A phosphatidylinositol 3-kinase/Akt/mTOR pathway mediates and PTEN antagonizes tumor necrosis factor inhibition of insulin signaling through insulin receptor substrate-1

Tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1) by the insulin receptor permits this docking protein to interact with signaling proteins that promote insulin action. Serine phosphorylation uncouples IRS-1 from the insulin receptor, thereby inhibiting its tyrosine phosphorylation and insulin signaling. For this reason, there is great interest in identifying serine/threonine kinases for which IRS-1 is a substrate. Tumor necrosis factor (TNF) inhibited insulin-promoted tyrosine phosphorylation of IRS-1 and activated the Akt/protein kinase B serine-threonine kinase, a downstream target for phosphatidylinositol 3-kinase (PI 3-kinase). The effect of TNF on insulin-promoted tyrosine phosphorylation of IRS-1 was blocked by inhibition of PI 3-kinase and the PTEN tumor suppressor, which dephosphorylates the lipids that mediate PI 3-kinase functions, whereas constitutively active Akt impaired insulin-promoted IRS-1 tyrosine phosphorylation. Conversely, TNF inhibition of IRS-1 tyrosine phosphorylation was blocked by kinase dead Akt. Inhibition of IRS-1 tyrosine phosphorylation by TNF was blocked by rapamycin, an inhibitor of the mammalian target of rapamycin (mTOR), a downstream target of Akt. mTOR induced the serine phosphorylation of IRS-1 (Ser-636/639), and such phosphorylation was inhibited by rapamycin. These results suggest that TNF impairs insulin signaling through IRS-1 by activation of a PI 3-kinase/Akt/mTOR pathway, which is antagonized by PTEN.     

Rescuing 3T3-L1 adipocytes from insulin resistance induced by stimulation of Akt-mammalian target of rapamycin/p70 S6 kinase (S6K1) pathway and serine phosphorylation of insulin receptor substrate-1: effect of reduced expression of p85alpha subunit of phosphatidylinositol 3-kinase and S6K1 kinase

Phosphorylation of insulin receptor substrate-1 (IRS-1) on serine residues has been recognized as a mechanism responsible for a diminution of insulin action and insulin resistance. Potential approaches to improve insulin sensitivity may include interference with and/or reduction in expression of certain signaling intermediates that participate in the pathogenesis of insulin resistance. In this study, we transduced fully differentiated 3T3-L1 adipocytes with a constitutively active myristoylated Akt that led to hyperactivation of mammalian target of rapamycin and p70 S6 kinase (S6K1), increased serine phosphorylation of IRS-1, and reduction in insulin-stimulated phosphatidylinositol (PI) 3-kinase activity and glucose transport. We then reduced expression of the PI 3-kinase regulatory subunit, p85alpha, or expression of S6K1 kinase using small interfering RNA transfections, which led to a reduction in p85alpha expression of 70% at 48 h (P < 0.05) and S6K1 of 49% (P < 0.05). Reduction in expression of either p85alpha or S6K1 achieved with small interfering RNA in the presence of myristoylated Akt rescued 3T3-L1 adipocytes from the insulin resistance induced by serine phosphorylation of IRS-1 and completely restored insulin-stimulated activation of PI 3-kinase and glucose uptake. We conclude that reduction in expression of p85alpha or S6K1 could represent therapeutic targets to mitigate insulin resistance.     

Insulin signaling through insulin receptor substrate 1 and 2 in normal liver development

BACKGROUND & AIMS: The insulin growth factor signal transduction pathway is an important regulator of adult hepatocyte proliferation. The purpose of this study was to determine the roles of the insulin receptor substrate (IRS-1 and IRS-2)-mediated growth cascades in rapidly growing fetal rat liver. METHODS: We determined the expression and tyrosyl phosphorylation of the insulin receptor beta subunit (IRbeta), IRS-1 and IRS-2, the binding of phosphatidylinositol 3-kinase (PI3K), and activation of the mitogen-activated protein kinase (MAPK) pathway in the presence or absence of insulin stimulation in vivo during development and in the adult liver. In addition, activation of other downstream components including PI3K, Akt, GSK3beta, Bad, and p70S6 kinase was studied. RESULTS: We observed reduced expression and tyrosyl phosphorylation of IRS-1 in the fetal liver compared with the adult liver. These developmental changes resulted in a lack of sensitivity to insulin stimulation and subsequent downstream activation of the PI3K and MAPK cascades until the postneonatal period. In contrast, there was a high level of IRS-2 expression and insulin-stimulated tyrosyl phosphorylation as early as embryonic day 15 with robust PI3K binding and activation, which may enhance hepatocyte survival during the rapid growth phase of the liver. CONCLUSIONS: The IRS-1 signal transduction pathway does not play a major role in fetal liver growth because IRS-2 functions as the major insulin responsive molecule in early development. However, insulin-mediated IRS-1/MAPK cascade activation contributes to growth in the adult.     

