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Show 1 SCHOOL OF MEDICINE AND HEALTH SCIENCES UNDERGRADUATE RESEARCH ABSTRACTS MEASURING CELLULAR CERAMIDE ACCRUAL USING IMMUNOFLUORESCENCE Anindita Ravindran (John David Symons) Division of Endocrinology, Diabetes & Metabolism University of Utah Obesity predisposes individuals with Type II Diabetes to cardiovascular complications like impaired blood vessel function. Due to the elevation of free fatty acids (FFAs) in obese individuals, ceramide, a lipid metabolite, accumulates and might contribute to the inability of a blood vessel to constrict or relax appropriately. Vessel dysfunction is partly caused by the inability of the endothelium to synthesize nitric oxide (NO). Our data indicate that ceramide impairs endothelial N O synthase (eNOS), the enzyme that synthesizes NO. In order to study mechanisms whereby ceramide might impair eNOS, it is important to measure cellular ceramide production in response to physiological, pharmacological, and genetic manipulations. Previously w e used P-32 radioactive assays to measure ceramide accumulation. However, the use of radioactivity is expensive, hazardous, and an environmental concern. Therefore, w e imported a cost-effective, non-hazardous, yet accurate technique of measuring ceramide production by immunofluorescence where ceramide is tagged with a primary antibody which can be detected by a secondary antibody conjugated with a fluorescent dye. I observed that 250, 500, and 750 pM palmitate (pal) incubation for 3 h increases (p<0.05) endothelial cell ceramide in a dose-dependent manner in Bovine Aortic Endothelial Cells (BAECs). Further, a FFA-independent method to alter ceramide accrual i.e., 3 h incubation of cells with 250 p M N-oleoylethanolamine, also elevates (p<0.05) ceramide production. Importantly, I have shown that 500 p M pal-induced ceramide accrual can be prevented (p<0.05) by two structurally dissimilar inhibitors (10 p M myriocin, 1 m M L-cycloser-ine) of the rate-limiting enzyme responsible for ceramide biosynthesis i.e., serine palmitoyl transferase (SPT). None of these inhibitors impairs cell viability. I also performed a genetic knockdown of SPT using silencing RNA to verify that ceramide does not accumulate in response to a genetic intervention (p<0.05). W e have shown that Protein Phosphatase 2A (PP2A) binds with eNOS and prevents eNOS from synthesizing NO. I treated BAECs with LB1 to inhibit PP2A to ensure that ceramide accumulation is not influenced by LB1 (p<0.05). Neither SPT knockdown nor LB1 treatment evoked significant cell death.These data indicate that IF is an accurate and reproducible method whereby ceramide accrual can be quantified in endothelial cell systems. John David Symons |
