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

Glucocorticoids are required for the development of all forms of experimental obesity. Studies on Zucker fa/fa rats have revealed that the obese Zucker rat is hyper-responsive to the effects of glucocorticoids. This excessive response may result from an alteration in level or activity of a transcription factor in nuclei of obese fa/fa rat. Evidence from gel retardation assays using DNA fragments from the upstream region of glucocorticoid regulated tyrosine aminotransferase (TAT) gene and the nuclear extracts from obese and lean rats showed in this study that an increased amount of DNA fragment was shifted by nuclear extracts from obese rats in comparison to that from lean rats, suggesting that an alteration in levels or activity of a transcription factor or factors was present in the nuclei of obese rats. Three proteins which have approximate molecular weight of 55, 59 and 62 KDa were purified from the specific DNA-protein complex which showed a difference between lean and obese rats. These three proteins were able to reconstitute the specific DNA-protein complexes in gel retardation assays. Antibody against glucocorticoid receptor did not alter the gel retardation pattern, while anti-HNF3$\alpha$ (hepatic nuclear factor 3$\alpha)$ antibody caused a super-shift of DNA-protein complex, suggesting that HNF3$\alpha$ was one of the three proteins which formed the DNA-protein complexes. Northern blot analysis showed no significant difference in mRNA levels of HNF3$\alpha$ gene between lean and obese rats, suggesting that the increased formation of DNA-protein complex in obese rat may not reflect an increased amount of protein but may result from an increased affinity of HNF3$\alpha$ or an increased level or affinity of the other two nuclear proteins in the nuclei of obese rats. Dephosphorylation of the nuclear proteins altered the gel retardation pattern, suggesting that phosphorylation regulated the specific DNA-protein complex formation. Although the glucocorticoid receptor was not a part of the DNA-protein complex, the formation of the specific DNA-protein complexes changes in response to the change of glucocorticoid status, suggesting that the transcription factors that are responsible for the specific DNA-protein complex formation may be modulated directly or indirectly by glucocorticoids.

Pages

132

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