Mechanisms of liver and muscle insulin resistance induced by chronic high-fat feeding

To elucidate cellular mechanisms of insulin resistance induced by excess dietary fat, we studied conscious chronically high-fat-fed (HFF) and control chow diet-fed rats during euglycemic-hyperinsulinemic (560 pmol/l plasma insulin) clamps. Compared with chow diet feeding, fat feeding significantly impaired insulin action (reduced whole body glucose disposal rate, reduced skeletal muscle glucose metabolism, and decreased insulin suppressibility of hepatic glucose production [HGP]). In HFF rats, hyperinsulinemia significantly suppressed circulating free fatty acids but not the intracellular availability of fatty acid in skeletal muscle (long chain fatty acyl-CoA esters remained at 230% above control levels). In HFF animals, acute blockade of beta-oxidation using etomoxir increased insulin-stimulated muscle glucose uptake, via a selective increase in the component directed to glycolysis, but did not reverse the defect in net glycogen synthesis or glycogen synthase. In clamp HFF animals, etomoxir did not significantly alter the reduced ability of insulin to suppress HGP, but induced substantial depletion of hepatic glycogen content. This implied that gluconeogenesis was reduced by inhibition of hepatic fatty acid oxidation and that an alternative mechanism was involved in the elevated HGP in HFF rats. Evidence was then obtained suggesting that this involves a reduction in hepatic glucokinase (GK) activity and an inability of insulin to acutely lower glucose-6-phosphatase (G-6-Pase) activity. Overall, a 76% increase in the activity ratio G-6-Pase/GK was observed, which would favor net hepatic glucose release and elevated HGP in HFF rats. Thus in the insulin-resistant HFF rat 1) acute hyperinsulinemia fails to quench elevated muscle and liver lipid availability, 2) elevated lipid oxidation opposes insulin stimulation of muscle glucose oxidation (perhaps via the glucose-fatty acid cycle) and suppression of hepatic gluconeogenesis, and 3) mechanisms of impaired insulin-stimulated glucose storage and HGP suppressibility are not dependent on concomitant lipid oxidation; in the case of HGP we provide evidence for pivotal involvement of G-6-Pase and GK in the regulation of HGP by insulin, independent of the glucose source.     

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.     

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.     

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.     

Evidence for involvement of protein kinase C (PKC)-zeta and noninvolvement of diacylglycerol-sensitive PKCs in insulin-stimulated glucose transport in L6 myotubes

We examined the question of whether insulin activates protein kinase C (PKC)-zeta in L6 myotubes, and the dependence of this activation on phosphatidylinositol (PI) 3-kinase. We also evaluated a number of issues that are relevant to the question of whether diacylglycerol (DAG)-dependent PKCs or DAG-insensitive PKCs, such as PKC-zeta, are more likely to play a role in insulin-stimulated glucose transport in L6 myotubes and other insulin-sensitive cell types. We found that insulin increased the enzyme activity of immunoprecipitable PKC-zeta in L6 myotubes, and this effect was blocked by PI 3-kinase inhibitors, wortmannin and LY294002; this suggested that PKC-zeta operates downstream of PI 3-kinase during insulin action. We also found that treatment of L6 myotubes with 5 microM tetradecanoyl phorbol-13-acetate (TPA) for 24 h led to 80-100% losses of all DAG-dependent PKCs (alpha, beta1, beta2, delta, epsilon) and TPA-stimulated glucose transport (2-deoxyglucose uptake); in contrast, there was full retention of PKC-zeta, as well as insulin-stimulated glucose transport and translocation of GLUT4 and GLUT1 to the plasma membrane. Unlike what has been reported in BC3H-1 myocytes, TPA treatment did not elicit increases in PKCbeta2 messenger RNA or protein in L6 myotubes, and selective retention of this PKC isoform could not explain the retention of insulin effects on glucose transport after prolonged TPA treatment. Of further interest, TPA acutely activated membrane-associated PI 3-kinase in L6 myotubes, and acute effects of TPA on glucose transport were inhibited, not only by the PKC inhibitor, LY379196, but also by both wortmannin and LY294002; this suggested that DAG-sensitive PKCs activate glucose transport through cross-talk with phosphatidylinositol (PI) 3-kinase, rather than directly through PKC. Also, the cell-permeable, myristoylated PKC-zeta pseudosubstrate inhibited insulin-stimulated glucose transport both in non-down-regulated and PKC-depleted (TPA-treated) L6 myotubes; thus, the PKC-zeta pseudosubstrate appeared to inhibit a protein kinase that is required for insulin-stimulated glucose transport but is distinct from DAG-sensitive PKCs. In keeping with the latter dissociation of DAG-sensitive PKCs and insulin-stimulated glucose transport, LY379196, which inhibits PKC-beta (preferentially) and other DAG-sensitive PKCs at relatively low concentrations, inhibited insulin-stimulated glucose transport only at much higher concentrations, not only in L6 myotubes, but also in rat adipocytes, BC3H-1 myocytes, 3T3/L1 adipocytes and rat soleus muscles. Finally, stable and transient expression of a kinase-inactive PKC-zeta inhibited basal and insulin-stimulated glucose transport in L6 myotubes. Collectively, our findings suggest that, whereas PKC-zeta is a reasonable candidate to participate in insulin stimulation of glucose transport, DAG-sensitive PKCs are unlikely participants.     

