Overexpressing Insulin Receptors Research Articles

Improvement of Lipotoxicity-Induced Islet β Cellular Insulin Secretion Disorder by Osteocalcin.

Background Osteocalcin (OCN) has been proved to be closely related with the development of type 2 diabetes mellitus (T2DM). We aimed to study if OCN could improve the disorder of islet cell caused by lipotoxicity. Methods Alizarin red staining was used to investigate the mineralization. Western blotting and ELISA methods were used to measure protein expression. Immunofluorescence staining was used to investigate the protein nuclear transfer. Results High glucose and high fat inhibited the differentiation of osteoblast precursors. Overexpression of insulin receptor (InsROE) significantly promoted the Runx2 and OCN expression. The increase of insulin, Gprc6a, and Glut2 by osteoblast culture medium overexpressing insulin receptor was reversed by osteocalcin neutralizing antibody. Undercarboxylated osteocalcin (ucOC) suppressed the lipotoxic islet β-cell damage caused by palmitic acid. The FOXO1 from intranuclear to extranuclear was also significantly increased after ucOC treatment compared with the group PA. Knockdown of Gprc6a or suppression of PI3K/AKT signal pathway could reverse the upregulation of GPRC6A/PI3K/AKT/FoxO1/Pdx1 caused by ucOC. Conclusion OCN could activate the FOXO1 signaling pathway to regulate GLUT2 expression and improve the insulin secretion disorder caused by lipotoxicity.

Characterization of viral insulins reveals white adipose tissue-specific effects in mice

ObjectiveMembers of the insulin/insulin-like growth factor (IGF) superfamily are well conserved across the evolutionary tree. We recently showed that four viruses in the Iridoviridae family possess genes that encode proteins highly homologous to human insulin/IGF-1. Using chemically synthesized single-chain (sc), i.e., IGF-1-like, forms of the viral insulin/IGF-1-like peptides (VILPs), we previously showed that they can stimulate human receptors. Because these peptides possess potential cleavage sites to form double chain (dc), i.e., more insulin-like, VILPs, in this study, we have characterized dc forms of VILPs for Grouper iridovirus (GIV), Singapore grouper iridovirus (SGIV) and Lymphocystis disease virus-1 (LCDV-1) for the first time. MethodsThe dcVILPs were chemically synthesized. Using murine fibroblast cell lines overexpressing insulin receptor (IR-A or IR-B) or IGF1R, we first determined the binding affinity of dcVILPs to the receptors and characterized post-receptor signaling. Further, we used C57BL/6J mice to study the effect of dcVILPs on lowering blood glucose. We designed a 3-h dcVILP in vivo infusion experiment to determine the glucose uptake in different tissues. ResultsGIV and SGIV dcVILPs bind to both isoforms of human insulin receptor (IR-A and IR-B) and to the IGF1R, and for the latter, show higher affinity than human insulin. These dcVILPs stimulate IR and IGF1R phosphorylation and post-receptor signaling in vitro and in vivo. Both GIV and SGIV dcVILPs stimulate glucose uptake in mice. In vivo infusion experiments revealed that while insulin (0.015 nmol/kg/min) and GIV dcVILP (0.75 nmol/kg/min) stimulated a comparable glucose uptake in heart and skeletal muscle and brown adipose tissue, GIV dcVILP stimulated 2-fold higher glucose uptake in white adipose tissue (WAT) compared to insulin. This was associated with increased Akt phosphorylation and glucose transporter type 4 (GLUT4) gene expression compared to insulin in WAT. ConclusionsOur results show that GIV and SGIV dcVILPs are active members of the insulin superfamily with unique characteristics. Elucidating the mechanism of tissue specificity for GIV dcVILP will help us to better understand insulin action, design new analogs that specifically target the tissues and provide new insights into their potential role in disease.

Muscle-Specific Insulin Receptor Overexpression Protects Mice From Diet-Induced Glucose Intolerance but Leads to Postreceptor Insulin Resistance.

