The physiological relevance of phosphoinositide 3-kinase (PI 3-K) signaling in the liver to fuel homeostasis was investigated. Systemic infusion of an adenovirus encoding a dominant negative mutant of PI 3-K ((Delta)p85) resulted in liver-specific expression of this protein and in inhibition of the insulin-induced activation of PI 3-K in the liver within 3 days, without affecting insulin signaling in skeletal muscle. Hepatic expression of (Delta)p85 led to hyperinsulinemia and to a marked increase in blood glucose concentration in response to oral glucose intake. The increases in both glycogen and glucose 6-phosphate content, as well as in Akt and glycogen synthase activities in the liver, that were induced by glucose intake were markedly impaired in mice expressing (Delta)p85. Despite an upregulation of mRNAs for gluconeogenic enzymes apparent in the liver of these animals, the fasting blood glucose concentration was increased only slightly, and the serum concentrations of gluconeogenic precursors were reduced. However, administration of pyruvate, a substrate for gluconeogenesis, resulted in an exaggerated increase in blood glucose concentration. In the fasted state, the mass of adipose tissue of the mice was about 1.5 times that in control mice. The mice also exhibited marked decreases in the serum concentrations of FFAs and triglyceride and suppression of insulin-induced PI 3-K activation in adipose tissue, probably due to the associated hyperinsulinemia. PI 3-K activity in the liver is thus essential for normal carbohydrate and lipid metabolism in living animals.