Impaired insulin-stimulated nonoxidative glucose metabolism in pancreas-kidney transplant recipients. Dose-response effects of insulin on glucose turnover

Insulin resistance is a characteristic feature in recipients of a pancreas transplant, but the relative contribution of the liver and peripheral tissues to this abnormality within a spanning range of insulin concentrations is unknown. To assess the impact of insulin action on glucose metabolism after pancreas transplantation, a euglycemic-hyperinsulinemic clamp with sequential insulin infusions (5, 40, and 200 mU.m-2.min-1 for 120 min each), combined with isotopic determinations of the rates of hepatic glucose production and extrahepatic glucose uptake, indirect calorimetry, and measurements of glycogen synthase and hexokinase activities in vastus lateralis muscle, were performed in six pancreas-kidney transplant recipients (Px group) and compared with those performed in six nondiabetic kidney transplant recipients with similar immunosuppression (Kx group) and six nondiabetic control subjects. The overall effects of insulin on whole-body glucose metabolism, determined as the glucose infusion rates versus the corresponding steady-state serum insulin concentrations, demonstrated a rightward shift in the dose-response curves of the transplanted groups compared with those of normal subjects. The dose-response curve for glucose disposal rates (Rd) was shifted to the right in the Px and Kx groups, and the maximal glucose disposal rate was reduced by 40% in the Px group (11.7 +/- 1.1 mg.kg-1 fat-free mass.min-1) and 30% in the Kx group (13.9 +/- 1.2 mg.kg-1 fat-free mass.min-1) compared with that in control subjects (19.1 +/- 2.2 mg.kg-1 fat-free mass.min-1) (P < 0.05). The dose-response curve for suppression of hepatic glucose output rates was similar at increasing hepatic sinusoidal insulin concentrations. Glucose oxidation rates were similar in all groups, whereas nonoxidative glucose rates were reduced by 50% in the Px group and by 30% in the Kx group compared with those in the control group (P < 0.05). In the Px group, an impaired activation of the fractional velocity and absent decrease in the half-maximal stimulation of muscle glycogen synthase occurred during the insulin infusion. However, this finding could only explain in part the degree of impairment in nonoxidative glucose metabolism. No differences were found in total hexokinase activity in muscle between normal subjects and the transplant groups at basal insulinemia or after insulin stimulation. During hyperinsulinemia, glucagon and nonesterified fatty acids were not suppressed as much in the transplanted groups as they were in normal control subjects (P < 0.05). In conclusion, pancreas transplantation causes impaired peripheral action of insulin as compared with that in normal subjects and kidney transplant recipients. The main course of insulin resistance in the two transplant groups is explained by the immunosuppressive treatment, but the augmented insulin resistance in pancreas transplant recipients could partly be explained by the chronic peripheral hyperinsulinemia. The principal site of insulin resistance was a reduced insulin-stimulated nonoxidative glucose metabolism of peripheral tissues, which resulted in decreased capacity to store glucose as glycogen. The impaired peripheral insulin action could only partly be explained by a reduced activation of the glycogen synthase enzyme in skeletal muscle.     

