Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.7.11.26 (GSK)
 6,788 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Molecular scanning of insulin receptor substrate-1 (IRS-1) revealed several amino acid substitutions. The most common IRS-1 variant, a Gly to Arg972 change, is more prevalent among type 2 diabetic patients. In this study we overexpressed wild-type and Arg972IRS-1 variant in L6 skeletal muscle cells and examined the functional consequences of this polymorphism on insulin metabolic signaling. L6 cells expressing Arg972-IRS-1 (L6-Arg972) showed a decrease in insulin-stimulated IRS-1-associated phosphatidylinositol 3-kinase (PI 3-kinase) activity compared with L6 cells expressing wild-type IRS-1 (L6-WT) as a consequence of decreased binding of p85 subunit of PI 3-kinase to IRS-1. L6-Arg972 exhibited a decrease in both basal and insulin-stimulated glucose transport due to a reduction in the amount of both GLUT1 and GLUT4 translocated to the plasma membrane. Both basal and insulin-stimulated Akt phosphorylations were decreased in L6-Arg972 compared with L6-WT. Basal glycogen synthase kinase-3 (GSK-3) activity was increased in L6-Arg972 compared with L6-WT, and insulin-induced inactivation of GSK-3 was also reduced in L6-Arg972. This change was associated with a significant decrease in insulin-stimulated glucose incorporation into glycogen and glycogen synthase activity in L6-Arg972 compared with L6-WT. These results indicate that the Arg972-IRS-1 polymorphism impairs the ability of insulin to stimulate glucose transport, glucose transporter translocation, and glycogen synthesis by affecting the PI 3-kinase/Akt/GSK-3 signaling pathway. The present data indicate that the polymorphism at codon 972 of IRS-1 may contribute to the in vivo insulin resistance observed in carriers of this variant.
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PMID:The Gly-->Arg972 amino acid polymorphism in insulin receptor substrate-1 affects glucose metabolism in skeletal muscle cells. 1084 89

Muscle glucose uptake, glycogen synthase activity, and insulin signaling were investigated in response to a physiological hyperinsulinemic (600 pmol/l)-euglycemic clamp in young healthy subjects. Four hours before the clamp, the subjects performed one-legged exercise for 1 h. In the exercised leg, insulin more rapidly activated glucose uptake (half activation time [t1/2] = 11 vs. 34 min) and glycogen synthase activity (t1/2 = 8 vs. 17 min), and the magnitude of increase was two- to fourfold higher compared with the rested leg. However, prior exercise did not result in a greater or more rapid increase in insulin-induced receptor tyrosine kinase (IRTK) activity (t1/2 = 50 min), serine phosphorylation of Akt (t1/2 = 1-2 min), or serine phosphorylation of glycogen synthase kinase-3 (GSK-3) (t1/2 = 1-2 min) or in a larger or more rapid decrease in GSK-3 activity (t1/2 = 3-8 min). Thirty minutes after cessation of insulin infusion, glucose uptake, glycogen synthase activity, and signaling events were partially reversed in both the rested and the exercised leg. We conclude the following: 1) physiological hyperinsulinemia induces sustained activation of insulin-signaling molecules in human skeletal muscle; 2) the more distal insulin-signaling components (Akt, GSK-3) are activated much more rapidly than the proximal signaling molecules (IRTK as well as insulin receptor substrate 1 and phosphatidylinositol 3-kinase [Wojtaszewski et al., Diabetes 46:1775-1781, 1997]); and 3) prior exercise increases insulin stimulation of both glucose uptake and glycogen synthase activity in the absence of an upregulation of signaling events in human skeletal muscle.
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PMID:Insulin signaling and insulin sensitivity after exercise in human skeletal muscle. 1086 52

In insulin-sensitive L6 myocytes, insulin stimulated glycogen synthesis in a dose-dependent manner and lithium further stimulated glycogen synthesis at all insulin concentrations. Lithium alone at 20 mM stimulated glycogen synthesis to the degree similar to the maximal insulin response. Effects of lithium and insulin were fully additive for both glycogen synthesis and glycogen synthase activity. In L6 myocytes, insulin increased phosphorylation of Akt1 and glycogen synthase kinase-3 alpha and beta (GSK-3 alpha and beta), resulting in its activation and inactivation, respectively. Unlike insulin, lithium directly inhibited GSK-3 (both alpha and beta) without affecting phosphorylation of GSK-3. Moreover, lithium in vitro could further inhibit enzyme activity of GSK-3 (both alpha and beta) that was isolated from insulin-stimulated cells (thus already phosphorylated and inactivated by insulin). In summary, insulin increases glycogen synthesis by the Akt1/GSK-3/glycogen synthase pathway, but lithium increases glycogen synthesis by direct inhibition of GSK-3 in L6 myocytes. Inhibitory effects of lithium and insulin on GSK-3 (both alpha and beta) were additive, which may account, at least in part, for their additive effects on glycogen synthase activity and glycogen synthesis in L6 myocytes.
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PMID:Effects of lithium and insulin on glycogen synthesis in L6 myocytes: additive effects on inactivation of glycogen synthase kinase-3. 1091 20

