Prolonged exposure of 3T3-L1 adipocytes to micromolar concentrations of H2O2 results in an impaired response to the acute metabolic effects of insulin. In this study, we further characterized the mechanisms by which oxidative stress impairs insulin stimulation of glucose transport activity. Although insulin induced a 2.5-fold increase in plasma membrane GLUT4 content and a 50% reduction in its abundance in the low-density microsomal (LDM) fraction in control cells, oxidation completely prevented these responses. The net effect of insulin on 2-deoxyglucose uptake activity was reduced in oxidized cells and could be attributed to GLUT1 translocation. Insulin stimulation of insulin receptor substrate (IRS) 1 tyrosine phosphorylation and the association of IRS-1 with phosphatidylinositol (PI) 3-kinase were not impaired by oxidative stress. However, a 1.9-fold increase in the LDM content of the p85 subunit of PI 3-kinase after insulin stimulation was observed in control, but not in oxidized, cells. Moreover, although insulin induced an increase in IRS-1-associated PI 3-kinase activity in the LDM in control cells, this effect was prevented by oxidation. These findings suggest that prolonged low-grade oxidative stress impairs insulin-stimulated GLUT4 translocation, potentially by interfering with compartment-specific activation of PI 3-kinase.

The insulin receptor (IR)1 is a heterotetrameric transmembrane glycoprotein composed of two extracellular ␣ subunits and two transmembrane ␤ subunits linked by disulfide bonds. The ␣ subunits contain the insulin-binding domain, while the transmembrane ␤ subunits function as Tyr-specific kinases (insulin receptor kinases). Insulin signaling utilizes the Tyr kinase activity of the receptor to phosphorylate docking proteins on multiple Tyr residues and further propagate insulin action (1). The major substrates of insulin receptor kinase are Shc (2) and the IRS proteins, IRS-1 (3), IRS-2 (4), IRS-3 (5), and IRS-4 (6). IRS proteins contain a conserved pleckstrin homology domain (7, 8) located at the amino terminus, adjacent to a phosphotyrosine binding (PTB) domain. The PTB domain is present in a number of signaling molecules (9) and shares 75% sequence identity between IRS-1 and IRS-2 (10). This domain interacts with the NPXY motif of the juxtamembrane (JM) region of IR and promotes IR/IRS-1 interactions (11, 12). The C-terminal region of IRS proteins is poorly conserved. It contains multiple Tyr phosphorylation motifs that serve as docking sites for SH2 domain-containing proteins like the p85␣ regulatory subunit of PI3K, Grb2, Nck, Crk, Fyn, SHP-2, and others, which mediate the metabolic and growth-promoting functions of insulin (1, 13).The signaling pathways regulated by IRS proteins control glucose uptake and lipogenesis, protein synthesis, and cell survival (1, 13). The relative roles of the different IRS proteins in mediating insulin action are still unclear; however, studies of gene disruption revealed that IRS-2 compensates for the absence of IRS-1 in hepatocytes of IRS-1 null mice, while IRS-3 provides the major alternative pathway to PI3K activation in skeletal muscle and adipocytes of these animals (14 -17). In contrast, IRS-2 null mice develop both insulin resistance and beta cell failure, which leads to their death (18). These data implicate different IRS proteins as mediators of insulin action in different tissues.IRS proteins contain over 30 potential Ser/Thr phosphorylation sites for kinases like protein kinase A, PKC, and mitogenactivated protein kinase (3,4,19). In previous studies, we have demonstrated that Ser/Thr phosphorylation of IRS-1 and IRS-2 significantly reduces their ability to interact with the JM region of IR. Such impaired interactions abolish the ability of IRS-1 and IRS-2 to undergo insulin-induced Tyr phosphorylation and further propagate insulin signaling, thus providing a possible molecular mechanism for the induction of an insulinresistant state (20,21). Ser/Thr phosphorylation of IRS pro-

Tumor necrosis factor-␣ (TNF) has been suggested to be the mediator of insulin resistance in infection, tumor cachexia, and obesity. We have previously shown that TNF diminishes insulin-induced tyrosine phosphorylation of insulin receptor substrate 1 (IRS-1). The current work examines potential mechanisms that mediate this event. TNF effect on IRS-1 in Fao hepatoma cells was not associated with a significant reduction in insulin receptor tyrosine kinase activity as measured in vitro but impaired the association of IRS-1 with phosphatidylinositol 3-kinase, localizing TNF impact to IRS-1. TNF did not increase protein-tyrosine phosphatase activity and protein-tyrosine phosphatase inhibition by vanadate did not change TNF effect on IRS-1 tyrosine phosphorylation, suggesting that protein-tyrosine phosphatases are not involved in this TNF effect. In contrast, TNF increased IRS-1 phosphorylation on serine residues, leading to a decrease in its electrophoretic mobility. TNF effect on IRS-1 tyrosine phosphorylation was not abolished by inhibiting protein kinase C using staurosporine, while inactivation of Ser/Thr phosphatases by calyculin A and okadaic acid mimicked it. Our data suggest that TNF induces serine phosphorylation of IRS-1 through inhibition of serine phosphatases or activation of serine kinases other than protein kinase C. This increased serine phosphorylation interferes with insulin-induced tyrosine phosphorylation of IRS-1 and impairs insulin action.