Fatty acid induced insulin resistance in rat-1 fibroblasts overexpressing human insulin receptors: impaired insulin-stimulated mitogen-activated protein kinase activity

Saturated fatty acids cause insulin resistance but the underlying molecular mechanism is still unknown. We examined the effect of saturated nonesterified fatty acids on insulin binding and action in transfected Rat-1 fibroblasts, which over-expressed human insulin receptors. Incubation with 1.0 mmol/l palmitate for 1-4 h did not affect insulin binding, insulin receptor autophosphorylation, insulin-stimulated tyrosine kinase activity toward poly(Glu4:Tyr1), pp185 and Shc phosphorylation and PI3-kinase activity in these cells. However, the dose response curve of insulin-stimulated glucose transport was right-shifted. Palmitate inhibited the maximally insulin-stimulated mitogen activated protein (MAP) kinase activity toward synthetic peptide to 7% that of control. The palmitate treatment influenced neither cytosolic protein kinase A activity nor cAMP levels. These results suggested that 1) palmitate did not inhibit the early steps of insulin action from insulin binding to pp185 or Shc phosphorylation but inhibited insulin-stimulated MAP kinase, and that 2) palmitate decreased insulin sensitivity as manifested by inhibited insulin-stimulated glucose uptake. In conclusion, the mechanism of saturated non-esterified fatty acid induced insulin resistance in glucose uptake may reside at post PI3-kinase or Shc steps, including the level of MAP kinase activation.     

Primary sites of actions of staurosporine and H-7 in the cascade of insulin action to glucose transport in rat adipocytes

The insulin-stimulated glucose transporter in rat adipocytes was inhibited by two protein kinase inhibitors, staurosporine (SSP) and H-7 (1-(5-isoquinolinylsulfonyl)-2-methylpiperazine). However, whereas SSP (10 microM) blocked the insulin-dependent translocation of glucose transporter, H-7 (3 mM) did not. The latter inhibited glucose transporter activity not only in cells, but also in reconstituted liposomes. On the other hand, SSP blocked both the action of insulin and the insulinomimetic action of GTP gamma S (Guanosine 5'-O-(3-thiotriphosphate)). GTP gamma S had distinct effects on the glucose transport and cAMP phosphodiesterase (PDE) activities. It is suggest that H-7 may inhibit glucose transport activity per se; a SSP sensitive protein kinases (protein kinase C isoforms?) may be involved in cascade of the insulin action on glucose transporter as modulated by GTP gamma S; and glucose transport and PDE activities may be regulated by distinct GTP gamma S-sensitive factors.     

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.     

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.     

Interleukin-1 beta induces prostaglandin G/H synthase-2 (cyclooxygenase-2) in primary murine astrocyte cultures

Activation of glial cells and the consequent release of cytokines, proteins, and other intercellular signaling molecules is a well-recognized phenomenon in brain injury and neurodegenerative disease. We and others have previously described an inducible prostaglandin G/H synthase, known as PGHS-2 or cyclooxygenase-2, that is up-regulated in many cell systems by cytokines and growth factors and down-regulated by glucocorticoid hormones. In cultured mouse astrocytes we observed increased production of prostaglandin E2 (PGE2) after stimulation with either interleukin-1 beta (IL-1 beta) or the protein kinase C activator phorbol 12-myristate 13-acetate (TPA). This increase in PGE2 content was blocked by pretreatment with dexamethasone and correlated with increases in cyclooxygenase activity measured at 4 h. Northern blots revealed concomitant increases in PGHS-2 mRNA levels that peaked at 2 h and were dependent on the dosage of IL-1 beta. Dexamethasone inhibited this induction of PGHS-2 mRNA by IL-1 beta. TPA, basic fibroblast growth factor, and the proinflammatory factors tumor necrosis factor alpha and lipopolysaccharide, but not interleukin-6, also stimulated PGHS-2 mRNA expression. Relative to IL-1 beta, the greater increases in PGE2 production and cyclooxygenase activity caused by TPA correlated with a greater induction of PGHS-2 mRNA. Furthermore NS-398, a specific inhibitor of cyclooxygenase-2, blocked > 80% of the cyclooxygenase activity in TPA-treated astrocytes. These findings indicate that increased expression of PGHS-2 contributes to prostaglandin production in cultured astrocytes exposed to cytokines and other factors.     

