Direct comparison of inositol phosphoglycan with prostaglandylinositol cyclic phosphate, two potential mediators of insulin action

Though insulin signalling is thought by many groups to function without second messenger action, others have provided evidence for the existence and action of such regulators. Chemically quite different compounds, however, have been proposed as mediators, such as various inositol phosphoglycans and prostaglandylinositol cyclic phosphate (cyclic PIP). In spite of marked structural differences, these compounds are reported to have the same regulatory properties, i.e. to activate protein ser/thr phosphatases and to inhibit protein kinase A. In order to clarify this discrepancy, the regulatory potency of these different compounds was assayed under identical conditions. It was found that in contrast to cyclic PIP, the synthetic inositol phosphoglycan PIG41 neither directly inhibited protein kinase A nor activated protein ser/thr phosphatases. However, when added to intact cells, such as primary adipocytes, PIG41 inhibited isoproterenol-stimulated lipolysis. This effect most likely results from tyrosine phosphorylation of insulin receptor substrates (IRSs) by PIG41. This tyrosine phosphorylation is not carried out by the insulin receptor tyrosine kinase but by cytosolic tyrosine kinases. This indicates that cyclic PIP, an intracellular regulator, which primarily acts on protein kinase A and on protein ser/thr phosphatases, operates more downstream in the signal transduction cascade as compared to the inositol phosphoglycan PIG41. Thus, cyclic PIP appears to be a suitable candidate to close the gap between IRSs and the protein kinases/phosphatases involved in the signal transduction of insulin.     

Control of glycogen synthesis in cultured human muscle cells

The regulation of glycogen synthesis and associated enzymes was studied in human myoblasts and myotubes maintained in culture. Both epidermal growth factor (EGF) and insulin stimulated glycogen synthesis approximately 2-fold, this stimulation being accompanied by a rapid and stable activation of the controlling enzyme glycogen synthase (GS). EGF also caused inhibition of glycogen synthase kinase 3 (GSK-3) and activation of the alpha isoform of protein kinase B (PKB) with the time-course and magnitude of its effects being similar to those induced by insulin. An inhibitor of the mitogen-activated protein (MAP) kinase pathway did not prevent stimulation of GS by EGF, suggesting that this pathway is not essential for the effect. A partial decrease in the fold activation of GS was, however, observed when p70(S6k) activation was blocked with rapamycin, suggesting a contribution of this pathway to the control of GS by either hormone. Wortmannin, a selective inhibitor of phosphatidylinositol 3'-kinase (PI-3 kinase) completely blocked the effects of both EGF and insulin in these cells. These results demonstrate that EGF, like insulin, activates glycogen synthesis in muscle, acting principally via the PKB/GSK-3 pathway but with a contribution from a rapamycin-sensitive component that lies downstream of PI-3 kinase.     

Interleukin 1beta and interleukin 6, but not tumor necrosis factor alpha, inhibit insulin-stimulated glycogen synthesis in rat hepatocytes

Recent evidence indicates that inflammatory cytokines are involved in changes of blood glucose concentrations and hepatic glucose metabolism in infectious diseases, including sepsis. However, little is known regarding how cytokines interact with glucoregulatory hormones such as insulin. The objective of the present study is to investigate if and how cytokines influence insulin-stimulated glycogen metabolism in the liver. Interleukin 1beta (IL-1beta) and interleukin 6 (IL-6) markedly inhibited the increase of glycogen deposition stimulated by insulin in primary rat hepatocyte cultures; however, tumor necrosis factor alpha had no effect. Labeling experiments revealed that both cytokines counteracted insulin action by decreasing [14C]-glucose incorporation into glycogen and by increasing [14C]-glycogen degradation. Furthermore, it was discovered that IL-1beta and IL-6 inhibited glycogen synthase activity and, in contrast, accelerated glycogen phosphorylase activity. In experiments with kinase inhibitors, serine/threonine kinase inhibitor K252a blocked IL-1beta- and IL-6-induced inhibitions of glycogen deposition, as well as glycogen synthase activity, whereas another kinase inhibitor staurosporine blocked only IL-6-induced inhibition. Tyrosine kinase inhibitor herbimycin A blocked only IL-1beta-induced inhibition. These results indicate that IL-1beta and IL-6 regulate insulin-stimulated glycogen synthesis through different pathways involving protein phosphorylation in hepatocytes. They may mediate the change of hepatic glucose metabolism under pathological and even physiological conditions by modifying insulin action in vivo.     

