OBJECTIVEIncreased oxidative stress (OS) and impaired anti-OS defenses are important in the development and persistence of insulin resistance (IR). Several anti-inflammatory and cell-protective mechanisms, including advanced glycation end product (AGE) receptor-1 (AGER1) and sirtuin (silent mating-type information regulation 2 homolog) 1 (SIRT1) are suppressed in diabetes. Because basal OS in type 2 diabetic patients is influenced by the consumption of AGEs, we examined whether AGE consumption also affects IR and whether AGER1 and SIRT1 are involved.RESEARCH DESIGN AND METHODSThe study randomly assigned 36 subjects, 18 type 2 diabetic patients (age 61 ± 4 years) and 18 healthy subjects (age 67 ± 1.4 years), to a standard diet (>20 AGE equivalents [Eq]/day) or an isocaloric AGE-restricted diet (<10 AGE Eq/day) for 4 months. Circulating metabolic and inflammatory markers were assessed. Expression and activities of AGER1 and SIRT1 were examined in patients’ peripheral blood mononuclear cells (PMNC) and in AGE-stimulated, AGER1-transduced (AGER1+), or AGER1-silenced human monocyte-like THP-1 cells.RESULTSInsulin and homeostasis model assessment, leptin, tumor necrosis factor-α and nuclear factor-κB p65 acetylation, serum AGEs, and 8-isoprostanes decreased in AGE-restricted type 2 diabetic patients, whereas PMNC AGER1 and SIRT1 mRNA, and protein levels normalized and adiponectin markedly increased. AGEs suppressed AGER1, SIRT-1, and NAD+ levels in THP-1 cells. These effects were inhibited in AGER1+ but were enhanced in AGER1-silenced cells.CONCLUSIONSFood-derived pro-oxidant AGEs may contribute to IR in clinical type 2 diabetes and suppress protective mechanisms, AGER1 and SIRT1. AGE restriction may preserve native defenses and insulin sensitivity by maintaining lower basal OS.

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...

Reduction of AGEs in normal diets may lower oxidant stress/inflammation and restore levels of AGER1, an antioxidant, in healthy and aging subjects and CKD-3 patients. AGE intake has implications for health outcomes and costs and warrants further testing.

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...

Background: The general increase in reactive oxygen species generated from glucose-derived advanced glycation endproducts (AGEs) is among the key mechanisms implicated in tissue injury due to diabetes. AGE-rich foods could exacerbate diabetic injury, at least by raising the endogenous AGE. Materials and Methods: Herein, we tested whether, prior to ingestion, diet-derived AGEs contain species with cell activating (TNF␣), chemical (cross-linking) or cell oxidative properties, similar to native AGEs. Glutathione (GSH) and GSH peroxidase (GPx) were assessed after exposure of human umbilical vein endothelial cell (HUVECs) to affinity-purified food-AGE extracts, each exposed to 250ЊC, for 10 min, along with synthetic AGEs. Results: Animal product-derived AGE, like synthetic methylglyoxal-bovine serum albumin (MG-BSA