A protocol was developed in 3T3-L1 adipocytes that resulted in the specific desensitization of glycogen synthase activation by insulin. Cells were pretreated for 15 min with 100 nm insulin, and then recovered for 1.5 h in the absence of hormone. Subsequent basal and insulin-induced phosphorylation of the insulin receptor, IRS-1, MAPK, Akt kinase, and GSK-3 were similar in control and pretreated cells. Additionally, enhanced glucose transport and incorporation into lipid in response to insulin were unaffected. However, pretreatment reduced insulin-stimulated glycogen synthesis by over 50%, due to a nearly complete inhibition of glycogen synthase activation. Removal of extracellular glucose during the recovery period blocked the increase in glycogen levels, and restored insulin-induced glycogen synthase activation. Furthermore, incubation of pretreated 3T3-L1 adipocytes with glycogenolytic agents reversed the desensitization event. Separation of cellular lysates on sucrose gradients revealed that glycogen synthase was primarily located in the dense pellet fraction, with lesser amounts in the lighter fractions. Insulin induced glycogen synthase translocation from the lighter to the denser glycogen-containing fractions. Interestingly, insulin preferentially activated translocated enzyme while having little effect on the majority of glycogen synthase activity in the pellet fraction. In insulin-pretreated cells, glycogen synthase did not return to the lighter fractions during recovery, and thus did not move in response to the second insulin exposure. These results suggest that, in 3T3-L1 adipocytes, the translocation of glycogen synthase may be an important step in the regulation of glycogen synthesis by insulin. Furthermore, intracellular glycogen levels can regulate glycogen synthase activation, potentially through modulation of enzymatic localization.
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PMID:Specific desensitization of glycogen synthase activation by insulin in 3T3-L1 adipocytes. Connection between enzymatic activation and subcellular localization. 1101 39

The effect of insulin on glycogen synthesis and key enzymes of glycogen metabolism, glycogen phosphorylase and glycogen synthase, was studied in HepG2 cells. Insulin stimulated glycogen synthesis 1.83-3.30 fold depending on insulin concentration in the medium. Insulin caused a maximum of 65% decrease in glycogen phosphorylase 'a' and 110% increase in glycogen synthase activities in 5 min. Although significant changes in enzyme activities were observed with as low as 0.5 nM insulin level, the maximum effects were observed with 100 nM insulin. There was a significant inverse correlation between activities of glycogen phosphorylase 'a' and glycogen synthase 'a' (R2= 0.66, p < 0.001). Addition of 30 mM glucose caused a decrease in phosphorylase 'a' activity in the absence of insulin and this effect was additive with insulin up to 10 nM concentration. The inactivation of phosphorylase 'a' by insulin was prevented by wortmannin and rapamycin but not by PD98059. The activation of glycogen synthase by insulin was prevented by wortmannin but not by PD98059 or rapamycin. In fact, PD98059 slightly stimulated glycogen synthase activation by insulin. Under these experimental conditions, insulin decreased glycogen synthase kinase-3beta activity by 30-50% and activated more than 4-fold particulate protein phosphatase- activity and 1.9-fold protein kinase B activity; changes in all of these enzyme activities were abolished by wortmannin. The inactivation of GSK-3beta and activation of PKB by insulin were associated with their phosphorylation and this was also reversed by wortmannin. The addition of protein phosphatase-1 inhibitors, okadaic acid and calyculin A, completely abolished the effects of insulin on both enzymes. These data suggest that stimulation of glycogen synthase by insulin in HepG2 cells is mediated through the PI-3 kinase pathway by activating PKB and PP-1G and inactivating GSK-3beta. On the other hand, inactivation of phosphorylase by insulin is mediated through the PI-3 kinase pathway involving a rapamycin-sensitive p70(s6k) and PP-1G. These experiments demonstrate that insulin regulates glycogen phosphorylase and glycogen synthase through (i) a common signaling pathway at least up to PI-3 kinase and bifurcates downstream and (ii) that PP-1 activity is essential for the effect of insulin.
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PMID:Reciprocal regulation of glycogen phosphorylase and glycogen synthase by insulin involving phosphatidylinositol-3 kinase and protein phosphatase-1 in HepG2 cells. 1105 55

