The protein-tyrosine kinase fer associates with signaling complexes containing insulin receptor substrate-1 and phosphatidylinositol 3-kinase

In a screen for 3T3-F442A adipocyte proteins that bind SH2 domains, we isolated a cDNA encoding Fer, a nonreceptor protein-tyrosine kinase of the Fes/Fps family that contains a functional SH2 domain. A truncated splicing variant, iFer, was also cloned. iFer is devoid of both the tyrosine kinase domain and a functional SH2 domain but displays a unique 42-residue C terminus and retains the ability to form oligomers with Fer. Expression of both Fer and iFer proteins are strikingly increased upon differentiation of 3T3-L1 fibroblasts to adipocytes. Platelet-derived growth factor treatment of the cultured adipocytes caused rapid tyrosine phosphorylation of Fer and its recruitment to complexes containing platelet-derived growth factor receptor and the p85 regulatory subunit of phosphatidylinositol (PI) 3-kinase. Insulin treatment of 3T3-L1 adipocytes stimulated association of Fer with complexes containing tyrosine phosphorylated IRS-1 and PI 3-kinase but did not stimulate tyrosine phosphorylation of Fer. PI 3-kinase activity in anti-Fer immunoprecipitates was also acutely activated by insulin treatment of cultured adipocytes. These data demonstrate the presence of Fer tyrosine kinase in insulin signaling complexes, suggesting a role of Fer in insulin action.     

Differences in signaling properties of the cytoplasmic domains of the insulin receptor and insulin-like growth factor receptor in 3T3-L1 adipocytes.

Insulin and insulin-like growth factors (IGFs) elicit distinct but overlapping biological effects in vivo. To investigate whether differences in intrinsic signaling capacity of receptors contribute to biological specificity, we constructed chimeric receptors containing the extracellular portion of the neurotrophin receptor TrkC fused to the intracellular portion of the insulin or IGF-I receptors. Chimeras were stably expressed in 3T3-L1 adipocytes at levels comparable to endogenous insulin receptors and were efficiently activated by neurotrophin-3. The wild-type insulin receptor chimera mediated approximately 2-fold greater phosphorylation of insulin receptor substrate 1 (IRS-1), association of IRS-1 with phosphoinositide 3-kinase, stimulation of glucose uptake, and GLUT4 translocation, compared with the IGF-I receptor chimera. In contrast, the IGF-I receptor chimera mediated more effective Shc phosphorylation, association of Shc with Grb2, and activation of mitogen-activated protein kinase compared with the insulin receptor chimera. The two receptors elicited similar activation of protein kinase B, p70S6 kinase, and glycogen synthesis. We conclude that the insulin receptor mediates some aspects of metabolic signaling in adipocytes more effectively than the IGF-I receptor, as a consequence of more efficient phosphorylation of IRS-1 and greater recruitment/activation of phosphoinositide 3-kinase.     

Pertussis toxin-sensitive and insensitive intracellular signalling pathways in undifferentiated 3T3-L1 cells stimulated by insulin converge with phosphatidylinositol 3-kinase upstream of the Ras mitogen-activated protein kinase cascade.

We have previously reported that pertussis toxin (PTX)-sensitive GTP binding protein (G-protein) and phosphatidylinositol 3-kinase (PI 3-K) are involved in adipocyte differentiation of 3T3-L1 cells induced by insulin/dexamethasone/methylisobutyl xanthine. The aim of this study was to examine the effect of PTX on the tyrosine kinase cascade stimulated by insulin acting through insulin-like growth factor-I (IGF-I) receptors in undifferentiated 3T3-L1 cells. A high level of mitogen-activated protein kinase (MAPK) activation was sustained for up to 4 h after insulin treatment, and mobility shifted and tyrosine phosphorylated MAPK was also detected. MAPK kinase activity measured by the incorporation of 32P into kinase-negative recombinant MAPK was enhanced by insulin treatment. We previously discovered that insulin activates Ras and that this is mediated by wortmannin-sensitive PI 3-K. Tyrosine-phosphorylation of IRS-1 and Shc also occurred in response to insulin. Subsequently, we investigated the effects of PTX on the activation of these proteins by insulin. Interestingly, treating 3T3-L1 cells with PTX attenuates the activation by insulin of both the Ras-MAPK cascade and PI 3-K. In contrast, neither tyrosine-phosphorylation of IRS-1 and Shc nor the interaction between IRS-1 and PI 3-K is sensitive to PTX. However, activation of the Ras-MAPK cascade and tyrosine-phosphorylation of Shc by epidermal growth factor are insensitive to PTX. These results indicate that there is another pathway which regulates PI 3-K and Ras-MAPK, independent of the pathway mediated by IGF-I receptor kinase. These findings suggest that in 3T3-L1 fibroblasts, PTX-sensitive G-proteins cross-talk with the Ras-MAPK pathway via PI 3-K by insulin acting via IGF-I receptors.     