Insulin-stimulated glycogen synthesis in cultured hepatoma cells: differential effects of inhibitors of insulin signaling molecules

In rat HTC hepatoma cells overexpressing human insulin receptors, insulin stimulated glycogen synthesis by 55-70%. To study postreceptor signaling events leading to insulin-stimulated glycogen synthesis in these cells, we have employed pathway-specific chemical inhibitors such as LY294002, rapamycin and PD98059 to inhibit phosphatidylinositol-3-kinase (PI3K), p70 ribosomal S6 kinase and mitogen-activated protein kinase (MAPK) kinase/MAPK, respectively. LY294002 (50 microM) completely abolished insulin-stimulated glycogen synthesis whereas rapamycin (2-20 nM) partially inhibited it. Neither LY294002 nor rapamycin significantly affected the basal glycogen synthesis. However, PD98059 (100 microM) significantly inhibited the basal glycogen synthesis without affecting insulin-stimulated glycogen synthesis. In these cells, insulin at 100 nM decreased glycogen synthase kinase 3 alpha (GSK3 alpha) activity by 30-35%. LY294002, but neither rapamycin nor PD98059, abolished insulin-induced inactivation of GSK3 alpha. These data suggest that insulin-stimulated glycogen synthesis in rat HTC hepatoma cells is mediated mainly by PI3K-dependent mechanism. In these cells, inactivation of GSK3 alpha, downstream of PI3K, may play a role in insulin-stimulated glycogen synthesis.     

Lesions of the midline thalamic nuclei impair classical conditioning under partial but not continuous reinforcement conditions

Protein Phosphatase-1 (PP-1) appears to be the key component of the insulin signalling pathway which is responsible for bridging the initial insulin-simulated phosphorylation cascade with the ultimate dephosphorylation of insulin sensitive substrates. Dephosphorylations catalyzed by PP-1 activate glycogen synthase (GS) and simultaneously inactivate phosphorylase a and phosphorylase kinase promoting glycogen synthesis. Our in vivo studies using L6 rat skeletal muscle cells and freshly isolated adipocytes indicate that insulin stimulates PP-1 by increasing the phosphorylation status of its regulatory subunit (PP-1G). PP-1 activation is accompanied by an inactivation of Protein Phosphatase-2A (PP-2A) activity. To gain insight into the upstream kinases that mediate insulin-stimulated PP-1G phosphorylation, we employed inhibitors of the ras/MAPK, PI3-kinase, and PKC signalling pathways. These inhibitor studies suggest that PP-1G phosphorylation is mediated via a complex, cell type specific mechanism involving PI3-kinase/PKC/PKB and/or the ras/MAP kinase/Rsk kinase cascade. cAMP agonists such as SpcAMP (via PKA) and TNF-alpha (recently identified as endogenous inhibitor of insulin action via ceramide) block insulin-stimulated PP-1G phosphorylation with a parallel decrease of PP-1 activity, presumably due to the dissociation of the PP-1 catalytic subunit from the regulatory G-subunit. It appears that any agent or condition which interferes with the insulin-induced phosphorylation and activation of PP-1, will decrease the magnitude of insulin's effect on downstream metabolic processes. Therefore, regulation of the PP-1G subunit by site-specific phosphorylation plays an important role in insulin signal transduction in target cells. Mechanistic and functional studies with cell lines expressing PP-1G subunit site-specific mutations will help clarify the exact role and regulation of PP-1G site-specific phosphorylations on PP-1 catalytic function.     

