Src homology 2 domains of protein tyrosine phosphatase are associated in vitro with both the insulin receptor and insulin receptor substrate-1 via different phosphotyrosine motifs

To clarify the role of protein tyrosine phosphatase containing Src homology 2 (SH2) regions on insulin signaling, we investigated the interactions among the insulin receptor, a pair of SH2 domains of SH-PTP2 coupled to glutathione-S-transferase (GST) and insulin receptor substrate-1 (IRS-1)-GST fusion proteins (amino-portion, IRS-1N; carboxyl portion, IRS-1C). GST-SH2 protein of SH-PTP2 bound to the wild type insulin receptor, but not to that with a carboxyl-terminal mutation (Y/F2). Furthermore, even though Y/F2 receptors were used, the SH2 protein was also co-immunoprecipitated with IRS-1C, but not with IRS-1N. These results indicate that SH2 domains of SH-PTP2 can directly associate with the Y¹³²²TXM motif on the carboxyl terminus of insulin receptors and also may bind to the carboxyl portion of IRS-1, possibly via the V¹¹⁷²IDL motif in vitro.     

Effect of insulin nitration by peroxynitrite on its biological activity

Insulin may be a potential target of ONOO- during conditions involving accelerated rates of oxygen radical and nitric oxide generation in pancreatic islet beta-cells. To evaluate the effect of protein tyrosine nitration on insulin function, the porcine insulin was nitrated by ONOO-. Insulin nitration was confirmed by spectral changes, Native-PAGE, and mass spectrometry. The bioassay results show that mono-nitro-insulin affected its receptor binding and hypoglycemic capacities to a certain extent, but still hold high biological activities.     

Bidirectional regulation of insulin receptor autophosphorylation and kinase activity by peroxynitrite

Accumulating evidence suggests that enhanced peroxynitrite formation occurs during diabetes. This report describes the effect of peroxynitrite on insulin receptor (IR) function. Addition of peroxynitrite to purified IR resulted in concentration-dependent tyrosine nitration and thiol oxidation. Interestingly, the basal and insulin-stimulated IR autophosphorylation and tyrosine kinase activity were upregulated at low peroxynitrite concentrations, but downregulated at high peroxynitrite concentrations. Concomitantly, peroxynitrite dramatically reduced ¹²⁵I-insulin binding capacity and phosphotyrosine phosphatase activity of IR preparations. Moreover, SIN-1 administration decreased blood glucose levels in normal mice via upregulation of IR/IRS-1 tyrosine phosphorylation. In contrast, SIN-1 markedly increased blood glucose levels in diabetic mice concomitant with downregulation of IR/IRS-1 tyrosine phosphorylation. Taken together, these data provide new insights regarding how peroxynitrite influences IR function in vitro and in vivo, suggesting that peroxynitrite plays a dual role in regulation of IR autophosphorylation and tyrosine kinase activity, and SIN-1 has hyperglycemic effect in diabetic mice.     

Modulation of lysozyme function and degradation after nitration with peroxynitrite

Background

Peroxynitrite (PN) is formed from superoxide and nitric oxide, both of which are increased during hepatic ethanol metabolism. Peroxynitrite forms adducts with proteins, causing structural and functional alterations. Here, we investigated PN-induced alterations in lysozyme structure and function, and whether they altered the protein's susceptibility to proteasome-catalyzed degradation.

Methods

Hen egg lysozyme was nitrated using varying amounts of either PN or the PN donor, 3-morpholinosydnonimine (SIN-1). The activity, nitration status and the susceptibility of lysozyme to proteasome-catalyzed degradation were assessed.

Results

Lysozyme nitration by PN or SIN-1 caused dose-dependent formation of 3-nitrotyrosine-lysozyme adducts, causing decreased catalytic activity, and enhanced susceptibility to degradation by the 20S proteasome. Kinetic analyses revealed an increased affinity by the 20S proteasome toward nitrated lysozyme compared with the native protein.

Conclusion

Lysozyme nitration enhances the affinity of the modified enzyme for degradation by the proteasome, thereby increasing its susceptibility to proteolysis.

