Protein kinase C-zeta phosphorylates insulin receptor substrate-1 and impairs its ability to activate phosphatidylinositol 3-kinase in response to insulin

Protein kinase C-zeta (PKC-zeta) is a serine/threonine kinase downstream from phosphatidylinositol 3-kinase in insulin signaling pathways. However, specific substrates for PKC-zeta that participate in the biological actions of insulin have not been reported. In the present study, we identified insulin receptor substrate-1 (IRS-1) as a novel substrate for PKC-zeta. Under in vitro conditions, wild-type PKC-zeta (but not kinase-deficient mutant PKC-zeta) significantly phosphorylated IRS-1. This phosphorylation was reversed by treatment with the serine-specific phosphatase, protein phosphatase 2A. In addition, the overexpression of PKC-zeta in NIH-3T3(IR) cells caused significant phosphorylation of cotransfected IRS-1 as demonstrated by [(32)P]orthophosphate labeling experiments. In rat adipose cells, endogenous IRS-1 coimmunoprecipitated with endogenous PKC-zeta, and this association was increased 2-fold upon insulin stimulation. Furthermore, the overexpression of PKC-zeta in NIH-3T3(IR) cells significantly impaired insulin-stimulated tyrosine phosphorylation of cotransfected IRS-1. Importantly, this was accompanied by impaired IRS-1-associated phosphatidylinositol 3-kinase activity. Taken together, our results raise the possibility that IRS-1 is a novel physiological substrate for PKC-zeta. Because PKC-zeta is located downstream from IRS-1 and phosphatidylinositol 3-kinase in established insulin signaling pathways, PKC-zeta may participate in negative feedback pathways to IRS-1 similar to those described previously for Akt and GSK-3.     

Modulation of insulin-stimulated degradation of human insulin receptor substrate-1 by Serine 312 phosphorylation

Ser/Thr phosphorylation of insulin receptor substrate-1 (IRS-1) is a negative regulator of insulin signaling. One potential mechanism for this is that Ser/Thr phosphorylation decreases the ability of IRS-1 to be tyrosine-phosphorylated by the insulin receptor. An additional mechanism for modulating insulin signaling is via the down-regulation of IRS-1 protein levels. Insulin-induced degradation of IRS-1 has been well documented, both in cells as well as in patients with diabetes. Ser/Thr phosphorylation of IRS-1 correlates with IRS-1 degradation, yet the details of how this occurs are still unknown. In the present study we have examined the potential role of different signaling cascades in the insulin-induced degradation of IRS-1. First, we found that inhibitors of the phosphatidylinositol 3-kinase and mammalian target of rapamycin block the degradation. Second, knockout cells lacking one of the key effectors of this cascade, the phosphoinositide-dependent kinase-1, were found to be deficient in the insulin-stimulated degradation of IRS-1. Conversely, overexpression of this enzyme potentiated insulin-stimulated IRS-1 degradation. Third, concurrent with the decrease in IRS-1 degradation, the inhibitors of the phosphatidylinositol 3-kinase and mammalian target of rapamycin also blocked the insulin-stimulated increase in Ser(312) phosphorylation. Most important, an IRS-1 mutant in which Ser(312) was changed to alanine was found to be resistant to insulin-stimulated IRS-1 degradation. Finally, an inhibitor of c-Jun N-terminal kinase, SP600125, at 10 microm did not block IRS-1 degradation and IRS-1 Ser(312) phosphorylation yet completely blocked insulin-stimulated c-Jun phosphorylation. Further, insulin-stimulated c-Jun phosphorylation was not blocked by inhibitors of the phosphatidylinositol 3-kinase and mammalian target of rapamycin, indicating that c-Jun N-terminal kinase is unlikely to be the kinase phosphorylating IRS-1 Ser(312) in response to insulin. In summary, our results indicate that the insulin-stimulated degradation of IRS-1 via the phosphatidylinositol 3-kinase pathway is in part dependent upon the Ser(312) phosphorylation of IRS-1.     