Insulin-stimulated oocyte maturation requires insulin receptor substrate 1 and interaction with the SH2 domains of phosphatidylinositol 3-kinase.

Xenopus oocytes from unprimed frogs possess insulin-like growth factor I (IGF-I) receptors but lack insulin and IGF-I receptor substrate 1 (IRS-1), the endogenous substrate of this kinase, and fail to show downstream responses to hormonal stimulation. Microinjection of recombinant IRS-1 protein enhances insulin-stimulated phosphatidylinositol (PtdIns) 3-kinase activity and restores the germinal vesicle breakdown response. Activation of PtdIns 3-kinase results from formation of a complex between phosphorylated IRS-1 and the p85 subunit of PtdIns 3-kinase. Microinjection of a phosphonopeptide containing a pYMXM motif with high affinity for the src homology 2 (SH2) domain of PtdIns 3-kinase p85 inhibits IRS-1 association with and activation of the PtdIns 3-kinase. Formation of the IRS-1-PtdIns 3-kinase complex and insulin-stimulated PtdIns 3-kinase activation are also inhibited by microinjection of a glutathione S-transferase fusion protein containing the SH2 domain of p85. This effect occurs in a concentration-dependent fashion and results in a parallel loss of hormone-stimulated oocyte maturation. These inhibitory effects are specific and are not mimicked by glutathione S-transferase fusion proteins expressing the SH2 domains of ras-GAP or phospholipase C gamma. Moreover, injection of the SH2 domains of p85, ras-GAP, and phospholipase C gamma do not interfere with progesterone-induced oocyte maturation. These data demonstrate that phosphorylation of IRS-1 plays an essential role in IGF-I and insulin signaling in oocyte maturation and that this effect occurs through interactions of the phosphorylated YMXM/YXXM motifs of IRS-1 with SH2 domains of PtdIns 3-kinase or some related molecules.     

Extracellular matrix induced by TGFβ impairs insulin signal transduction in 3T3-L1 preadipose cells

When 3T3-L1 preadipose cells are exposed to transforming growth factor β (TGFβ), they synthesize more extracellular matrix (ECM) and resist differentiation-inducing stimuli. The mechanism by which ECM suppresses adipose cell differentiation (adipogenesis) remains unknown. Since adipogenesis is an insulin/insulin-like growth factor-1 (IGF-1)-dependent process, we investigated whether TGFβ-induced ECM inhibits insulin signaling. When preadipose cells were pretreated overnight with TGFβ, we observed a 75% decrease in insulin-stimulated tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1) compared to that in control cells. Culturing 3T3-L1 preadipose cells on fibronectin, a component of the ECM induced by TGFβ, also inhibited insulin-dependent IRS-1 tyrosine phosphorylation and adipogenesis, supporting a role for ECM in mediating TGFβ's inhibitory effect on insulin signaling. Since the insulin-stimulated association of phosphoinositide (PI) 3-kinase with IRS-1 depends on IRS-1 tyrosine phosphorylation, we measured the presence of the PI 3-kinase 85 kDa regulatory subunit in anti-IRS-1 immunoprecipitates. Following insulin stimulation, PI 3-kinase-IRS-1 association was reduced by 70% in TGFβ pretreated vs. control preadipose cells. However, insulin-stimulated cellular production of PI(3,4,5)P3 was unaltered by TGFβ pretreatment. This suggests that IRS-1-associated p85-type PI 3-kinase may represent a particular subset of total cellular PI 3-kinase that is specifically inhibited by TGFβ. Reduction of insulin-stimulated association of IRS-1 with p85-type PI 3-kinase by TGFβ may be one potential mechanism through which TGFβ blocks 3T3-L1 adipose cell differentiation. J. Cell. Physiol. 175:370–378, 1998. © 1998 Wiley-Liss, Inc.     

