Insulin-stimulated Phosphatidylinositol 3-kinase Research Articles

Vav2 catalysis-dependent pathways contribute to skeletal muscle growth and metabolic homeostasis

Skeletal muscle promotes metabolic balance by regulating glucose uptake and the stimulation of multiple interorgan crosstalk. We show here that the catalytic activity of Vav2, a Rho GTPase activator, modulates the signaling output of the IGF1- and insulin-stimulated phosphatidylinositol 3-kinase pathway in that tissue. Consistent with this, mice bearing a Vav2 protein with decreased catalytic activity exhibit reduced muscle mass, lack of proper insulin responsiveness and, at much later times, a metabolic syndrome-like condition. Conversely, mice expressing a catalytically hyperactive Vav2 develop muscle hypertrophy and increased insulin responsiveness. Of note, while hypoactive Vav2 predisposes to, hyperactive Vav2 protects against high fat diet-induced metabolic imbalance. These data unveil a regulatory layer affecting the signaling output of insulin family factors in muscle.

Adipocyte Overexpression of p55alpha Subunit of Pi3k Improves Systemic Glucose Metabolism in Lean Mice

Phosphatidylinositol 3-kinase (PI3K), a heterodimeric enzyme composed of a regulatory subunit (p85α/β, p55α/γ, p50α) and a catalytic subunit (p110α/β), is an essential component of the canonical insulin signaling cascade in adipose tissue. Insulin-stimulated PI3K activity is decreased in obese adipose tissue in parallel with a 4-fold increase in p55α abundance; heterozygous deletion of Pik3r1 limits regulatory subunit expression and enhances adipocyte insulin signaling. Additionally, genetic deletion of p55α/p50α in mice has been shown to increase adipocyte glucose uptake and systemic insulin sensitivity. Therefore, we hypothesized that adipocyte-specific p55α overexpression (OX) would reduce adipocyte insulin signaling and decrease systemic glucose tolerance. To test this hypothesis, we generated mice with p55α knock-in at the endogenous ROSA26 locus. The recombination cassette was made by flanking the p55α cDNA and a floxed 4x SV40 polyA STOP signal with FRT and F3. The insertion of Pik3r1 at the ROSA26 locus mediated by Flp-recombinase in ES cells targeted with FRT-PGK-neo-F3 at the ROSA26 locus. The polyA STOP signal was removed by crossing the p55α flox (Fl/Fl) mice with mice carrying Adipoq-cre recombinase (OX). At 12 weeks of age, the metabolic phenotype, glucose tolerance and adipocyte insulin signaling were measured in Fl/FL and OX mice. In adipose tissue, p55α abundance was 7-fold higher in OX compared to FL/FL controls with no difference detected in p85α abundance. Body weight and adiposity was not different between FL/FL and OX mice. Food intake, energy expenditure and the respiratory quotient were not different in OX vs. FL/FL, despite a decrease in total activity in OX mice. Contrary to our hypothesis, glucose AUC calculated from oral glucose tolerance tests was 40% lower (P=0.005) in OX vs. FL/FL. Overall these data suggest that increased expression of the p55α regulatory subunit, in contrast to p85α, enhances PI3K activity and insulin signaling in adipocytes. Disclosure Z.S. Clayton: None. C. McCurdy: None.

Inducible heterozygous knockout of Pik3r1 in adipocytes reverses glucose intolerance and enhances adipocyte insulin signaling in obese mice