Grb10 is a member of a family of adapter proteins that binds to tyrosine-phosphorylated receptors including the insulin receptor kinase (IRK). In this study recombinant adenovirus was used to over-express hGrb10zeta, a new Grb10 isoform, in primary rat hepatocytes and the consequences for insulin signaling were evaluated. Over-expression of hGrb10zeta resulted in 50% inhibition of insulin-stimulated IRK autophosphorylation and activation. Analysis of downstream events showed that hGrb10zeta over-expression specifically inhibits insulin-stimulated glycogen synthase (GS) activity and glycogen synthesis without affecting insulin-induced IRS1/2 phosphorylation, PI3-kinase activation, insulin like growth factor binding protein-1 (IGFBP-1) mRNA expression, and ERK1/2 MAP kinase activity. The classical pathway from PI3-kinase through Akt-PKB/GSK-3 leading to GS activation by insulin was also not affected by hGrb10zeta over-expression. These results indicate that hGrb10zeta inhibits a novel and presently unidentified insulin signaling pathway leading to GS activation in liver.
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PMID:Specific inhibition by hGRB10zeta of insulin-induced glycogen synthase activation: evidence for a novel signaling pathway. 1122 74

Phosphatidylinositol (PI) 3-kinase plays an important role in various metabolic actions of insulin including glucose uptake and glycogen synthesis. Although PI 3-kinase primarily functions as a lipid kinase which preferentially phosphorylates the D-3 position of phospholipids, the effect of hydrolysis of the key PI 3-kinase product PI 3,4,5-triphosphate [PI(3,4,5)P3] on these biological responses is unknown. We recently cloned rat SH2-containing inositol phosphatase 2 (SHIP2) cDNA which possesses the 5'-phosphatase activity to hydrolyze PI(3,4,5)P3 to PI 3,4-bisphosphate [PI(3,4)P2] and which is mainly expressed in the target tissues of insulin. To study the role of SHIP2 in insulin signaling, wild-type SHIP2 (WT-SHIP2) and 5'-phosphatase-defective SHIP2 (Delta IP-SHIP2) were overexpressed in 3T3-L1 adipocytes by means of adenovirus-mediated gene transfer. Early events of insulin signaling including insulin-induced tyrosine phosphorylation of the insulin receptor beta subunit and IRS-1, IRS-1 association with the p85 subunit, and PI 3-kinase activity were not affected by expression of either WT-SHIP2 or Delta IP-SHIP2. Because WT-SHIP2 possesses the 5'-phosphatase catalytic region, its overexpression marked by decreased insulin-induced PI(3,4,5)P3 production, as expected. In contrast, the amount of PI(3,4,5)P3 was increased by the expression of Delta IP-SHIP2, indicating that Delta IP-SHIP2 functions in a dominant-negative manner in 3T3-L1 adipocytes. Both PI(3,4,5)P3 and PI(3,4)P2 were known to possibly activate downstream targets Akt and protein kinase C lambda in vitro. Importantly, expression of WT-SHIP2 inhibited insulin-induced activation of Akt and protein kinase C lambda, whereas these activations were increased by expression of Delta IP-SHIP2 in vivo. Consistent with the regulation of downstream molecules of PI 3-kinase, insulin-induced 2-deoxyglucose uptake and Glut4 translocation were decreased by expression of WT-SHIP2 and increased by expression of Delta IP-SHIP2. In addition, insulin-induced phosphorylation of GSK-3beta and activation of PP1 followed by activation of glycogen synthase and glycogen synthesis were decreased by expression of WT-SHIP2 and increased by the expression of Delta IP-SHIP2. These results indicate that SHIP2 negatively regulates metabolic signaling of insulin via the 5'-phosphatase activity and that PI(3,4,5)P3 rather than PI(3,4)P2 is important for in vivo regulation of insulin-induced activation of downstream molecules of PI 3-kinase leading to glucose uptake and glycogen synthesis.
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PMID:Overexpression of SH2-containing inositol phosphatase 2 results in negative regulation of insulin-induced metabolic actions in 3T3-L1 adipocytes via its 5'-phosphatase catalytic activity. 1123