Impaired IRS-1/PI3-kinase signaling in patients with HCV: a mechanism for increased prevalence of type 2 diabetes

Patients with hepatitis C virus (HCV) infection have a greater risk of developing type 2 diabetes mellitus. However, the mechanism of this association is unclear. In this study, we examined the potential defects in upstream insulin signaling pathways in liver specimens obtained from nonobese/nondiabetic subjects with HCV infection. Fasting liver biopsy specimens were obtained from 42 HCV-infected subjects and 10 non-HCV-infected subjects matched for age and body mass index. Liver tissues were exposed to insulin and examined for the contents and phosphorylation/activation status of the upstream insulin signaling molecules by immunoprecipitation and Western blot analysis. HCV infection resulted in a trend toward a 2-fold to 3-fold increase in insulin receptor (IR) and insulin receptor substrate (IRS)-1 contents when compared with non-HCV. In contrast, insulin-stimulated IRS-1 tyrosine phosphorylation was decreased by 2-fold in HCV-infected subjects compared with non-HCV-infected subjects (P <.05). The observed reductions in IRS-1 tyrosine phosphorylation were accompanied by a 3.4-fold decrease in IRS-1/p85 phosphatidylinositol 3-kinase (PI3-kinase) association and a 2.5-fold decrease in IRS-1-associated PI3-kinase enzymatic activity (P <.05 vs. non-HCV). This was accompanied by a marked reduction in insulin-stimulated Akt phosphorylation without any alterations in mitogen-activated protein kinase (MAPK) phosphorylation. Cellular contents of the hepatic p85 subunit of PI3-kinase were comparable between HCV-infected and non-HCV-infected subjects. In conclusion, we found that (1). HCV infection leads to a postreceptor defect in IRS-1 association with the IR and (2). insulin signaling defects in hepatic IRS-1 tyrosine phosphorylation and PI3-kinase association/activation may contribute to insulin resistance, which leads to the development of type 2 diabetes mellitus in patients with HCV infection.     

Angiotensin II impairs the insulin signaling pathway promoting production of nitric oxide by inducing phosphorylation of insulin receptor substrate-1 on Ser312 and Ser616 in human umbilical vein endothelial cells

It has been suggested that serine (Ser) phosphorylation of insulin receptor substrate-1 (IRS-1) decreases the ability of IRS-1 to be phosphorylated on tyrosine, thereby attenuating insulin signaling. There is evidence that angiotensin II (AII) may impair insulin signaling to the IRS-1/phosphatydilinositol 3-kinase (PI 3-kinase) pathway by enhancing Ser phosphorylation. Insulin stimulates NO production by a pathway involving IRS-1/PI3-kinase/Akt/endothelial NO synthase (eNOS). We addressed the question of whether AII affects insulin signaling involved in NO production in human umbilical vein endothelial cells and tested the hypothesis that the inhibitory effect of AII on insulin signaling was caused by increased site-specific Ser phosphorylation in IRS-1. Exposure of human umbilical vein endothelial cells to AII resulted in inhibition of insulin-stimulated production of NO. This event was associated with impaired IRS-1 phosphorylation at Tyr612 and Tyr632, two sites essential for engaging the p85 subunit of PI3-kinase, resulting in defective activation of PI 3-kinase, Akt, and eNOS. This inhibitory effect of AII was reversed by the type 1 receptor antagonist losartan. AII increased c-Jun N-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK) 1/2 activity, which was associated with a concomitant increase in IRS-1 phosphorylation at Ser312 and Ser616, respectively. Inhibition of JNK and ERK1/2 activity reversed the negative effects of AII on insulin-stimulated NO production. Our data suggest that AII, acting via the type 1 receptor, increases IRS-1 phosphorylation at Ser312 and Ser616 via JNK and ERK1/2, respectively, thus impairing the vasodilator effects of insulin mediated by the IRS-1/PI 3-kinase/Akt/eNOS pathway.     

