Peculiarities of the osmotic response in dehydrated erythrocytes

The regulation of glycogen synthase (GS) and glycogen phosphorylase (GP) activity by phosphorylation/ dephosphorylation has been proposed to be via changes in activities of several different protein (serine/threonine) phosphatases and kinases, including protein phosphatase (PP) 1/2A, PP2C, and cAMP-dependent protein kinase (PKA). In order to determine whether PP1/2A, PP2C, and/or PKA activities are related to GS and/or GP activities, these enzymes were measured in freeze-clamped liver biopsies obtained under basal fasting conditions from 16 obese monkeys. Four monkeys were normoglycemic and normoinsulinemic, five were hyperinsulinemic, and seven had type 2 diabetes (NIDDM). Liver glycogen and glucose 6-phosphate (G6P) contents were also determine. Basal enzyme activities and basal substrate concentrations were not significantly different between the three group of obese monkeys; however, there were several significant linear relationships observed when the monkeys were treated as one group. Therefore, multiple regression was used to determine the correlation between key variables. GS fractional activity was correlated to GP fractional activity (p < 0.05) and to PP2C activity (p = 0.005) (adjusted R2, 53%). GP independent activity was correlated to GS independent activity (p < 0.07) and to PKA fractional activity (p = 0.005) (adjusted R2, 64%). PP2C activity was correlated to GS fractional activity (p < 0.0005) and to PP1/2A activity (p < 0.0001) (adjusted R2, 83%). PKA fractional activity was correlated to GP total activity (p < 0.0005) and to age (p = 0.001) (adjusted R2, 82%). G6P content was correlated to glycogen content (p < 0.05) and to PP2C activity (p = 0.0005) (adjusted R2, 73%). In conclusion, PP2C and PKA are involved in the regulation of GS and GP activity in the basal state in liver of obese monkeys with a wide range of glucose tolerance.     

Chiroinositol deficiency and insulin resistance. I. Urinary excretion rate of chiroinositol is directly associated with insulin resistance in spontaneously diabetic rhesus monkeys

Previously, we demonstrated that nondiabetic insulin-resistant monkeys had reduced covalent insulin activation of muscle glycogen synthase (GS) compared to normal monkeys and that covalent insulin activation of adipose tissue GS was absent in these monkeys. Covalent insulin activation of muscle and adipose tissue GS in monkeys with impaired glucose tolerance and noninsulin-dependent diabetes (NIDDM) was also absent. As in humans, monkeys with NIDDM have a lower urinary excretion rate of chiroinositol (CI), a component of a putative mediator of insulin action, compared to normal monkeys. To determine whether the urinary excretion rate of CI was related to insulin resistance, which develops naturally in many obese rhesus monkeys, we examined the relationships between 24-h urinary CI excretion rate and 1) whole body insulin-mediated glucose disposal rates (M) and insulin-mediated changes in 2) the skeletal muscle GS activity ratio (sm delta GSAR), 3) the skeletal muscle glycogen phosphorylase activity ratio, and 4) the adipose tissue GS activity ratio (at delta GSAR) in 27 monkeys ranging from normal (n = 12) to insulin resistant (n = 8) to overtly diabetic (n = 7). The urinary CI excretion rate was significantly correlated with M (r = 0.47; P < 0.02), sm delta GSAR (r = 0.38; P < 0.05), skeletal muscle glycogen phosphorylase activity ratio (r = -0.49; P < 0.01), and at delta GSAR (r = 0.46; P < 0.02). The urinary CI excretion rate was also correlated with glucose tolerance (r = 0.39; P < 0.05). There was a wide range of urinary CI excretion rates (0.42-5.17 mumol/day) in monkeys with normal fasting plasma glucose concentrations. However, of the 7 diabetic monkeys, 6 had a urinary CI excretion rate below 2.0 mumol/day, and in the subgroup of 16 monkeys with a urinary CI excretion rate less than 2.0 mumol/day, the associations of urinary CI with M rate (r = 0.65; P < 0.005), glucose tolerance (r = 0.63; P < 0.01), and sm delta GSAR (r = 0.73; P < 0.001) increased in strength and significance. We propose that the urinary CI excretion rate may be 1) a biochemical indicator of both in vivo and in vitro insulin resistance and 2) a noninvasive diagnostic tool with potential for the identification of those individuals at risk for NIDDM and other related diseases with insulin resistance.     

