Semester

Spring

Date of Graduation

1999

Document Type

Dissertation

Degree Type

PhD

College

School of Medicine

Department

Biochemistry

Committee Chair

Lisa M. Salati.

Abstract

The goal of this research is to understand the molecular mechanism(s) by which polyunsaturated fatty acids inhibit gene expression of the lipogenic enzymes. The model enzyme which we are using to study this phenomenon is liver glucose-6-phosphate dehydrogenase (G6PD). In order to determine if regulation of G6PD occurred at a transcriptional or a posttranscriptional step in rat hepatocytes, enzyme activity, mRNA accumulation and transcriptional activity of the G6PD gene were measured in the presence and absence of a polyunsaturated fatty acid, arachidonic acid. Insulin and glucose stimulated a 5- to 7-fold increase in G6PD activity and mRNA. This increase was attenuated by 60% due to the addition of arachidonic acid to the medium of primary rat hepatocytes in culture. Transcriptional activity of the G6PD gene was measured using nuclear run-on assays. The changes in mRNA accumulation occurred in the absence of changes in transcription. To more precisely define the regulated step, the abundance of G6PD mRNA both in the nucleus and in total RNA was measured using a ribonuclease protection assay with probes designed to cross intronexon boundaries. Amounts of precursor mRNAs for G6PD in the nucleus changed in parallel with the amount of mature mRNA. The decrease in pre-mRNA accumulation was only caused by long chain polyunsaturated fatty acids not monounsaturated fatty acids. Further, this decrease was not due to toxicity of the cells as a result of fatty acid oxidation. Thus, regulation occurs at a nuclear posttranscriptional step and represents a novel form of regulation by fatty acids. Our hypothesis is that regulation of G6PD occurs very early in the processing pathway through the interaction of a trans-acting factor with a cis-acting element within the precursor mRNA for the gene. Using transient transfection analysis of a mouse G6PD cDNA construct in rat hepatocytes, a putative fatty acid response element has been localized within 2.3 kb of the G6PD mature sequences. Identification of the pathway involved in the inhibition of de novo fatty acid synthesis by polyunsaturated fat will help understand the molecular basis for diseases associated with the consumption of fat, such as diabetes, obesity and atherosclerotic disease.

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