Skeletal muscle insulin resistance is a prominent early feature in the pathogenesis of type 2 diabetes. In attempt to overcome this defect, we generated mice overexpressing insulin receptors (IR) specifically in skeletal muscle (IRMOE). On normal chow, IRMOE mice have body weight similar to that of controls but an increase in lean mass and glycolytic muscle fibers and reduced fat mass. IRMOE mice also show higher basal phosphorylation of IR, IRS-1, and Akt in muscle and improved glucose tolerance compared with controls. When challenged with high-fat diet (HFD), IRMOE mice are protected from diet-induced obesity. This is associated with reduced inflammation in fat and liver, improved glucose tolerance, and improved systemic insulin sensitivity. Surprisingly, however, in both chow and HFD-fed mice, insulin-stimulated Akt phosphorylation is significantly reduced in muscle of IRMOE mice, indicating postreceptor insulin resistance. RNA sequencing reveals downregulation of several postreceptor signaling proteins that contribute to this resistance. Thus, enhancing early insulin signaling in muscle by overexpression of the IR protects mice from diet-induced obesity and its effects on glucose metabolism. However, chronic overstimulation of this pathway leads to postreceptor desensitization, indicating the critical balance between normal signaling and hyperstimulation of the insulin signaling pathway.

Insulin decreases atherosclerosis by inducing endothelin receptor B expression.

Endothelial cell (EC) insulin resistance and dysfunction, caused by diabetes, accelerates atherosclerosis. It is unknown whether specifically enhancing EC-targeted insulin action can decrease atherosclerosis in diabetes. Accordingly, overexpressing insulin receptor substrate-1 (IRS1) in the endothelia of Apoe-/- mice (Irs1/Apoe-/-) increased insulin signaling and function in the aorta. Atherosclerosis was significantly reduced in Irs1/ApoE-/- mice on diet-induced hyperinsulinemia and hyperglycemia. The mechanism of insulin's enhanced antiatherogenic actions in EC was related to remarkable induction of NO action, which increases endothelin receptor B (EDNRB) expression and intracellular [Ca2+]. Using the mice with knockin mutation of eNOS, which had Ser1176 mutated to alanine (AKI), deleting the only known mechanism for insulin to activate eNOS/NO pathway, we observed that IRS1 overexpression in the endothelia of Aki/ApoE-/- mice significantly decreased atherosclerosis. Interestingly, endothelial EDNRB expression was selectively reduced in intima of arteries from diabetic patients and rodents. However, endothelial EDNRB expression was upregulated by insulin via P13K/Akt pathway. Finally EDNRB deletion in EC of Ldlr-/- and Irs1/Ldlr-/- mice decreased NO production and accelerated atherosclerosis, compared with Ldlr-/- mice. Accelerated atherosclerosis in diabetes may be reduced by improving insulin signaling selectively via IRS1/Akt in the EC by inducing EDNRB expression and NO production.

Preparation and characterization of zedoary turmeric oil–loaded insulin-modified sterically stabilized liposomes

The poor selectivity of anticancer drugs often leads to their multiplicate dose-limiting toxicities in humans, which severely restricts their clinical application. In this study, a novel liposomal formulation of zedoary turmeric oil (ZTO) targeting the insulin receptor (IR) was prepared by covalently conjugating insulin to the terminal of the polyethylene glycol (PEG) chain of sterically stabilized liposomes. In vitro assays indicated that a higher uptake of insulin-modified sterically stabilized liposomes (ISSLs) was observed in SMMC-7721 hepatocarcinoma cells overexpressing insulin receptors. IC50 values of ISSLs, NTLs (nontargeted liposomes), and ZTO injection (free ZTO) against SMMC-7721, determined by MTT assays, were 157.2, 256.7, and 43.3 μg·ml−1, respectively. Plasma-clearance profiles of ZTO in the liposomal formulations were then compared with that of ZTO injection. The liposomal formulations showed much longer terminal half-lives (11.24 and 14.73 hours for ISSLs and NTLs, respectively) than that of ZTO injection (1.45 hours). All results above indicated the ISSLs were potentially useful for the treatment of IR (+) tumors and are worthy of further investigation.

Abstract CN05-03: Energy balance in cancer patients: Are we ready to intervene?

