SH2-containing inositol phosphatase 2 negatively regulates insulin-induced glycogen synthesis in L6 myotubes

Aims/hypothesis: PI(3,4,5)P3 produced by PI3-kinase seems to be a key mediator for insulin's metabolic actions. We have recently cloned rat SHIP2 cDNA which is abundantly expressed in target tissues of insulin. Here, we clarify the role of SHIP2 possessing 5'-phosphatase activity toward PI(3,4,5)P3 in insulin signalling in the skeletal muscle. Methods: The role of SHIP2 in insulin-induced glycogen synthesis was studied by expressing wild-type (WT)-SHIP2 and a 5'-phosphatase defective (ΔIP)-SHIP2 into L6 myotubes by means of adenovirus mediated gene transfer. Results: The early events of insulin signalling including tyrosine phosphorylation of the insulin receptor and IRS-1, IRS-1 association with the p85 subunit, and PI3-kinase activity were not affected by expression of WT- and ΔIP-SHIP2. Although PI(3,4,5)P3 and PI(3,4)P2 are known to possibly activate a downstream molecule of PI3-kinase Akt in vitro, overexpression of WT-SHIP2 inhibited insulin-induced phosphorylation and activation of Akt. Conversely, Akt activity was increased by expression of ΔIP-SHIP2. GSK3β located downstream of Akt is an important molecule to further transmit insulin signal for glycogen synthesis in skeletal muscles. In accordance with the results of Akt, insulin-induced phosphorylation and inactivation of GSK3β, subsequent activation of glycogen synthase and glycogen synthesis were decreased by expression of WT-SHIP2, whereas these events were increased by expression of ΔIP-SHIP2. Conclusion/interpretation: Our results indicate that SHIP2 plays a negative regulatory role via the 5'-phosphatase activity in insulin signalling, and that PI(3,4,5)P3 rather than PI(3,4)P2 is important for in vivo regulation of insulin-induced Akt activation leading to glycogen synthesis in L6 myotubes. [Diabetologia (2001) 44: 1258–1267] Received: 5 February 2001 and in revised form: 25 June 2001     

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.     

Regulation of insulin signalling, glucose uptake and metabolism in rat skeletal muscle cells upon prolonged exposure to resistin

Aims/hypothesis Debate exists regarding the role of resistin in the pathophysiology of insulin resistance. The aim of this study was to directly assess the effects of resistin (0–24 h) on basal and insulin-stimulated glucose uptake and metabolism in skeletal muscle cells and to investigate the mechanisms responsible for the effects of resistin. Methods We used L6 rat skeletal muscle cells and examined [³H]2-deoxyglucose uptake, GLUT4 translocation and GLUT protein content. We assessed glucose metabolism by measuring the incorporation of D-[U-¹⁴C]glucose into glycogen, ¹⁴CO² and lactate production, as well as the phosphorylation level and total protein content of insulin signalling proteins, including insulin receptor β-subunit (IRβ), insulin receptor substrate (IRS), Akt and glycogen synthase kinase-3β (GSK-3β). Results Treatment of L6 rat skeletal muscle cells with recombinant resistin (50 nmol/l, 0–24 h) reduced levels of basal and insulin-stimulated 2-deoxyglucose uptake and decreased insulin-stimulated GLUT4myc content at the cell surface, with no alteration in the production of GLUT4 or GLUT1. Resistin also decreased glycogen synthesis and GSK-3β phosphorylation. Insulin-stimulated oxidation of glucose via the Krebs cycle was reduced by resistin, whereas lactate production was unaltered. Although insulin receptor protein level and phosphorylation were unaltered by resistin, production of IRS-1, but not IRS-2, was downregulated and a decreased tyrosine phosphorylation of IRS-1 was detected. Reduced phosphorylation of Akt on T308 and S473 was observed, while total Akt and Akt1, but not Akt2 or Akt3, production was decreased. Conclusions/interpretation Our data show that resistin regulates the function of IRS-1 and Akt1 and decreases GLUT4 translocation and glucose uptake in response to insulin. Selective decreases in insulin-stimulated glucose metabolism via oxidation and conversion to glycogen were also induced by resistin. These observations highlight the potential role of resistin in the pathophysiology of type 2 diabetes in obesity.     