Intracellular Localization of Phosphatidylinositide 3-kinase and Insulin Receptor Substrate-1 in Adipocytes: Potential Involvement of a Membrane Skeleton

Phosphatidylinositide (PI) 3-kinase binds to tyrosyl-phosphorylated insulin receptor substrate-1 (IRS-1) in insulin-treated adipocytes, and this step plays a central role in the regulated movement of the glucose transporter, GLUT4, from intracellular vesicles to the cell surface. PDGF, which also activates PI 3-kinase in adipocytes, has no significant effect on GLUT4 trafficking in these cells. We propose that this specificity may be mediated by differential localization of PI 3-kinase in response to insulin versus PDGF activation. Using subcellular fractionation in 3T3-L1 adipocytes, we show that insulin- and PDGF-stimulated PI 3-kinase activities are located in an intracellular high speed pellet (HSP) and in the plasma membrane (PM), respectively. The HSP is also enriched in IRS-1, insulin-stimulated tyrosyl-phosphorylated IRS-1 and intracellular GLUT4-containing vesicles. Using sucrose density gradient sedimentation, we have been able to segregate the HSP into two separate subfractions: one enriched in IRS-1, tyrosyl-phosphorylated IRS-1, PI 3-kinase as well as cytoskeletal elements, and another enriched in membranes, including intracellular GLUT4 vesicles. Treatment of the HSP with nonionic detergent, liberates all membrane constituents, whereas IRS-1 and PI 3-kinase remain insoluble. Conversely, at high ionic strength, membranes remain intact, whereas IRS-1 and PI 3-kinase become freely soluble. We further show that this IRS-1–PI 3-kinase complex exists in CHO cells overexpressing IRS-1 and, in these cells, the cytosolic pool of IRS-1 and PI 3-kinase is released subsequent to permeabilization with Streptolysin-O, whereas the particulate fraction of these proteins is retained. These data suggest that IRS-1, PI 3-kinase, as well as other signaling intermediates, may form preassembled complexes that may be associated with the actin cytoskeleton. This complex must be in close apposition to the cell surface, enabling access to the insulin receptor and presumably other signaling molecules that somehow confer the absolute specificity of insulin signaling in these cells.     

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.     

Hyperosmotic stress inhibits insulin receptor substrate-1 function by distinct mechanisms in 3T3-L1 adipocytes

In 3T3-L1 adipocytes, hyperosmotic stress was found to inhibit insulin signaling, leading to an insulin-resistant state. We show here that, despite normal activation of insulin receptor, hyperosmotic stress inhibits both tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1) and IRS-1-associated phosphoinositide 3 (PI 3)-kinase activity in response to physiological insulin concentrations. Insulin-induced membrane ruffling, which is dependent on PI 3-kinase activation, was also markedly reduced. These inhibitory effects were associated with an increase in IRS-1 Ser307 phosphorylation. Furthermore, the mammalian target of rapamycin (mTOR) inhibitor rapamycin prevented the osmotic shock-induced phosphorylation of IRS-1 on Ser307. The inhibition of mTOR completely reversed the inhibitory effect of hyperosmotic stress on insulin-induced IRS-1 tyrosine phosphorylation and PI 3-kinase activation. In addition, prolonged osmotic stress enhanced the degradation of IRS proteins through a rapamycin-insensitive pathway and a proteasome-independent process. These data support evidence of new mechanisms involved in osmotic stress-induced cellular insulin resistance. Short-term osmotic stress induces the phosphorylation of IRS-1 on Ser307 by an mTOR-dependent pathway. This, in turn, leads to a decrease in early proximal signaling events induced by physiological insulin concentrations. On the other hand, prolonged osmotic stress alters IRS-1 function by inducing its degradation, which could contribute to the down-regulation of insulin action.     