Insulin resistance is a central and defining feature of the metabolic diseases associated with obesity. Phosphatidylinositol 3‐kinase (PI3K), a heterodimeric enzyme composed of a regulatory subunit (p85α/β, p55α/γ, p50α) and a catalytic subunit (p110α/β), is an essential component of the conical insulin signaling cascade in adipose tissue. PI3K activity is decreased in obese adipose tissue (AT); however, a global heterozygous (HZ) deletion of Pik3r1 (encodes p85/p55/050α) prevents the decrease in insulin‐stimulated PI3K activity, and reduces AT inflammation and systemic insulin resistance relative to obese control mice. Therefore, we hypothesized that adipocyte‐specific deletion of Pik3r1 in already obese mice would reverse obesity‐induced systemic insulin resistance by increasing insulin signaling downstream of PI3K in adipocytes. Inducible adipocyte‐specific Pik3r1 heterozygous (HZ) or homozygous (KO) knockout mice were generated by crossing Pik3r1 flox mice (LoxP sites flanking exon7 of the Pik3r1 gene) with Adipoq‐CreERT2 mice, which utilize tamoxifen‐dependent recombination. Pik3r1 flox/flox (FL/FL) mice were used as controls. At 8 weeks of age, mice were fed a high‐fat diet (HFD; 45% fat) or sucrose‐matched control diet (10% fat) for 10 weeks. At week 11, tamoxifen was delivered by oral gavage (100μL of 10 mg/mL) for 5 days and allowed to recover for 1 week) glucose tolerance was measured before and 1–2 weeks post tamoxifen. Total activity, energy expenditure and respiratory exchange ratio (RER) were collected for 3 days following one‐week acclimation and recovery. Isolated adipocytes from lean and obese mice were stimulated +/− 1.2nM insulin and signaling proteins were measured by immunoassay. There were differences in body weight, body composition before or after gene knockout with tamoxifen in HFD vs. CON, for WT, HZ and KO. Total activity (WT: 90±6; HZ: 115±12; KO: 106±10), energy expenditure (WT: 143±11; HZ: 122±12; KO: 134±12), and RER (WT: 0.73±0.001; HZ: 0.775±0.03; KO: 0.81±0.024) were not different for WT, HZ, and KO during HFD. Glucose AUC calculated from oral glucose tolerance test was significantly higher in all groups with HFD vs. CON. We find that after tamoxifen dosing, glucose (g) AUC was reduced by 59% in obese HZ mice as compared to pre‐tamoxifen levels. No changes in gAUC were found in obese WT or KO. Insulin‐stimulated AKT(S473) phosphorylation was decreased by 41% and 63% in adipocytes from obese WT and KO, respectively, compared to lean; however, only 17% in obese HZ. Taken together, these data show that adipocyte Pik3r1 plays a critical role in mediating systemic glucose metabolism and that novel treatments that target Pik3r1expression may be successful in improving insulin sensitivity in individuals with obesity.Support or Funding InformationSupported by Eugene & Clarissa Evonuk Memorial Fellowship, University of Oregon Graduate Student Food Studies Grant (Z.S.C) and National Institute of Diabetes Digestive and Kidney Diseases under Award Number RO1DK095926 (C.E.M)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Impaired insulin-stimulated glucose transport in ATM-deficient mouse skeletal muscle

There are reports that ataxia telangiectasia mutated (ATM) plays a role in insulin-stimulated Akt phosphorylation, although this is not the case in some cell types. Because Akt plays a key role in insulin signaling, which leads to glucose transport in skeletal muscle, the predominant tissue in insulin-stimulated glucose disposal, we examined whether insulin-stimulated Akt phosphorylation and (or) glucose transport would be decreased in skeletal muscle of mice lacking functional ATM, compared with muscle from wild-type mice. We found that in vitro insulin-stimulated Akt phosphorylation was normal in soleus muscle from mice with 1 nonfunctional allele of ATM (ATM+/-) and from mice with 2 nonfunctional alleles (ATM-/-). However, insulin did not stimulate glucose transport or the phosphorylation of AS160 in ATM-/- soleus. ATM protein level was markedly higher in wild-type extensor digitorum longus (EDL) than in wild-type soleus. In EDL from ATM-/- mice, insulin did not stimulate glucose transport. However, in contrast to findings for soleus, insulin-stimulated Akt phosphorylation was blunted in ATM-/- EDL, concomitant with a tendency for insulin-stimulated phosphatidylinositol 3-kinase activity to be decreased. Together, the findings suggest that ATM plays a role in insulin-stimulated glucose transport at the level of AS160 in muscle comprised of slow and fast oxidative-glycolytic fibers (soleus) and at the level of Akt in muscle containing fast glycolytic fibers (EDL).

Insulin receptor and IRS-1 co-immunoprecipitation with SOCS-3, and IKKα/β phosphorylation are increased in obese Zucker rat skeletal muscle