General significance

Increased levels of peroxynitrite have been detected in tissues of ethanol-fed animals. The damaging effects from excessive peroxynitrite in the cell increase hepatotoxicity and cellular death by protein modification due to nitration. Cellular defenses against such changes include enhanced proteolysis by the proteasome in order to maintain protein quality control.     

Discoidin domain receptor 2 impairs insulin-stimulated insulin receptor substrate-1 tyrosine phosphorylation and glucose uptake in 3T3-L1 adipocytes.

Differentiation of preadipocytes into functional adipocytes depends on early proliferative events (mitotic clonal expansion) and extracellular matrix interactions. We report that discoidin domain receptor (DDR) 2, a novel adhesion receptor, is expressed in 3T3-L1 preadipocytes and is downregulated during the early phase of adipogenesis. DDR2 overexpression (DDR2-L1 preadipocytes) reduced subconfluent proliferation by 56% (p<0.001) and insulin-stimulated tyrosine phosphorylation of insulin receptor substrate (IRS)-1 by 34% (p<0.05). The mitotic clonal expansion phase of differentiating confluent DDR2-L1 preadipocytes was impaired by approximately 25% (p<0.05). Although induction of peroxisome proliferator-activated receptor gamma, fatty acid synthase, and adiponectin was not altered, the resulting adipocytes were 55% larger (p<0.05), and contained 66% more triacylglycerol (p<0.01). The induction of CCAAT/enhancer binding protein alpha was reduced by 37% (p<0.05), correlating with a similar reduction in insulin-stimulated IRS-1 tyrosine phosphorylation and glucose transport in DDR2-L1 adipocytes (decreases of 22% and 27%, respectively; p<0.05 for both). Our data show that DDR2 is expressed in adipose cells and that its overexpression leads to insulin resistance.     

Polycystic ovary syndrome is associated with genetic polymorphism in the insulin signaling gene IRS-1 but not ENPP1 in a Japanese population

Recent studies indicate that insulin resistance resulting from altered post-receptor signaling is associated with polycystic ovary syndrome (PCOS). We hypothesized that insulin receptor substrate-1 (IRS-1) Gly972Arg polymorphism and/or ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) Lys121Gln polymorphism predisposes women to PCOS and that these polymorphisms also affect anthropometric variables, glucose metabolism and androgen synthesis. To test those ideas, we studied the genotypes, indexes of insulin resistance, and hormone profiles in 123 Japanese women with PCOS and 380 healthy Japanese controls. We found that there were significantly more IRS-1 972Arg carriers among the PCOS patients than among the healthy controls (10.6% vs. 4.8%, p=0.029), which is consistent with our finding that women carrying the IRS-1 972Arg allele had a significantly increased risk of developing PCOS (odds ratio: 3.31, 95% confidence interval: 1.49-7.35). By contrast, the ENPP1 Lys121Arg polymorphism was distributed equally among PCOS patients and controls. In addition, neither of these polymorphisms studied affected the anthropometric variables, metabolic parameters or androgen levels of women with PCOS. We conclude that the IRS-1 Gly972Arg polymorphism is associated with PCOS in the Japanese population.     

Glucose-modulated tyrosine nitration in beta cells: Targets and consequences

Hyperglycemia, key factor of the pre-diabetic and diabetic pathology, is associated with cellular oxidative stress that promotes oxidative protein modifications. We report that protein nitration is responsive to changes in glucose concentrations in islets of Langerhans and insulinoma beta cells. Alterations in the extent of tyrosine nitration as well as the cellular nitroproteome profile correlated tightly with changing glucose concentrations. The target proteins we identified function in protein folding, energy metabolism, antioxidant capacity, and membrane permeability. Nitration of heat shock protein 60 in vitro was found to decrease its ATP hydrolysis and interaction with proinsulin, suggesting a mechanism by which protein nitration could diminish insulin secretion. This was supported by our finding of a decrease in stimulated insulin secretion following glycolytic stress in cultured cells. Our results reveal that protein tyrosine nitration may be a previously unrecognized factor in beta-cell dysfunction and the pathogenesis of diabetes.     