Insulin signaling in the yeast Saccharomyces cerevisiae. 1. Stimulation of glucose metabolism and Snf1 kinase by human insulin

Effects of human insulin on glucose metabolism in the yeast Saccharomyces cerevisiae were studied in this report. Under two conditions of growth limitation (glucose-grown cells during transition to stationary phase or spheroplasts during incubation in synthetic glucose medium), human insulin (10 and 1 microM, respectively) enhanced glycogen accumulation and glycogen synthase activity by 40-60% compared to control cells. Glycogen phosphorylase activity was also increased under the same conditions, but this stimulation was diminished by 35-45% in insulin-treated compared to control cells. Thus, under growth limitation, insulin causes glycogen phosphorylase and glycogen synthase to become more sensitive to inactivation and activation, respectively. In glucose-induced spheroplasts, insulin (1 microM), in addition to glycogen accumulation, led to about 2-fold increases of the rates of ethanol production and glucose oxidation compared to control cells, and the maximal concentration of hexose 6-phosphate was increased by 30-40%. In contrast, glucose transport as well as the levels of the allosteric regulators, fructose 2,6-bisphosphate and cAMP, were not altered at all. Snf1 kinase is assumed to be involved in the regulation of glycogen metabolism in yeast, although it does not seem to be modulated directly by the glucose concentration. Snf1 kinase activity was elevated 5-10-fold in response to insulin both during glucose induction of yeast spheroplasts and during transition to stationary phase of glucose-grown cells. We conclude that Saccharomyces cerevisiae and insulin-sensitive mammalian cells share some parts of the signaling cascades regulating oxidative and nonoxidative glucose metabolism in response to glucose and insulin.     

Axin, a negative regulator of the Wnt signaling pathway, forms a complex with GSK-3beta and beta-catenin and promotes GSK-3beta-dependent phosphorylation of beta-catenin

Glycogen synthase kinase-3 (GSK-3) mediates epidermal growth factor, insulin and Wnt signals to various downstream events such as glycogen metabolism, gene expression, proliferation and differentiation. We have isolated here a GSK-3beta-interacting protein from a rat brain cDNA library using a yeast two-hybrid method. This protein consists of 832 amino acids and possesses Regulators of G protein Signaling (RGS) and dishevelled (Dsh) homologous domains in its N- and C-terminal regions, respectively. The predicted amino acid sequence of this GSK-3beta-interacting protein shows 94% identity with mouse Axin, which recently has been identified as a negative regulator of the Wnt signaling pathway; therefore, we termed this protein rAxin (rat Axin). rAxin interacted directly with, and was phosphorylated by, GSK-3beta. rAxin also interacted directly with the armadillo repeats of beta-catenin. The binding site of rAxin for GSK-3beta was distinct from the beta-catenin-binding site, and these three proteins formed a ternary complex. Furthermore, rAxin promoted GSK-3beta-dependent phosphorylation of beta-catenin. These results suggest that rAxin negatively regulates the Wnt signaling pathway by interacting with GSK-3beta and beta-catenin and mediating the signal from GSK-3beta to beta-catenin.     

Abnormal expression of epiligrin and alpha 6 beta 4 integrin in basal cell carcinoma

Lipoprotein lipase (LPL) hydrolyzes the triacylglycerol component of circulating lipoprotein particles, mediating the uptake of fatty acids into adipose tissue and muscle. Insulin is the principal factor responsible for regulating LPL activity in adipose tissue, yet the mechanisms whereby insulin controls LPL expression are unknown. The current studies used wortmannin, a specific inhibitor of phosphatidylinositol (PI) 3-kinase, and rapamycin, a specific inhibitor of activation of phosphoprotein 70 ribosomal protein S6 kinase (p70s6k), to explore some of the components of the insulin signaling pathway controlling LPL activity in adipose cells. Preincubation of isolated rat adipose cells with wortmannin completely abrogated the stimulation of LPL activity by insulin, while preincubation with rapamycin caused approximately a 60% inhibition of insulin-stimulated LPL activity. Thus, the current studies show that the regulation of adipose tissue LPL by insulin is mediated via a wortmannin-sensitive pathway, most likely PI 3-kinase, and that a rapamycin-sensitive pathway, most likely p705s6k, constitutes an important downstream component in the insulin signaling pathway through which LPL is regulated.     