AimsWe evaluated if selected pro-inflammatory cytokines and/or the protein suppressor of cytokine signaling 3 (SOCS-3) could account for decreased insulin-stimulated phosphatidylinositol 3-kinase (PI3-K) activity in the skeletal muscle of the obese Zucker rat. Main methodsEight lean and eight obese Zucker rats ~4weeks of age were obtained and allowed to feed ad libitum for 4weeks before undergoing hind limb perfusion in the presence of 500μU/ml insulin. Key findingsInsulin-stimulated skeletal muscle PI3-K activity and 3-O-methylglucose transport rates were reduced (P<0.05) in obese compared to lean animals. IRS-1 concentration remained unchanged although IRS-1 tyrosine phosphorylation was decreased (P<0.05), and IRS-1 serine phosphorylation (pS) was increased (P<0.05) in obese animals compared to lean animals. IKKα/β pS and JNK theronine/tyrosine phosphorylation was increased (P<0.05) in the obese animals. IκBα concentration was decreased (P<0.05) and IκBα pS was increased (P<0.05) in the obese compared to lean Zucker animals. SOCS-3 concentration and SOCS-3 co-immunoprecipitation with both insulin receptor β-subunit (IR-β) and IRS-1 were elevated (P<0.05) in obese compared to lean animals. IRS-1 co-immunoprecipitation with IR-β was reduced 56% in the obese animals. SignificanceIncreased IKKα/β and JNK serine phosphorylation may contribute to increasing IRS-1 serine phosphorylation, while concurrent co-localization of SOCS-3 with both IR-β and IRS-1 may prevent IRS-1 from interacting with IR-β. These two mechanisms thusly may independently contribute to impairing insulin-stimulated PI3-K activation in the skeletal muscle of the obese Zucker rat.

Regulation of Glucose Transport by ROCK1 Differs from That of ROCK2 and Is Controlled by Actin Polymerization

A role of Rho-associated coiled-coil-containing protein kinase (ROCK)1 in regulating whole-body glucose homeostasis has been reported. However, cell-autonomous effects of ROCK1 on insulin-dependent glucose transport in adipocytes and muscle cells have not been elucidated. To determine the specific role of ROCK1 in glucose transport directly, ROCK1 expression in 3T3-L1 adipocytes and L6 myoblasts was biologically modulated. Here, we show that small interfering RNA-mediated ROCK1 depletion decreased insulin-induced glucose transport in adipocytes and myoblasts, whereas adenovirus-mediated ROCK1 expression increased this in a dose-dependent manner, indicating that ROCK1 is permissive for glucose transport. Inhibition of ROCK1 also impaired glucose transporter 4 translocation in 3T3-L1 adipocytes. Importantly, the ED₅₀ of insulin for adipocyte glucose transport was reduced when ROCK1 was expressed, leading to hypersensitivity to insulin. These effects are dependent on actin cytoskeleton remodeling, because inhibitors of actin polymerization significantly decreased ROCK1's effect to promote insulin-stimulated glucose transport. Unlike ROCK2, ROCK1 binding to insulin receptor substrate (IRS)-1 was not detected by immunoprecipitation, although cell fractionation demonstrated both ROCK isoforms localize with IRS-1 in low-density microsomes. Moreover, insulin's ability to increase IRS-1 tyrosine 612 and serine 632/635 phosphorylation was attenuated by ROCK1 suppression. Replacing IRS-1 serine 632/635 with alanine reduced insulin-stimulated phosphatidylinositol 3-kinase activation and glucose transport in 3T3-L1 adipocytes, indicating that phosphorylation of these serine residues of IRS-1, which are substrates of the ROCK2 isoform in vitro, are crucial for maximal stimulation of glucose transport by insulin. Our studies identify ROCK1 as an important positive regulator of insulin action on glucose transport in adipocytes and muscle cells.

Disrupted Anabolic and Catabolic Processes May Contribute to Alcohol-Accentuated SAIDS-Associated Wasting

Alcohol abuse is a comorbid factor in many human immunodeficiency virus (HIV)-infected patients. Previously, we demonstrated that chronic binge alcohol accentuates loss of body mass at terminal stage of simian immunodeficiency virus (SIV) infection. The purpose of this study was to investigate changes in pathways that may contribute to muscle wasting in chronic binge alcohol-fed SIV-infected macaques. The impact of chronic binge alcohol during SIV infection on insulin signaling and the ubiquitin (Ub)-proteasome system-regulators of protein synthesis and degradation-was examined in SIV-infected macaques. SIV infection induced an inflammatory and pro-oxidative milieu in skeletal muscle, which was associated with decreased insulin-stimulated phosphatidylinositol 3-kinase (PI-3k) activity and upregulated gene expression of mTOR and atrogin-1, and protein expression of Ub-proteasome system 19S base. Chronic binge alcohol accentuated the skeletal muscle pro-oxidative milieu and 19S base expression. Additionally, chronic binge alcohol increased skeletal muscle protein expression of protein-tyrosine phosphatase 1B (a negative regulator of insulin signaling) and 19S proteasome regulator non-ATPase (Rpn) 6 subunit and Rpn12, and suppressed PI-3K activity. Animals that were alcohol-fed and SIV-infected for >15 months had increased Ub-proteasome system activity. These data suggest negative modulation of insulin signaling coupled with enhanced Ub-proteasome system activity may be central mechanisms underlying chronic binge alcohol-induced accentuation of SIV-associated muscle wasting.