Insulin signals to prenyltransferases via the Shc branch of intracellular signaling

We assessed the roles of insulin receptor substrate-1 (IRS-1) and Shc in insulin action on farnesyltransferase (FTase) and geranylgeranyltransferase I (GGTase I) using Chinese hamster ovary (CHO) cells that overexpress wild-type human insulin receptors (CHO-hIR-WT) or mutant insulin receptors lacking the NPEY domain (CHO-DeltaNPEY) or 3T3-L1 fibroblasts transfected with adenoviruses that express the PTB or SAIN domain of IRS-1 and Shc, the pleckstrin homology (PH) domain of IRS-1, or the Src homology 2 (SH2) domain of Shc. Insulin promoted phosphorylation of the alpha-subunit of FTase and GGTase I in CHO-hIR-WT cells, but was without effect in CHO-DeltaNPEY cells. Insulin increased FTase and GGTase I activities and the amounts of prenylated Ras and RhoA proteins in CHO-hIR-WT (but not CHO-DeltaNPEY) cells. Overexpression of the PTB or SAIN domain of IRS-1 (which blocked both IRS-1 and Shc signaling) prevented insulin-stimulated phosphorylation of the FTase and GGTase I alpha-subunit activation of FTase and GGTase I and subsequent increases in prenylated Ras and RhoA proteins. In contrast, overexpression of the IRS-1 PH domain, which impairs IRS-1 (but not Shc) signaling, did not alter insulin action on the prenyltransferases, but completely inhibited the insulin effect on the phosphorylation of IRS-1 and on the activation of phosphatidylinositol 3-kinase and Akt. Finally, overexpression of the Shc SH2 domain completely blocked the insulin effect on FTase and GGTase I activities without interfering with insulin signaling to MAPK. These data suggest that insulin signaling from its receptor to the prenyltransferases FTase and GGTase I is mediated by the Shc pathway, but not the IRS-1/phosphatidylinositol 3-kinase pathway. Shc-mediated insulin signaling to MAPK may be necessary (but not sufficient) for activation of prenyltransferase activity. An additional pathway involving the Shc SH2 domain may be necessary to mediate the insulin effect on FTase and GGTase I.     

Insulin-dependent formation of a complex containing an 85-kDa subunit of phosphatidylinositol 3-kinase and tyrosine-phosphorylated insulin receptor substrate 1.

Monoclonal antibodies raised against the 85-kDa subunit (p85) of bovine phosphatidylinositol (PI) 3-kinase were found to recognize uncomplexed p85 or p85 in the active PI 3-kinase. Immunoprecipitation studies of Chinese hamster ovary cells, which overexpress the human insulin receptor when treated with insulin, showed increased amounts of p85 and PI 3-kinase activity immunoprecipitable with monoclonal anti-p85 antibody and no increase in the tyrosine phosphorylation of p85. Insulin also induced an association of p85 with the tyrosine-phosphorylated insulin receptor substrate 1 (IRS-1) and other phosphorylated proteins ranging in size from 100 to 170 kDa but not with the activated insulin receptor. In vitro reconstitution studies were used to show p85 in the active PI 3-kinase associated with the tyrosine-phosphorylated IRS-1 but not with the activated insulin receptor. Competition studies using synthetic phosphopeptides corresponding to potential tyrosine phosphorylation sites of IRS-1 revealed that phosphopeptides containing YMXM motifs inhibited this association with different potencies, whereas nonphosphorylated analogues and a phosphopeptide containing the EYYE motif had no effect. Src homology region 2 domains of p85 expressed as glutathione S-transferase fusion proteins also bound to tyrosine-phosphorylated IRS-1. These results suggest that insulin causes the association of PI 3-kinase with IRS-1 via phosphorylated YMXM motifs of IRS-1 and Src homology region 2 domains of p85.     

The Drosophila insulin receptor activates multiple signaling pathways but requires insulin receptor substrate proteins for DNA synthesis.

The Drosophila insulin receptor (DIR) contains a 368-amino-acid COOH-terminal extension that contains several tyrosine phosphorylation sites in YXXM motifs. This extension is absent from the human insulin receptor but resembles a region in insulin receptor substrate (IRS) proteins which binds to the phosphatidylinositol (PI) 3-kinase and mediates mitogenesis. The function of a chimeric DIR containing the human insulin receptor binding domain (hDIR) was investigated in 32D cells, which contain few insulin receptors and no IRS proteins. Insulin stimulated tyrosine autophosphorylation of the human insulin receptor and hDIR, and both receptors mediated tyrosine phosphorylation of Shc and activated mitogen-activated protein kinase. IRS-1 was required by the human insulin receptor to activate PI 3-kinase and p70s6k, whereas hDIR associated with PI 3-kinase and activated p70s6k without IRS-1. However, both receptors required IRS-1 to mediate insulin-stimulated mitogenesis. These data demonstrate that the DIR possesses additional signaling capabilities compared with its mammalian counterpart but still requires IRS-1 for the complete insulin response in mammalian cells.     

