hepatic cDNA libraries. The glucocorticoid responsiveness of these genes will be studied. The possibility that abnormal protein phosphorylation causes the changes in GR translocation, protein binding to DNA and transcriptional responses will be studied with the use of protein kinase and phosphatase activators and inhibitors. The role of glucocorticoids in modulating these changes in phosphorylation will be studied. The relationship of the observed changes in these response systems to the defective fa gene and the early development of obesity will be studied in Zucker X Brown Norway rat crosses that can be genotyped for the fa gene. We shall also use confocal scanning laser (CSL) immunochemistry and CSL fluorescence in situ hybridization to show that these changes in GR transcription and nuclear localization are present in brain tissue as well as in hepatocytes. Obesity, the major nutritional disorder in the Western World, is a major risk factor for diabetes, cardiovascular disease, hypertension and several other clinical conditions. A fuller understanding of the factors which cause and maintain obesity will promote new approaches to the treatment and prevention of the obese state and contribute to an improvement in the health of Western populations.
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