Abstract There is growing evidence that aspects of energy balance, including overweight/obesity and physical inactivity are associated with poor outcomes in some cancers, notably breast and colorectal. In breast cancer, evidence is strongest for overweight/obesity; recent evidence suggests physical activity may have important independent associations with outcomes. In colorectal cancer, evidence is strongest for physical activity. Clinical mechanisms that have been postulated for these associations include presentation of cancer at a more advanced stage or receipt of less than optimal therapy in overweight or obese individuals. Potential biologic mechanisms have included sex hormones, insulin and related IGFs, adipocytokines and inflammatory markers. In breast cancer there is growing evidence that insulin (and associated insulin resistance) may play a key role in mediating these prognostic associations - high insulin levels have been associated with increased risk of breast cancer recurrence or death. Breast cancer cells commonly overexpress insulin receptors (frequently a fetal form of the receptor that may hybridize with the IGF-1 receptor) that are not downregulated by circulating insulin; as a result, cancer cell growth may be stimulated by high circulating insulin levels. Observational clinical studies linking energy balance to prognosis of breast and colorectal cancer will be reviewed, along with early intervention work demonstrating the feasibility, short-term benefits (e.g. on QOL) and biologic (mechanistic) changes associated with lifestyle interventions in breast and colon cancer survivors. The impact of the available evidence on clinical practice and the role of large scale clinical trials of energy balance modification with recurrence or survival endpoints (completed, ongoing and planned) will be reviewed, with an emphasis on issues relating to feasibility, cost and the need for embedded correlative research to investigate biologic mechanisms for any survival effects that are identified. Related metformin trials (which target insulin and AMPK/mTOR signaling), notably NCIC MA.32, an adjuvant trial in early breast cancer that will be activated early in 2010, will also be reviewed. Citation Information: Cancer Prev Res 2010;3(1 Suppl):CN05-03.

Obesity, Insulin Resistance and Insulin.

Abstract There is growing acceptance that overweight and obesity are associated with poor outcomes in early stage breast cancer. This effect has been reported in pre-and postmenopausal women, regardless of hormone receptor status. Postulated mechanisms have included presentation of cancer at a more advanced stage, underdosing of chemotherapy and higher levels of endogenous estrogens in overweight and obese women. Recent research has provided strong evidence that insulin may play a key role in mediating these prognostic effects. Overweight and obesity are associated with insulin resistance (and resulting hyperinsulinemia) in both the general population and in breast cancer patients; both insulin resistance and high insulin levels have been associated with increased risk of breast cancer recurrence or death, independent of traditional prognostic factors. Breast cancer cells commonly overexpress insulin receptors (frequently a fetal form of the receptor that may hybridize with the IGF-1 receptor), providing a biologic basis for a prognostic effect of insulin. These receptors are not downregulated by circulating insulin; as a result, cancer cell growth may be stimulated by high circulating insulin levels, leading to poor prognosis. Preclinical work provides strong evidence for a mitogenic role of insulin in breast cancer. Taken together, these observations have led to the development of lifestyle interventions targeting insulin and/or insulin resistance in breast cancer patients. Interventions include weight loss (which reduces insulin levels in the general population) and physical activity (which has been shown to reduce insulin in breast cancer patients). Randomized trials of weight loss are ongoing and planned. One completed trial (Women's Intervention Nutrition Study, J Natl Cancer Inst 2006;98:1767-1776) reported improved relapse free survival in breast cancer patients randomized to a reduced fat diet (that was associated with significant weight loss). Effects were greatest in hormone receptor negative breast cancer. Recent interest has focused on the use of metformin, a biguanide commonly used to treat type 2 diabetes. Metformin reduces insulin resistance and lowers insulin levels in many populations, including early stage breast cancer patients. In addition to this indirect (insulin lowering) effect, metformin may also exert direct anti-tumor effects via mTOR inhibition. Studies using metformin in the neoadjuvant setting in breast cancer are ongoing. The effect of metformin on breast cancer outcomes (with embedded correlative studies that will examine direct and indirect effects of the drug) will be examined in NCIC MA.32, a randomized trial of metformin versus placebo involving 3582 women with early breast cancer that will be activated in early 2010. Together, these lifestyle and pharmacologic studies will provide important information on the effect of modification of a key host factor (obesity with associated hyperinsulinemia) on breast cancer outcomes. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr MS2-1.