Tyrosine phosphatase inhibitors, vanadate and pervanadate, stimulate glucose transport and GLUT translocation in muscle cells by a mechanism independent of phosphatidylinositol 3-kinase and protein kinase C

Vanadate and pervanadate (pV) are protein tyrosine phosphatase (PTP) inhibitors that mimic insulin to stimulate glucose transport. To determine whether phosphatidylinositol (PI) 3-kinase is required for vanadate and pV, as it is for insulin, cultured L6 myotubes were treated with vanadate and pV. The two compounds stimulated glucose transport to levels similar to those stimulated by insulin; however, while PI 3-kinase activity and the increase in the lipid products PI 3,4-bisphosphate and PI 3,4,5-trisphosphate were inhibited by wortmannin after stimulation by all three agents--insulin, vanadate, and pV--wortmannin blocked glucose transport stimulated by insulin but not vanadate or pV. Vanadate and pV stimulated the translocation of GLUTs from an intracellular compartment to the plasma membrane; this stimulation was not blocked by wortmannin, but insulin-induced GLUT translocation was inhibited. Similar results were obtained in cultured H9c2 cardiac muscle cells in which wortmannin did not inhibit glucose transport or the vanadate-induced translocation of GLUT4 in c-myc-GLUT4 transfected cells. The ser/thr kinase PKB (Akt/PKB/RAC-PK) is activated by insulin, lies downstream of PI 3-kinase, and has been implicated in signaling of glucose transport. Insulin and pV stimulated PKB activity, and both were inhibited by wortmannin. In contrast, vanadate, at concentrations that maximally stimulated glucose transport, did not significantly increase PKB activity. To determine the potential role of protein kinase C (PKC), L6 cells were incubated chronically with phorbol myristate acetate (PMA) or acutely with the PKC inhibitors calphostin C and bisindolylmaleimide. There was no inhibition of glucose transport stimulation by insulin, vanadate, or pV, and a combination of wortmannin and PKC inhibitors also failed to block the effect of vanadate and pV. In contrast, disassembly of the actin network with cytochalasin D blocked the stimulation of glucose transport by all three agents. In conclusion, vanadate and pV are able to stimulate glucose transport and GLUT translocation by a mechanism independent of PI 3-kinase and PKC. Similar to that by insulin, glucose transport stimulation by vanadate and pV requires the presence of an intact actin network.     

Insulin increases liver protein phosphatase-1 and protein phosphatase-2C activities in lean, young adult rhesus monkeys

Liver glycogen synthase activity is increased, and glycogen phosphorylase activity and glucose 6-phosphate content reduced by in vivo insulin during a euglycemic hyperinsulinemic clamp in lean young adult rhesus monkeys. To examine the mechanism of dephosphorylation of liver glycogen synthase and glycogen phosphorylase, the enzyme activities of protein phosphatase-1, protein phosphatase-2C, cAMP-dependent protein kinase, glycogen synthase kinase-3, protein kinase C and protein tyrosine kinase were determined before and after three hours of in vivo insulin in these same monkeys. The bioactivity of an inositol phosphoglycan insulin mediator (pH 2.0) and cAMP concentrations were also measured in the liver before and after insulin administration. Insulin caused significant increases in protein phosphatase-1 (p = 0.005) and in protein phosphatase-2C activities (p = 0.001). Insulin-stimulated minus basal bioactivity of the pH 2.0 insulin mediator was strongly inversely related to the insulin-stimulated minus basal glucose 6-phosphate content (r = -0.93, p < 0.0001). These findings suggest that protein phosphatase-1 and protein phosphatase-2C may be involved in the mechanism of in vivo insulin activation of liver glycogen synthase and inactivation of liver glycogen phosphorylase.     