Overexpression of Fer increases the association of tyrosine-phosphorylated IRS-1 with P85 phosphatidylinositol kinase via SH2 domain of Fer in transfected cells

We have reported that the protein-tyrosine kinase Fer is associated with signaling complexes containing insulin receptor substrate-1 (IRS-1) and phosphatidylinositol 3-kinase (PI-3 kinase) in insulin-stimulated 3T3-L1 adipocytes [J. Biol. Chem. 275 (50) (2000) 38995]. We examined the subcellular localization of this complex in 3T3-L1 adipocytes and performed transfection study to know how this complex is formed. Interestingly we have detected that this complex is formed in LDM of insulin-stimulated 3T3-L1 adipocytes, which may be important for specific biological insulin effect. Based on transfection study, we have demonstrated that overexpression of both Fer and IRS-1 can induce Fer/IRS-1/P85 complexes without insulin stimulation and SH2 domain of Fer is essential for this complex. We have also demonstrated that Fer was an efficient substrate for insulin receptor kinase. Taken together, these data suggested that Fer may play a critically important role to form Fer/IRS-1/P85 complex in LDM of insulin-stimulated adipocytes and elicit biological effect through PI-3 kinase activity in LDM.     

Involvement of phosphoinositide 3-kinase in insulin- or IGF-1-induced membrane ruffling.

Insulin, IGF-1 or EGF induce membrane ruffling through their respective tyrosine kinase receptors. To elucidate the molecular link between receptor activation and membrane ruffling, we microinjected phosphorylated peptides containing YMXM motifs or a mutant 85 kDa subunit of phosphoinositide (PI) 3-kinase (delta p85) which lacks a binding site for the catalytic 110 kDa subunit of PI 3-kinase into the cytoplasm of human epidermoid carcinoma KB cells. Both inhibited the association of insulin receptor substrate-1 (IRS-1) with PI 3-kinase in a cell-free system and also inhibited insulin- or IGF-1-induced, but not EGF-induced, membrane ruffling in KB cells. Microinjection of nonphosphorylated analogues, phosphorylated peptides containing the EYYE motif or wild-type 85 kDa subunit (Wp85), all of which did not inhibit the association of IRS-1 with PI 3-kinase in a cell-free system, did not inhibit membrane ruffling in KB cells. In addition, wortmannin, an inhibitor of PI 3-kinase activity, inhibited insulin- or IGF-1-induced membrane ruffling. These results suggest that the association of IRS-1 with PI 3-kinase followed by the activation of PI 3-kinase are required for insulin- or IGF-1-induced, but not for EGF-induced, membrane ruffling.     

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.     

Overexpression of SH2-containing inositol phosphatase 2 results in negative regulation of insulin-induced metabolic actions in 3T3-L1 adipocytes via its 5'-phosphatase catalytic activity

Phosphatidylinositol (PI) 3-kinase plays an important role in various metabolic actions of insulin including glucose uptake and glycogen synthesis. Although PI 3-kinase primarily functions as a lipid kinase which preferentially phosphorylates the D-3 position of phospholipids, the effect of hydrolysis of the key PI 3-kinase product PI 3,4,5-triphosphate [PI(3,4,5)P3] on these biological responses is unknown. We recently cloned rat SH2-containing inositol phosphatase 2 (SHIP2) cDNA which possesses the 5'-phosphatase activity to hydrolyze PI(3,4,5)P3 to PI 3,4-bisphosphate [PI(3,4)P2] and which is mainly expressed in the target tissues of insulin. To study the role of SHIP2 in insulin signaling, wild-type SHIP2 (WT-SHIP2) and 5'-phosphatase-defective SHIP2 (Delta IP-SHIP2) were overexpressed in 3T3-L1 adipocytes by means of adenovirus-mediated gene transfer. Early events of insulin signaling including insulin-induced tyrosine phosphorylation of the insulin receptor beta subunit and IRS-1, IRS-1 association with the p85 subunit, and PI 3-kinase activity were not affected by expression of either WT-SHIP2 or Delta IP-SHIP2. Because WT-SHIP2 possesses the 5'-phosphatase catalytic region, its overexpression marked by decreased insulin-induced PI(3,4,5)P3 production, as expected. In contrast, the amount of PI(3,4,5)P3 was increased by the expression of Delta IP-SHIP2, indicating that Delta IP-SHIP2 functions in a dominant-negative manner in 3T3-L1 adipocytes. Both PI(3,4,5)P3 and PI(3,4)P2 were known to possibly activate downstream targets Akt and protein kinase C lambda in vitro. Importantly, expression of WT-SHIP2 inhibited insulin-induced activation of Akt and protein kinase C lambda, whereas these activations were increased by expression of Delta IP-SHIP2 in vivo. Consistent with the regulation of downstream molecules of PI 3-kinase, insulin-induced 2-deoxyglucose uptake and Glut4 translocation were decreased by expression of WT-SHIP2 and increased by expression of Delta IP-SHIP2. In addition, insulin-induced phosphorylation of GSK-3beta and activation of PP1 followed by activation of glycogen synthase and glycogen synthesis were decreased by expression of WT-SHIP2 and increased by the expression of Delta IP-SHIP2. These results indicate that SHIP2 negatively regulates metabolic signaling of insulin via the 5'-phosphatase activity and that PI(3,4,5)P3 rather than PI(3,4)P2 is important for in vivo regulation of insulin-induced activation of downstream molecules of PI 3-kinase leading to glucose uptake and glycogen synthesis.     