Phosphorylation of PDE3B by phosphatidylinositol 3-kinase associated with the insulin receptor

Phosphatidylinositol 3-kinase mediates several actions of insulin including its antilipolytic effect. This effect is elicited by the insulin-stimulated serine phosphorylation and activation of cGMP-inhibited phosphodiesterase (PDE3B). In human adipocytes, we found that insulin differentially stimulated phosphatidylinositol 3-kinase activity; the lipid kinase activity was associated with IRS-1, whereas the serine kinase activity was associated with the insulin receptor and phosphorylated a number of proteins including p85, p110, and a 135-kDa protein identified as PDE3B. PDE3B phosphorylation was associated with enzyme activation, thus initiating the antilipolytic effect of insulin. These results show a novel pathway for intracellular signaling through the insulin receptor leading to the serine phosphorylation of key proteins involved in insulin action.     

Sequential phosphorylation of insulin receptor substrate-2 by glycogen synthase kinase-3 and c-Jun NH2-terminal kinase plays a role in hepatic insulin signaling

Serine phosphorylation of insulin receptor substrate (IRS) proteins is a potential inhibitory mechanism in insulin signaling. Here we show that IRS-2 is phosphorylated by glycogen synthase kinase (GSK)-3. Phosphorylation by GSK-3 requires prior phosphorylation of its substrates, prompting us to identify the "priming kinase." It was found that the stress activator anisomycin enhanced the ability of GSK-3 to phosphorylate IRS-2. Use of a selective c-Jun NH(2)-terminal kinase (JNK) inhibitor and cells overexpressing JNK implicated JNK as the priming kinase. This allowed us to narrow down the number of potential GSK-3 phosphorylation sites within IRS-2 to four regions that follow the motif SXXXSP. IRS-2 deletion mutants enabled us to localize the GSK-3 and JNK phosphorylation sites to serines 484 and 488, respectively. Mutation at serine 488 reduced JNK phosphorylation of IRS-2, and mutation of each site separately abolished GSK-3 phosphorylation of IRS-2. Treatment of H4IIE liver cells with anisomycin inhibited insulin-induced tyrosine phosphorylation of IRS-2; inhibition was reversed by pretreatment with the JNK and GSK-3 inhibitors. Moreover, overexpression of JNK and GSK-3 in H4IIE cells reduced insulin-induced tyrosine phosphorylation of IRS-2 and its association with the p85 regulatory subunit of phosphatidylinositol 3-kinase. Finally, both GSK-3 and JNK are abnormally upregulated in the diabetic livers of ob/ob mice. Together, our data indicate that IRS-2 is sequentially phosphorylated by JNK and GSK-3 at serines 484/488 and provide evidence for their inhibitory role in hepatic insulin signaling.     

Insulin and IGF-I signaling through the insulin receptor substrate 1

The insulin and insulin-like growth factor-I (IGF-I) receptors are tyrosine kinases. Consequently, an approach to investigating signaling pathways from these receptors is to characterize proteins rapidly phosphorylated on tyrosine in response to insulin and IGF-I. In many cell types the most prominent phosphotyrosine (Ptyr) protein, in addition to the receptors themselves, is a protein of ?160 kD, now known as the insulin receptor substrate 1 (IRS-1). We have purified IRS-1 from mouse 3T3-L1 adipocytes, obtained the sequences of tryptic peptides, and cloned its cDNA based on this information. Mouse IRS-1 is a protein of 1,231 amino acids. It contains 12 tyrosine residues in sequence contexts typical for tyrosine phosphorylation sites. Six of these begin the sequence motif YMXM and two begin the motif YXXM. Recent studies have shown that the enzyme phosphatidylinositol 3-kinase (PI 3-kinase) binds tightly to the activated platelet-derived growth factor (PDGF) and colony-stimulating factor-1 (CSF-1) receptors, through interaction of the src homology 2 (SH2) domains on the 85 kD subunit of PI 3-kinase with Ptyr in one of these motifs on the receptors. We have found that, upon insulin treatment of 3T3-L1 adipocytes, a portion of the Ptyr form of IRS-1 becomes tightly complexed with PI 3-kinase. Since IRS-1 binds to fusion proteins containing the SH2 domains of PI 3-kinase, association most likely occurs through this domain. The association of IRS-1 with PI 3-kinase activates the enzyme about fivefold. Thus, one signaling pathway from the insulin and IGF-I receptors probably proceeds as follows: tyrosine phosphorylation of IRS-1, tight association of IRS-1 with PI 3-kinase with accompanying activation of the kinase, elevation of the PI 3-phosphates. © 1993 Wiley-Liss, Inc.     