Control of glucose utilization in working perfused rat heart

Metabolic control analyses of glucose utilization were performed for four groups of working rat hearts perfused with Krebs-Henseleit buffer containing 10 mM glucose only, or with the addition of 4 mM D-beta-hydroxybutyrate/1 mM acetoacetate, 100 nM insulin (0.05 unit/ml), or both. Net glycogen breakdown occurred in the glucose group only and was converted to net glycogen synthesis in the presence of all additions. The flux of [2-3H]glucose through P-glucoisomerase (EC 5.3.1.9) was reduced with ketones, elevated with insulin, and unchanged with the combination. Net glycolytic flux was reduced in the presence of ketones and the combination. The flux control coefficients were determined for the portion of the pathway involving glucose transport to the branches of glycogen synthesis and glycolysis. Major control was divided between the glucose transporter and hexokinase (EC 2.7.1.1) in the glucose group. The distribution of the control was slightly shifted to hexokinase with ketones, and control at the glucose transport step was abolished in the presence of insulin. Analysis of the pathway from 3-P-glycerate to pyruvate determined that the major control was shared by enolase (EC 4.2.1.1) and pyruvate kinase (EC 2.7.1.40) in the glucose group. Addition of ketones, insulin, or the combination shifted the control to P-glycerate mutase (EC 5.4.2.1) and pyruvate kinase. These results illustrate that the control of the metabolic flux in glucose metabolism of rat heart is not exerted by a single enzyme but variably distributed among enzymes depending upon substrate availability, hormonal stimulation, or other changes of conditions.     

Functional consequences of the Ser334-->Pro mutation in a human factor X variant (factor XMarseille)

Phosphorylase b kinase from rabbit skeletal muscle can be phosphorylated and activated by a cyclic nucleotide- and Ca2(+)-independent protein kinase previously identified as an autophosphorylation-dependent multifunctional protein kinase (auto-kinase) from brain and liver (Yang et al., J. Biol. Chem. 262, 7034-7040 (1987) and Yang et al. J. Biol. Chem. 262, 9421-9427 (1987)). This independent kinase phosphorylates both alpha and beta subunits of phosphorylase b kinase and results in a approximately 5-fold activation of the kinase when 0.55 and 0.5 mol of phosphate are incorporated into the alpha and beta subunits, respectively. Activation of phosphorylase b kinase catalyzed by auto-kinase is about 70% of that observed with cAMP-dependent protein kinase. Analysis of phosphopeptide maps of alpha and beta subunits further reveals that both kinases phosphorylate almost the same sites on both alpha and beta subunits, suggesting that activation of phosphorylase b kinase by the two kinases may be through a common molecular action mechanism. Taken together with the previous result that auto-kinase can inactivate glycogen synthase, the present study provides initial evidence that a coordinate control mechanism for simultaneous regulation of glycogenolysis and glycogenesis can be modulated by autophosphorylation-dependent protein kinase in a cAMP- and Ca2(+)-independent pathway, representing a new mode of control mechanism for the regulation of glycogen metabolism in cells.     

Vanadyl sulfate-stimulated glycogen synthesis is associated with activation of phosphatidylinositol 3-kinase and is independent of insulin receptor tyrosine phosphorylation

Salts of the trace element vanadium, such as sodium orthovanadate and vanadyl sulfate (VS), exhibit a myriad of insulin-like effects, including stimulation of glycogen synthesis and improvement of glucose homeostasis in type I and type II animal models of diabetes mellitus. However, the cellular mechanism by which these effects are mediated remains poorly characterized. We have shown earlier that different vanadium salts stimulate the MAP kinase pathway and ribosomal-S-6-kinase (p70s6k) in chinese hamster ovary cells overexpressing human insulin receptor (CHO-HIR cells) [Pandey, S. K., Chiasson, J.-L., and Srivastava, A. K. (1995) Mol. Cell. Biochem. 153, 69-78]. In the present studies, we have investigated if similar to insulin, VS also activates phosphatidylinositol 3-kinase (PI3-k) activity, and whether VS-induced activation of the PI3-k, MAP kinase, and p70s6k pathways contributes to glycogen synthesis. Treatment of CHO-HIR cells with VS resulted in increased glycogen synthesis and PI3-k activity which were blocked by pretreatment of the cells with wortmannin and LY294002, two specific inhibitors of PI3-k. On the other hand, PD98059 and rapamycin, specific inhibitors of the MAP kinase pathway and p70s6k, respectively, were unable to inhibit VS-stimulated glycogen synthesis. Moreover, VS-stimulated glycogen synthesis and PI3-k were observed without any change in the tyrosine phosphorylation of insulin receptor (IR) beta-subunit but were associated with increased tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1). In addition, PI3-k activation was detected in IRS-1 immunoprecipitates from VS-stimulated cells, indicating that tyrosine-phosphorylated IRS-1 was able to interact and thereby activate PI3-k in response to VS. Taken together, these results provide evidence that tyrosine phosphorylation of IRS-1 and activation of PI3-k play a key role in mediating the insulinomimetic effect of VS on glycogen synthesis independent of IR-tyrosine phosphorylation.     