The role of glucose 6-phosphate in the control of glycogen synthase

Elevated blood glucose concentrations result in increased intracellular levels of glucose 6-phosphate in liver, skeletal muscle, and adipose tissue. In liver, blood glucose concentrations are the main factor in control of the synthesis of glycogen; insulin has only a potentiating effect. In skeletal muscle and adipocytes, glucose alone has little effect on the activity of glycogen synthase, the limiting enzyme in glycogen synthesis. However, insulin released as a result of elevated blood glucose stimulates the translocation of specific glucose transporters to the cell membrane, increases the uptake of glucose, and causes the covalent, dephosphorylation-mediated activation of glycogen synthase. We present evidence that elevated intracellular contents of glucose 6-phosphate provoke the activation of glycogen synthase in liver, muscle, and adipose tissue. In addition, glucose 6-phosphate may inhibit the phosphorylation of glycogen synthase by cyclic AMP-stimulated protein kinase. We show that the stimulated glucose uptake and phosphorylation appear to play a major role in the control by insulin of the enzymes involved in glycogen synthesis.     

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 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.     

Unchanged gene expression of glycogen synthase in muscle from patients with NIDDM following sulphonylurea-induced improvement of glycaemic control

We have previously shown that the mRNA expression of muscle glycogen synthase is decreased in non-insulin-dependent diabetic (NIDDM) patients; the objective of the present protocol was to examine whether the gene expression of muscle glycogen synthase in NIDDM is affected by chronic sulphonylurea treatment. Ten obese patients with NIDDM were studied before and after 8 weeks of treatment with a weight-maintaining diet in combination with the sulphonylurea gliclazide. Gliclazide treatment was associated with significant reductions in HbA1C (p=0.001) and fasting plasma glucose (p=0.005) as well as enhanced beta-cell responses to an oral glucose load. During euglycaemic, hyperinsulinaemic clamp (2 mU x kg-1 x min-1) in combination with indirect calorimetry, a 35% (p=0.005) increase in whole-body insulin-stimulated glucose disposal rate, predominantly due to an increased non-oxidative glucose metabolism (p=0.02) was demonstrated in teh gliclazide-treated patients when compared to pre-treatment values. In biopsies obtained from vastus lateralis muscle during insulin infusion, the half-maximal activation of glycogen synthase was achieved at a significantly lower concentration of the allosteric activator glucose 6-phosphate (p=0.01). However, despite significant increases in both insulin-stimulated non-oxidative glucose metabolism and muscle glycogen synthase activation in gliclazide-treated patients no changes were found in levels of glycogen synthase mRNA or immunoreactive protein in muscle. In conclusion, improved blood glucose control in gliclazide-treated obese NIDDM patients has no impact on the gene expression of muscle glycogen synthase.     

Conditional, tissue-specific expression of Q205L Galphai2 in vivo mimics insulin activation of c-Jun N-terminal kinase and p38 kinase

Deficiency of the G-protein subunit Galphai2 impairs insulin action (Moxham, C. M., and Malbon, C. C. (1996) Nature 379, 840-844). By using the promoter for the phosphoenolpyruvate carboxykinase gene, conditional, tissue-specific expression of the constitutively active mutant form (Q205L) of Galphai2 was achieved in mice harboring the transgene. Expression of Q205L Galphai2 was detected in skeletal muscle, liver, and adipose tissue of transgenic mice. Whereas the Galphai2-deficient mice displayed blunted insulin action, the Q205L Galphai2-expressing mice displayed enhanced insulin-like effects. Glycogen synthase in skeletal muscle was found to be activated in Q205L Galphai2-expressing mice, in the absence of the administration of insulin. Analysis of members of mitogen-activated protein kinase family revealed that both c-Jun N-terminal kinase and p38 are constitutively activated in vivo in the mice that express the Q205L Galphai2. ERK1,2, in contrast, are unaffected in the Q205L Galphai2-expressing mice. Insulin, like expression of Q205L Galphai2, activates both p38 and c-Jun N-terminal kinases as well as glycogen synthase. Activation of c-Jun N-terminal and p38 kinases in vivo with anisomycin, however, was insufficient to activate glycogen synthase. Much like Galphai2 deficiency provokes insulin resistance, expression of Q205L constitutively active Galphai2 mimics insulin action in vivo, sharing with insulin the activation of two mitogen-activated protein kinase members, p38 and c-Jun N-terminal kinases.     