Protein-tyrosine phosphatase-1B negatively regulates insulin signaling in l6 myocytes and Fao hepatoma cells

Insulin signaling is regulated by tyrosine phosphorylation of the signaling molecules, such as the insulin receptor and insulin receptor substrates (IRSs). Therefore, the balance between protein-tyrosine kinases and protein-tyrosine phosphatase activities is thought to be important in the modulation of insulin signaling in insulin-resistant states. We thus employed the adenovirus-mediated gene transfer technique, and we analyzed the effect of overexpression of a wild-type protein-tyrosine phosphatase-1B (PTP1B) on insulin signaling in both L6 myocytes and Fao cells. In both cells, PTP1B overexpression blocked insulin-stimulated tyrosine phosphorylation of the insulin receptor and IRS-1 by more than 70% and resulted in a significant inhibition of the association between IRS-1 and the p85 subunit of phosphatidylinositol 3-kinase and Akt phosphorylation as well as mitogen-activated protein kinase phosphorylation. Moreover, insulin-stimulated glycogen synthesis was also inhibited by PTP1B overexpression in both cells. These effects were specific for insulin signaling, because platelet-derived growth factor (PDGF)-stimulated PDGF receptor tyrosine phosphorylation and Akt phosphorylation were not inhibited by PTP1B overexpression. The present findings demonstrate that PTP1B negatively regulates insulin signaling in L6 and Fao cells, suggesting that PTP1B plays an important role in insulin resistance in muscle and liver.     

Phosphorylation of insulin receptor substrate-1 (IRS-1) by protein kinase B positively regulates IRS-1 function.

Incubation of cells with insulin leads to a transient rise in Tyr phosphorylation of insulin receptor substrate (IRS) proteins, accompanied by elevation in their Ser(P)/Thr(P) content and their dissociation from the insulin receptor (IR). Wortmannin, a phosphatidylinositol 3-kinase inhibitor, selectively prevented the increase in Ser(P)/Thr(P) content of IRS-1, its dissociation from IR, and the decrease in its Tyr(P) content following 60 min of insulin treatment. Four conserved phosphorylation sites within the phosphotyrosine binding/SAIN domains of IRS-1 and IRS-2 served as in vitro substrates for protein kinase B (PKB), a Ser/Thr kinase downstream of phosphatidylinositol 3-kinase. Furthermore, PKB and IRS-1 formed stable complexes in vivo, and overexpression of PKB enhanced Ser phosphorylation of IRS-1. Overexpression of PKB did not affect the acute Tyr phosphorylation of IRS-1; however, it significantly attenuated its rate of Tyr dephosphorylation following 60 min of treatment with insulin. Accordingly, overexpression of IRS-1(4A), lacking the four potential PKB phosphorylation sites, markedly enhanced the rate of Tyr dephosphorylation of IRS-1, while inclusion of vanadate reversed this effect. These results implicate a wortmannin-sensitive Ser/Thr kinase, different from PKB, as the kinase that phosphorylates IRS-1 and acts as the feedback control regulator that turns off insulin signals by inducting the dissociation of IRS proteins from IR. In contrast, insulin-stimulated PKB-mediated phosphorylation of Ser residues within the phosphotyrosine binding/SAIN domain of IRS-1 protects IRS-1 from the rapid action of protein-tyrosine phosphatases and enables it to maintain its Tyr-phosphorylated active conformation. These findings implicate PKB as a positive regulator of IRS-1 functions.     