Growth factor receptor-binding protein 10 (Grb10) as a partner of phosphatidylinositol 3-kinase in metabolic insulin action

The regulation of the metabolic insulin response by mouse growth factor receptor-binding protein 10 (Grb10) has been addressed in this report. We find mouse Grb10 to be a critical component of the insulin receptor (IR) signaling complex that provides a functional link between IR and p85 phosphatidylinositol (PI) 3-kinase and regulates PI 3-kinase activity. This regulatory mechanism parallels the established link between IR and p85 via insulin receptor substrate (IRS) proteins. A direct association was demonstrated between Grb10 and p85 but was not observed between Grb10 and IRS proteins. In addition, no effect of mouse Grb10 was observed on the association between IRS-1 and p85, on IRS-1-associated PI 3-kinase activity, or on insulin-mediated activation of IR or IRS proteins. A critical role of mouse Grb10 was observed in the regulation of PI 3-kinase activity and the resulting metabolic insulin response. Dominant-negative Grb10 domains, in particular the SH2 domain, eliminated the metabolic response to insulin in differentiated 3T3-L1 adipocytes. This was consistently observed for glycogen synthesis, glucose and amino acid transport, and lipogenesis. In parallel, the same metabolic responses were substantially elevated by increased levels of Grb10. A similar role of Grb10 was confirmed in mouse L6 cells. In addition to the SH2 domain, the Pro-rich amino-terminal region of Grb10 was implicated in the regulation of PI 3-kinase catalytic activity. These regulatory roles of Grb10 were extended to specific insulin mediators downstream of PI 3-kinase including PKB/Akt, glycogen synthase kinase, and glycogen synthase. In contrast, a regulatory role of Grb10 in parallel insulin response pathways including p70 S6 kinase, ubiquitin ligase Cbl, or mitogen-activated protein kinase p38 was not observed. The dissection of the interaction of mouse Grb10 with p85 and the resulting regulation of PI 3-kinase activity should help elucidate the complexity of the IR signaling mechanism.     

Role of IRS-3 in the insulin signaling of IRS-1-deficient brown adipocytes

Insulin receptor substrate-1 (IRS-1) plays an essential role in mediating the insulin signals that trigger mitogenesis, lipid synthesis, and uncoupling protein-1 gene expression in mouse brown adipocytes. Expression of IRS-3 is restricted mainly to white adipose tissue; expression of this IRS protein is virtually absent in brown adipocytes. We have tested the capacity of IRS-3 to mediate insulin actions in IRS-1-deficient brown adipocytes. Thus, we expressed exogenous IRS-3 in immortalized IRS-1-/- brown adipocytes at a level comparable with that of endogenous IRS-3 in white adipose tissue. Under these conditions, IRS-3 signaling in response to insulin was observed, as revealed by tyrosine phosphorylation of IRS-3, and the activation of phosphatidylinositol (PI) 3-kinase associated with this recombinant protein. However, although insulin promoted the association of Grb-2 with recombinant IRS-3 in IRS-1-/- cells, the exogenous expression of this IRS family member failed to activate p42/44 MAPK and mitogenesis in brown adipocytes lacking IRS-1. Downstream of PI 3-kinase, IRS-3 expression restored insulin-induced Akt phosphorylation, which is impaired by the lack of IRS-1 signaling. Whereas the generation of IRS-3 signals enhanced adipocyte determination and differentiation-dependent factor 1/sterol regulatory element-binding protein (ADD-1/SREBP-1c) and fatty acid synthase mRNA and protein expression, activation of this pathway was unable to reconstitute CCAAT/enhancer-binding protein alpha and uncoupling protein-1 transactivation and gene expression in response to insulin. Similar results were obtained following insulin-like growth factor-I stimulation. In brown adipocytes expressing the IRS-3F4 mutant, the association of the p85alpha regulatory subunit via Src homology 2 binding was lost, but insulin nevertheless induced PI 3-kinase activity and Akt phosphorylation in a wortmannin-dependent manner. In contrast, activation of IRS-3F4 signaling failed to restore the induction of ADD-1/SREBP-1c and fatty acid synthase gene expression in IRS-1-deficient brown adipocytes. These studies demonstrate that recombinant IRS-3 may reconstitute some, but not all, of the signals required for insulin action in brown adipocytes. Thus, our data further implicate a unique role for IRS-1 in triggering insulin action in brown adipocytes.     

