Ting Ruan, University of Utah
Physical Sciences
Cardiovascular complications are more prevalent in patients with diet-induced obesity and type 2 diabetes. Both of these conditions are associated with elevated levels of free fatty acids (FFAs). Elevated FFAs might precipitate cardiovascular complications by disrupting endothelial nitric oxide (NO) synthase (eNOS) enzyme function. The physiologically abundant saturated FFA palmitate decreases eNOS phosphorylation at serine 1177 (p-eNOS S1177) in a ceramide and protein-phosphatase 2A (PP2A) -dependent manner. p-eNOS S1177 is a positive regulatory site on the eNOS enzyme. As such, p-eNOS S1177 to total eNOS can be used as an estimate of eNOS enzyme function. We sought to determine the extent to which two other phosphatases that are abundant in the cytosol i.e., protein phosphatase 1 (PP1) and protein phosphatase 2B (PP2B) might contribute to palmitate-induced reductions in p-eNOS S1177 to total eNOS. Bovine aortic endothelial cells (BAECs) were treated for 3 hours with 500 uM palmitate or vehicle in the absence and presence of the PP1 inhibitor tautomycin (3 uM). p-eNOS S1177 to total eNOS was assessed using immunoblotting procedures. Palmitate-induced reductions (30±3%, p<0.05, n=3) in p-eNOS to total eNOS were similar in the absence and presence of tautomycin. These data indicate that PP1 does not contribute to palmitate-induced disruption of eNOS enzyme function. Next, BAECs were treated for 3 hours with 500 uM palmitate or vehicle in the absence and presence of the PP2B inhibitor cyclosporine (100nM). Palmitate-induced reductions (31±4%, p<0.05, n=3) in p-eNOS to total eNOS were similar in the absence and presence of cyclosporine. Taken together, these data suggest that neither PP1 nor PP2B contribute to palmitate-induced reductions in p-eNOS S1177 to total eNOS.