Action of insulin receptor substrate-3 (IRS-3) and IRS-4 to stimulate translocation of GLUT4 in rat adipose cells

The insulin receptor initiates insulin action by phosphorylating multiple intracellular substrates. Previously, we have demonstrated that insulin receptor substrates (IRS)-1 and -2 can mediate insulin's action to promote translocation of GLUT4 glucose transporters to the cell surface in rat adipose cells. Although IRS-1, -2, and -4 are similar in overall structure, IRS-3 is approximately 50% shorter and differs with respect to sites of tyrosine phosphorylation. Nevertheless, as demonstrated in this study, both IRS-3 and IRS-4 can also stimulate translocation of GLUT4. Rat adipose cells were cotransfected with expression vectors for hemagglutinin (HA) epitope-tagged GLUT4 (GLUT4-HA) and human IRS-1, murine IRS-3, or human IRS-4. Overexpression of IRS-1 led to a 2-fold increase in cell surface GLUT4-HA in cells incubated in the absence of insulin; overexpression of either IRS-3 or IRS-4 elicited a larger increase in cell surface GLUT4-HA. Indeed, the effect of IRS-3 in the absence of insulin was approximately 40% greater than the effect of a maximally stimulating concentration of insulin in cells not overexpressing IRS proteins. Because phosphatidylinositol (PI) 3-kinase is essential for insulin-stimulated translocation of GLUT4, we also studied a mutant IRS-3 molecule (IRS-3-F4) in which Phe was substituted for Tyr in all four YXXM motifs (the phosphorylation sites predicted to bind to and activate PI 3-kinase). Interestingly, overexpression of IRS-3-F4 did not promote translocation of GLUT4-HA, but actually inhibited the ability of insulin to stimulate translocation of GLUT4-HA to the cell surface. Our data suggest that IRS-3 and IRS-4 are capable of mediating PI 3-kinase-dependent metabolic actions of insulin in adipose cells, and that IRS proteins play a physiological role in mediating translocation of GLUT4.     

Insulin receptor substrate-2-dependent interleukin-4 signaling in macrophages is impaired in two models of type 2 diabetes mellitus

We have shown previously that hyperinsulinemia inhibits interferon-alpha-dependent activation of phosphatidylinositol 3-kinase (PI3-kinase) through mammalian target of rapamycin (mTOR)-induced serine phosphorylation of insulin receptor substrate (IRS)-1. Here we report that chronic insulin and high glucose synergistically inhibit interleukin (IL)-4-dependent activation of PI3-kinase in macrophages via the mTOR pathway. Resident peritoneal macrophages (PerMPhis) from diabetic (db/db) mice showed a 44% reduction in IRS-2-associated PI3-kinase activity stimulated by IL-4 compared with PerMPhis from heterozygote (db/+) control mice. IRS-2 from db/db mouse PerMPhis also showed a 78% increase in Ser/Thr-Pro motif phosphorylation without a difference in IRS-2 mass. To investigate the mechanism of this PI3-kinase inhibition, 12-O-tetradecanoylphorbol-13-acetate-matured U937 cells were treated chronically with insulin (1 nm, 18 h) and high glucose (4.5 g/liter, 48 h). In these cells, IL-4-stimulated IRS-2-associated PI3-kinase activity was reduced by 37.5%. Importantly, chronic insulin or high glucose alone did not impact IL-4-activated IRS-2-associated PI3-kinase. Chronic insulin + high glucose did reduce IL-4-dependent IRS-2 tyrosine phosphorylation and p85 association by 54 and 37%, respectively, but did not effect IL-4-activated JAK/STAT signaling. When IRS-2 Ser/Thr-Pro motif phosphorylation was examined, chronic insulin + high glucose resulted in a 92% increase in IRS-2 Ser/Thr-Pro motif phosphorylation without a change in IRS-2 mass. Pretreatment of matured U937 cells with rapamycin blocked chronic insulin + high glucose-dependent IRS-2 Ser/Thr-Pro motif phosphorylation and restored IL-4-dependent IRS-2-associated PI3-kinase activity. Taken together these results indicate that IRS-2-dependent IL-4 signaling in macrophages is impaired in models of type 2 diabetes mellitus through a mechanism that relies on insulin/glucose-dependent Ser/Thr-Pro motif serine phosphorylation mediated by the mTOR pathway.     

