Regulation of stearoyl-CoA desaturases by oleate

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DESCRIPTION: (Scanned from the applicant's abstract) The long term objective is to understand the mechanisms by which C18 fatty acids regulate the synthesis of cholesterol and fatty acids necessary for the synthesis of membranes during cellular proliferation. The fatty acid component of plasma membranes is comprised of an approximately equal mixture of saturated and unsaturated fatty acids. This ratio of unsaturated to saturated fatty acids is one of the major factors controlling the fluidity and function of plasma membranes, and had been associated with a number of disease states such as diabetes, obesity, cancer, hypertension and atherosclerosis. Since saturated fatty acids are precursors for unsaturated fatty acids, the enzymes that catalyze this conversion, fatty acyl-CoA desaturases, must be tightly regulated to maintain the proper fatty acid ratios in the membrane. The most common unsaturated fatty acid of the plasma membrane is oleate. Oleate is produced from the saturated fatty acid, stearate, by the activity of stearoyl-CoA desaturases (SCD). One potential SCD regulatory mechanism is end-product feedback inhibition by oleate. It has been well demonstrated that in lipogenic cells, oleate is not a regulator of SCD. However, the primary physiological fate of the product of SCD activity in lipogenic cells is not the plasma membrane as it is in nonlipogenic cells, thus the regulation of SCD in lipogenic cells is likely to be different. Our preliminary data shows this to be the case as oleate strongly inhibits SCO activity in CHO fibroblasts, represses the mRNA levels and transcription of the two known SCD genes, SCD1 and SCD2. The main objective of this proposal is to test the hypothesis that in proliferating cells (as opposed to lipogenic cells) the end product of stearoyl-C0A desaturase activity, oleate, represses the level of SCD mRNAs by inhibiting transcription of the SCD genes. SCD1 and SCD2 are known to be regulated transcriptionally by sterols via sterol response element binding proteins (SREBPs) that bind to sterol response elements (SREs) present in the promoters of both genes. Since we have shown that oleate can repress transcription other promoters containing SREs, we speculate that transcriptional regulation of SCD by oleate will involve SREBPs. The specific aims of the proposal are 1) to identify the role of SREs in oleate regulation of SCD transcription. 2) To determine what trans-acting factors are necessary for oleate regulation of SCD transcription. 3) To determine the mechanism by which oleate represses transcription of SRE containing promoters. Transfection of CHO fibroblasts with SCD reporter genes containing various mutations in the promoters will be used to determine the location of the cis-acting elements that regulate SCD expression in response to oleate. Ectotopic expression of SREBPs or other trans-acting factors will be used to determine their contribution to oleate regulation of SCD transcription.