Spontaneous hypoglycaemia in the presence of both anti-insulin antibody and anti-insulin receptor antibody

Beside insulinoma, alternative causes of hyperinsulinaemic hypoglycaemia include the rare autoimmune syndrome related to spontaneous autoantibodies either to insulin or to insulin receptor. We describe a case of hypoglycaemia with high insulinemia in which insulinoma could not be evidenced. Surprisingly, we found in the patient's serum both insulin autoantibodies and insulin receptor autoantibodies. Available data eventually supported the predominant role of insulin autoantibodies rather than insulin receptor autoantibodies in the mechanism of hypoglycaemia of this patient. Insulin antibodies were present in high titre. Most of the insulin in serum was bound to the insulin antibodies and free insulin was slightly increased. HLA typing displayed DR4 haplotype, known to be strongly linked to the insulin autoimmune syndrome. The patient's serum was able to inhibit insulin binding to its receptor in a cultured cell line overexpressing insulin receptors both in experiments with native serum and with serum depleted from insulin antibodies. However, we could not demonstrate that the insulin receptor antibodies had insulin mimicking effect. We have no obvious explanation for the presence of these two antibodies in the same patient. Possible hypotheses might involve an idiotype-anti-idiotype mechanism or a poly-autoimmune disease.

Identification of elongation factor 1α as a potential associated binding partner for Akt2

Akt protein kinase has been shown to play a pivotal role in diverse cell functions, including motility, apoptosis, growth and metabolism. How it differentially regulates these diverse functions is of significant interest. Three isoforms have been well characterized, Akt1, 2, and 3, encoded by separate genes, but showing high homology over the entire coding sequence (> 80%). An area of variability between the three isoforms is the C-terminal tail. To find potentially regulating binding partners of Akt2, the isoform implicated in metabolic control, we used a glutathione-S-transferase (GST) fusion protein expressing the C-terminal 75 residues of Akt2 (GST-Akt2 tail) to screen for proteins that specifically bound the Akt2 tail. Elongation factor 1alpha (EF1alpha) and beta-tubulin were identified as binding partners for the Akt2 tail by peptide mass fingerprinting. These two proteins have themselves been previously identified as interacting partners (Nakazawa et al.FEBS Lett. 453,29-34, 1999). Using CHOT cells that overexpress insulin receptors and HA-tagged Akt2, we showed that EF1alpha co-immunoprecipitated with HA-tagged Akt2. It is thus possible that these proteins colocalise as part of a regulatory signaling complex with the cytoskeleton directing them to sites of cell activity.

Insulin Glulisine—A Comprehensive Preclinical Evaluation

Receptor binding and signaling and the mitogenic potential of insulin glulisine (glulisine), regular human insulin (RHI), and Asp(B10) were compared in vivo and in vitro. Insulin and insulin-like growth factor 1 (IGF-1) receptor binding was studied with human insulin receptors (293HEK cells) and the human osteosarcoma-derived cell line B10. Insulin receptor-mediated signaling was assessed in rat-1 fibroblasts overexpressing insulin receptors. Activation of insulin receptor substrates 1 and 2 (IRS-1/ IRS-2) was studied in rat and human myoblasts and rat cardiomyocytes. DNA synthesis induction was assessed by [3H] thymidine incorporation in the human epithelial breast cell line MCF10. Interaction with the IGF-1 receptor, DNA synthesis, and intracellular signal transduction were assessed in cardiac K6 myoblasts. Immunohistochemical examination of Sprague-Dawley rat tissue treated with glulisine for 6 months (n = 40), and glulisine and RHI for 12 months (n = 60), was performed. Steady-state insulin receptor binding affinity was slightly lower for glulisine versus RHI (approximately 0.70). IGF-1 receptor binding affinity was lower (four- to fivefold) for glulisine, but significantly higher (four-fold) for Asp(B10) versus RHI. Glulisine, Asp(B10), and RHI showed similar insulin receptor-association kinetics; however, Asp(B10) revealed increased insulin receptor affinity. Glulisine and RHI showed similar insulin receptor-mediated phosphorylation and IRS-2 activation. Activation of IRS-1 was 6- to 10-fold lower with glulisine; glulisine was less potent and Asp(B10) slightly more potent in stimulating DNA synthesis versus RHI. Stimulation of DNA synthesis was comparable for glulisine and RHI in K6 myoblasts. At 12 months, there was no significant difference between glulisine and RHI in proliferative activity. This preclinical evaluation suggests that structural changes in glulisine versus RHI are not associated with any safety issues.