Impaired muscle glycogen synthase in type 2 diabetes is associated with diminished phosphatidylinositol 3-kinase activation

Insulin signaling pathways potentially involved in regulation of skeletal muscle glycogen synthase were compared in differentiated human muscle cell cultures from nondiabetic and type 2 diabetic patients. Insulin stimulation of glycogen synthase activity as well as phosphorylation of MAPK, p70 S6 kinase, and protein kinase B (Akt) were blocked by the phosphatidylinositol 3-kinase inhibitors wortmannin (50 nM) and LY294002 (10 microM). In contrast to lean and obese nondiabetic subjects, where there were minimal effects (15-20% inhibition), insulin stimulation of glycogen synthase in muscle cultures from diabetic subjects was greatly diminished ( approximately 75%) by low concentrations of wortmannin (25 nM) or LY294002 (2 microM). This increased sensitivity of diabetic muscle to impairment of insulin-stimulated glycogen synthase activity occurs together with diminished insulin-stimulation (by 40%) of IRS-1-associated phosphatidylinositol 3-kinase activity in the same cells. Protein expression of IRS-1, p85, p110, Akt, p70 S6 kinase, and MAPK were normal in diabetic cells, as was insulin-stimulated phosphorylation of Akt, p70 S6 kinase, and MAPK. These studies indicate that, despite prolonged growth and differentiation of diabetic muscle under normal metabolic culture conditions, defects of insulin-stimulated phosphatidylinositol 3-kinase and glycogen synthase activity in diabetic muscle persist, consistent with intrinsic (rather than acquired) defects of insulin action.     

Human triglyceride-rich lipoproteins impair glucose metabolism and insulin signalling in L6 skeletal muscle cells independently of non-esterified fatty acid levels

AIMS/HYPOTHESIS: Elevated fasting and postprandial plasma levels of triglyceride-rich lipoproteins (TGRLs), i.e. VLDL/remnants and chylomicrons/remnants, are a characteristic feature of insulin resistance and are considered a consequence of this state. The aim of this study was to investigate whether intact TGRL particles are capable of inducing insulin resistance. METHODS: We studied the effect of highly purified TGRLs on glycogen synthesis, glycogen synthase activity, glucose uptake, insulin signalling and intramyocellular lipid (IMCL) content using fully differentiated L6 skeletal muscle cells. RESULTS: Incubation with TGRLs diminished insulin-stimulated glycogen synthesis, glycogen synthase activity, glucose uptake and insulin-stimulated phosphorylation of Akt and glycogen synthase kinase 3. Insulin-stimulated tyrosine phosphorylation of IRS-1, and IRS-1- and IRS-2-associated phosphatidylinositol 3-kinase (PI3K) activity were not impaired by TGRLs, suggesting that these steps were not involved in the lipoprotein-induced effects on glucose metabolism. The overall observed effects were time- and dose-dependent and paralleled IMCL accumulation. NEFA concentration in the incubation media did not increase in the presence of TGRLs indicating that the effects observed were solely due to intact lipoprotein particles. Moreover, co-incubation of TGRLs with orlistat, a potent active-site inhibitor of various lipases, did not alter TGRL-induced effects, whereas co-incubation with receptor-associated protein (RAP), which inhibits interaction of TGRL particles with members of the LDL receptor family, reversed the TGRL-induced effects on glycogen synthesis and insulin signalling. CONCLUSIONS/INTERPRETATION: Our data suggest that the accumulation of TGRLs in the blood stream of insulin-resistant patients may not only be a consequence of insulin resistance but could also be a cause for it.     