Insulin-like growth factor-1 potentiates platelet activation via the IRS/PI3Kalpha pathway

As insulin-like growth factor-1 (IGF-1) is present in the alpha granules of platelets and its receptor is expressed on the platelet surface, it may contribute to the amplification of platelet responses and pathogenesis of cardiovascular disease. The functional and signaling pathways that are involved in IGF-1 modulation of platelet function, however, are presently unknown. Here, I report that IGF-1 stimulation of platelets results in dose-dependent phosphorylation of the IGF receptor in the range of 1 to 100 nM. Phosphorylation of the IGF receptor is rapid and sustained, with maximal phosphorylation reached within 1 minute. Furthermore, IGF-1 stimulates tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1) and IRS-2 and their association with the p85 subunit of phosphoinositide-3 kinase (PI3K). IGF-1-stimulated tyrosine phosphorylation of IRS-1 and IRS-2 and subsequent p85 binding is transient and precedes phosphorylation of protein kinase B (PKB) on Ser473. PAR-1-mediated platelet aggregation is potentiated by IGF-1 and this potentiation, together with PKB phosphorylation, is abolished by the PI3Kalpha inhibitors PI-103 and PIK-75. Importantly, the IGF receptor inhibitor NVP-AEW541 and the neutralization antibody alphaIR3 inhibit SFLLRN-stimulated aggregation, implicating IGF-1 in autocrine regulation of platelet function. These results demonstrate that IGF-1 activates the IGF receptor/IRS/PI3K/PKB pathway, and that PI3Kalpha is essential for the potentiatory effect of IGF-1 on platelet responses.     

Regulation of IRS-2 tyrosine phosphorylation in fasting and diabetes

Intracellular insulin signaling involves a series of alternative and complementary pathways created by the multiple substrates of the insulin receptor (IRS) and the various isoforms of the SH2 domain signaling molecules that can interact with substrate. In this study we investigated IRS-1 and IRS-2 tyrosine phosphorylation, their association with PI3-kinase and the phosphorylation of Akt, a serine-threonine kinase situated downstream to PI 3-kinase, in liver and muscle of two animal models of insulin resistance: 72 h of fasting and STZ-diabetic rats. There was an upregulation in insulin-induced IRS-1 and IRS-2 tyrosine phosphorylation and association with PI3-kinase in liver and muscle of both animal models of insulin resistance. However, Akt phosphorylation showed different regulation, increasing in fasting and decreasing in STZ-diabetic rats. Since an important difference between these two animal models of insulin resistance is the plasma glucose levels, we can suggest that in STZ diabetic rats, the reduction in Akt phosphorylation is probably related to hyperglycemia and may certainly contribute to the molecular mechanism of insulin resistance observed in these animals.     

Development of whole-body and skeletal muscle insulin resistance after one day of hindlimb suspension

Hindlimb suspension (HS) of rats is a model of simulated weightlessness and induces dynamic alterations in insulin action. In the present study, the effect of acute (1-day) HS on whole-body glucose tolerance and insulin action on skeletal muscle glucose transport was assessed in juvenile, female Sprague-Dawley rats. Compared to weight-bearing control rats, 1-day HS animals displayed significantly decreased glucose tolerance and diminished whole-body insulin sensitivity. Glucose transport activity in the 1-day unweighted soleus muscle was significantly decreased (P <.05) compared to weight-bearing control muscles both in the absence and presence of insulin (2 mU/mL). Insulin-mediated glucose transport activity in the extensor digitorum longus (EDL) muscles also tended (P =.09) to be lower. There was no change in the protein expression of insulin receptor beta-subunit (IR-beta), insulin receptor substrate-1 (IRS-1), IRS-2, the p85 subunit of phosphatidylinositol-3 kinase (PI3-kinase), Akt, and glucose transporter protein 4 (GLUT-4). The activities of these proteins were also unchanged, as insulin-stimulated IR-beta tyrosine phosphorylation, IRS-1 tyrosine phosphorylation, IRS-1-associated p85, and Akt serine phosphorylation were similar to controls. However, basal Akt phosphorylation was significantly depressed (P <.05) in the 1-day HS soleus. In addition, the protein expression and basal phosphorylation of the stress-activated p38 mitogen-activated protein kinase (p38 MAPK) were significantly elevated (P <.05) in the 1-day unweighted soleus. These results indicate that the development of insulin resistance in the 1-day unweighted soleus is not due to impaired functionality of elements involved in the IR/IRS-1/PI3-kinase/Akt signaling pathway. However, activation of p38 MAPK may play a role in this response.     

