Insulin stimulation of target cells elicits a burst of H 2 O 2 that enhances tyrosine phosphorylation of the insulin receptor and its cellular substrate proteins as well as distal signaling events in the insulin action cascade. The molecular mechanism coupling the insulin receptor with the cellular oxidant-generating apparatus has not been elucidated. Using reverse transcription-PCR and Northern blot analyses, we found that Nox4, a homolog of gp91phox, the phagocytic NAD(P)H oxidase catalytic subunit, is prominently expressed in insulin-sensitive adipose cells. Adenovirus-mediated expression of Nox4 deletion constructs lacking NAD(P)H or FAD/NAD(P)H cofactor binding domains acted in a dominant-negative fashion in differentiated 3T3-L1 adipocytes and attenuated insulin-stimulated H 2 O 2 generation, insulin receptor (IR) and IRS-1 tyrosine phosphorylation, activation of downstream serine kinases, and glucose uptake. Transfection of specific small interfering RNA oligonucleotides reduced Nox4 protein abundance and also inhibited the insulin signaling cascade. Overexpression of Nox4 also significantly reversed the inhibition of insulin-stimulated IR tyrosine phosphorylation induced by coexpression of PTP1B by inhibiting PTP1B catalytic activity. These data suggest that Nox4 provides a novel link between the IR and the generation of cellular reactive oxygen species that enhance insulin signal transduction, at least in part via the oxidative inhibition of cellular protein-tyrosine phosphatases (PTPases), including PTP1B, a PTPase that has been previously implicated in the regulation of insulin action.

The insulin signaling pathway is activated by tyrosine phosphorylation of the insulin receptor and key postreceptor substrate proteins and balanced by the action of specific protein-tyrosine phosphatases (PTPases). PTPase activity, in turn, is highly regulated in vivo by oxidation/reduction reactions involving the cysteine thiol moiety required for catalysis. Here we show that insulin stimulation generates a burst of intracellular H 2 O 2 in insulin-sensitive hepatoma and adipose cells that is associated with reversible oxidative inhibition of up to 62% of overall cellular PTPase activity, as measured by a novel method using strictly anaerobic conditions. The specific activity of immunoprecipitated PTP1B, a PTPase homolog implicated in the regulation of insulin signaling, was also strongly inhibited by up to 88% following insulin stimulation. Catalase pretreatment abolished the insulin-stimulated production of H 2 O 2 as well as the inhibition of cellular PTPases, including PTP1B, and was associated with reduced insulin-stimulated tyrosine phosphorylation of its receptor and high M r insulin receptor substrate (IRS) proteins. These data provide compelling new evidence for a redox signal that enhances the early insulin-stimulated cascade of tyrosine phosphorylation by oxidative inactivation of PTP1B and possibly other tyrosine phosphatases. Protein-tyrosine phosphatases (PTPases)1 play a key role in the regulation of reversible tyrosine phosphorylation in the insulin action pathway. Insulin signaling is initiated by the phosphorylation of specific tyrosyl residues of the cell surface insulin receptor, which activates its exogenous kinase activity and promotes the phosphorylation of IRS proteins on specific tyrosine residues (1). These activation steps are balanced, in turn, by specific cellular PTPases that dephosphorylate and inactivate the receptor kinase and reverse the adapter function of the receptor substrate proteins (2). The cellular role of PTPases is apparent from the observation that highly purified insulin receptors and IRS proteins retain their tyrosine phosphorylation and activation state in vitro (3, 4), while in intact or permeabilized cells, receptor activation and substrate tyrosine phosphorylation are rapidly reversed (5-7).Since PTPases are high turnover number enzymes, physiological suppression of PTPase catalytic activity has been postulated to be a key feature of their regulation within the cellular environment to allow tyrosine phosphorylation to proceed in a balanced manner (8). PTPases have in common a conserved ϳ230-amino acid domain that contains the cysteine residue that catalyzes the hydrolysis of protein phosphotyrosine residues by the formation of a cysteinyl-phosphate intermediate (9, 10). Several laboratories have recently provided evidence that reactive oxygen species, including H 2 O 2 , can oxidize and inactivate PTPases in vivo (11,12). Since only the reduced form of the catalytic site is enzymatically active, stepwise and progressively irreversible oxidative inhibition is em...

9-␤-D-arabinofuranoside and compound C) completely abrogated the increase in glucose uptake stimulated by globular adiponectin, indicating that AMP kinase is integrally involved in the adiponectin signal transduction pathway. Coupled with recent evidence that the effects of adiponectin are mediated via AMP kinase activation in liver and skeletal muscle, the findings reported here provide an important mechanistic link in the signaling effects of adiponectin in diverse metabolically responsive tissues.