Accumulation of endogenous methylglyoxal impaired insulin signaling in adipose tissue of fructose-fed rats

Increased accumulation of methylglyoxal (MG) has been linked to different insulin resistance states including diabetes and hypertension. In this study, the effects of MG on insulin signaling pathway were investigated. Following 9 weeks of fructose treatment, an insulin resistance state was developed in Sprague-Dawley (SD) rats, demonstrated as increased triglyceride and insulin levels, high blood pressure, and decreased insulin-stimulated glucose uptake by adipose tissue. More importantly, we observed a close correlation between the development of insulin resistance and elevated MG level in serum and adipose tissue. Both insulin resistance state and the elevated MG level were reversed by the MG scavenger, N-acetyl cysteine (NAC). When 3T3-L1 adipocytes were treated directly with MG, the impaired insulin signaling was also observed, indicated by decreased insulin-induced insulin-receptor substrate-1 (IRS-1) tyrosine phosphorylation and the decreased kinase activity of phosphatidylinositol (PI) 3-kinase (PI3K). The ability of NAC to block MG-impairment of PI3K activity and IRS-1 phosphorylation further confirmed the role of MG in the development of insulin resistance. In conclusion, the increase in endogenous MG accumulation impairs insulin-signaling pathway and decreases insulin-stimulated glucose uptake in adipose tissue, which may contribute to the development of insulin resistance.     

Insulin receptor substrates form high-molecular-mass complexes that modulate their availability to insulin/insulin-like growth factor-I receptor tyrosine kinases

Insulin receptor substrates (IRSs) are phosphorylated by activated insulin/insulin-like growth factor (IGF)-I receptor tyrosine kinases. Phosphotyrosyl IRSs are recognized by signaling molecules possessing src homology region 2 (SH2) domains, which mediate various insulin/IGF bioactivities. However, we have shown that IRSs are also associated with other proteins by a phosphotyrosine-independent mechanism. Here, we demonstrated that IRSs form high-molecular-mass complexes (we named these complexes IRSomes) with various proteins and we elucidated their possible roles. Blue native-polyacrylamide gel electrophoresis of cell lysates revealed IRSome formation. Some proteins associated with IRSs in IRS-isoform-, cell-type-, or stimulus-specific manners. Results of the in vitro tyrosine phosphorylation assay indicated that tyrosine phosphorylation of IRS-1 by insulin receptor was decreased when IRS-1 was contained in IRSomes prepared from 3T3-L1 adipocytes treated with TNF-α. Also, tyrosine phosphorylation of IRS-2 by IGF-I receptor was increased when IRS-2 was contained in IRSomes prepared from FRTL-5 thyrocytes treated with dibutyryl cAMP. These results demonstrated that cytokine/hormone-induced formation of IRSomes modulates availability of IRSs to receptor tyrosine kinases.     

Adenovirus-mediated overexpression of IRS-1 interacting domains abolishes insulin-stimulated mitogenesis without affecting glucose transport in 3T3-L1 adipocytes.