Altered subcellular distribution of estrogen receptor alpha is implicated in estradiol-induced dual regulation of insulin signaling in 3T3-L1 adipocytes

We investigated the mechanisms by which estrogen alters insulin signaling in 3T3-L1 adipocytes. Treatment with 17beta-estradiol (E2) did not affect insulin-induced tyrosine phosphorylation of insulin receptor. E2 enhanced insulin-induced tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1), IRS-1/p85 association, phosphorylation of Akt, and 2-deoxyglucose uptake at 10(-8) m, but inhibited these effects at 10(-5) m. A concentration of 10(-5) m E2 enhanced insulin-induced phosphorylation of IRS-1 at Ser(307), which was abolished by treatment with a c-Jun NH(2)-terminal kinase inhibitor. In addition, the effect of E2 was abrogated by pretreatment with a specific estrogen receptor antagonist, ICI182,780. Membrane-impermeable E2, E2-BSA, did not affect the insulin-induced phosphorylation of Akt at 10(-8) m, but inhibited it at 10(-5) m. Furthermore, E2 decreased the amount of estrogen receptor alpha at the plasma membrane at 10(-8) m, but increased it at 10(-5) m. In contrast, the subcellular distribution of estrogen receptor beta was not altered by the treatment. These results indicate that E2 affects the metabolic action of insulin in a concentration-specific manner, that high concentrations of E2 inhibit insulin signaling by modulating phosphorylation of IRS-1 at Ser(307) via a c-Jun NH(2)-terminal kinase-dependent pathway, and that the subcellular redistribution of estrogen receptor alpha in response to E2 may explain the dual effect of E2.     

Persistent activation of phosphatidylinositol 3-kinase causes insulin resistance due to accelerated insulin-induced insulin receptor substrate-1 degradation in 3T3-L1 adipocytes

Recently, we have reported that the overexpression of a membrane-targeted phosphatidylinositol (PI) 3-kinase (p110CAAX) stimulated p70S6 kinase, Akt, glucose transport, and Ras activation in the absence of insulin but inhibited insulin-stimulated glycogen synthase activation and MAP kinase phosphorylation in 3T3-L1 adipocytes. To investigate the mechanism of p110CAAX-induced cellular insulin resistance, we have now studied the effect of p110CAAX on insulin receptor substrate (IRS)-1 protein. Overexpression of p110CAAX alone decreased IRS-1 protein levels to 63+/-10% of control values. Insulin treatment led to an IRS-1 gel mobility shift (most likely caused by serine/threonine phosphorylation), with subsequent IRS-1 degradation. Moreover, insulin-induced IRS-1 degradation was enhanced by expression of p110CAAX (61+/-16% vs. 13+/-15% at 20 min, and 80+/-8% vs. 41+/-12% at 60 min, after insulin stimulation with or without p110CAAX expression, respectively). In accordance with the decreased IRS-1 protein, the insulin-stimulated association between IRS-1 and the p85 subunit of PI 3-kinase was also decreased in the p110CAAX-expressing cells, and IRS-1-associated PI 3-kinase activity was decreased despite the fact that total PI 3-kinase activity was increased. Five hours of wortmannin pretreatment inhibited both serine/threonine phosphorylation and degradation of IRS-1 protein. These results indicate that insulin treatment leads to serine/threonine phosphorylation of IRS-1, with subsequent IRS-1 degradation, through a PI 3-kinase-sensitive mechanism. Consistent with this, activated PI 3-kinase phosphorylates IRS-1 on serine/threonine residues, leading to IRS- 1 degradation. The similar finding was observed in IRS-2 as well as IRS-1. These results may also explain the cellular insulin-resistant state induced by chronic p110CAAX expression.     