Tumor necrosis factor-alpha induces insulin resistance in endothelial cells via a p38 mitogen-activated protein kinase-dependent pathway

Chronic inflammation contributes to vascular insulin resistance and endothelial dysfunction. Systemic infusion of TNF-alpha abrogates insulin's action to enhance skeletal muscle microvascular perfusion. In skeletal muscle TNF-alpha induces insulin resistance via the p38 MAPK pathway. To examine whether p38 MAPK also regulates TNF-alpha-induced vascular insulin resistance, bovine aortic endothelial cells (bAECs) were incubated+/-TNF-alpha (5 ng/ml) for 6 h in the presence or absence of SB203580 (p38 MAPK specific inhibitor, 10 microM) after serum starvation for 10 h. For the last 30 min, cells were treated+/-1 nM insulin, and insulin receptor substrate (IRS)-1, Akt, endothelial nitric oxide synthase (eNOS), p38 MAPK, ERK1/2, c-Jun N-terminal kinase, and AMP-activated protein kinase (AMPK) phosphorylation, and eNOS activity were measured. TNF-alpha increased p38 MAPK phosphorylation, potently stimulated IRS-1 serine phosphorylation, and blunted insulin-stimulated IRS-1 tyrosine and Akt phosphorylation and eNOS activity. TNF-alpha also potently stimulated the phosphorylation of ERK1/2 and AMPK. Treatment with SB203580 decreased p38 MAPK phosphorylation back to the baseline and restored insulin sensitivity of IRS-1 tyrosine and Akt phosphorylation and eNOS activity in TNF-alpha-treated bAECs without affecting TNF-alpha-induced ERK1/2 and AMPK phosphorylation. We conclude that in cultured bAECs, TNF-alpha induces insulin resistance in the phosphatidylinositol 3-kinase/Akt/eNOS pathway via a p38 MAPK-dependent mechanism and enhances ERK1/2 and AMPK phosphorylation independent of the p38 MAPK pathway. This differential modulation of TNF-alpha's actions by p38 MAPK suggests that p38 MAPK plays a key role in TNF-alpha-mediated vascular insulin resistance and may contribute to the generalized endothelial dysfunction seen in type 2 diabetes mellitus and the cardiometabolic syndrome.     

In vivo administration of leptin activates signal transduction directly in insulin-sensitive tissues: overlapping but distinct pathways from insulin

To determine whether leptin signal transduction is exerted directly upon insulin-sensitive tissues in vivo, we examined the ability of iv leptin to acutely stimulate phosphorylation of STAT3, STAT1, and MAPK, and activities of PI 3-kinase and Akt, in insulin-sensitive tissues of normal rats. Both leptin (1 mg/kg iv x 3 min) and insulin (10 U/kg iv x 3 min) stimulated tyrosine phosphorylation of STAT3 5.6- to 6.0-fold and of STAT1 4.0-fold in adipose tissue. Leptin tended to increase STAT3 phosphorylation in liver and muscle. Both hormones also increased MAPK phosphorylation: leptin increased it 3.2- to 3.8-fold in adipose tissue and liver, whereas insulin stimulated MAPK phosphorylation 5.0-fold in adipose tissue, 6.8-fold in liver, and 2.5-fold in muscle. Leptin was much less effective than insulin at stimulating IRS pathways. Leptin increased IRS-1-associated PI 3-kinase activity in adipose tissue only 2.0-fold (P < 0.01) compared with the 10-fold effect of insulin. IRS-2-associated PI 3-kinase activity was increased 1.7-fold (P < 0.01) by leptin in liver and 6-fold by insulin. Akt phosphorylation and activity were not changed by leptin but increased with insulin. Lower concentrations of leptin (10 and 50 microg/kg) also stimulated STAT3 phosphorylation in fat. These effects appear to be direct because 3 min after leptin intracerebroventricular injection, phosphorylation of STAT3, STAT1, and MAPK were not stimulated in hypothalamus or adipose tissue. Furthermore, leptin activated STAT3 and MAPK in adipose tissue explants ex vivo and in 3T3-L1 adipocytes. Leptin did not activate STAT3 or MAPK in adipose tissue of db/db mice. Thus, leptin rapidly activates signaling pathways directly at the level of insulin sensitive tissues through the long-form leptin receptor, and these pathways overlap with, but are distinct from, those engaged by insulin.     