Proteomic analysis to investigate the pathogenesis of diabetes using 2D DIGE

Two-dimensional differential in-gel electrophoresis technology (2D DIGE) technique is highly useful for differential analysis of protein spots in two-dimensional differential gels. Utilizing this technique, we attempted to search for diabetes-related drug targets and biomarkers. In human hepatoma cell line, HepG2, we analyzed secretome in the presence or absence of a Liver X receptor agonist, TO-901317, and identified one of the up-regulated proteins in response to LXR activation as apolipoprotein E. We also evaluated nuclear proteome of cultured cells overexpressing insulin receptor substrate proteins, in which insulin-stimulated cell cycle progression is differentially regulated, and the gel pattern indicated that insulin-induced phosphorylation of a nuclear protein may be impaired in cells overexpressing cell cycle-suppressive insulin receptor substrate-3. In addition, to search for urinary markers of diabetic nephropathy using 2D DIGE, we analyzed urine samples in which most abundant proteins were removed by immunoaffinity depletion. These findings indicate that the 2D DIGE-based approach is useful for the discovery of disease-specific drug targets and diagnostic biomarkers.

Protein Kinase C-α Regulates Insulin Action and Degradation by Interacting with Insulin Receptor Substrate-1 and 14-3-3ϵ

Protein kinase C (PKC)-alpha exerts a regulatory function on insulin action. We showed by overlay blot that PKCalpha directly binds a 180-kDa protein, corresponding to IRS-1, and a 30-kDa molecular species, identified as 14-3-3epsilon. In intact NIH-3T3 cells overexpressing insulin receptors (3T3-hIR), insulin selectively increased PKCalpha co-precipitation with IRS-1, but not with IRS-2, and with 14-3-3epsilon, but not with other 14-3-3 isoforms. Overexpression of 14-3-3epsilon in 3T3-hIR cells significantly reduced IRS-1-bound PKCalpha activity, without altering IRS-1/PKCalpha co-precipitation. 14-3-3epsilon overexpression also increased insulin-stimulated insulin receptor and IRS-1 tyrosine phosphorylation, followed by increased activation of Raf1, ERK1/2, and Akt/protein kinase B. Insulin-induced glycogen synthase activity and thymidine incorporation were also augmented. Consistently, selective depletion of 14-3-3epsilon by antisense oligonucleotides caused a 3-fold increase of IRS-1-bound PKCalpha activity and a similarly sized reduction of insulin receptor and IRS-1 tyrosine phosphorylation and signaling. In turn, selective inhibition of PKCalpha expression by antisense oligonucleotides reverted the negative effect of 14-3-3epsilon depletion on insulin signaling. Moreover, PKCalpha inhibition was accompanied by a >2-fold decrease of insulin degradation. Similar results were also obtained by overexpressing 14-3-3epsilon. Thus, in NIH-3T3 cells, insulin induces the formation of multimolecular complexes, including IRS-1, PKCalpha, and 14-3-3epsilon. The presence of 14-3-3epsilon in the complex is not necessary for IRS-1/PKCalpha interaction but modulates PKCalpha activity, thereby regulating insulin signaling and degradation.

Cross-talk between the Two Divergent Insulin Signaling Pathways Is Revealed by the Protein Kinase B (Akt)-mediated Phosphorylation of Adapter Protein APS on Serine 588

The APS adapter protein is recruited to the autophosphorylated kinase domain of the insulin receptor and initiates the phosphatidylinositol 3-kinase (PI3K)-independent pathway of insulin-stimulated glucose transport by recruiting CAP and c-Cbl. In this study, we have identified APS as a novel substrate for protein kinase B/Akt using an antibody that exhibits insulin-dependent immunoreactivity with a phosphospecific antibody raised against the protein kinase B substrate consensus sequence RXRXX(pS/pT) and a phosphospecific antibody that recognizes serine 21/9 of glycogen synthase kinase-3alpha/beta. This phosphorylation of APS is observed in both 3T3-L1 adipocytes and transfected cells. The insulin-stimulated serine phosphorylation of APS was inhibited by a PI3-kinase inhibitor, LY290004, a specific protein kinase B (PKB) inhibitor, deguelin, and knockdown of Akt. Serine 588 of APS is contained in a protein kinase B consensus sequence for phosphorylation conserved in APS across multiple species but not found in other members of this family, including SH2-B and Lnk. Mutation of serine 588 to alanine abolished the insulin-stimulated serine phosphorylation of APS and prevented the localization of APS to membrane ruffles. A glutathione S-transferase fusion protein containing amino acids 534-621 of APS was phosphorylated by purified PKB in vitro, and mutation of serine 588 abolished the PKB-mediated phosphorylation of APS in vitro. Taken together, this study identifies APS as a novel physiological substrate for PKB and the first serine phosphorylation site on APS. These data therefore reveal the molecular cross-talk between the insulin-activated PI3-kinase-dependent and -independent pathways previously thought to be distinct and divergent.