Cardiotrophin-1 phosphorylates akt and BAD, and prolongs cell survival via a PI3K-dependent pathway in cardiac myocytes

Growth factors and cytokines trigger survival signaling in a wide variety of cell systems, including cardiac myocytes. Participation of the phosphatidylinositol 3-OH kinase (PI3K)/Akt pathway in survival signaling has already been described in some cell types, but its involvement in the survival of cardiac myocytes is as yet unknown. Recently, CT-1, an interleukin 6-related cytokine, was shown to have survival-promoting, anti-apoptotic effects on cultured cardiac myocytes. However, roles of PI3K-dependent pathways in this signaling have not been elucidated. In the present study, therefore, we examined the participation of the PI3K/Akt pathway in CT-1-induced, survival-promoting signaling in cultured ventricular myocytes. It was found that CT-1 phosphorylated and activated Akt, and the effect was blocked by the PI3K inhibitors LY294002 and wortmannin. CT-1 also phosphorylated the pro-apoptotic factor, BAD, and the BAD phosphorylation was inhibited by LY294002, suggesting that phosphorylation of BAD is one of the key events by which the PI3K/Akt pathway mediates CT-1-induced survival signaling. Further, CT-1 PI3K-dependently prolonged the survival of serum-starved ventricular myocytes by preventing apoptosis. In summary, our findings show that PI3K-dependent survival signals contribute to CT-1-mediated ventricular myocyte survival. In vivo, the death of ventricular myocytes leads to heart failure, and downregulation of survival signals and/or augmentation of pro-apoptotic signals are likely to be important components of disease processes. Thus, the extent to which CT-1 and the PI3K/Akt pathway mitigate such pathological processes, in vivo, is an important question for the future.     

Increased insulin sensitivity in mice lacking p85β subunit of phosphoinositide 3-kinase

On the basis of ex vivo studies using insulin-responsive cells, activation of a Class IA phosphoinositide 3-kinase (PI3K) seems to be required for a wide variety of cellular responses downstream of insulin. The Class IA PI3K enzymes are heterodimers of catalytic and regulatory subunits. In mammals, insulin-responsive tissues express both the p85alpha and p85beta isoforms of the regulatory subunit. Surprisingly, recent studies have revealed that disruption of the p85alpha gene in the mouse (p85alpha(-/-) mice) results in hypoglycemia with decreased plasma insulin, and the p85alpha(+/-) mice exhibit significantly increased insulin sensitivity. These results suggest either that p85alpha negatively regulates insulin signaling, or that p85beta, which mediates the major fraction of Class IA PI3K signaling in the absence of p85alpha, is more efficient than p85alpha in mediating insulin responses. To address this question, we have generated mice in which the p85beta gene is deleted (p85beta(-/-) mice). As with the p85alpha(-/-) mice, the p85beta(-/-) mice showed hypoinsulinemia, hypoglycemia, and improved insulin sensitivity. At the molecular level, PI3K activity associated with phosphotyrosine complexes was preserved despite a 20-30% reduction in the total protein level of the regulatory subunits. Moreover, insulin-induced activation of AKT was significantly up-regulated in muscle from the p85beta(-/-) mice. In addition, insulin-dependent tyrosine phosphorylation of insulin receptor substrate-2 was enhanced in the p85beta(-/-) mice, a phenotype not observed in the p85alpha(-/-) mice. These results indicate that in addition to their roles in recruiting the catalytic subunit of PI3K to the insulin receptor substrate proteins, both p85alpha and p85beta play negative roles in insulin signaling.     