Blockade of IRS1 in isolated rat pancreatic islets improves glucose-induced insulin secretion

Several neural, hormonal and biochemical inputs actively participate in the balance of insulin secretion induced by blood glucose fluctuations. The exact role of insulin as an autocrine and paracrine participant in the control of its own secretion remains to be determined, mostly due to insufficient knowledge about the molecular phenomena that govern insulin signaling in pancreatic islets. In the present experiments we demonstrate that higher insulin receptor and insulin receptor substrates-1 and -2 (IRS1 and IRS2) concentrations are predominantly encountered in cells of the periphery of rat pancreatic islets, as compared to centrally located cells, and that partial blockade of IRS1 protein expression by antisense oligonucleotide treatment leads to improved insulin secretion induced by glucose overload, which is accompanied by lower steady-state glucagon secretion and blunted glucose-induced glucagon fall. These data reinforce the inhibitory role of insulin upon its own secretion in isolated, undisrupted pancreatic islets.     

Insulin renders diabetic rats resistant to acute ischemic stroke by arresting nitric oxide reaction with superoxide to form peroxynitrite

Background

The functions of free radicals on the effects of insulin that result in protection against cerebral ischemic insult in diabetes remain undefined. This present study aims to explain the contradiction among nitric oxide (NO)/superoxide/peroxynitrite of insulin in amelioration of focal cerebral ischemia–reperfusion (FC I/R) injury in streptozotocin (STZ)-diabetic rats and to delineate the underlying mechanisms. Long-Evans male rats were divided into three groups (age-matched controls, diabetic, and diabetic treated with insulin) with or without being subjected to FC I/R injury.

Results

Hyperglycemia exacerbated microvascular functions, increased cerebral NO production, and aggravated FC I/R-induced cerebral infarction and neurological deficits. Parallel with hypoglycemic effects, insulin improved microvascular functions and attenuated FC I/R injury in STZ-diabetic rats. Diabetes decreased the efficacy of NO and superoxide production, but NO and superoxide easily formed peroxynitrite in diabetic rats after FC I/R injury. Insulin treatment significantly rescued the phenomenon.

Conclusions

These results suggest that insulin renders diabetic rats resistant to acute ischemic stroke by arresting NO reaction with superoxide to form peroxynitrite.     

Chemerin enhances insulin signaling and potentiates insulin-stimulated glucose uptake in 3T3-L1 adipocytes

To explore a novel adipokine, we screened adipocyte differentiation-related gene and found that TIG2/chemerin was strongly induced during the adipocyte differentiation. Chemerin was secreted by the mature 3T3-L1 adipocytes and expressed abundantly in adipose tissue in vivo as recently described. Intriguingly, the expression of chemerin was differently regulated in the liver and adipose tissue in db/db mice. In addition, serum chemerin concentration was decreased in db/db mice. Chemerin and its receptor/ChemR23 were expressed in mature adipocytes, suggesting its function in autocrine/paracrine fashion. Finally, chemerin potentiated insulin-stimulated glucose uptake concomitant with enhanced insulin signaling in the 3T3-L1 adipocytes. These data establish that chemerin is a novel adipokine that regulates adipocyte function.     

Peptidyl-prolyl cis/trans isomerase NIMA-interacting 1 associates with insulin receptor substrate-1 and enhances insulin actions and adipogenesis

Peptidyl-prolyl cis/trans isomerase NIMA-interacting 1 (Pin1) is a unique enzyme that associates with the pSer/Thr-Pro motif and catalyzes cis-trans isomerization. We identified Pin1 in the immunoprecipitates of overexpressed IRS-1 with myc and FLAG tags in mouse livers and confirmed the association between IRS-1 and Pin1 by not only overexpression experiments but also endogenously in the mouse liver. The analysis using deletion- and point-mutated Pin1 and IRS-1 constructs revealed the WW domain located in the N terminus of Pin1 and Ser-434 in the SAIN (Shc and IRS-1 NPXY binding) domain of IRS-1 to be involved in their association. Subsequently, we investigated the role of Pin1 in IRS-1 mediation of insulin signaling. The overexpression of Pin1 in HepG2 cells markedly enhanced insulin-induced IRS-1 phosphorylation and its downstream events: phosphatidylinositol 3-kinase binding with IRS-1 and Akt phosphorylation. In contrast, the treatment of HepG2 cells with Pin1 siRNA or the Pin1 inhibitor Juglone suppressed these events. In good agreement with these in vitro data, Pin1 knock-out mice exhibited impaired insulin signaling with glucose intolerance, whereas adenoviral gene transfer of Pin1 into the ob/ob mouse liver mostly normalized insulin signaling and restored glucose tolerance. In addition, it was also demonstrated that Pin1 plays a critical role in adipose differentiation, making Pin1 knock-out mice resistant to diet-induced obesity. Importantly, Pin1 expression was shown to be up-regulated in accordance with nutrient conditions such as food intake or a high-fat diet. Taken together, these observations indicate that Pin1 binds to IRS-1 and thereby markedly enhances insulin action, essential for adipogenesis.     