Active Rho kinase (ROK-alpha ) associates with insulin receptor substrate-1 and inhibits insulin signaling in vascular smooth muscle cells.

Recent studies from our laboratory have shown that insulin stimulates myosin-bound phosphatase (MBP) in vascular smooth muscle cells (VSMCs) by decreasing site-specific phosphorylation of the myosin-bound subunit (MBS) of MBP via nitric oxide/cGMP-mediated Rho/Rho kinase inactivation. Here we tested potential interactions between Rho kinase and insulin signaling pathways. In control VSMCs, insulin inactivates ROK-alpha, the major Rho kinase isoform in VSMCs, and inhibits thrombin-induced increase in ROK-alpha association with the insulin receptor substrate-1 (IRS-1). Hypertension (in spontaneous hypertensive rats) or expression of an active RhoA(V14) up-regulates Rho kinase activity and increases ROK-alpha/IRS-1 association resulting in IRS-1 serine phosphorylation that leads to inhibition of both insulin-induced IRS-1 tyrosine phosphorylation and phosphatidylinositol 3-kinase (PI3-kinase) activation. In contrast, expression of dominant negative RhoA or cGMP-dependent protein kinase type I alpha inactivates Rho kinase, abolishes ROK-alpha/IRS-1 association, and potentiates insulin-induced tyrosine phosphorylation and PI3-kinase activation leading to decreased MBS(T695) phosphorylation and decreased MBP inhibition. Collectively, these results suggest a novel function for ROK-alpha in insulin signal transduction at the level of IRS-1 and potential cross-talk between cGMP-dependent protein kinase type I alpha, Rho/Rho kinase signaling, and insulin signaling at the level of IRS-1/PI3-kinase.     

In vivo insulin signaling through PI3-kinase is impaired in skeletal muscle of adult rat offspring exposed to ethanol in utero.

It is now known that prenatal ethanol (EtOH) exposure is associated with impaired glucose tolerance and insulin resistance in rat offspring, but the underlying mechanism(s) is not known. To test the hypothesis that in vivo insulin signaling through phosphatidylinositol 3 (PI3)-kinase is reduced in skeletal muscle of adult rat offspring exposed to EtOH in utero, we gave insulin intravenously to these rats and probed steps in the PI3-kinase insulin signaling pathway. After insulin treatment, EtOH-exposed rats had decreased tyrosine phosphorylation of the insulin receptor beta-subunit and of insulin receptor substrate-1 (IRS-1), as well as reduced IRS-1-associated PI3-kinase in the gastrocnemius muscle compared with control rats. There was no significant difference in basal or insulin-stimulated Akt activity between EtOH-exposed rats and controls. Insulin-stimulated PKC isoform zeta phosphorylation and membrane association were reduced in EtOH-exposed rats compared with controls. Muscle insulin binding and peptide contents of insulin receptor, IRS-1, p85 subunit of PI3-kinase, Akt/PKB, and atypical PKC isoform zeta were not different between EtOH-exposed rats and controls. Thus insulin resistance in rat offspring exposed to EtOH in utero may be explained, at least in part, by impaired insulin signaling through the PI3-kinase pathway in skeletal muscle.     

Insulin receptor substrate-1 and phosphoinositide-dependent kinase-1 are required for insulin-stimulated production of nitric oxide in endothelial cells