Exercise-induced overexpression of key regulatory proteins involved in glucose uptake and metabolism in tetraplegic persons: molecular mechanism for improved glucose homeostasis

Complete spinal cord lesion leads to profound metabolic abnormalities and striking changes in muscle morphology. Here we assess the effects of electrically stimulated leg cycling (ESLC) on whole body insulin sensitivity, skeletal muscle glucose metabolism, and muscle fiber morphology in five tetraplegic subjects with complete C5-C7 lesions. Physical training (seven ESLC sessions/wk for 8 wk) increased whole body insulin-stimulated glucose uptake by 33+/-13%, concomitant with a 2.1-fold increase in insulin-stimulated (100 microU/ml) 3-O-methylglucose transport in isolated vastus lateralis muscle. Physical training led to a marked increase in protein expression of GLUT4 (378+/-85%), glycogen synthase (526+/-146%), and hexokinase II (204+/-47%) in vastus lateralis muscle, whereas phosphofructokinase expression (282+/-97%) was not significantly changed. Hexokinase II activity was significantly increased, whereas activity of phosphofructokinase, glycogen synthase, and citrate synthase was not changed after training. Muscle fiber type distribution and fiber area were markedly altered compared to able-bodied subjects before ESLC training, with no change noted in either parameter after ECSL training. In conclusion, muscle contraction improves insulin action on whole body and cellular glucose uptake in cervical cord-injured persons through a major increase in protein expression of key genes involved in the regulation of glucose metabolism. Furthermore, improvements in insulin action on glucose metabolism are independent of changes in muscle fiber type distribution.     

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.     

Bacterial lipopolysaccharide increases interleukin-6 and prostaglandin release in rat cortical type I astrocytes by different mechanisms: role of anti-inflammatory agents

LPS stimulated IL-6 release in a concentration-dependent manner from rat cortical type I astrocytes. This stimulatory action was completely abolished by Dexamethasone (DEX), but was not affected by indomethacin (IND), a 5-cyclooxigenase inhibitor. LPS-induced IL-6 release was partially inhibited by BW 4AC, a 5-lipoxygenase inhibitor. LPS concentration-dependently increased the release of PGE2 from type I astrocytes, an effect completely inhibited by IND. To rule out the possibility that DEX was inhibiting LPS-induced IL-6 release by blocking IL-6 gene expression, we tested the effect of DEX on interleukin 1beta(IL-1)-induced IL-6 release. DEX slightly inhibited IL-1-induced IL-6 release, while IL-1 releasing action on IL-6 was significantly reduced by IND. The involvement of nitric oxide (NO) generation on LPS-induced IL-6 release was also studied. We found that L-NO-arginine, an inhibitor of nitric oxide synthase, concentration-dependently reduced LPS-induced IL-6 release in astrocytes. In conclusion, we provide evidence that LPS action on IL-6 and PGE2 release can be ascribed to the activation of different transduction mechanisms, which can be pharmacologically dissected with the aid of anti-inflammatory drugs.     

Regulation of insulin-stimulated glucose transporter GLUT4 translocation and Akt kinase activity by ceramide

