Glimepiride, a third-generation sulfonylurea (SU), exerts its effects mainly by stimulating insulin secretion but has also been shown to have pleiotropic effects. Recent clinical studies showed glimepiride to enhance insulin sensitivity. In the present study, to clarify the mechanism by which insulin resistance is improved, we investigated the effects of glimepiride on AMP-activated protein kinase (AMPK) and peroxisome proliferator-activated receptor-gamma (PPAR gamma) activity, using cultured adipocytes and muscle cells. When we treated fully differentiated 3T3-L1 adipocytes with 1 microM glimepiride, endogenous PPAR gamma transcriptional activity was significantly elevated, while AICAR-induced phosphorylation of AMPK was not affected in differentiated C2C12 myoblasts. The maximum PPAR gamma activity enhancing effect of glimepiride is approximately 20% that of 1 microM pioglitazone. In contrast, this mild PPAR gamma-stimulatory effect was not observed under the same conditions with a 2nd generation SU, glibenclamide. Furthermore, with glimepiride treatment, transcriptional levels of aP2, the adipogenic marker gene, were increased 2.4- and 3.7-fold in 3T3-L1 adipocytes and fibroblasts, respectively. Analysis of triglyceride contents revealed glimepiride to promote differentiation of 3T3-L1 adipocytes. These results indicate that glimepiride has the potential to induce PPAR gamma activity, thereby improving insulin resistance.