The present study aimed to investigate the molecular mechanism(s) of insulin action on angiotensinogen (ANG) secretion and gene expression in kidney proximal tubular cells exposed to high levels of glucose. Immortalized rat proximal tubular cells (IRPTC) were cultured in monolayer. The levels of rat ANG and ANG messenger RNA in the IRPTC were quantified by a specific RIA for rat ANG (RIA-rANG) and by an RT-PCR assay. Insulin inhibited the stimulatory effect of a high level of glucose (25 mM) and phorbol 12-myristate 13-acetate, an activator of protein kinase C) on the secretion of ANG and the expression of the ANG messenger RNA in IRPTC. This inhibitory action of insulin on the ANG secretion and gene expression was blocked by PD98059 (an inhibitor of mitogen-activated protein kinase kinase) but not by Wortmannin (an inhibitor of phosphatidylinositol-3-kinase). PD98059 was effective in inhibiting the phosphorylation of MEK 1/2 and p44/42 MAP kinase in IRPTC stimulated by insulin. These studies demonstrate that insulin prevents the stimulatory effect of high levels of glucose on the expression of the renal ANG gene in IRPTC, at least in part, via the MAPK kinase signal transduction pathway, subsequently inhibiting the activation of the local renal renin-angiotensin system.