Propelled by the identification of a small family of NADPH oxidase (Nox) enzyme homologs that produce superoxide in response to cellular stimulation with various growth factors, renewed interest has been generated in characterizing the signaling effects of reactive oxygen species (ROS) in relation to insulin action. Two key observations made >30 years ago-that oxidants can facilitate or mimic insulin action and that H 2 O 2 is generated in response to insulin stimulation of its target cells-have led to the hypothesis that ROS may serve as second messengers in the insulin action cascade. Specific molecular targets of insulin-induced ROS include enzymes whose signaling activity is modified via oxidative biochemical reactions, leading to enhanced insulin signal transduction. These positive responses to cellular ROS may seem "paradoxical" because chronic exposure to relatively high levels of ROS have also been associated with functional ␤-cell impairment and the chronic complications of diabetes. The best-characterized molecular targets of ROS are the protein-tyrosine phosphatases (PTPs) because these important signaling enzymes require a reduced form of a critical cysteine residue for catalytic activity. PTPs normally serve as negative regulators of insulin action via the dephosphorylation of the insulin receptor and its tyrosinephosphorylated cellular substrates. However, ROS can rapidly oxidize the catalytic cysteine of target PTPs, effectively blocking their enzyme activity and reversing their inhibitory effect on insulin signaling. Among the cloned Nox homologs, we have recently provided evidence that Nox4 may mediate the insulin-stimulated generation of cellular ROS and is coupled to insulin action via the oxidative inhibition of PTP1B, a PTP known to be a major regulator of the insulin signaling cascade. Further characterization of the molecular components of this novel signaling cascade, including the mechanism of ROS generated by insulin and the identification of various oxidation-sensitive signaling targets in insulin-sensitive cells, may provide a novel means of facilitating insulin action in states of insulin resistance.

In a variety of cell types, insulin stimulation elicits the rapid production of H 2 O 2 , which causes the oxidative inhibition of protein-tyrosine phosphatases and enhances the tyrosine phosphorylation of proteins in the early insulin action cascade (Mahadev, K., Zilbering, A., Zhu, L., and Goldstein, B. J. (2001) J. Biol. Chem. 276, 21938 -21942). In the present work, we explored the potential role of insulin-induced H 2 O 2 generation on downstream insulin signaling using diphenyleneiodonium (DPI), an inhibitor of cellular NADPH oxidase that blocks insulin-stimulated cellular H 2 O 2 production. DPI completely inhibited the activation of phosphatidylinositol (PI) 3-kinase activity by insulin and reduced the insulin-induced activation of the serine kinase Akt by up to 49%; these activities were restored when H 2 O 2 was added back to cells that had been pretreated with DPI. Interestingly, the H 2 O 2 -induced activation of Akt was entirely mediated by upstream stimulation of PI 3-kinase activity, since treatment of 3T3-L1 adipocytes with the PI 3-kinase inhibitors wortmannin or LY294002 completely blocked the subsequent activation of Akt by exogenous H 2 O 2 . Preventing oxidant generation with DPI also blocked insulin-stimulated glucose uptake and GLUT4 translocation to the plasma membrane, providing further evidence for an oxidant signal in the regulation of the distal insulin-signaling cascade. Finally, in contrast to the cellular mechanism of H 2 O 2 generation by other growth factors, such as plateletderived growth factor, we also found that insulin-stimulated cellular production of H 2 O 2 may occur through a unique pathway, independent of cellular PI 3-kinase activity. Overall, these data provide insight into the physiological role of insulin-dependent H 2 O 2 generation, which is not only involved in the regulation of tyrosine phosphorylation events in the early insulin signaling cascade but also has important effects on the regulation of downstream insulin signaling, involving the activation of PI 3-kinase, Akt, and ultimately cellular glucose transport in response to insulin.Major advances in our understanding of the regulation of the insulin action pathway have focused on the key role of tyrosine phosphorylation of the insulin receptor and its cellular substrate proteins (1). Insulin binding leads to autophosphorylation of specific residues of the transmembrane insulin receptor and activation of the intrinsic tyrosine kinase activity of its intracellular domains (2). The insulin signal is then transmitted further into the cell through the tyrosine phosphorylation of specific sites on cellular substrate proteins (e.g. IRS 1 and Shc), which act as docking sites for the binding and activation of a variety of Src homology 2 domain-containing signaling proteins (3). Much of insulin's downstream signaling to metabolic events involves the activation of phosphatidylinositol (PI) 3Ј-kinase activity by the docking of its p85 subunit to tyrosinephosphorylated IRS-1 and IRS-2 (4 -6), which is linked to a number...