Adipose Triglyceride Lipase Deficiency Causes Tissue-specific Changes in Insulin Signaling

Triacylglycerol accumulation in insulin target tissues is associated with insulin resistance. Paradoxically, mice with global targeted deletion of adipose triglyceride lipase (ATGL), the rate-limiting enzyme in triacylglycerol hydrolysis, display improved glucose tolerance and insulin sensitivity despite triacylglycerol accumulation in multiple tissues. To determine the molecular mechanisms for this phenotype, ATGL-deficient (ATGL(-/-)) and wild-type mice were injected with saline or insulin (10 units/kg, intraperitoneally), and then phosphorylation and activities of key insulin-signaling proteins were determined in insulin target tissues (liver, adipose tissue, and muscle). Insulin signaling and/or glucose transport was also evaluated in isolated adipocytes and skeletal muscle ex vivo. In ATGL(-/-) mice, insulin-stimulated phosphatidylinositol 3-kinase and Akt activities as well as phosphorylation of critical residues of IRS1 (Tyr(P)-612) and Akt (Ser(P)-473) were increased in skeletal muscle in vivo. Insulin-stimulated phosphatidylinositol 3-kinase activity and total insulin receptor and insulin receptor substrate 1, but not other parameters, were also increased in white adipose tissue in vivo. In contrast, in vivo measures of insulin signaling were decreased in brown adipose tissue and liver. Interestingly, the enhanced components of insulin signaling identified in skeletal muscle and white adipose tissue in vivo and their expected downstream effects on glucose transport were not present ex vivo. ATGL deficiency altered intramyocellular lipids as well as serum factors known to influence insulin sensitivity. Thus, skeletal muscle, rather than other tissues, primarily contributes to enhanced insulin sensitivity in ATGL(-/-) mice in vivo despite triacylglycerol accumulation, and both local and systemic factors contribute to tissue-specific effects of global ATGL deficiency on insulin action.

Targeted Disruption of ROCK1 Causes Insulin Resistance in Vivo

Insulin signaling is essential for normal glucose homeostasis. Rho-kinase (ROCK) isoforms have been shown to participate in insulin signaling and glucose metabolism in cultured cell lines. To investigate the physiological role of ROCK1 in the regulation of whole body glucose homeostasis and insulin sensitivity in vivo, we studied mice with global disruption of ROCK1. Here we show that, at 16-18 weeks of age, ROCK1-deficient mice exhibited insulin resistance, as revealed by the failure of blood glucose levels to decrease after insulin injection. However, glucose tolerance was normal in the absence of ROCK1. These effects were independent of changes in adiposity. Interestingly, ROCK1 gene ablation caused a significant increase in glucose-induced insulin secretion, leading to hyperinsulinemia. To determine the mechanism(s) by which deletion of ROCK1 causes insulin resistance, we measured the ability of insulin to activate phosphatidylinositol 3-kinase and multiple distal pathways in skeletal muscle. Insulin-stimulated phosphatidylinositol 3-kinase activity associated with IRS-1 or phospho-tyrosine was also reduced approximately 40% without any alteration in tyrosine phosphorylation of insulin receptor in skeletal muscle. Concurrently, serine phosphorylation of IRS-1 at serine 632/635, which is phosphorylated by ROCK in vitro, was also impaired in these mice. Insulin-induced phosphorylation of Akt, AS160, S6K, and S6 was also decreased in skeletal muscle. These data suggest that ROCK1 deficiency causes systemic insulin resistance by impairing insulin signaling in skeletal muscle. Thus, our results identify ROCK1 as a novel regulator of glucose homeostasis and insulin sensitivity in vivo, which could lead to new treatment approaches for obesity and type 2 diabetes.

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 p85α 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.

Protein kinase C-zeta phosphorylates insulin receptor substrate-1, -3, and -4 but not -2: isoform specific determinants of specificity in insulin signaling.