Structure and regulation of the activity of muscle phosphorylase kinase

Phosphorylase kinase is the key enzyme in the control of glycogen metabolism in skeletal muscles, the heart and the liver. The quaternary structure of the enzyme, the primary structure of the enzyme subunits as well as the kinetic properties and regulation of the skeletal muscle enzyme activity by covalent modification, phosphorylation and some physiological effectors (Ca2+, calmodulin, troponin C) are reviewed.     

Requirement for activation of the serine-threonine kinase Akt (protein kinase B) in insulin stimulation of protein synthesis but not of glucose transport

A wide variety of biological activities including the major metabolic actions of insulin is regulated by phosphatidylinositol (PI) 3-kinase. However, the downstream effectors of the various signaling pathways that emanate from PI 3-kinase remain unclear. Akt (protein kinase B), a serine-threonine kinase with a pleckstrin homology domain, is thought to be one such downstream effector. A mutant Akt (Akt-AA) in which the phosphorylation sites (Thr308 and Ser473) targeted by growth factors are replaced by alanine has now been shown to lack protein kinase activity and, when overexpressed in CHO cells or 3T3-L1 adipocytes with the use of an adenovirus vector, to inhibit insulin-induced activation of endogenous Akt. Akt-AA thus acts in a dominant negative manner in intact cells. Insulin-stimulated protein synthesis, which is sensitive to wortmannin, a pharmacological inhibitor of PI 3-kinase, was abolished by overexpression of Akt-AA without an effect on amino acid transport into the cells, suggesting that Akt is required for insulin-stimulated protein synthesis. Insulin activation of p70 S6 kinase was inhibited by approximately 75% in CHO cells and approximately 30% in 3T3-L1 adipocytes, whereas insulin-induced activation of endogenous Akt was inhibited by 80 to 95%, by expression of Akt-AA. Thus, Akt activity appears to be required, at least in part, for insulin stimulation of p70 S6 kinase. However, insulin-stimulated glucose uptake in both CHO cells and 3T3-L1 adipocytes was not affected by overexpression of Akt-AA, suggesting that Akt is not required for this effect of insulin. These data indicate that Akt acts as a downstream effector in some, but not all, of the signaling pathways downstream of PI 3-kinase.     

The activation of glycogen synthase by insulin switches from kinase inhibition to phosphatase activation during adipogenesis in 3T3-L1 cells

The effects of insulin and platelet-derived growth factor (PDGF) on glycogen synthase activation were compared in 3T3-L1 fibroblasts and adipocytes. In the fibroblasts, PDGF elicited a stronger phosphorylation of mitogen-activated protein kinase (MAPK) and AKT than did insulin. Both agents caused a comparable stimulation of receptor autophosphorylation, MAPK, and phosphatidylinositol 3-kinase (PI3-K) activation in the adipocytes. However, adipogenesis resulted in the uncoupling of PI3-K activation by PDGF from subsequent AKT phosphorylation. The relative contributions of glycogen synthase kinase-3 (GSK-3) inactivation and protein phosphatase-1 (PP1) activation in the regulation of glycogen synthase in both cell types were evaluated. Insulin and PDGF caused a small increase in glycogen synthase a activity in the fibroblasts. Additionally, both agents caused a similar inhibition of GSK-3, while having no effect on PP1 activity. Following differentiation, insulin treatment resulted in a 5-fold stimulation of glycogen synthase, whereas PDGF was without effect. Both agents caused a comparable inhibition of GSK-3 activity in the adipocytes, whereas only insulin activated PP1. Finally, wortmannin completely blocked the stimulation of PP1 by insulin in 3T3-L1 adipocytes, indicating that PI3-K inhibition can impinge on PP1 activation. Cumulatively these results suggest that the weak activation of glycogen synthase in 3T3-L1 fibroblasts is mediated by GSK-3 inactivation, whereas in the more metabolically active adipocytes, the insulin-specific activation of glycogen synthase is mediated by PP1 activation.