Reported discrepancies in the effects of tumor necrosis factor (TNF)-alpha in modulating insulin sensitivity of cultured cells may relate both to cell types studied and to the time course of exposure to the cytokine. Additionally, the relationship of effects on glucose metabolism to changes in the insulin signaling pathway cannot be assumed. For in vitro study, the cell type most relevant to insulin resistance in humans is the cultured human muscle cell. In the present study, TNF brought about no change in the rate of glycogen synthesis in cultured human muscle cells unless present during differentiation. The presence of TNF (5 ng/ml) during the process of differentiation of myoblasts into mature myotubes diminished the response of glycogen synthesis to acute insulin stimulation. This finding was associated with an impairment of differentiation-dependent increases in total cellular glycogen synthase (GS) activity. Under the same conditions of TNF exposure, there was no effect on the response to acute insulin stimulation of the fractional activity of GS. Similarly, there was no effect on the insulin stimulation of protein kinase B (PKB) and inhibition of glycogen synthase kinase 3 (GSK-3). Acute insulin stimulation brought about a 4.08 +/- 0.44-fold stimulation of activity of PKB in the absence of TNF, with 4.81 +/- 0.70-fold stimulation in cells exposed to TNF. GSK-3 activity decreased to 74.0 +/- 5.8% of basal after insulin stimulation without TNF and 78.3 +/- 5.0% after TNF exposure. However, differentiation of myocytes, as defined by an increase in the acetylcholine receptor, myogenin, and mature creatine kinase isoform expression, was impaired in TNF-treated cells. These studies demonstrate that TNF, if present during differentiation, decreases insulin-stimulated rates of storage of glucose as glycogen and total GS activity but does not downregulate the insulin-signaling system to GS. More generally, TNF also inhibits differentiation of human muscle cells in culture.
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PMID:Effects of tumor necrosis factor-alpha on insulin action in cultured human muscle cells. 1133 14

Glycogen synthase kinase 3 (GSK-3) has previously been shown to play an important role in the regulation of apoptosis. However, the nature of GSK-3 effector pathways that are relevant to neuroprotection remains poorly defined. Here, we have compared neuroprotection resulting from modulation of GSK-3 activity in PC12 cells using either selective small molecule ATP-competitive GSK-3 inhibitors (SB-216763 and SB-415286), or adenovirus overexpressing frequently rearranged in advanced T-cell lymphomas 1 (FRAT1), a protein proposed as a negative regulator of GSK-3 activity towards Axin and beta-catenin. Our data demonstrate that cellular overexpression of FRAT1 is sufficient to confer neuroprotection and correlates with inhibition of GSK-3 activity towards Tau and beta-catenin, but not modulation of glycogen synthase (GS) activity. By comparison, treatment with SB-216763 and SB-415286 proved more potent in terms of neuroprotection, and correlated with inhibition of GSK-3 activity towards GS in addition to Tau and beta-catenin.
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PMID:GSK-3 inhibition by adenoviral FRAT1 overexpression is neuroprotective and induces Tau dephosphorylation and beta-catenin stabilisation without elevation of glycogen synthase activity. 1169 57

Experimental data support a role for FAK, an important component of the integrin signaling pathway, in insulin action. To test the hypothesis that FAK plays a regulatory role in hepatic insulin action, we overexpressed wild type (WT), a kinase inactive (KR), or a COOH-terminal focal adhesion targeting (FAT) sequence-truncated mutant of FAK in HepG2 hepatoma cells. In control untransfected (NON) and vector (CMV2)- and WT-transfected cells, insulin stimulated an expected 54 +/- 13, 37 +/- 4, and 47 +/- 12 increase in [U-(14)C]glucose incorporation into glycogen, respectively. This was entirely abolished in the presence of either KR (-1 +/- 7%) or FAT mutants (0 +/- 8%, n = 5, p < 0.05 for KR or FAT versus other groups), and this was associated with a significant attenuation of incremental insulin-stimulated glycogen synthase (GS) activity. Insulin-stimulated serine phosphorylation of Akt/protein kinase B was significantly impaired in mutant-transfected cells. Moreover, the ability of insulin to inactivate GS kinase-3beta (GSK-3beta), the regulatory enzyme immediately upstream of GS, by serine phosphorylation (308 +/- 16, 321 +/- 41, and 458 +/- 34 optical densitometric units (odu) in NON, CMV2, and WT, respectively, p < 0.02 for WT versus CMV2) was attenuated in the presence of either FAT (205 +/- 14, p < 0.01) or KR (189 +/- 4, p < 0.005) mutants. FAK co-immunoprecipitated with GSK-3beta, but only in cells overexpressing the KR (374 +/- 254 odu) and FAT (555 +/- 308) mutants was this association stimulated by insulin compared with NON (-209 +/- 92), CMV2 (-47 +/- 70), and WT (-39 +/- 31 odu). This suggests that FAK and GSK-3beta form both a constitutive association and a transient complex upon insulin stimulation, the dissociation of which requires normal function and localization of FAK. We conclude that FAK regulates the activity of Akt/protein kinase B and GSK-3beta and the association of GSK-3beta with FAK to influence insulin-stimulated glycogen synthesis in hepatocytes. Insulin action may be subject to regulation by the integrin signaling pathway, ensuring that these growth and differentiation-promoting pathways act in a coordinated and/or complementary manner.
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PMID:Focal adhesion kinase (FAK) regulates insulin-stimulated glycogen synthesis in hepatocytes. 1180 46


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