The sphingomyelin derivative ceramide is a signaling molecule implicated in numerous physiological events. Recently published reports indicate that ceramide levels are elevated in insulin-responsive tissues of diabetic animals and that agents which trigger ceramide production inhibit insulin signaling. In the present series of studies, the short-chain ceramide analog C2-ceramide inhibited insulin-stimulated glucose transport by approximately 50% in 3T3-L1 adipocytes, with similar reductions in hormone-stimulated translocation of the insulin-responsive glucose transporter (GLUT4) and insulin-responsive aminopeptidase. C2-ceramide also inhibited phosphorylation and activation of Akt, a molecule proposed to mediate multiple insulin-stimulated metabolic events. C2-ceramide, at concentrations which antagonized activation of both glucose uptake and Akt, had no effect on the tyrosine phosphorylation of insulin receptor substrate 1 (IRS-1) or the amounts of p85 protein and phosphatidylinositol kinase activity that immunoprecipitated with anti-IRS-1 or antiphosphotyrosine antibodies. Moreover, C2-ceramide also inhibited stimulation of Akt by platelet-derived growth factor, an event that is IRS-1 independent. C2-ceramide did not inhibit insulin-stimulated phosphorylation of mitogen-activated protein kinase or pp70 S6-kinase, and it actually stimulated phosphorylation of the latter in the absence of insulin. Various pharmacological agents, including the immunosuppressant rapamycin, the protein synthesis inhibitor cycloheximide, and several protein kinase C inhibitors, were without effect on ceramide's inhibition of Akt. These studies demonstrate ceramide's capacity to inhibit activation of Akt and imply that this is a mechanism of antagonism of insulin-dependent physiological events, such as the peripheral activation of glucose transport and the suppression of apoptosis.     

In the rat, endogenous nitric oxide modulates the response of the hypothalamic-pituitary-adrenal axis to interleukin-1 beta, vasopressin, and oxytocin

Nitric oxide (NO) synthase (NOS), the enzyme responsible for NO formation, is found in hypothalamic neurons containing oxytocin (OT), vasopressin (VP), and to a lesser extent corticotropin-releasing factor (CRF). Because NO is reported to modulate endocrine activity, we have investigated the hypothesis that endogenous NO participates in ACTH released by various secretagogues in the rat. In the adult male rat, the intravenous injection of interleukin-1 beta (IL-1 beta; 0.2-0.3 micrograms/kg), VP (0.3-0.9 micrograms/kg), and OT (30 micrograms/kg) significantly increased plasma ACTH and corticosterone levels. Pretreatment with the L-form, but not the D-form, of N omega nitro-L-arginine-methylester (L-NAME; a specific inhibitor of NOS) markedly augmented the effects of these secretagogues whether it was injected acutely or over a 4 d period. Blockade of NOS activity also caused significant (P < 0.01) extensions of the duration of action of IL-1 beta, VP, and OT. In contrast, L-NAME did not significantly alter the stimulatory action of peripherally injected CRF, or centrally administered IL-1 beta. Administration of L-arginine, but not D-arginine (100 mg/kg), used as a substrate for basal NO synthesis and which did not by itself alter the activity of the hypothalamic-pituitary-adrenal (HPA) axis, blunted IL-1-induced ACTH secretion, and reversed the interaction between L-NAME and IL-1 beta. The stimulatory action of endotoxin, a lipopolysaccharide that releases endogenous cytokines, was also augmented by inhibition of NO formation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interleukin-8 differentially modulates interleukin-4- and interleukin-2-induced human B cell growth

The effect of interleukin-8 (IL)-8 on human B cell growth, as determined by thymidine uptake and viable cell numbers was studied. IL-8 inhibited IL-4-induced growth of B cells costimulated with anti-mu antibodies (Ab) or Staphylococcus aureus Cowan strain I (SAC) in a dose-dependent fashion. In contrast, IL-8 did not inhibit IL-2-induced growth of B cells. The IL-8-mediated inhibition was specific, since it was blocked by anti-IL-8 mAb but not by control IgG1. Moreover, anti-tumor necrosis factor-alpha (anti-TNF-alpha) Ab blocked IL-8-mediated inhibition. On the other hand, TNF-alpha, but not other cytokines including IL-1 beta, IL-3, IL-5, IL-6, interferon-alpha (IFN-alpha) or IFN-gamma, inhibited IL-4-mediated growth, and inhibition by TNF-alpha was blocked by anti-TNF-alpha Ab but not by control IgG. IL-4 had no effect on TNF-alpha binding by B cells while it decreased TNF-alpha production by B cells. IL-8 had no effect in binding of IL-4, IL-2 or TNF-alpha by B cells, however, it enhanced TNF-alpha production by B cells. These results indicate that IL-8 inhibited IL-4-induced human B cell growth by enhancement of endogenous TNF-alpha production.