Vasodilator actions of insulin are mediated by signaling pathways involving phosphatidylinositol 3-kinase (PI 3-kinase) and Akt that lead to activation of endothelial nitric oxide synthase (eNOS) in endothelium. Signaling molecules immediately upstream and downstream from PI 3-kinase involved with production of NO in response to insulin have not been previously identified. In this study, we evaluated roles of insulin receptor substrate 1 (IRS-1) and phosphoinositide-dependent kinase 1 (PDK-1) in production of NO. The fluorescent dye 4,5-diamine fluorescein diacetate was used to directly measure NO in NIH-3T3(IR) cells transiently cotransfected with eNOS and various IRS-1 or PDK-1 constructs. In control cells, transfected with only eNOS, insulin stimulated a rapid dose-dependent increase in NO. Overexpression of wild-type IRS-1 increased the maximal insulin response 3-fold. Overexpression of IRS1-F6 (mutant that does not bind PI 3-kinase) or an antisense ribozyme against IRS-1 substantially inhibited insulin-stimulated production of NO. Likewise, overexpression of wild-type PDK-1 enhanced insulin-stimulated production of NO, whereas a kinase-inactive mutant PDK-1 inhibited this action of insulin. Qualitatively similar results were observed in vascular endothelial cells. Production of NO by a calcium-dependent mechanism in response to lysophosphatidic acid was unaffected by either wild-type or mutant IRS-1 and PDK-1. We conclude that IRS-1 and PDK-1 play necessary roles in insulin-signaling pathways leading to activation of eNOS. Furthermore, classical Ca2+-mediated pathways for activation of eNOS are separable from IRS-1- and PDK-1-dependent insulin-signaling pathways.     

Combination of insulinomimetic agents H2O2 and vanadate enhances insulin receptor mediated tyrosine phosphorylation of IRS-1 leading to IRS-1 association with the phosphatidylinositol 3-kinase

To analyze the mechanism of action of the insulinomimetic agents H₂O₂, vanadate, and pervanadate (H₂O₂ and vanadate), CHO cells or CHO cells that overexpress wild-type or mutant insulin receptor and/or the insulin receptor substrate (IRS-1) were used. H₂O₂ or vanadate treatment alone had little or no effect on tyrosine phosphorylation of cellular proteins; however, pevanadate treatment dramatically enhanced tyrosine phosphorylation of a number of proteins including the insulin receptor and IRS-1. However, the insulin receptor and IRS-1 coimmunoprecipitate from insulin-treated but not from pervanadate-treated cells. Pervanadate-induced tyrosine phosphorylation of the insulin receptor led to an increase in insulin receptor tyrosine kinase activity toward IRS-1 in vivo and IRS-1 peptides in vitro equal to that induced by insulin treatment. Pervanadate-enhanced phosphorylation of IRS-1 led to a fifteenfold increase in IRS-1–associated phosphatidylinositol (Ptdlns) 3-kinase activity. However, insulin receptor–associated Ptdlns 3-kinase activity from pervanadate-treated cells was not detectable, while insulin receptor–associated Ptdlns 3-kinase activity from insulin-treated cells was 20% of the IRS-1-associated activity. Thus, pervanadate but not H₂O₂ or vanadate alone under these conditions mimics many of insulin actions, but pervanadate treatment does not induce insulin receptor/IRS-1 association.     

Met326Ile aminoacid polymorphism in the human p85 alpha gene has no major impact on early insulin signaling in type 2 diabetes.

Class I alpha phosphatidylinositol (PI) 3-kinase is an important enzyme in the early insulin signaling cascade, and plays a key role in insulin-mediated glucose transport. Despite extensive investigation, the genes responsible for the development of the common forms of type 2 diabetes remain unknown. This study was performed to identify variants in the coding region of p85 alpha, the regulatory subunit of PI 3-kinase. Fibroblasts from skin biopsies from type 2 diabetics and controls were established to address this issue. P85 alpha cDNA was sequenced, and a single point mutation at codon 326 was found. This mutation resulted in a homozygous missense amino acid change Met --> Ile in one subject with type 2 diabetes and heterozygous variant in two other diabetic patients and one with severe insulin resistance. Interestingly, those patients revealed an impaired insulin-mediated insulin receptor substrate (IRS)-1 binding to p85 alpha without any alteration in IRS-2/p85 alpha association. Furthermore, IRS-1, IRS-2, p85 alpha and MAPK protein contents were not significantly changed, and neither were MAPK or Akt phosphorylation. We conclude from our data that this variant may have only minor impact on signaling events; however, in combination with variants in other genes encoding signaling proteins, this may have a functional impact on early insulin signaling.     