Glycogen synthase regulation in hyperinsulinemic/obese progeny of rats fed a high carbohydrate formula in their infancy

OBJECTIVE: To evaluate the effects of chronic hyperinsulinemia/obesity on the proximal events leading to the activation of glycogen synthase. DESIGN: 100 d old second generation of chronically hyperinsulinemic/obese rats born to mothers which were artificially reared on a high carbohydrate (HC) milk formula in their infancy were used for this study and compared with mother-fed (MF) controls. MEASUREMENTS: Glycogen, glycogen synthase, protein phosphatase-1 (PP-1), mitogen-activated protein kinase (MAPK), insulin-stimulated protein kinase (ISPK) and protein kinase A (PKA) were measured in liver and muscle of both MF and HC rats. RESULTS: Glycogen content, glycogen synthase and PP-1 activities were significantly reduced in liver and muscle of HC rats compared to MF controls while trypsin released PP-1 activity was elevated. The activities of both MAPK and ISPK were also decreased in the HC rats. In contrast PKA activity was increased. CONCLUSIONS: Glycogen synthase activity in the basal state may be impaired in the hyperinsulinemic HC rats in two ways: (i) by a decrease in the activities of the kinases that presumably activate PP-1 and (ii) by increased activity of PKA which inactivates glycogen synthase directly by phosphorylation and indirectly by its effects on PP-1.     

Mechanisms of insulin resistance during acute endotoxemia

We characterized the mechanisms underlying acute endotoxin-induced alterations in glucose metabolism and determined the extent to which catecholamines mediate these changes. Acute endotoxemia was induced in chronically catheterized awake rats by a bolus injection of lipopolysaccharide (LPS; 1 mg/kg; LD10). Basal glucose turnover (Rt; infusion of [5-3H]glucose), in vivo insulin action on overall glucose utilization (euglycemic clamp), glycolysis, and glycogen synthesis were determined in four groups of rats. These groups received 1) LPS (LPS rats; n = 6), 2) saline (control rats; n = 6), 3) LPS and alpha beta-blockade (alpha beta-blockade and LPS rats; n = 9), or 4) saline and alpha beta-blockade (alpha beta-blockade control rats; n = 9). In the basal state, LPS induced hypotension and transient hyperglycemia. These changes were associated with glycogen depletion in both skeletal muscle and liver, and increased Rt. During hyperinsulinemia, whole body glucose disposal was 37% decreased (105 vs. 166 mumol/kg.min; P < 0.01). This whole body insulin resistance was characterized by decreased glycogen synthesis and glycogen synthase activity, but not by altered whole body glycolysis. alpha beta-Blockade abolished transient hyperglycemia, increased Rt, and accelerated basal liver glycogen depletion (45 vs. 105 mmol/kg dry, LPS and alpha beta-blockade rats vs. LPS rats; P < 0.05), but inhibited muscle glycogenolysis. alpha beta-Blockade did not reverse the insulin resistance induced by endotoxin. These data suggest that catecholamines counteract the LPS-induced increase in basal glucose turnover and stimulate muscle glycogenolysis during acute endotoxemia. These effects might explain the better preservation of hepatic glycogen in the absence than in the presence of alpha beta-blockade and serve as a defense mechanism against hypoglycemia. Catecholamines do not seem to be the immediate causes of insulin resistance during acute endotoxemia.     