Interactive effects of insulin-like growth factor-1 and beta-estradiol on endothelial nitric oxide synthase activity in rat aortic endothelial cells

Insulin-like growth factor-1 (IGF-1) and beta-estradiol (E2) have vasodilatory effects, in part, through stimulation of vascular nitric oxide (NO) production. However, their interactive effects on endothelial nitric oxide synthase (eNOS) and NO production have not been previously studied in endothelial cells (EC). Employing rat aortic EC (RAEC), the effects of acute (20 and 30 minutes) and prolonged (4 hours) stimulation with 100 nmol/L IGF-1 and 1 nmol/L E2 (alone or in combination) were assessed with respect to protein levels and enzymatic activities for phosphatidyl inositol 3-kinase (PI3K) and serine/threonine kinase Akt (Akt), enzymes involved in eNOS activation. Exposure to IGF-1 for 30 minutes or E2 for 20 minutes increased insulin receptor substrate-1 (IRS-1) association with the regulatory (p85) subunit of PI3K, enhanced tyrosine phosphorylation of p85, and increased PI3K activity. Combined treatment had a greater effect on p85 phosphorylation and PI3K activity then either agonist alone. Moreover, IGF-1 and E2 enhanced Akt Ser(473) phosphorylation, with the effect of IGF-1 being much greater. Acute expose to both E2 (20 minutes) and IGF-1 (30 minutes) were associated with an increase in eNOS activity. Prolonged exposure (4 hours) to either IGF-1 or E2 increased expression of the p85 subunit as well as eNOS activity. Pretreatment with PI3K antagonist wortmannin (WT) prevented this increase in eNOS activity. The results suggest that IGF-1 and E2 may interact through PI3K/Akt-related pathways to increase eNOS activity.     

Wortmannin-sensitive pathway is required for insulin-stimulated phosphorylation of inhibitor kappaBalpha.

The aim of this study was to examine the signaling pathways by which insulin promotes activation of nuclear factor kappaB (NFkappaB) through the regulation of inhibitor kappaBalpha (IkappaBalpha). We show here that although insulin increased kappaB-dependent reporter gene expression and augmented nuclear translocation of the p65/RelA subunit of NFkappaB and its DNA binding, it was able to induce a time-dependent accumulation of phosphorylated and ubiquitinated IkappaBalpha without its proteolytic degradation. In contrast, cell stimulation with the cytokine TNFalpha allowed activation of NFkappaB through phosphorylation, ubiquitination, and subsequent degradation of IkappaBalpha. Immunofluorescence studies revealed the presence of a large pool of phosphorylated IkappaBalpha in the nucleus of unstimulated and insulin-treated cells. IkappaB kinase alpha and beta, central players in the phosphorylation of IkappaBalpha, were rapidly induced following exposure to TNFalpha but not insulin. Furthermore, insulin-stimulated IkappaBalpha phosphorylation did not depend on activation of the Ras/ERK cascade. Expression of a dominant-negative mutant of Akt1 or class I PI3K inhibited the insulin stimulation of PI3K/Akt1 signaling without affecting phosphorylation of IkappaBalpha. Interestingly, the PI3K inhibitors wortmannin and LY294002 blocked insulin-stimulated class I PI3K-dependent events at much lower doses than that required to inhibit phosphorylation of IkappaBalpha. These data demonstrate that insulin regulates IkappaBalpha function through a distinct low-affinity wortmannin-sensitive pathway.     

The p110beta isoform of phosphoinositide 3-kinase signals downstream of G protein-coupled receptors and is functionally redundant with p110gamma

The p110 isoforms of phosphoinositide 3-kinase (PI3K) are acutely regulated by extracellular stimuli. The class IA PI3K catalytic subunits (p110alpha, p110beta, and p110delta) occur in complex with a Src homology 2 (SH2) domain-containing p85 regulatory subunit, which has been shown to link p110alpha and p110delta to Tyr kinase signaling pathways. The p84/p101 regulatory subunits of the p110gamma class IB PI3K lack SH2 domains and instead couple p110gamma to G protein-coupled receptors (GPCRs). Here, we show, using small-molecule inhibitors with selectivity for p110beta and cells derived from a p110beta-deficient mouse line, that p110beta is not a major effector of Tyr kinase signaling but couples to GPCRs. In macrophages, both p110beta and p110gamma contributed to Akt activation induced by the GPCR agonist complement 5a, but not by the Tyr kinase ligand colony-stimulating factor-1. In fibroblasts, which express p110beta but not p110gamma, p110beta mediated Akt activation by the GPCR ligands stromal cell-derived factor, sphingosine-1-phosphate, and lysophosphatidic acid but not by the Tyr kinase ligands PDGF, insulin, and insulin-like growth factor 1. Introduction of p110gamma in these cells reduced the contribution of p110beta to GPCR signaling. Taken together, these data show that p110beta and p110gamma can couple redundantly to the same GPCR agonists. p110beta, which shows a much broader tissue distribution than the leukocyte-restricted p110gamma, could thus provide a conduit for GPCR-linked PI3K signaling in the many cell types where p110gamma expression is low or absent.     

