PI3K inhibitors reverse the suppressive actions of insulin on CYP2E1 expression by activating stress-response pathways in primary rat hepatocytes

Insulin-associated signaling pathways are critical in the regulation of hep atic physiology. Recent inhibitor-based studies have implicated a mechanist ic role for phosphatidylinositol 3' kinase (PI3K) in the insulin-mediated s uppression of CYP2E1 mRNA levels in hepatocytes. We investigated the dose d ependence for this response and for the effects of insulin and extracellula r matrix on PI3K signaling and CYP2E1 mRNA expression levels using a highly defined rat primary hepatocyte culture system. The PI3K inhibitors wortman nin and LY294002 stimulated stress-activated protein kinase/c-Jun NH2-termi nal kinase (SAPK/JNK) and p38 mitogen-activated protein kinase (MAPK) phosp horylation in a rapid and concentration-dependent manner that paralleled th e inhibition of protein kinase B (PKB) phosphorylation. Although PI3K inhib itors reversed the suppressive effects of insulin on CYP2E1 expression, the se effects only occurred at concentrations well in excess of those required to achieve complete inhibition of PKB phosphorylation. These same concentr ations produced cytotoxic responses as evidenced by perturbed cellular morp hology and elevated release of lactate dehydrogenase. Wortmannin-mediated a ctivation of the SAPK/JNK and p38 MAPK pathways also resulted in the mobili zation of activator protein-1 complex to the nuclear compartment. We conclu de that the suppression of CYP2E1 mRNA expression by insulin is not directl y associated with PI3K-dependent pathway activation, but rather is linked t o a cytotoxic response stemming from acute challenge with PI3K inhibitors.