Effects of dichloroacetate on glycogen metabolism in B6C3F1 mice

Dichloroacetate (DCA) is a by-product of drinking water chlorination. Administration of DCA in drinking water results in accumulation of glycogen in the liver of B6C3F1 mice. To investigate the processes affecting liver glycogen accumulation, male B6C3F1 mice were administered DCA in drinking water at levels varying from 0.1 to 3 g/l for up to 8 weeks. Liver glycogen synthase (GS) and glycogen phosphorylase (GP) activities, liver glycogen content, serum glucose and insulin levels were analyzed. To determine whether effects were primary or attributable to increased glycogen synthesis, some mice were fasted and administered a glucose challenge (20 min before sacrifice). DCA treatments in drinking water caused glycogen accumulation in a dose-dependent manner. The DCA treatment in drinking water suppressed the activity ratio of GS measured in mice sacrificed at 9:00 AM, but not at 3:00 AM. However, net glycogen synthesis after glucose challenge was increased with DCA treatments for 1-2 weeks duration, but the effect was no longer observed at 8 weeks. Degradation of glycogen by fasting decreased progressively as the treatment period was increased, and no longer occurred at 8 weeks. A shift of the liver glycogen-iodine spectrum from DCA-treated mice was observed relative to that of control mice, suggesting a change in the physical form of glycogen. These data suggest that DCA-induced glycogen accumulation at high doses is related to decreases in the degradation rate. When DCA was administered by single intraperitoneal (i.p.) injection to na?ve mice at doses of 2-200 mg/kg at the time of glucose challenge, a biphasic response was observed. Doses of 10-25 mg/kg increased both plasma glucose and insulin concentrations. In contrast, very high i.p. doses of DCA (> 75 mg/kg) produced progressive decreases in serum glucose and glycogen deposition in the liver. Since the blood levels of DCA produced by these higher i.p. doses were significantly higher than observed with drinking water treatment, we conclude that apparent differences with data of previous investigations is related to substantial differences in systemic dose and/or dose-time relations.     

Mechanism of impaired metabolic signaling by a truncated human insulin receptor. Decreased activation of protein phosphatase 1 by insulin

Previous studies have shown that a human insulin receptor lacking the COOH-terminal 43-amino acid domain (HIR delta CT) displays a compromised ability to stimulate glucose transport and glycogen synthase, whereas mitogenic signaling and stimulation of the insulin receptor tyrosine kinase activity remain intact (Maegawa, H., McClain, D. A., Freidenberg, G., Olefsky, J. M., Napier, M., Lipari, T., Dull, T. J., Lee, J., and Ullrich, A. (1988) J. Biol. Chem. 263, 8912-8917). In this study, we examined the effect of insulin on protein phosphatase 1 (PP-1) activity and phosphorylation in cells expressing wild-type human insulin receptor (HIRc) and HIR delta CT cells using phosphorylase alpha as substrate in the presence of 3 nM okadaic acid. Basal PP-1 activity was significantly lower in HIR delta CT than in HIRc cells (p < 0.05). Insulin stimulated PP-1 activity in HIRc cells (25-30% increase over basal activity) in a time- and dose-dependent manner. Insulin failed to stimulate PP-1 activity in HIR delta CT cells. Western blotting with the catalytic subunit antibody and the regulatory subunit antibody revealed similar amounts of the 37-kDa band (catalytic subunit) and the 160-kDa band (presumed regulatory subunit) in HIRc and HIR delta CT cells. We conclude that the COOH-terminal domain of the insulin receptor is an important element in mediating the effect of insulin on PP-1 and suggest that activation of PP-1 may be linked to signaling insulin's metabolic actions.     