The cross-talk between angiotensin and insulin differentially affects phosphatidylinositol 3-kinase- and mitogen-activated protein kinase-mediated signaling in rat heart: implications for insulin resistance

Insulin and angiotensin II (AngII) may act through overlapping intracellular pathways to promote cardiac myocyte growth. In this report insulin and AngII signaling, through the phosphatidylinositol 3-kinase (PI 3-kinase) and MAPK pathways, were compared in cardiac tissues of control and obese Zucker rats. AngII induced Janus kinase 2 tyrosine phosphorylation and coimmunoprecipitation with insulin receptor substrate 1 (IRS-1) and IRS-2 as well as an increase in tyrosine phosphorylation of IRS and its association with growth factor receptor-binding protein 2. Simultaneous treatment with both hormones led to marked increases in the associations of IRS-1 and -2 with growth factor receptor-binding protein 2 and in the dual phosphorylation of ERK1/2 compared with the administration of AngII or insulin alone. In contrast, an acute inhibition of both basal and insulin-stimulated PI 3-kinase activity was induced by both hormones. Insulin stimulated the phosphorylation of MAPK equally in lean and obese rats. Conversely, insulin-induced phosphorylation of Akt in heart was decreased in obese rats. Pretreatment with losartan did not change insulin-induced activation of ERK1/2 and attenuated the reduction of Akt phosphorylation in the heart of obese rats. Thus, the imbalance between PI 3-kinase-Akt and MAPK signaling pathways in the heart may play a role in the development of cardiovascular abnormalities observed in insulin-resistant states, such as in obese Zucker rats.     

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.     

Glucose modifies the cross-talk between insulin and the beta-adrenergic signalling system in vascular smooth muscle cells

BACKGROUND: Abnormalities in the vascular function of insulin are observed in insulin resistance, and hyperglycaemia is one of the important factors inducing insulin resistance. OBJECTIVE: To investigate the role of glucose in the interaction of insulin and beta-adrenergic signalling systems in vascular smooth muscle cells (VSMC). METHODS: After cells were treated with D-glucose (525 mmol/l) and insulin (100 nmol/l), adenylyl cyclase activity was measured in the presence of isoproterenol, forskolin, and cholera toxin. Assays for insulin-induced activities of insulin receptor substrate (IRS)-1, phosphoinositide 3-kinase (PI3-K) and mitogen-activated protein kinase (MAPK) were performed. RESULTS: In the presence of low glucose concentrations (5 mmol/l), insulin enhanced isoproterenol-, forskolin- and cholera toxin-stimulated adenylyl cyclase activities. This stimulatory effect was abolished by PI3-K inhibitors, wortmannin, or LY294002. In contrast, in the presence of high glucose concentrations (25 mmol/l), insulin attenuated isoproterenol-stimulated activity but not cholera toxin- or forskolin-stimulated activity. Insulin-stimulated activities of IRS-1 and PI3-K, but not MAPK activity, were also attenuated in the presence of high concentrations of glucose. The MAPK kinase inhibitor, PD98059, abolished the inhibitory effect of insulin on the beta-adrenergic signalling system. Troglitazone and pioglitazone prevented this inhibitory effect of insulin by restoring IRS-1 and PI3-K activities. CONCLUSIONS: In the presence of low glucose concentrations, insulin stimulates the beta-adrenergic signalling system through the IRS-1/PI3-K pathway. However, in the presence of high glucose concentrations, the effect of insulin is switched to an inhibitory one, through the MAPK pathway. Our finding suggests that high glucose concentrations modify the cross-talk between insulin and the beta-adrenergic signalling systems in VSMC.     

Leptin activates PI-3 kinase in C2C12 myotubes via janus kinase-2 (JAK-2) and insulin receptor substrate-2 (IRS-2) dependent pathways

Summary We have recently shown that leptin mimicks insulin effects on glucose transport and glycogen synthesis through a phosphatidylinositol-3 (PI) kinase dependent pathway in C₂C₁₂ myotubes. The aim of the present study was to identify the signalling path from the leptin receptor to the PI-3 kinase. We stimulated C₂C₁₂ myotubes with insulin (100 nmol/l, 5 min) or leptin (0.62 nmol/l, 10 min) and determined PI-3 kinase activity in immunoprecipitates with specific non-crossreacting antibodies against insulin-receptor substrate (IRS 1/IRS 2) and against janus kinase (JAK 1 and JAK 2). While insulin-stimulated PI-3 kinase activity is detected in IRS-1 and IRS-2 immunoprecipitates, leptin-stimulated PI-3 kinase activity is found only in IRS-2 immunoprecipitates, suggesting that the leptin signal to PI-3 kinase occurs via IRS-2 and not IRS-1. Leptin-, but not insulin-stimulated PI-3 kinase activity is also detected in immunoprecipitates with antibodies against JAK-2, but not JAK-1. The data suggest that JAK-2 and IRS-2 couple the leptin signalling pathway to the insulin signalling chain. Since we have also detected leptin-stimulated tyrosine phosphorylation of JAK-2 and IRS-2 in C₂C₁₂ myotubes it can be assumed that leptin activates JAK-2 which induces tyrosine phosphorylation of IRS-2 leading to activation of PI-3 kinase. As we could not detect the long leptin receptor isoform in C₂C₁₂ myotubes we conclude that this signalling pathway is activated by a short leptin receptor isoform. [Diabetologia (1997) 40: 1358–1362] Received: 4 August 1997     

