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 insulinomimetic agent, englitazone, on functional end points and putative mediators of insulin action in 3T3-L1 adipocytes. Cells were incubated with englitazone for 48 h or with insulin for 10 or 30 min, or both, and 2-deoxy-D-[3H]glucose (2DG) uptake and lipogenesis (from [14C]glucose) were measured. Tyrosine phosphorylation of the insulin receptor (IR), insulin receptor substrates 1 and 2 (IRS-1 and IRS-2), and pp60, and phosphatidylinositol (PI) 3-kinase activity (using PI as substrate) and mitogen-activated protein kinase (MAPK) activity were assayed in cell lysates. Englitazone increased 2DG uptake in a concentration-dependent (10-100 micromol/l) manner by up to sixfold, and preincubation with englitazone significantly enhanced insulin-stimulated 2DG uptake. However, englitazone had a biphasic effect on lipogenesis (163 +/- 13% basal at 10 micromol/l vs. 96 +/- 14% at 100 micromol/l), but when acetate was used as substrate, only concentration-dependent inhibition of lipogenesis occurred. In addition, englitazone decreased insulin-stimulated lipogenesis in a concentration-dependent manner. Englitazone did not increase IR, IRS-1/IRS-2, pp60, or MAPK phosphorylation, nor did it enhance insulin's stimulation of these parameters. Although englitazone alone did not activate PI 3-kinase, it did enhance the stimulation of the enzyme produced by a submaximally effective insulin concentration. Significant (63%) inhibition of insulin-stimulated lipogenesis occurred at a concentration of englitazone (30 micromol/l) that did not affect MAPK activation, which suggests that the drug's inhibitory effect 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 englitazone via peroxisome proliferator activated receptor-gamma activation or by an alternate pathway. Thus englitazone stimulates 2DG uptake without affecting PI 3-kinase, but it can enhance both insulin-stimulated 2DG uptake and PI 3-kinase activity. However, englitazone inhibits insulin-stimulated lipogenesis without inhibiting PI 3-kinase activity. Assuming activation of PI 3-kinase mediates insulin-stimulated 2-DG and lipogenesis, then the signaling pathways for each process diverge beyond PI 3-kinase.