Protein kinase C-zeta, a downstream effector of phosphatidylinositol 3-kinase (PI3K), phosphorylates insulin receptor substrate (IRS)-1 on serine residues impairing activation of PI3K in response to insulin. Because IRS-1 is upstream from PI3K, this represents a negative feedback mechanism that may contribute to signal specificity in insulin action. To determine whether similar feedback pathways exist for other IRS isoforms, we evaluated IRS-2, -3, and -4 as substrates for PKC-zeta. In an in vitro kinase assay, purified recombinant PKC-zeta phosphorylated IRS-1, -3 and -4 but not IRS-2. Similar results were obtained with an immune-complex kinase assay demonstrating that wild-type, but not kinase-deficient mutant PKC-zeta, phosphorylated IRS-1, -3, and -4 but not IRS-2. We evaluated functional consequences of serine phosphorylation of IRS isoforms by PKC-zeta in NIH-3T3(IR) cells cotransfected with epitope-tagged IRS proteins and either PKC-zeta or empty vector control. Insulin-stimulated IRS tyrosine phosphorylation was impaired by overepxression of PKC-zeta for IRS-1, -3, and -4 but not IRS-2. Significant insulin-stimulated increases in PI3K activity was coimmunoprecipitated with all IRS isoforms. In cells overexpressing PKC-zeta there was marked inhibition of insulin-stimulated PI3K activity associated with IRS-1, -3 and -4 but not IRS-2. That is, PI3K activity associated with IRS-2 in response to insulin was similar in control cells and cells overexpressing PKC-zeta. We conclude that IRS-3 and -4 are novel substrates for PKC-zeta that may participate in a negative feedback pathway for insulin signaling similar to IRS-1. The inability of PKC-zeta to phosphorylate IRS-2 may help determine specific functional roles for IRS-2.

Increased p85/55/50 Expression and Decreased Phosphotidylinositol 3-Kinase Activity in Insulin-Resistant Human Skeletal Muscle

Insulin resistance is predominantly characterized by decreased insulin-stimulated glucose uptake into skeletal muscle. In the current study, we have assessed various aspects of the phosphatidylinositol (PI) 3-kinase pathway in skeletal muscle biopsies obtained from normal, obese nondiabetic, and type 2 diabetic subjects, before and after a 5-h insulin infusion. We found a highly significant inverse correlation between in vivo insulin sensitivity (as measured by the glucose infusion rate) and increased protein expression of p85/55/50, protein kinase C (PKC)-theta activity, levels of pSer307 insulin receptor substrate (IRS)-1 and p-Jun NH2-terminal kinase (JNK)-1, and myosin heavy chain IIx fibers. Increased basal phosphorylation of Ser307 IRS-1 in the obese and type 2 diabetic subjects corresponds with decrease in insulin-stimulated IRS-1 tyrosine phosphorylation, PI 3-kinase activity, and insulin-induced activation of Akt and, more prominently, PKC-zeta/lambda. In summary, increased expression of the PI 3-kinase adaptor subunits p85/55/50, as well as increased activity of the proinflammatory kinases JNK-1, PKC-theta, and, to a lesser extent, inhibitor of kappaB kinase-beta, are associated with increased basal Ser307 IRS-1 phosphorylation and decreased PI 3-kinase activity and may follow a common pathway to attenuate in vivo insulin sensitivity in insulin-resistant subjects. These findings demonstrate interacting mechanisms that can lead to impaired insulin-stimulated PI 3-kinase activity in skeletal muscle from obese and type 2 diabetic subjects.

Phosphatidylinositol 4,5-Bisphosphate Reverses Endothelin-1–Induced Insulin Resistance via an Actin-Dependent Mechanism

Phosphatidylinositol (PI) 4,5-bisphosphate (PIP(2)) plays a pivotal role in insulin-stimulated glucose transport as an important precursor to PI 3,4,5-trisphosphate (PIP(3)) and a key regulator of actin polymerization. Since endothelin (ET)-1 impairs insulin sensitivity and PIP(2) is a target of ET-1-induced signaling, we tested whether a change in insulin-stimulated PIP(3) generation and signaling, PIP(2)-regulated actin polymerization, or a combination of both accounted for ET-1-induced insulin resistance. Concomitant with a time-dependent loss of insulin sensitivity, ET-1 caused a parallel reduction in plasma membrane PIP(2). Despite decreased insulin-stimulated PI 3-kinase activity and PIP(3) generation, ET-1 did not diminish downstream signaling to Akt-2. Furthermore, addition of exogenous PIP(2), but not PIP(3), restored insulin-regulated GLUT4 translocation and glucose transport impaired by ET-1. Microscopic and biochemical analyses revealed a PIP(2)-dependent loss of cortical filamentous actin (F-actin) in ET-1-treated cells. Restoration of insulin sensitivity by PIP(2) add-back occurred concomitant with a reestablishment of cortical F-actin. The corrective effect of exogenous PIP(2) in ET-1-induced insulin-resistant cells was not present in cells where cortical F-actin remained experimentally depolymerized. These data suggest that ET-1-induced insulin resistance results from reversible changes in PIP(2)-regulated actin polymerization and not PIP(2)-dependent signaling.