Mechanism by which glucose and insulin inhibit net hepatic glycogenolysis in humans

13C NMR spectroscopy was used to assess flux rates of hepatic glycogen synthase and phosphorylase in overnight-fasted subjects under one of four hypoglucagonemic conditions: protocol I, hyperglycemic (approximately 10 mM) -hypoinsulinemia (approximately 40 pM); protocol II, euglycemic (approximately 5 mM) -hyperinsulinemia (approximately 400 pM); protocol III, hyperglycemic (approximately 10 mM) -hyperinsulinemia (approximately 400 pM); and protocol IV; euglycemic (approximately 5 mM) -hypoinsulinemia (approximately 40 pM). Inhibition of net hepatic glycogenolysis occurred in both protocols I and II compared to protocol IV but via a different mechanism. Inhibition of net hepatic glycogenolysis occurred in protocol I mostly due to decreased glycogen phosphorylase flux, whereas in protocol II inhibition of net hepatic glycogenolysis occurred exclusively through the activation of glycogen synthase flux. Phosphorylase flux was unaltered, resulting in extensive glycogen cycling. Relatively high rates of net hepatic glycogen synthesis were observed in protocol III due to combined stimulation of glycogen synthase flux and inhibition of glycogen phosphorylase flux. In conclusion, under hypoglucagonemic conditions: (a) hyperglycemia, per se, inhibits net hepatic glycogenolysis primarily through inhibition of glycogen phosphorylase flux; (b) hyperinsulinemia, per se, inhibits net hepatic glycogenolysis primarily through stimulation of glycogen synthase flux; (c) inhibition of glycogen phosphorylase and the activation of glycogen synthase are not necessarily coupled and coordinated in a reciprocal fashion; and (d) promotion of hepatic glycogen cycling may be the principal mechanism by which insulin inhibits net hepatic glycogenolysis and endogenous glucose production in humans under euglycemic conditions.     

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.     

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.     

Properties and phosphorylation sites of baculovirus-expressed nuclear inhibitor of protein phosphatase-1 (NIPP-1)

NIPP-1 is the RNA-binding subunit of a major species of protein phosphatase-1 in the nucleus. We have expressed nuclear inhibitor of protein phosphatase-1 (NIPP-1) in Sf9 cells, using the baculovirus-expression system. The purified recombinant protein was a potent (Ki = 9.9 +/- 0.3 pM) and specific inhibitor of protein phosphatase-1 and was stoichiometrically phosphorylated by protein kinases A and CK2. At physiological ionic strength, phosphorylation by these protein kinases drastically decreased the inhibitory potency of free NIPP-1. Phosphorylation of NIPP-1 in a heterodimeric complex with the catalytic subunit of protein phosphatase-1 resulted in an activation of the holoenzyme without a release of NIPP-1. Sequencing and phosphoamino acid analysis of tryptic phosphopeptides enabled us to identify Ser178 and Ser199 as the phosphorylation sites of protein kinase A, whereas Thr161 and Ser204 were phosphorylated by protein kinase CK2. These residues all conform to consensus recognition sites for phosphorylation by protein kinases A or CK2 and are clustered near a RVXF sequence that has been identified as a motif that interacts with the catalytic subunit of protein phosphatase-1.     

N-glycoprotein biosynthesis in plants: recent developments and future trends

This review summarizes the evolution of ideas concerning insulin signal transduction, the current information on protein ser/thr kinase cascades as signalling intermediates, and their status as participants in insulin regulation of energy metabolism. Best characterized is the Ras-MAPK pathway, whose input is crucial to cell fate decisions, but relatively dispensable in metabolic regulation. By contrast the effectors downstream of PI-3 kinase, although less well elucidated, include elements indispensable for the insulin regulation of glucose transport, glycogen and cAMP metabolism. Considerable information has accrued on PKB/cAkt, a protein kinase that interacts directly with Ptd Ins 3'OH phosphorylated lipids, as well as some of the elements further downstream, such as glycogen synthase kinase-3 and the p70 S6 kinase. Finally, some information implicates other erk pathways (e.g. such as the SAPK/JNK pathway) and Nck/cdc42-regulated PAKs (homologs of the yeast Ste 20) as participants in the cellular response to insulin. Thus insulin recruits a broad array of protein (ser/thr) kinases in its target cells to effectuate its characteristic anabolic and anticatabolic programs.     

