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...

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...

Insulin signal transduction in adipocytes is accompanied by a burst of cellular hydrogen peroxide (H 2 O 2 facilitates insulin signaling by inhibiting thiol-dependent protein-tyrosine phosphatases (PTPs)) that are negative regulators of insulin action. As hyperglycemia is associated with increased cellular reactive oxygen species, we postulated that high glucose conditions might potentiate the H 2 O 2 generated by insulin and modulate insulin-stimulated protein phosphorylation. Basal H 2 O 2 generation was increased threefold in differentiated 3T3-L1 adipocytes by growth in 25 mM glucose versus 5 mM glucose. High glucose increased the sensitivity of the insulin-stimulated H 2 O 2 signal to lower concentrations of insulin. Basal endogenous total PTP activity and the activity of PTP1B, a PTP implicated in the negative regulation of insulin signaling, were reduced in high glucose conditions, and their further reduction by insulin stimulation was more enhanced in high versus low glucose medium. Phosphorylation of the insulin receptor, IRS-1, and Akt in response to insulin was also significantly enhanced in high glucose conditions, especially at submaximal insulin concentrations. In primary rat adipocytes, high glucose increased insulin-stimulated H 2 O 2 the oxidative inhibition of total PTP production and potentiated and PTP1B activity; however, insulin signaling was not enhanced in the primary cells in high glucose apparently due to cross-regulation of insulin-stimulated protein phosphorylation by activation of protein kinase C (PKC). These studies indicate that high glucose can enhance insulin-stimulated H 2 O 2 generation and augment oxidative PTP inhibition in cultured and primary adipocytes, but the overall balance of insulin signal transduction is determined by additional signal effects in high glucose, including the activation of PKC.

Compared with the sc depot, omental (om) adipose tissue is relatively resistant to the metabolic actions of insulin. Protein-tyrosine phosphatases (PTPases) modulate receptor kinase activation and signal transduction in insulin-sensitive tissues, and their activity is dependent on the reduced state of the cysteine thiol required for catalysis. Using a novel anaerobic technique to avoid air oxidation, we found that the mean endogenous PTPase activity was 2.1-fold higher in om compared with paired samples of sc adipose tissue (P < 0.003). The specific activity of PTP1B isolated under anaerobic conditions was also 41% higher in om adipose tissue (P < 0.001). Interestingly, the total PTPase activity from both adipose depots and the specific activity of PTP1B was increased by 42-71% after reduction in vitro with dithiothreitol, indicating that a major fraction of the cellular PTPase activity can be reactivated by sulfhydryl reduction. The mass of the insulin receptor beta-subunit and the PTPases PTP1B and leukocyte antigen related was not significantly different between the two adipose depots. These studies provide the first demonstration that endogenous PTPase activity, including PTP1B, is increased in om adipose tissue and may contribute to the relative insulin resistance of this fat depot. The finding that a substantial fraction of PTPase activity in human adipose tissue is present in a latent, oxidized form also suggests a potential means of in vivo regulation of these important cellular enzymes that modulate the insulin signaling cascade.

Protein-tyrosine phosphatases (PTPases) have a common cysteine residue whose reduced state is integral to their phosphocysteine-mediated reaction mechanism. The catalytic cysteine thiol can be oxidized or conjugated during cellular redox reactions, which provides an important means of PTPase regulation in vivo. Because exposure to air can artifactually oxidize this reactive thiol, PTPase assays have typically used potent reducing agents such as dithiothreitol to reactivate the enzymes present. However, this approach does not allow for the measurement of endogenous PTPase activity as directly isolated from the in vivo cellular environment. Here we show that sample processing and assay in an anaerobic chamber by using deoxygenated buffers can preserve the overall activity of PTPases in subcellular fractions of 3T3-L1 adipocytes, HepG2 hepatoma cells, and human adipose tissue, as well as with PTP1B, specifically isolated by immunoprecipitation. Cell lysis into air reduced the PTPase activity to as low as 20% of the level observed with sample handling in the anaerobic environment, which was variably restored towards the activity in the anaerobic samples by treatment with dithiothreitol. The approach reported here provides a new framework for characterizing the activity of PTPases as isolated from the intracellular milieu, which more closely reflects the endogenous reactivity and potential impact of these PTPases on signal transduction pathways involving reversible protein-tyrosine phosphorylation.

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Various molecular forms of chromium have been implicated in the regulation of glucose metabolism, and chromium deficiency can be associated with insulin resistance and impaired glucose tolerance. Protein-tyrosine phosphatases (PTPases), which negatively regulate signaling through the insulin receptor, are potential targets of chromium action, since this transition metal may inhibit catalysis at the thiol-dependent active sites of these enzymes. Treatment of cultured rat hepatoma cells with 0.1 mM CrCl 3 for 16 h increased the insulin-stimulated tyrosine phosphorylation of high M r insulin receptor substrate (IRS) proteins by 49% to 7.3-fold over basal (n ‫ס‬ 7; P ‫ס‬ 0.03), without altering basal insulin receptor or IRS tyrosine phosphorylation or insulin-stimulated receptor autophosphorylation, suggesting a post-receptor effect of chromium on signal transduction. PTPase activity in cell extracts of CrCl 3 -treated hepatoma cells before or after insulin stimulation was unchanged, indicating that if chromium acted via cellular PTPases, the effect was reversible and limited to the in vivo state. Chromium (Cr +3 ) ion and two organic derivatives, an oligopeptide chromium complex from bovine liver (Cr-pep), and a synthetic multinuclear complex of chromium with carboxylate ligands (Sm-Cr) were also tested for their direct in vitro inhibition of the enzymatic activity of LAR and PTP1B, two structurally variant PTPases that have been implicated in regulation of the insulin signaling pathway. PTP1B (rat and human) was strongly inhibited by CrCl 3 to 21-33% of control (n ‫ס‬ 4-6; P < 0.001). In contrast, LAR activity was actually enhanced by CrCl 3 to 47% above the control value (n ‫ס‬ 12; P < 0.001). The Cr-pep and Sm-Cr complexes had no effect on PTP1B and LAR activity at the tested concentrations using the pNPP assay. These data suggest that the metabolic effects of chromium may be mediated by inhibition of PTP1B, a PTPase that negatively modulates insulin signaling, consistent with other recent studies implicating PTP1B in the regulation of the dephosphorylation of post-insulin receptor substrate proteins.