Chronic aerobic exercise enhances components of the classical and novel insulin signalling cascades in Sprague–Dawley rat skeletal muscle

The aim of this study was to provide a more extensive evaluation of the effects of chronic aerobic exercise on various components of the insulin signalling cascade in normal rodent skeletal muscle because of the limited body of literature that exists in this area of investigation. Male Sprague-Dawley rats were assigned to either control (n = 7) or chronic aerobic exercise (n = 7) groups. Aerobic exercise animals were run 3 day week(1) for 45 min on a motor-driven treadmill (32 m min(1), 15% grade) for a 12 week period. Following the training period, all animals were subjected to hind limb perfusion in the presence of 500 microU mL(1) insulin to determine what effect chronic aerobic training had on various components of the insulin signalling cascade, c-Cbl protein concentration and c-Cbl phosphorylation. Twelve weeks of aerobic training did not alter skeletal muscle Akt 1/2 protein concentration, Akt Ser 473 phosphorylation, Akt Thr 308 phosphorylation, Akt 1 activity, aPKC-zeta protein concentration, aPKC-lambda protein concentration or c-Cbl protein concentration. In contrast, chronic aerobic exercise increased insulin-stimulated phosphatidylinositol 3-kinase, Akt 2 kinase and aPKC-zeta/lambda kinase activities, as well as c-Cbl tyrosine phosphorylation, in a fibre type specific response to aerobic training. In addition, chronic aerobic exercise enhanced insulin-stimulated plasma membrane glucose transporter 4 (GLUT4) protein concentration. Collectively, these findings suggest that chronic aerobic exercise enhances components of both the classical and novel insulin signalling cascades in normal rodent skeletal muscle, which may contribute to an increased insulin-stimulated plasma membrane GLUT4 protein concentration.

α1A-Adrenoceptors Activate Glucose Uptake in L6 Muscle Cells through a Phospholipase C-, Phosphatidylinositol-3 Kinase-, and Atypical Protein Kinase C-Dependent Pathway

The role of alpha1-adrenoceptor activation on glucose uptake in L6 cells was investigated. The alpha1-adrenoceptor agonist phenylephrine [pEC50 (-log10 EC50), 5.27 +/- 0.30] or cirazoline (pEC50, 5.00 +/- 0.23) increased glucose uptake in a concentration-dependent manner, as did insulin (pEC50, 7.16 +/- 0.21). The alpha2-adrenoceptor agonist clonidine was without any stimulatory effect on glucose uptake. The stimulatory effect of cirazoline was inhibited by the alpha1-adrenoceptor antagonist prazosin, but not by the beta-adrenoceptor antagonist propranolol. RT-PCR showed that the alpha1A-adrenoceptor was the sole alpha1-adrenoceptor subtype expressed in L6 cells. Cirazoline- or insulin-mediated glucose uptake was inhibited by the phosphatidylinositol-3 kinase inhibitor LY294002, suggesting a possible interaction between the alpha1-adrenoceptor and insulin pathways. Cirazoline or insulin stimulated phosphatidylinositol-3 kinase activity, but alpha1-adrenoceptor activation did not phosphorylate Akt. Both cirazoline- and insulin-mediated glucose uptake were inhibited by protein kinase C (PKC), phospholipase C, and p38 kinase inhibitors, but not by Erk1/2 inhibitors (despite both treatments being able to phosphorylate Erk1/2). Insulin and cirazoline were able to activate and phosphorylate p38 kinase. The phorbol ester 12-O-tetradecanoylphorbol-13-acetate and the calcium ionophore A23187 produced significant increases in glucose uptake, indicating roles for PKC and calcium in glucose uptake. Down-regulation of conventional PKC isoforms inhibited glucose uptake mediated by 12-O-tetradecanoylphorbol-13-acetate, but not by insulin or cirazoline. This study demonstrates that alpha1-adrenoceptors mediate increases in glucose uptake in L6 muscle cells. This effect appears to be related to activation of phospholipase C, phosphatidylinositol-3 kinase, p38 kinase, and PKC.