Selective impairment of insulin signalling in the hypothalamus of obese Zucker rats

Aim/hypothesis By acting in the brain, insulin suppresses food intake. However, little is known with regard to insulin signalling in the hypothalamus in insulin-resistant states.Methods Western blotting, immunohistochemistry and polymerase chain reaction assays were combined to compare in vivo hypothalamic insulin signalling through the PI3-kinase and MAP kinase pathways between lean and obese Zucker rats.Results Intracerebroventricular insulin infusion reduced food intake in lean rats to a greater extent than that observed in obese rats, and pre-treatment with PI3-kinase inhibitors prevented insulin-induced anorexia. The relative abundance of IRS-2 was considerably higher than that of IRS-1 in hypothalamus of both lean and obese rats. Insulin-stimulated phosphorylation of IR, IRS-1/2, the associations of PI 3-kinase to IRS-1/2 and phosphorylation of Akt in hypothalamus were decreased in obese rats compared to lean rats. These effects seem to be mediated by increased phosphoserine content of IR, IRS-1/2 and decreased protein levels of IRS-1/2 in obese rats. In contrast, insulin stimulated the phosphorylation of MAP kinase equally in lean and obese rats.Conclusion/interpretation This study provides direct measurements of insulin signalling in hypothalamus, and documents selective resistance to insulin signalling in hypothalamus of Zucker rats. These findings provide support for the hypothesis that insulin could have anti-obesity actions mediated by the PI3-kinase pathway, and that impaired insulin signalling in hypothalamus could play a role in the development of obesity in this animal model of insulin-resistance.Abbreviations ERK extracellular signal-regulated kinase - Grb2 growth factor receptor binding protein 2 - IR insulin receptor - IRS insulin receptor substrate - MAPK mitogen-activated protein kinase - PI 3-kinase phosphatidylinositol 3-kinase - PKC Protein kinase C - Shc Src-homology and collagen homology - SHP2 Src-homology phosphatase 2 - TNF- Tumor-necrosis factor-     

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.     

Crosstalk between insulin and angiotensin II signalling systems.

Insulin resistance and hypertension commonly occur together. Pharmacological inhibition of the renin-angiotensin system has been found to reduce not only hypertension, but also insulin resistance. This raises the possibility that the renin-angiotensin system may interact with insulin signalling. We have investigated the relationship between insulin and angiotensin II (AII) intracellular signalling in vivo using an intact rat heart model, and in vitro using rat aorta smooth muscle cells (RASMC). Results generated in the in vivo studies indicate that, like insulin, AII stimulates tyrosine phosphorylation of the insulin receptor substrates IRS-1 and IRS-2. This leads to binding of IRS-1 and IRS-2 to PI3-kinase. However, in contrast to the effect of insulin. IRS-1- and IRS-2-associated PI3-kinase activity is inhibited by AII in a dose-dependent manner. Moreover, AII inhibits insulin-stimulated IRS-1/IRS-2-associated PI3-kinase activity. The in vivo effects of AII are mediated via the AT1 receptor. The results of the in vitro studies indicate that AII inhibits insulin-stimulated, IRS-1-associated PI3-kinase activity by interfering with the docking of IRS-1 with the p85 regulatory subunit of PI3-kinase. It appears that AII achieves this effect by stimulating serine phosphorylation of the insulin receptor beta-subunit IRS-1, and the p85 regulatory subunit of PI3-kinase. These actions result in the inhibition of normal interactions between the insulin signalling pathway components. Thus, we believe that AII negatively modulates insulin signalling by stimulating multiple serine phosphorylation events in the early components of the insulin signalling cascade. Overactivity of the renin-angiotensin system is likely to impair insulin signalling and contribute to insulin resistance observed in essential hypertension.     