Serine 332 Phosphorylation of Insulin Receptor Substrate-1 by Glycogen Synthase Kinase-3 Attenuates Insulin Signaling

The ability of glycogen synthase kinase-3 (GSK-3) to phosphorylate insulin receptor substrate-1 (IRS-1) is a potential inhibitory mechanism for insulin resistance in type 2 diabetes. However, the serine site(s) phosphorylated by GSK-3 within IRS-1 had not been yet identified. Using an N-terminal deleted IRS-1 mutant and two IRS-1 fragments, PTB-1 1-320 and PTB-2 1-350, we localized GSK-3 phosphorylation site(s) within amino acid sequence 320-350. Mutations of serine 332 or 336, which lie in the GSK-3 consensus motif (SXXXS) within PTB-2 or IRS-1, to alanine abolished their phosphorylation by GSK-3. This suggested that Ser332 is a GSK-3 phosphorylation site and that Ser336 serves as the "priming" site typically required for GSK-3 action. Indeed, dephosphorylation of IRS-1 prevented GSK-3 phosphorylation. Furthermore, the phosphorylated peptide derived from the IRS-1 sequence was readily phosphorylated by GSK-3, in contrast to the nonphosphorylated peptide, which was not phosphorylated by the enzyme. When IRS-1 mutants S332A(IRS-1), S336A(IRS-1), or S332A/336A(IRS-1) were expressed in Chinese hamster ovary cells overexpressing insulin receptors, their insulin-induced tyrosine phosphorylation levels increased compared with that of wild-type (WT) IRS-1. This effect was stronger in the double mutant S332A/336A(IRS-1) and led to enhanced insulin-mediated activation of protein kinase B. Finally, immunoblot analysis with polyclonal antibody directed against IRS-1 phosphorylated at Ser332 confirmed IRS-1 phosphorylation in cultured cells. Moreover, treatment with the GSK-3 inhibitor lithium reduced Ser332 phosphorylation, whereas overexpression of GSK-3 enhanced this phosphorylation. In summary, our studies identify Ser332 as the GSK-3 phosphorylation target in IRS-1, indicating its physiological relevance and demonstrating its novel inhibitory role in insulin signaling.

Regulation of the insulin receptor by protein kinase C isoenzymes: preferential interaction with β isoenzymes and interaction with the catalytic domain of βII

We analysed the effects of high glucose in rat1 cells overexpressing insulin receptor. High (25 mM) glucose inhibited insulin-stimulated tyrosine kinase activity completely at insulin concentrations of 1 and 5 ng/ml. Decapeptides modelled on insulin receptor sequences surrounding serines 1035 and 1270 were found to inhibit protein kinase C activity in vitro and after microinjection into cells blocked the inhibition of mitogenesis induced by glucose. Purification of receptor from 3T3L1 adipocytes revealed that only the isoenzymes β1, βII and δ were detected. The site of the interaction was mapped to the catalytic domain of βII. These results demonstrate that the inhibition of insulin receptor tyrosine kinase activity can be ameliorated using insulin receptor peptide sequences and there is constitutive and differential interaction of individual PKC isoenzymes with the insulin receptor, and in the case of βII, this interaction maps to the catalytic domain rather than the regulatory domain.

Membrane Localization of Src Homology 2-Containing Inositol 5′-Phosphatase 2 via Shc Association Is Required for the Negative Regulation of Insulin Signaling in Rat1 Fibroblasts Overexpressing Insulin Receptors