Atypical protein kinase C (PKCzeta/lambda) is a convergent downstream target of the insulin-stimulated phosphatidylinositol 3-kinase and TC10 signaling pathways.

Insulin stimulation of adipocytes resulted in the recruitment of atypical PKC (PKCζ/λ) to plasma membrane lipid raft microdomains. This redistribution of PKCζ/λ was prevented by Clostridium difficile toxin B and by cholesterol depletion, but was unaffected by inhibition of phosphatidylinositol (PI) 3-kinase activity. Expression of the constitutively active GTP-bound form of TC10 (TC10Q/75L), but not the inactive GDP-bound mutant (TC10/T31N), targeted PKCζ/λ to the plasma membrane through an indirect association with the Par6–Par3 protein complex. In parallel, insulin stimulation as well as TC10/Q75L resulted in the activation loop phosphorylation of PKCζ. Although PI 3-kinase activation also resulted in PKCζ/λ phosphorylation, it was not recruited to the plasma membrane. Furthermore, insulin-induced GSK-3β phosphorylation was mediated by both PI 3-kinase–PKB and the TC10–Par6–atypical PKC signaling pathways. Together, these data demonstrate that PKCζ/λ can serve as a convergent downstream target for both the PI 3-kinase and TC10 signaling pathways, but only the TC10 pathway induces a spatially restricted targeting to the plasma membrane.

High-fat diet and leptin treatment alter skeletal muscle insulin-stimulated phosphatidylinositol 3-kinase activity and glucose transport

The aim of this investigation was to evaluate if leptin treatment enhances insulin-stimulated glucose transport in normal (experimental group [EXP]-1) and insulin-resistant skeletal muscle (EXP-2) by altering components of the insulin-signaling cascade and/or glucose transport pathway. In EXP-1, Sprague Dawley rats were assigned to control-chow fed (CON-CF) or leptin treated-chow fed (LEP-CF) groups. Animals were implanted with miniosmotic pumps, which delivered 0.5 mg leptin/kg/d to the LEP-CF animals and vehicle to CON-CF animals for 14 days. For EXP-2, Sprague-Dawley rats consumed normal (CON) or high-fat diets for 3 months. After the dietary lead in, the high-fat diet group was further subdivided into high-fat (HF) and high-fat, leptin-treated (HF-LEP) animals. HF-LEP animals were injected with leptin (0.5 mg leptin/kg/d) for 12 days, while the CON and HF animals were injected with vehicle. After the treatment periods, all animals were prepared for and subjected to hind limb perfusion. In EXP-1, leptin treatment increased insulin-stimulated skeletal muscle glucose transporter (GLUT4) translocation, which appeared to be due to increased phosphatidylinositol 3-kinase (PI3-K) activation and Akt phosphorylation. In EXP-2, the high-fat diet reduced insulin-stimulated glucose transport, in part, by impairing insulin-stimulated PI3-K activation and glucose transporter translocation. Leptin treatment reversed high-fat-diet-induced insulin resistance in skeletal muscle by restoring insulin receptor substrate (IRS)-1-associated PI3-K activity, total GLUT4 protein concentration, and glucose transporter translocation. Collectively, these findings suggest that leptin treatment will enhance components of both the insulin-signaling cascade and glucose transport effector system in normal and insulin-resistant skeletal muscle.

The dwarf mutation decreases high dose insulin responses in skeletal muscle, the opposite of effects in liver

The in vivo status of the proximal components of the insulin signaling system was investigated in skeletal muscle of Ames ( Prop1 df / Prop1 df ) dwarf mice. The insulin-stimulated phosphorylation of the insulin receptor (IR) was reduced by 55% in Ames dwarf mice, while IR receptor protein content was not altered. Insulin-stimulated phosphorylation of IRS-1 and IRS-2 were decreased by 79 and 51%, respectively, while IRS-1 and IRS-2 protein levels were decreased by 66 and 43%. In addition, insulin-stimulated association of IRS-1 and IRS-2 with the p85 regulatory subunit of phosphatidylinositol (PI) 3-kinase was significantly reduced (by 80 and 41%, respectively), whereas insulin-stimulated PI 3-kinase activity was reduced by 66%. However, insulin-stimulated phosphorylation of Akt was slightly reduced (by 20%), suggesting that the attenuation of insulin signaling downstream PI 3-kinase may involve other signaling molecules. Our current results demonstrate that the Prop1 mutation decreases high dose insulin responses in skeletal muscle. This alteration is remarkable because these animals are hypersensitive to insulin and display an augmented response to insulin in liver at the same signaling steps. Reduced response to insulin in skeletal muscle could be important for the control of glucose homeostasis in these animals and could have implications in their extended longevity.