Activation of the hexosamine pathway leads to phosphorylation of insulin receptor substrate-1 on Ser307 and Ser612 and impairs the phosphatidylinositol 3-kinase/Akt/mammalian target of rapamycin insulin biosynthetic pathway in RIN pancreatic beta-cells

Many adverse effects of glucose were attributed to its increased routing through the hexosamine pathway (HBP). There is evidence for an autocrine role of the insulin signaling in beta-cell function. We tested the hypothesis that activation of the HBP induces defects in insulin biosynthesis by affecting the insulin-mediated protein translation signaling. Exposure of human pancreatic islets and RIN beta-cells to glucosamine resulted in reduction in glucose- and insulin-stimulated insulin biosynthesis, which in RIN beta-cells was associated with impairment in insulin-stimulated insulin receptor substrate-1 (IRS-1) phosphorylation at Tyr(608) and Tyr(628), which are essential for engaging phosphatidylinositol 3-kinase (PI 3-kinase). These changes were accompanied by impaired activation of PI 3-kinase, and activation of Akt/mammalian target of rapamycin/phosphorylated heat- and acid-stable protein-1/p70S6 kinase pathway. RIN beta-cells exposed to high glucose exhibited increased c-Jun N-terminal kinase (JNK) and ERK1/2 activity, which was associated with increased IRS-1 phosphorylation at serine (Ser)(307) and Ser(612), respectively, that inhibits coupling of IRS-1 to the insulin receptor and is upstream of the inhibition of IRS-1 tyrosine phosphorylation. Azaserine reverted the stimulatory effects of high glucose on JNK and ERK1/2 activity and IRS-1 phosphorylation at Ser(307) and Ser(612). Glucosamine mimicked the stimulatory effects of high glucose on JNK and ERK1/2 activity and IRS-1 phosphorylation at Ser(307) and Ser(612). Inhibition of JNK and MAPK kinase-1 activity reverted the negative effects of glucosamine on insulin-mediated protein synthesis. These results suggest that activation of the HBP accounts, in part, for glucose-induced phosphorylation at Ser(307) and Ser(612) of IRS-1 mediated by JNK and ERK1/2, respectively. These changes result in impaired coupling of IRS-1 and PI 3-kinase, and activation of the Akt/mammalian target of rapamycin/phosphorylated heat- and acid-stable protein-1/p70S6 kinase pathway.     

Selective interaction between leptin and insulin signaling pathways in a hepatic cell line

Leptin is a 16-kDa hormone secreted by adipocytes and plays an important role in control of feeding behavior and energy expenditure. In obesity, circulating levels of leptin and insulin are high because of the presence of increased body fat mass and insulin resistance. Recent reports have suggested that leptin can act through some of the components of the insulin signaling cascade, such as insulin receptor substrates (IRS-1 and IRS-2), phosphatidylinositol 3-kinase (PI 3-kinase), and mitogen-activated protein kinase, and can modify insulin-induced changes in gene expression in vitro and in vivo. Well differentiated hepatoma cells (Fao) possess both the long and short forms of the leptin receptor and respond to leptin with a stimulation of c-fos gene expression. In Fao cells, leptin alone had no effects on the insulin signaling pathway, but leptin pretreatment transiently enhanced insulin-induced tyrosine phosphorylation and PI 3-kinase binding to IRS-1, while producing an inhibition of tyrosine phosphorylation and PI 3-kinase binding to IRS-2. Leptin alone also induced serine phosphorylation of Akt and glycogen synthase kinase 3 but to a lesser extent than insulin, and the combination of these hormones was not additive. These results suggest complex interactions between the leptin and insulin signaling pathways that can potentially lead to differential modification of the metabolic and mitotic effects of insulin exerted through IRS-1 and IRS-2 and the downstream kinases that they activate.     

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.     