Synergistic depletion of astrocytic glutathione by glucose deprivation and peroxynitrite: correlation with mitochondrial dysfunction and subsequent cell death

Previously we reported that immunostimulated astrocytes were highly vulnerable to glucose deprivation. The augmented death was mimicked by the peroxynitrite (ONOO )-producing reagent 3-morpholinosydnonimine (SIN-1). Here we show that glucose deprivation and ONOO- synergistically deplete intracellular reduced glutathione (GSH) and augment the death of astrocytes via formation of cyclosporin A-sensitive mitochondrial permeability transition (MPT) pore. Astrocytic GSH levels were only slightly decreased by glucose deprivation or SIN-1 (200 microM) alone. In contrast, a rapid and large depletion of GSH was observed in glucose-deprived/ SIN-1-treated astrocytes. The depletion of GSH occurred before a significant release of lactate dehydrogenase (a marker of cell death). Superoxide dismutase and ONOO-scavengers completely blocked the augmented death, indicating that the reaction of nitric oxide with superoxide to form ONOO was implicated. Furthermore, nitrotyrosine immunoreactivity (a marker of ONOO-) was markedly enhanced in glucose-deprived/SIN-1 -treated astrocytes. Mitochondrial transmembrane potential (MTP) was synergistically decreased in glucose-deprived/SIN-1-treated astrocytes. The glutathione synthase inhibitor L-buthionine-(S,R)-sulfoximine markedly decreased the MTP and increased lactate dehydrogenase (LDH) releases in SIN-1-treated astrocytes. Cyclosporin A, an MPT pore blocker, completely prevented the MTP depolarization as well as the enhanced LDH releases in glucose-deprived/SIN-1-treated astrocytes.     

Inhibition of uncoupling protein 2 by genipin reduces insulin-stimulated glucose uptake in 3T3-L1 adipocytes

Uncoupling protein 2 (UCP2) was reported to be involved in insulin-glucose homeostasis, based on well established event that inhibition of UCP2 stimulates insulin secretion in pancreatic β-cells. However, the role of UCP2 on insulin-stimulated glucose uptake in adipose tissue, which is an indispensable process in insulin-glucose homeostasis, remains unknown. In this study, UCP2 was inhibited by genipin in 3T3-L1 adipocytes, which increased mitochondrial membrane potential, intracellular ATP level and production of reactive oxygen species (ROS). Importantly, insulin-stimulated glucose uptake in 3T3-L1 adipocytes was largely impaired in the presence of genipin, and recovered by CCCP, a mitochondrial uncoupler. Furthermore, genipin leaded to suppression of insulin signal transduction through hyperactivation of c-Jun N-terminal kinase (JNK) and subsequent serine phosphorylation of insulin receptor substrate-1 (IRS-1). These results suggest that mitochondrial uncoupling in adipocytes positively regulates insulin-stimulated glucose uptake in adipocytes, and UCP2 may play an important role in insulin resistance.     

