In the heart, insulin stimulates a variety of kinase cascades and controls glucose utilization. Because insulin is able to activate Akt and inactivate AMP-activated protein kinase (AMPK) in the heart, we hypothesized that Akt can regulate the activity of AMPK. To address the potential existence of this novel signaling pathway, we used a number of experimental protocols to activate Akt in cardiac myocytes and monitored the activation status of AMPK. Mouse hearts perfused in the presence of insulin demonstrated accelerated glycolysis and glucose oxidation rates as compared with non-insulin-perfused hearts. In addition, insulin caused an increase in Akt phosphorylation and a decrease in AMPK phosphorylation at its major regulatory site (threonine 172 of the ␣ catalytic subunit). Transgenic mice overexpressing a constitutively active mutant form of Akt1 displayed decreased phosphorylation of cardiac ␣-AMPK. Isolated neonatal cardiac myocytes infected with an adenovirus expressing constitutively active mutant forms of either Akt1 or Akt2 also suppressed AMPK phosphorylation. However, Akt-dependent depression of ␣-AMPK phosphorylation could be overcome in the presence of the AMPK activator, metformin, suggesting that an override mechanism exists that can restore AMPK activity. Taken together, this study suggests that there is crosstalk between the AMPK and Akt pathways and that Akt activation can lead to decreased AMPK activity. In addition, our data suggest that the ability of insulin to inhibit AMPK may be controlled via an Akt-mediated mechanism.The insulin signaling cascade is an important signaling pathway responsible for controlling substrate preference in the heart. Indeed, insulin has been shown to stimulate myocardial glucose uptake (1, 2) and accelerate glycolysis (2, 3) and glucose oxidation rates (2, 4) as well as promote glycogen synthesis (5, 6). These effects of insulin on glucose metabolism are particularly important because a significant amount of glucose-derived ATP is used for maintaining proper cardiac function. In addition, the stimulation of myocardial glucose utilization and subsequent decrease in fatty acid oxidation has been proven to be efficacious in reducing ischemic injury (7-11). Moreover, insulin-stimulated glucose utilization may be a central component of the beneficial effects of glucose-insulin-potassium therapy on the ischemic heart (12). Two kinases that can be regulated by insulin are Akt and AMPK. 1 Akt is a serine/threonine protein kinase that can be activated by insulin via a multistep pathway involving a phosphatidylinositol 3-kinase-dependent mechanism (see Ref. 13 for review). Once Akt is phosphorylated and activated, it can promote glucose uptake and subsequent metabolism via translocation of glucose transporter (GLUT) 4 to the plasma membrane (14 -18). In addition, the phosphorylation and inhibition of glycogen synthase kinase (GSK) 3 by Akt activates glycogen synthase and thereby promotes glycogen synthesis (19). Recent evidence in heart indicates that the presence of...