Dietary cod protein restores insulin-induced activation of phosphatidylinositol 3-kinase/Akt and GLUT4 translocation to the T-tubules in skeletal muscle of high-fat-fed obese rats.

Diet-induced obesity is known to cause peripheral insulin resistance in rodents. We have recently found that feeding cod protein to high-fat-fed rats prevents the development of insulin resistance in skeletal muscle. In the present study, we have further explored the cellular mechanisms behind this beneficial effect of cod protein on skeletal muscle insulin sensitivity. Rats were fed a standard chow diet or a high-fat diet in which the protein source was either casein, soy, or cod proteins for 4 weeks. Whole-body and muscle glucose disposal were reduced by approximately 50% in rats fed high-fat diets with casein or soy proteins, but these impairments were not observed in animals fed cod protein. Insulin-induced tyrosine phosphorylation of the insulin receptor and insulin receptor substrate (IRS) proteins were similar in muscle of chow- and high-fat-fed rats regardless of the dietary protein source. However, IRS-1-associated phosphatidylinositol (PI) 3-kinase activity was severely impaired (-60%) in muscle of high-fat-fed rats consuming casein or soy protein. In marked contrast, feeding rats with cod protein completely prevented the deleterious effect of fat feeding on insulin-stimulated PI 3-kinase activity. The activation of the downstream kinase Akt/PKB by insulin, assessed by in vitro kinase assay and phosphorylation of GSK-3beta, were also impaired in muscle of high-fat-fed rats consuming casein or soy protein, but these defects were also fully prevented by dietary cod protein. However, no effect of cod protein was observed on atypical protein kinase C activity. Normalization of PI 3-kinase/Akt activation by insulin in rats fed high-fat diets with cod protein was associated with improved translocation of GLUT4 to the T-tubules but not to the plasma membrane. Taken together, these results show that dietary cod protein is a natural insulin-sensitizing agent that appears to prevent obesity-linked muscle insulin resistance by normalizing insulin activation of the PI 3-kinase/Akt pathway and by selectively improving GLUT4 translocation to the T-tubules.

Metformin enhances insulin signalling in insulin-dependent and-independent pathways in insulin resistant muscle cells.

1 Metformin lowers blood glucose levels in type 2 diabetic patients. To evaluate the insulin sensitizing action of metformin on skeletal muscle cells, we have used C2C12 skeletal muscle cells differentiated in chronic presence or absence of insulin. 2 Metformin was added during the last 24 h of differentiation of the C2C12 myotubes. Insulin-stimulated tyrosine phosphorylation of insulin receptor (IR) and insulin receptor substrate-1 (IRS-1) was determined. 3 Chronic insulin treatment resulted in 60 and 40% reduction in insulin-stimulated tyrosine phosphorylation of IR and IRS-1, respectively. Treatment with metformin was able to increase the tyrosine phosphorylation of IR and IRS-1 by 100 and 90% respectively. 4 Chronic insulin treatment drastically reduced (45%) insulin-stimulated phosphatidyl inositol 3-kinase (PI 3-kinase) activity. Metformin treatment restored PI 3-kinase activity in insulin-resistant myotubes. 5 Insulin-stimulated glucose uptake was impaired in chronically insulin-treated myotubes. Metformin increased basal glucose uptake to significant levels (P<0.05), but metformin did not increase insulin-stimulated glucose transport. 6 All the three mitogen-activated protein kinases (MAPK) were activated by insulin in sensitive myotubes. The activation of p38 MAPK was impaired in resistant myotubes, while ERK and JNK were unaffected. Treatment with metformin enhanced the basal activation levels of p38 in both sensitive and resistant myotubes, but insulin did not further stimulate p38 activation in metformin treated cells. 7 Treatment of cells with p38 inhibitor, SB203580, blocked insulin- and metformin-stimulated glucose uptake as well as p38 activation. 8 Since the effect of metformin on glucose uptake corresponded to p38 MAPK activation, this suggests the potential role p38 in glucose uptake. 9 These data demonstrate the direct insulin sensitizing action of metformin on skeletal muscle cells.