Lipid phosphatase SHIP2 [Src homology 2 (SH2)-containing inositol 5'-phosphatase 2] has been shown to be a physiologically critical negative regulator of insulin signaling. We investigated the molecular mechanism by which SHIP2 negatively regulates insulin-induced phosphorylation of Akt, a key downstream molecule of phosphatidylinositol 3-kinase important for the biological action of insulin. Overexpression of wild-type SHIP2 (WT-SHIP2) inhibited insulin-induced phosphorylation of Akt at both Thr(308) and Ser(473) in Rat1 fibroblasts expressing insulin receptors. The degree of inhibition was less in the cells expressing either a mutant SHIP2 with R47Q change (R/Q-SHIP2) in the SH2 domain, or a mutant SHIP2 with Y987F change (Y/F-SHIP2) in the C-terminal tyrosine phosphorylation site. However, on addition of a myristoylation signal, WT-SHIP2, R/Q-SHIP2, and Y/F-SHIP2 all efficiently inhibited insulin-induced Akt phosphorylation at both residues, whereas a 5'-phosphatase-defective mutant SHIP2 (deltaIP-SHIP2) with the myristoylation signal did not. Interestingly, the degree of inhibition of Akt phosphorylation by R/Q-SHIP2 and Y/F-SHIP2 is well correlated with the extent of their association with Shc. In addition, overexpression of WT-Shc increased the insulin-induced association of SHIP2 with Shc, whereas a decrease in the amount of Shc on expression of antisense Shc mRNA led to a reduction in the SHIP2-Shc association. Furthermore, the inhibitory effect on insulin-induced Akt phosphorylation by WT-SHIP2 was decreased in antisense-Shc cells. These results indicate that the membrane localization of SHIP2 with its 5'-phosphatase activity is required for negative regulation of insulin-induced Akt phosphorylation and that the localization is regulated, at least in part, by the association of SHIP2 with Shc in Rat1 fibroblasts.

Protein Phosphatase 2A Forms a Molecular Complex with Shc and Regulates Shc Tyrosine Phosphorylation and Downstream Mitogenic Signaling

Protein phosphatase 2A (PP2A) is a multimeric serine/threonine phosphatase that carries out multiple functions. Although numerous observations suggest that PP2A plays a major role in downregulation of the mitogen-activated protein (MAP) kinase pathway, the precise mechanisms are unknown. To clarify the role of PP2A in growth factor (insulin, epidermal growth factor [EGF], and insulin-like growth factor 1 [IGF-1]) stimulation of the Ras/MAP kinase pathway, simian virus 40 small t antigen was expressed in Rat-1 fibroblasts which overexpress insulin receptors. Small t antigen is known to specifically inhibit PP2A by binding to the A PP2A regulatory subunit, interfering with the ability of PP2A to bind to its cellular substrates. Overexpressed small t protein was coimmunoprecipitated with PP2A and inhibited cellular PP2A activity but did not inhibit protein phosphatase 1 (PP1) activity. Insulin, IGF-1, and EGF stimulation also inhibited PP2A activity. Growth factor-stimulated Ras, Raf-1, MAP kinase, and mitogen-activated extracellular-signal-regulated kinase kinase (MEK) activities were elevated in small-t-antigen-expressing cells. Furthermore, Shc tyrosine phosphorylation and its association with Grb2 were also elevated in small-t-antigen-expressing cells. Expression levels of Shc, Ras, MEK, or MAP kinase and phosphorylation of insulin, EGF, and IGF-1 receptors were not altered. Interestingly, we found that PP2A associated with Shc in the basal state and dissociated in response to insulin and EGF and that this dissociation was inhibited by 65% in small-t-antigen-expressing cells. In addition, we found that PP2A associates with the phosphotyrosine-binding domain (PTB domain) of Shc and that phosphorylation of tyrosine 317 of Shc was required for PP2A-Shc dissociation. We conclude (i) that PP2A negatively regulates the Ras/MAP kinase pathway by binding to Shc, inhibiting tyrosine phosphorylation; (ii) that the Shc-PP2A association is mediated by the Shc PTB domain but the interaction is independent of phosphotyrosine binding, indicating a new molecular function for the PTB domain; (iii) that growth factor stimulation, or small-t-antigen expression, causes dissociation of the PP2A-Shc complex, facilitating Shc phosphorylation and downstream activations of the Ras/MAP kinase pathway; and (iv) that this defines a new mechanism of small-t-antigen action to promote mitogenesis.

Insulin-Mediated Cell Proliferation and Survival Involve Inhibition of c-Jun N-terminal Kinases through a Phosphatidylinositol 3-Kinase- and Mitogen-Activated Protein Kinase Phosphatase-1-Dependent Pathway