Combined use of insulin and endothelin-1 causes decrease of protein expression of beta-subunit of insulin receptor, insulin receptor substrate-1, and insulin-stimulated glucose uptake in rat adipocytes

Previously, we reported that insulin-stimulated glucose uptake (ISGU) can be inhibited by endothelin (ET-1). However, the mechanism by which ET-1 impairs ISGU in adipocytes remains unclear. This study investigated the effects of ET-1 on insulin action in rat adipocytes in order to elucidate the molecular mechanism of action of ET-1 on ISGU. The results show that ISGU was increased fivefold after 3-h treatment with 1 nM insulin. Treatment with 100 nM ET-1 had no effect on basal glucose uptake. However, ET-1 inhibited approximately 25% of ISGU and 20% of insulin binding after 3-h treatment in the presence of 1 nM insulin. Expression of the beta-subunit of the insulin receptor (IRbeta) and the insulin receptor substrate-1 (IRS-1) in adipocytes was not significantly affected by 1 nM insulin or by 100 nM ET-1, even after 3-h treatment. However, expressions of IRbeta and IRS-1 were dramatically decreased in a dose- and time-dependent manner when adipocytes were treated with both insulin and ET-1. Approximately 50% of IRbeta and 65% of IRS-1 expression levels were suppressed when adipocytes were simultaneously treated with both 1 nM insulin and 100 nM ET-1 for 3 h. These results suggest that the inhibitory effect of ET-1 on ISGU may be mediated via the insulin receptor and suppression of IRbeta/IRS-1 expression.     

Phosphorylation of IRS1 at serine 307 and serine 312 in response to insulin in human adipocytes

Feedback control in insulin signaling involves serine phosphorylation of insulin receptor substrate-1 (IRS1). By analyzing the insulin-induced phosphorylation of IRS1 at serine 307, serine 312, and tyrosine in the same primary human adipocytes, we now report that negative feedback phosphorylation of serine 312 (corresponding to murine serine 307) required relatively high concentrations of insulin (EC(50)=3 nM) for a long time (t(1/2) ca. 30 min) and reduced the steady-state tyrosine phosphorylation, without affecting the cellular concentration, of IRS1. In contrast, positive feedback phosphorylation of serine 307 was a rapid (t(1/2) ca. 2 min) event at physiological concentrations of insulin (EC(50)=0.2 nM).     

Free fatty acid-induced muscle insulin resistance and glucose uptake dysfunction: Evidence for PKC activation and oxidative stress-activated signaling pathways

In the present study, we examined the effects of free fatty acids (FFAs) on insulin sensitivity and signaling cascades in the C2C12 skeletal muscle cell culture system. Our data clearly manifested that the inhibitory effects of PKC on insulin signaling may at least in part be explained by the serine/threonine phosphorylation of IRS-1. Both oleate and palmitate treatment were able to increase the Serine³⁰⁷ phosphorylation of IRS-1. IRS-1 Serine³⁰⁷ phosphorylation is inducible which causes the inhibition of IRS-1 tyrosine phosphorylation by either IκB-kinase (IKK) or c-jun N-terminal kinase (JNK) as seen in our proteomic kinases screen. Furthermore, our proteomic data have also manifested that the two FFAs activate the IKKα/β, the stress kinases S6 kinase p70 (p70SK), stress-activated protein kinase (SAPK), JNK, as well as p38 MAP kinase (p38MAPK). On the other hand, the antioxidant, Taurine at 10 mM concentrations was capable of reversing the oleate-induced insulin resistance in myocytes as manifested from the glucose uptake data. Our current data point out the importance of FFA-induced insulin resistance via multiple signaling mechanisms.     

Irreversible inactivation of magnesium-dependent neutral sphingomyelinase 1 (NSM1) by peroxynitrite,a nitric oxide-derived oxidant

Previous results have indicated that the generation of ceramide by hydrolysis of sphingomyelin by magnesium-dependent neutral sphingomyelinase 1 (NSM1) is reversibly inhibited by hydrogen peroxide (H2O2) and oxidized glutathione (GSSG). This redox-dependent reversible regulation of NSM1 activity has been shown to involve the reversible formation and breakage of disulfide bonds. In this paper, we show that peroxynitrite, a nitric oxide-derived oxidant generated by SIN1, inactivates dose-dependently the NSM1 activity in an irreversible manner. In addition, we show that, in contrast to the reversible inhibition of NSM1 by H2O2 or GSSG which involves the formation of disulfide bonds, irreversible inactivation of this enzyme by peroxynitrite generated from SIN1 is likely due to definitive oxidative thiol modification. These results suggest that depending on the nature of the oxidative stress, the enzymatic activity of NSM1 could be reversibly or irreversibly inactivated.