POSSIBILITY OF DISTINCT INSULIN-SIGNALING PATHWAYS BEYOND PHOSPHATIDYLINOSITOL 3-KINASE-MEDIATING GLUCOSE-TRANSPORT AND LIPOGENESIS

The aim of this study was to compare the effects of insulin and the in sulinomimetic agent, englitazone, on functional end points and putativ e mediators of insulin action in 3T3-L1 adipocytes. Cells were incubat ed with englitazone for 48 h or with insulin for 10 or 30 min, or both , and 2-deoxy-D-[H-3]glucose (2DG) uptake and lipogenesis (from [C-14] glucose) were measured. Tyrosine phosphorylation of the insulin recept or (IR), insulin receptor substrates I and 2 (IRS-1 and IRS-2), and pp 60, and phosphatidylinositol (PI) 3-kinase activity (using PI as subst rate) and mitogen-activated protein kinase (MAPK) activity were assaye d in cell lysates. Englitazone increased 2DG uptake in a concentration -dependent (10-100 mu mol/l) manner by up to sixfold, and preincubatio n with englitazone significantly enhanced insulin-stimulated 2DG uptak e. However, englitazone had a biphasic effect on lipogenesis (163 +/- 13% basal at 10 mu mol/l vs. 96 +/- 14% at 100 mu mol/l), but when ace tate was used as substrate, only concentration-dependent inhibition of lipogenesis occurred. In addition, englitazone decreased insulin-stim ulated lipogenesis in a concentration-dependent manner. Englitazone di d not increase IR, IRS-1/IRS-2, pp60, or MAPK phosphorylation, nor did it enhance insulin's stimulation of these parameters. Although englit azone alone did not activate PI 3-kinase, it did enhance the stimulati on of the enzyme produced by a submaximally effective insulin concentr ation. Significant (63%) inhibition of insulin-stimulated lipogenesis occurred at a concentration of englitazone (30 mu mol/l) that did not affect MAPK activation, which suggests that the drug's inhibitory effe ct on lipogenesis is not mediated by this pathway. Englitazone did not affect the expression of the peroxisome proliferator response element -containing fatty acyl CoA synthase gene, although it cannot be ruled out that expression of other Lipogenic enzymes are altered by englitaz one via peroxisome proliferator activated receptor-gamma activation or by an alternate pathway. Thus englitazone stimulates 2DG uptake witho ut affecting PI 3-kinase, but it can enhance both insulin-stimulated 2 DG uptake and PI 3-kinase activity. However, englitazone inhibits insu lin-stimulated lipogenesis without inhibiting PI 3-kinase activity. As suming activation of PI 3-kinase mediates insulin-stimulated 2-DG and lipogenesis, then the signaling pathways for each process diverge beyo nd PI 3-kinase.