Role of calcium-independent phospholipase A2 in insulin-stimulated glucose uptake in 3T3-L1 adipocytes
Type of Degreedissertation
Nutrition and Food Science
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Calcium-independent phospholipase A2 (iPLA2) is a member of the phospholipase A2 family that catalyzes the hydrolysis of glycerophospholipid at the sn-2 position to liberate free fatty acid and lysophospholipid. Recent studies have suggested that iPLA2 may be important for adipocyte biology. The purpose of this study was to determine the role of iPLA2 in insulin-stimulated actions on glucose uptake in 3T3-L1 adipocytes. The 3T3-L1 adipocytes were pretreated with bromoenol lactone (BEL, 10 or 50 µM), a chemical inhibitor of iPLA2, for 30 minutes. After insulin (100 nM) stimulation, glucose uptake was determined by measuring intracellular incorporation of [3H]-2-deoxyglucose. With 10 and 50 µM BEL treatment, insulin-stimulated glucose uptake was decreased by 30% and 45%, respectively, compared to DMSO vehicle-treated cells. Further investigations confirmed that iPLA2 activity was responsive to the BEL inhibitory effects, and that iPLA2-beta and iPLA2-gamma were both involved in this process. Additionally, exogenous arachidonic acid (AA, 100 µM) restored the reduction of insulin-stimulated glucose uptake induced by BEL treatment. Small interfering RNA (siRNA) technique was utilized to selectively inhibit iPLA2-beta and iPLA2-gamma gene expression in 3T3-L1 adipocytes. Consistent with the BEL treatment results, the 3T3-L1 adipocytes transfected with iPLA2-beta or iPLA2-gamma siRNA (50 nM) exhibited impaired glucose uptake upon insulin stimulation, showing approximate 40% inhibition compared to scrambled siRNA treated cells. To elucidate the underlying mechanism for the effects of iPLA2 inhibition on insulin-stimulated glucose uptake, insulin-stimulated phosphorylation of insulin receptor (IR) and Akt was assessed by Western blot analysis. There was no significant difference between BEL-treated and vehicle-treated cells in the phosphorylation levels of IR and Akt. Fraction analysis of insulin-responsive GLUT4 translocation was also performed in adipocytes. Compared to vehicle control, BEL treatment noticeably inhibited the incorporation of GLUT4 into plasma membrane and lipid raft fractions in response to insulin. These results demonstrate that insulin-stimulated glucose uptake was decreased by iPLA2 inhibition, and that this effect was mediated via attenuation of the insulin-dependent GLUT4 translocation into plasma membrane. However, the early steps of insulin signaling were not affected by iPLA2 inhibition. It is conclude that insulin-stimulated glucose uptake is mediated, at least in part, by iPLA2, and that iPLA2 may represent a novel therapeutic target for the treatment of insulin resistance related diseases, such as obesity, type 2 diabetes, and other metabolic diseases.