Date of Award

1995

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Veterinary Medical Sciences - Pathobiological Sciences

First Advisor

H. Douglas Braymer

Abstract

Adrenal glucocorticoids are essential for the development of all forms of experimental obesity. In the obese (fa/fa) Zucker rats, adrenalectomy prevents the further development of obesity, and glucocorticoid replacement restores the obese phenotype. We have hypothesized that this dependence on glucocorticoids is due to differences in transcriptional regulation of a group of genes that are normally regulated by glucocorticoids. We have chosen to study the DNA fragments from the upstream region of the malic enzyme (ME) gene, which is positively regulated by glucocorticoids. Hepatic nuclear extracts from lean and obese Zucker rats were used in band shift studies to look for differences in binding of nuclear proteins to the DNA. All of the upstream DNA fragments bind more obese nuclear protein than lean nuclear protein. Adrenalectomy decreases the amount of obese nuclear protein bound to the 222 bp fragment to the same level as the lean nuclear protein. Treatment of the nuclear proteins with alkaline phosphatase altered gel retardation patterns and suggests that phosphorylation state of the nuclear proteins is important for DNA binding. The hepatic nuclear extracts were also analyzed in 2-dimensional SDS PAGE. Two proteins were present in lean nuclear extract but absent from obese nuclear extract. The proteins were N-terminal blocked and could not be used for N-terminal sequencing. These data suggest that changes in nuclear proteins may be involved in the altered transcription of this glucocorticoid regulated gene. Our study suggests that differences between lean and obese Zucker rats in nuclear proteins binding to the upstream regions of ME gene may reflect changes in phosphorylation state and differential effects of glucocorticoids.

Pages

155

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