Tumor necrosis factor ␣ (TNF␣) or chronic hyperinsulinemia that induce insulin resistance trigger increased Ser/Thr phosphorylation of the insulin receptor (IR) and of its major insulin receptor substrates, IRS-1 and IRS-2. To unravel the molecular basis for this uncoupling in insulin signaling, we undertook to study the interaction of Ser/Thr-phosphorylated IRS-1 and IRS-2 with the insulin receptor. We could demonstrate that, similar to IRS-1, IRS-2 also interacts with the juxtamembrane (JM) domain (amino acids 943-984) but not with the carboxylterminal region (amino acids 1245-1331) of IR expressed in bacteria as His 6 fusion peptides. Moreover, incubation of rat hepatoma Fao cells with TNF␣, bacterial sphingomyelinase, or other Ser(P)/Thr(P)-elevating agents reduced insulin-induced Tyr phosphorylation of IRS-1 and IRS-2, markedly elevated their Ser(P)/Thr(P) levels, and significantly reduced their ability to interact with the JM region of IR. Withdrawal of TNF␣ for periods as short as 30 min reversed its inhibitory effects on IR-IRS interactions. Similar inhibitory effects were obtained when Fao cells were subjected to prolonged (20 -60 min) pretreatment with insulin. Incubation of the cell extracts with alkaline phosphatase reversed the inhibitory effects of insulin. These findings suggest that insulin resistance is associated with enhanced Ser/Thr phosphorylation of IRS-1 and IRS-2, which impairs their interaction with the JM region of IR. Such impaired interactions abolish the ability of IRS-1 and IRS-2 to undergo insulin-induced Tyr phosphorylation and further propagate the insulin receptor signal. Moreover, the reversibility of the TNF␣ effects and the ability to mimic its action by exogenously added sphingomyelinase argue against the involvement of a proteolytic cascade in mediating the acute inhibitory effects of TNF␣ on insulin action. The insulin receptor (IR)1 is an heterotetrameric transmembrane glycoprotein composed of two extracellular ␣ subunits and two transmembrane ␤ subunits linked by disulfide bonds. The ␣ subunits contain the insulin-binding domain while the transmembrane ␤ subunits function as a tyrosine-specific protein kinase (IRK) that undergoes autophosphorylation following insulin binding (reviewed in Ref. 1). Autophosphorylation activates the IRK (2) and enables it to phosphorylate endogenous protein substrates, including Shc (3) and the insulin receptor substrates IRS-1 (4) and IRS-2 (5), to further propagate the insulin signal. IRS-1 and IRS-2, two related protein substrates of IRK, have a highly conserved amino terminus, which contains a pleckstrin homology domain and a phosphotyrosinebinding (PTB) domain, and a poorly conserved carboxyl terminus with several tyrosine phosphorylation motifs. IRS-1 and IRS-2 also contain over 30 Ser/Thr residues in consensus phosphorylation sites (4, 5). The relative roles of IRS-1 and IRS-2 in mediating insulin action is presently unknown, although IRS-2 functions as an alternative substrate of IR in IRS-1 null mice (6), which manifest a mild form...

Adiponectin is an adipocyte-derived plasma protein with insulin-sensitizing and antiatherosclerotic properties. The aim of this study was to examine whether adiponectin is present in human fetal blood, to define its association with fetal birth weight, and to evaluate whether dynamic changes in adiponectin levels occur during the early neonatal period. Cord blood adiponectin levels were extremely high (71.0 +/- 21.0 microg/ml; n = 51) compared with serum levels in children and adults and positively correlated with fetal birth weights (r = 0.4; P < 0.01). No significant differences in adiponectin levels were found between female and male neonates. In addition, there was no correlation between cord adiponectin levels and maternal body mass index, cord leptin, or insulin levels. Cord adiponectin levels were significantly higher compared with maternal levels at birth (61.1 +/- 19.0 vs. 17.6 +/- 4.9 microg/ml; P < 0.001; n = 17), and no correlation was found between cord and maternal adiponectin levels. There were no significant differences between adiponectin levels at birth and 4 d postpartum (61.1 +/- 19.0 vs. 63.8 +/- 22.0 microg/ml; n = 17). These findings indicate that adiponectin in cord blood is derived from fetal and not from placental or maternal tissues. The high adiponectin levels in newborns compared with adults may be due to lack of negative feedback on adiponectin production resulting from lack of adipocyte hypertrophy, low percentage of body fat, or a different distribution of fat depots in the newborns.