Acute ethanol exposure inhibits insulin signaling in the liver

Chronic ethanol consumption may produce hepatic injury and impair the ability of the liver to regenerate principally through its action on insulin signaling. These effects are mediated by insulin receptor substrate-1 (IRS-1) via the mitogen-activated protein kinase/extracellular signal regulated kinase (MAPK/Erk) pathway and by survival signals through phosphatidylinositol-3 kinase (PI3K) and protein kinase B (Akt). Because a protein phosphatase, phosphatase tensin homolog deleted on chromosome 10 (PTEN), has been reported to block insulin signaling through PI3K, we explored acute ethanol effects on signaling in the context of PTEN function. We measured upstream components of the insulin signal transduction pathway and Akt phosphorylation as an indicator of signaling through PI3K, including the generation of survival signals via glycogen synthase kinase 3beta (GSK3beta) and Bcl-2-associated death promoter (BAD). In addition, the physical association between PTEN and PI3K regulatory (p85alpha) and catalytic (p110alpha) subunits was evaluated both in vitro and in vivo. In Huh-7 cells, there was no effect of acute ethanol exposure on tyrosyl phosphorylation of the insulin receptor, IRS-1, and the association of IRS-1 with PI3K. However, Akt phosphorylation was impaired. The association of PTEN with the PI3K p85alpha subunit was substantially increased and led to the inhibition of downstream insulin-mediated survival signals through Akt, GSK3beta, and BAD; the ethanol effect was reversed by PTEN knockdown with small interfering RNA. These results were confirmed in the liver. Conclusion: Short-term ethanol exposure rapidly attenuates insulin signaling. The major cellular mechanism involves the increased association of PTEN with the PI3K p85alpha subunit, which results in reduced phospho-Akt formation and impaired downstream survival signaling. These findings may have relevance to acute toxic effects of ethanol on the liver.     

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.     

Regulation of insulin signalling by hyperinsulinaemia: role of IRS-1/2 serine phosphorylation and the mTOR/p70 S6K pathway

Aim/hypothesis Several epidemiological studies have suggested an association between chronic hyperinsulinaemia and insulin resistance. However, the causality of this relationship remains uncertain.Methods We performed chronic hyperinsulinaemic–euglycaemic clamps and delineated, by western blotting, an IR/IRSs/phosphatidylinositol 3-kinase(PI[3]K)/Akt pathway in insulin-responsive tissues of hyperinsulinaemic rats. IRS-1/2 serine phosphorylation, IR/protein tyrosine phosphatase 1B (PTP1B) association, and mammalian target of rapamycin (mTOR)/p70 ribosomal S6 kinase (p70 S6K) activity were also evaluated in the liver, skeletal muscle and white adipose tissue of hyperinsulinaemic animals.Results We found that chronic hyperinsulinaemic rats have insulin resistance and reduced levels of glycogen content in liver and muscle. In addition, we demonstrated an impairment of the insulin-induced IR/IRSs/PI(3)K/Akt pathway in liver and muscle of chronic hyperinsulinaemic rats that parallels increases in IRS1/2 serine phosphorylation, IR/PTP1B association and mTOR activity. Despite a higher association of IR/PTP1B, there was an increase in white adipose tissue of chronic hyperinsulinaemic rats in IRS-1/2 protein levels, tyrosine phosphorylation and IRSs/PI(3)K association, which led to an increase in basal Akt serine phosphorylation. No increases in IRS-1/2 serine phosphorylation and mTOR activity were observed in white adipose tissue. Rapamycin reversed the insulin resistance and the changes induced by hyperinsulinaemia in the three tissues studied.Conclusions/interpretation Our data provide evidence that chronic hyperinsulinaemia itself, imposed on normal rats, appears to have a dual effect, stimulating insulin signalling in white adipose tissue, whilst decreasing it in liver and muscle. The underlying mechanism of these differential effects may be related to the ability of hyperinsulinaemia to increase mTOR/p70 S6K pathway activity and IRS-1/2 serine phosphorylation in a tissue-specific fashion. In addition, we demonstrated that inhibition of the mTOR pathway with rapamycin can prevent insulin resistance caused by chronic hyperinsulinaemia in liver and muscle. These findings support the hypothesis that defective and tissue-selective insulin action contributes to the insulin resistance observed in hyperinsulinaemic states.