Activated insulin receptor (IR) interacts with its substrates, IRS-1, IRS-2, and Shc via the NPXY motif centered at Y960. This interaction is important for IRS-1 phosphorylation. Studies using the yeast two-hybrid system and sequence identity analysis between IRS-1 and IRS-2 have identified two putative elements, the PTB and SAIN domains, between amino acids 108 and 516 of IRS-1 that are sufficient for receptor interaction. However, their precise function in mediating insulin's bioeffects is not understood. We expressed the PTB and SAIN domains of IRS-1 in HIRcB fibroblasts and 3T3-L1 adipocytes utilizing replication-defective adenoviral infection to investigate their role in insulin signalling. In both cell types, overexpression of either the PTB or the SAIN protein caused a significant decrease in insulin-induced tyrosine phosphorylation of IRS-1 and Shc proteins, IRS-1-associated phosphatidylinositol 3-kinase (PI 3-K) enzymatic activity, p70s6k activation, and p44 and p42 mitogen-activated protein kinase (MAPK) phosphorylation. However, epidermal growth factor-induced Shc and MAPK phosphorylation was unaffected by the overexpressed proteins. These findings were associated with a complete inhibition of insulin-stimulated cell cycle progression. In 3T3-L1 adipocytes, PTB or SAIN expression extinguished IRS-1 phosphorylation with a corresponding 90% decrease in IRS-1-associated PI 3-K activity. p70s6k is a downstream target of PI 3-K, and insulin-stimulated p70s6k was inhibited by PTB or SAIN expression. Interestingly, overexpression of either PTB or SAIN protein did not affect insulin-induced AKT activation or insulin-stimulated 2-deoxyglucose transport, even though both of these bioeffects are inhibited by wortmannin. Thus, interference with the IRS-1-IR interaction inhibits insulin-stimulated IRS-1 and Shc phosphorylation, PI 3-K enzymatic activity, p70s6k activation, MAPK phosphorylation and cell cycle progression. In 3T3-L1 adipocytes, interference with the IR-IRS-1 interaction did not cause inhibition of insulin-stimulated AKT activation or glucose transport. These results indicate a bifurcation or subcompartmentalization of the insulin signalling pathway whereby some targets of PI 3-K (i.e., p70s6k) are dependent on IRS-1-associated PI 3-K and other targets (i.e., AKT and glucose transport) are not. IR-IRS-1 interaction is not essential for insulin's effect on glucose transport, and alternate, or redundant, pathways exist in these cells.     

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.     

Phosphatidylinositol 3-Kinase (PI3K) Activity Bound to Insulin-like Growth Factor-I (IGF-I) Receptor, which Is Continuously Sustained by IGF-I Stimulation, Is Required for IGF-I-induced Cell Proliferation

Continuous stimulation of cells with insulin-like growth factors (IGFs) in G(1) phase is a well established requirement for IGF-induced cell proliferation; however, the molecular components of this prolonged signaling pathway that is essential for cell cycle progression from G(1) to S phase are unclear. IGF-I activates IGF-I receptor (IGF-IR) tyrosine kinase, followed by phosphorylation of substrates such as insulin receptor substrates (IRS) leading to binding of signaling molecules containing SH2 domains, including phosphatidylinositol 3-kinase (PI3K) to IRS and activation of the downstream signaling pathways. In this study, we found prolonged (>9 h) association of PI3K with IGF-IR induced by IGF-I stimulation. PI3K activity was present in this complex in thyrocytes and fibroblasts, although tyrosine phosphorylation of IRS was not yet evident after 9 h of IGF-I stimulation. IGF-I withdrawal in mid-G(1) phase impaired the association of PI3K with IGF-IR and suppressed DNA synthesis the same as when PI3K inhibitor was added. Furthermore, we demonstrated that Tyr(1316)-X-X-Met of IGF-IR functioned as a PI3K binding sequence when this tyrosine is phosphorylated. We then analyzed IGF signaling and proliferation of IGF-IR(-/-) fibroblasts expressing exogenous mutant IGF-IR in which Tyr(1316) was substituted with Phe (Y1316F). In these cells, IGF-I stimulation induced tyrosine phosphorylation of IGF-IR and IRS-1/2, but mutated IGF-IR failed to bind PI3K and to induce maximal phosphorylation of GSK3β and cell proliferation in response to IGF-I. Based on these results, we concluded that PI3K activity bound to IGF-IR, which is continuously sustained by IGF-I stimulation, is required for IGF-I-induced cell proliferation.