Binding of IRS proteins to calmodulin is enhanced in insulin resistance

The IRS proteins, major endogenous targets of the insulin receptor, bind to calmodulin in a Ca(2+)-dependent manner. Here, we have examined the interaction between these proteins in animal and cultured cell models of insulin resistance. Both IRS-1 and IRS-2 co-immunoprecipitate with calmodulin from insulin target tissues in rats. The interaction between calmodulin and IRS proteins in rat soleus muscle was enhanced when insulin resistance was induced in rats by treatment with dexamethasone for 5 days. Moreover, injection of angiotensin II into the inferior vena cava enhanced the binding in rat cardiac muscle. Similarly, increased binding between calmodulin and IRS-1 was observed in isolated cells incubated with tumor necrosis factor-alpha. Overexpression of calmodulin in Chinese hamster ovary cells reduced the tyrosine phosphorylation of IRS-1 induced by insulin, with a concomitant decrease in insulin-stimulated association of IRS-1 with the 85-kDa regulatory subunit of phosphatidylinositol 3-kinase. Insulin-stimulated phosphatidylinositol 3-kinase activity associated with IRS-1 was also reduced in cells overexpressing calmodulin, while this activity was increased in cells incubated with the cell-permeable calmodulin antagonist trifluoperazine. These data demonstrate an enhanced interaction between calmodulin and IRS proteins in models of insulin resistance and suggest a possible mechanism by which increased intracellular Ca(2+) concentrations may contribute to impaired insulin sensitivity.     

Up-regulation of phosphatidylinositol 3-kinase and glucose transporter 4 in muscle of rats subjected to maternal undernutrition

Early postnatal nutrition has been associated with the long-term effects on glucose homeostasis in adulthood. To elucidate the molecular mechanisms by which undernutrition during early life leads to changes in insulin sensitivity, we investigated the insulin signaling in skeletal muscle of rats during development. Offspring of dams fed with either protein-free or normal diets during the first 10 days of lactation were studied from lactation period until adulthood. Early maternal undernutrition impaired secretion of insulin but maintained normal blood glucose levels until adulthood. Insulin receptor (IR) activation after insulin stimulation was decreased during the period of protein restriction. In addition, glucose uptake, insulin receptor substrate 1 (IRS-1) phosphorylation and glucose transporter 4 (GLUT-4) translocation in muscle were reduced in response to insulin during suckling. In contrast, non- or insulin-stimulated glucose uptake and GLUT-4 translocation were found significantly increased in muscle of adult offspring. Finally, basal association of phosphatidylinositol 3-kinase (PI3-kinase) with IRS-1 was increased and was highly stimulated by insulin in muscle from adult rats. Our findings suggest that early postnatal undernutrition increases insulin sensitivity in adulthood, which appears to be directly related to changes in critical steps required for glucose metabolism.     

Insulin receptor substrate 3 (IRS-3) and IRS-4 impair IRS-1- and IRS-2-mediated signaling

To investigate the roles of insulin receptor substrate 3 (IRS-3) and IRS-4 in the insulin-like growth factor 1 (IGF-1) signaling cascade, we introduced these proteins into 3T3 embryonic fibroblast cell lines prepared from wild-type (WT) and IRS-1 knockout (KO) mice by using a retroviral system. Following transduction of IRS-3 or IRS-4, the cells showed a significant decrease in IRS-2 mRNA and protein levels without any change in the IRS-1 protein level. In these cell lines, IGF-1 caused the rapid tyrosine phosphorylation of all four IRS proteins. However, IRS-3- or IRS-4-expressing cells also showed a marked decrease in IRS-1 and IRS-2 phosphorylation compared to the host cells. This decrease was accounted for in part by a decrease in the level of IRS-2 protein but occurred with no significant change in the IRS-1 protein level. IRS-3- or IRS-4-overexpressing cells showed an increase in basal phosphatidylinositol 3-kinase activity and basal Akt phosphorylation, while the IGF-1-stimulated levels correlated well with total tyrosine phosphorylation level of all IRS proteins in each cell line. IRS-3 expression in WT cells also caused an increase in IGF-1-induced mitogen-activated protein kinase phosphorylation and egr-1 expression ( approximately 1.8- and approximately 2.4-fold with respect to WT). In the IRS-1 KO cells, the impaired mitogenic response to IGF-1 was reconstituted with IRS-1 to supranormal levels and was returned to almost normal by IRS-2 or IRS-3 but was not improved by overexpression of IRS-4. These data suggest that IRS-3 and IRS-4 may act as negative regulators of the IGF-1 signaling pathway by suppressing the function of other IRS proteins at several steps.