Endogenous angiotensin II suppresses insulin signaling in vascular smooth muscle cells from spontaneously hypertensive rats

BACKGROUND : Angiotensin II (Ang II) has been reported to inhibit insulin signaling at multiple levels in vascular smooth muscle cells (VSMC) in vitro. We have demonstrated that VSMC from spontaneously hypertensive rats (SHR) produce Ang II in a homogeneous culture. OBJECTIVE : In the current study, we investigated influences of endogenous Ang II on insulin signaling in VSMC from SHR. DESIGN AND METHODS : Phosphatidylinositol 3-kinase (PI3-kinase) activity, insulin receptor substrate-1 (IRS-1) associated tyrosine phosphorylation, and p85 subunit of PI3-kinase were measured in VSMC from SHR and normotensive Wistar-Kyoto (WKY) rats in the absence and presence of Ang II type 1 receptor antagonist RNH6270 and mitogen-activated protein kinase/extracellular signal-regulated kinase (MEK) inhibitor U0126. RESULTS : Insulin treatment increased PI3-kinase activity in VSMC from WKY rats in a dose-dependent manner. In contrast, insulin treatment of VSMC from SHR did not affect PI3-kinase activity. However, co-treatment of VSMC from SHR with RNH6270 and insulin, increased PI3-kinase activity. PI3-kinase activity, IRS-1-associated tyrosine phosphorylation and p85 subunit of PI3-kinase in VSMC from WKY rats decreased in response to treatment with Ang II and returned to control levels upon co-treatment with U0126. Basal levels of PI3-kinase activity, IRS-1-associated tyrosine phosphorylation, and p85 subunit of PI3-kinase were significantly lower in VSMC from SHR than in cells from WKY rats. U0126 treatment of VSMC from SHR significantly increased levels of PI3-kinase activity, IRS-1-associated tyrosine phosphorylation, and p85 subunit of PI3-kinase. CONCLUSION : These results indicate that endogenous Ang II suppresses insulin signaling in VSMC from SHR by activating extracellular signal-regulated kinase. These findings suggest that tissue Ang II may play a role in insulin resistance in hypertension.     

Ethanol Inhibition of Insulin Signaling in Hepatocellular Carcinoma Cells

Chronic ethanol toxicity impairs liver regeneration, inhibits DNA synthesis, and mutes cellular responses to growth factor stimulation. Previous studies demonstrated that the adverse effects of ethanol are mediated by inhibition of tyrosyi phosphorylation of the insulin receptor and the insulin receptor substrate-type 1 (IRS-1). However, overexpression of IRS-1 leads to increased DNA synthesis and cellular transformation due to constitutive activation of mitogen-activated protein (MAP) kinase. The present study examines the effects of ethanol on insulin signaling through IRS-1 in FOCUS hepatocellular carcinoma cells, which overexpress IRS-1, to determine whether such ceils were resistant to the inhibitory effects of ethanol. The results demonstrated that ethanol treatment (100 mM) caused 30 to 50% reductions in the levels of insulin-stimulated tyrosyi phosphorylation of the insulin receptor β-subunit, tyrosyi phosphorylation of IRS-1, phosphorylation of Erk2, association of phosphatidylinositol-3 kinase with tyrosyl-phosphorylated IRS-1, and MAP kinase and phosphatidylinositol-3 kinase activities. In contrast, ethanol treatment had no effect on epidermal growth factor-stimulated tyrosyi phosphorylation of She. Corresponding with the pronounced inhibition of MAP kinase, ethanol treatment resulted in 30 to 50% reductions in the expression levels of two important insulin-responsive genes: glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and proliferating cell nuclear antigen (PCNA). The findings suggest that, in FOCUS hepatocellular carcinoma cells, which overexpress IRS-1, ethanol treatment substantially inhibits IRS-1 and MAP kinase signaling and growth-associated gene expression, but has no effect on She phosphorylation, which activates p21^ras through an IRS-1 independent pathway.