We previously reported that long term treatment with insulin led to sustained inhibition of c-Jun N-terminal kinases (JNKs) in CHO cells overexpressing insulin receptors. Here we investigated the signaling molecules involved in insulin inhibition of JNKs, focusing on phosphatidylinositol 3-kinase (PI 3-K) and mitogen-activated protein kinase phosphatase-1 (MKP-1). In addition, we examined the relevance of JNK inhibition for insulin-mediated proliferation and survival. Insulin inhibition of JNKs was mediated by PI 3-K, as it was blocked by wortmannin and LY294002 and required the de novo synthesis of a phosphatase(s), as it was abolished by orthovanadate and actinomycin D. MKP-1 was a good candidate because 1) insulin stimulation of MKP-1 expression correlated with insulin inhibition of JNKs; 2) insulin stimulation of MKP-1 expression, like insulin inhibition of JNKs, was mediated by PI 3-K; and 3) the transient expression of an antisense MKP-1 RNA reduced the insulin inhibitory effect on JNKs. The overexpression of a dominant negative JNK1 mutant increased insulin stimulation of DNA synthesis and mimicked the protective effect of insulin against serum withdrawal-induced apoptosis. The overexpression of wild-type JNK1 or antisense MKP-1 RNA reduced the proliferative and/or antiapoptotic responses to insulin. Altogether, these results demonstrate that insulin inhibits JNKs through a PI 3-K- and MKP-1-dependent pathway and provide evidence for a key role for JNK inhibition in insulin regulation of proliferation and survival.

Insulin-mediated cell proliferation and survival involve inhibition of c-Jun N-terminal kinases through a phosphatidylinositol 3-kinase- and mitogen-activated protein kinase phosphatase-1-dependent pathway.

We previously reported that long term treatment with insulin led to sustained inhibition of c-Jun N-terminal kinases (JNKs) in CHO cells overexpressing insulin receptors. Here we investigated the signaling molecules involved in insulin inhibition of JNKs, focusing on phosphatidylinositol 3-kinase (PI 3-K) and mitogen-activated protein kinase phosphatase-1 (MKP-1). In addition, we examined the relevance of JNK inhibition for insulin-mediated proliferation and survival. Insulin inhibition of JNKs was mediated by PI 3-K, as it was blocked by wortmannin and LY294002 and required the de novo synthesis of a phosphatase(s), as it was abolished by orthovanadate and actinomycin D. MKP-1 was a good candidate because 1) insulin stimulation of MKP-1 expression correlated with insulin inhibition of JNKs; 2) insulin stimulation of MKP-1 expression, like insulin inhibition of JNKs, was mediated by PI 3-K; and 3) the transient expression of an antisense MKP-1 RNA reduced the insulin inhibitory effect on JNKs. The overexpression of a dominant negative JNK1 mutant increased insulin stimulation of DNA synthesis and mimicked the protective effect of insulin against serum withdrawal-induced apoptosis. The overexpression of wild-type JNK1 or antisense MKP-1 RNA reduced the proliferative and/or antiapoptotic responses to insulin. Altogether, these results demonstrate that insulin inhibits JNKs through a PI 3-K- and MKP-1-dependent pathway and provide evidence for a key role for JNK inhibition in insulin regulation of proliferation and survival.

Intracellular Signalling Pathways of Okadaic Acid Leading to Mitogenesis in Rat1 Fibroblast Overexpressing Insulin Receptors: Okadaic Acid Regulates Shc Phosphorylation by Mechanisms Independent of Insulin

Okadaic acid is an powerful inhibitor of serine/threonine protein phosphatases 1 and 2A. Although it is known as a potent tumour promoter, the intracellular mechanism by which okadaic acid mediates its mitogenic effect remains to be clarified. We investigated the effect of okadaic acid on the activation of mitogenesis in Rat1 fibroblasts overexpressing insulin receptors. As previously reported, insulin induced Shc phosphorylation, Shc•Grb2 association, MAP kinase activation, and BrdU incorporation. Okadaic acid also stimulated tyrosine phosphorylation of Shc and its subsequent association with Grb2 in a time- and dose-dependent manner without affecting tyrosine phosphorylation of insulin receptor β-subunit and IRS. However, to a lesser extent, okadaic acid stimulated MAP kinase activity and BrdU incorporation. Interestingly, preincubation of okadaic acid potentiated insulin stimulation of tyrosine phosphorylation of Shc (213% of control), Shc•Grb2 association (150%), MAP kinase activity (152%), and BrdU incorporation (148%). These results further confirmed the important role of Shc, but not IRS, in cell cycle progression in Rat1 fibroblasts. Furthermore, serine/threonine phosphorylation appears to be involved in the regulation of Shc tyrosine phosphorylation leading to mitogenesis by mechanisms independent of insulin signalling.