Peripheral but not hepatic insulin resistance in mice with one disrupted allele of the glucose transporter type 4 (GLUT4) gene

Glucose transporter type 4 (GLUT4) is insulin responsive and is expressed in striated muscle and adipose tissue. To investigate the impact of a partial deficiency in the level of GLUT4 on in vivo insulin action, we examined glucose disposal and hepatic glucose production (HGP) during hyperinsulinemic clamp studies in 4-5-mo-old conscious mice with one disrupted GLUT4 allele [GLUT4 (+/-)], compared with wild-type control mice [WT (+/+)]. GLUT4 (+/-) mice were studied before the onset of hyperglycemia and had normal plasma glucose levels and a 50% increase in the fasting (6 h) plasma insulin concentrations. GLUT4 protein in muscle was approximately 45% less in GLUT4 (+/-) than in WT (+/+). Euglycemic hyperinsulinemic clamp studies were performed in combination with [3-3H]glucose to measure the rate of appearance of glucose and HGP, with [U-14C]-2-deoxyglucose to estimate muscle glucose transport in vivo, and with [U-14C]lactate to assess hepatic glucose fluxes. During the clamp studies, the rates of glucose infusion, glucose disappearance, glycolysis, glycogen synthesis, and muscle glucose uptake were approximately 55% decreased in GLUT4 (+/-), compared with WT (+/+) mice. The decreased rate of in vivo glycogen synthesis was due to decreased stimulation of glucose transport since insulin's activation of muscle glycogen synthase was similar in GLUT4 (+/-) and in WT (+/+) mice. By contrast, the ability of hyperinsulinemia to inhibit HGP was unaffected in GLUT4 (+/-). The normal regulation of hepatic glucose metabolism in GLUT4 (+/-) mice was further supported by the similar intrahepatic distribution of liver glucose fluxes through glucose cycling, gluconeogenesis, and glycogenolysis. We conclude that the disruption of one allele of the GLUT4 gene leads to severe peripheral but not hepatic insulin resistance. Thus, varying levels of GLUT4 protein in striated muscle and adipose tissue can markedly alter whole body glucose disposal. These differences most likely account for the interindividual variations in peripheral insulin action.     

Inhibition of phosphatidylinositol 3-kinase activity by adenovirus-mediated gene transfer and its effect on insulin action

Phosphatidylinositol 3-kinase (PI 3-K) is implicated in cellular events including glucose transport, glycogen synthesis, and protein synthesis. It is activated in insulin-stimulated cells by binding of the Src homology 2 (SH2) domains in its 85-kDa regulatory subunit to insulin receptor substrate-1 (IRS-1), and, others. We have previously shown that IRS-1-associated PI 3-kinase activity is not essential for insulin-stimulated glucose transport in 3T3-L1 adipocytes, and that alternate pathways exist in these cells. We now show that adenovirus-mediated overexpression of the p85N-SH2 domain in these cells behaves in a dominant-negative manner, interfering with complex formation between endogenous PI 3-K and its SH2 binding targets. This not only inhibited insulin-stimulated IRS-1-associated PI 3-kinase activity, but also completely blocked anti-phosphotyrosine-associated PI 3-kinase activity, which would include the non-IRS-1-associated activity. This resulted in inhibition of insulin-stimulated glucose transport, glycogen synthase activity and DNA synthesis. Further, Ser/Thr phosphorylation of downstream molecules Akt and p70 S6 kinase was inhibited. However, co-expression of a membrane-targeted p110(C) with the p85N-SH2 protein rescued glucose transport, supporting our argument that the p85N-SH2 protein specifically blocks insulin-mediated PI 3-kinase activity, and, that the signaling pathways downstream of PI 3-kinase are intact. Unexpectedly, GTP-bound Ras was elevated in the basal state. Since p85 is known to interact with GTPase-activating protein in 3T3-L1 adipocytes, the overexpressed p85N-SH2 peptide could titrate out cellular GTPase-activating protein by direct association, such that it is unavailable to hydrolyze GTP-bound Ras. However, insulin-induced mitogen-activated protein kinase phosphorylation was inhibited. Thus, PI 3-kinase may be required for this action at a step independent of and downstream of Ras. We conclude that, in 3T3-L1 adipocytes, non-IRS-1-associated PI 3-kinase activity is crucial for insulin's metabolic signaling, and that overexpressed p85N-SH2 protein inhibits a variety of insulin's ultimate biological effects.     

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.