Stimulation of glycogen synthesis by insulin requires S6 kinase and phosphatidylinositol-3-kinase in HTC-IR cells

In order to study the role of phosphatidylinositol-3-kinase (PI3K), PKB, FRAP, S6 kinase, and MAP kinase in insulin-stimulated glycogen synthesis, we used a specific inhibitor of PI3K, LY294002, the immunosuppressant inhibitor of FRAP, rapamycin, and the inhibitor of MAPK kinase (MEK)/MAPK, PD98059, in rat HTC hepatoma cells overexpressing human insulin receptors. The PI3K inhibitor LY294002 completely blocks insulin-stimulated glycogen synthesis by inhibiting glycogen synthase, PKB (Akt-1), and FRAP (RAFT) autophosphorylation, as well as p70 S6 kinase activation, whereas insulin receptor substrates tyrosine phosphorylation and MEK activity were not affected. However, rapamycin only partially blocks insulin-stimulated glycogen synthesis by partial inhibition of glycogen synthase, whereas it completely blocks S6 kinase activation and FRAP autophosphorylation, but does not affect either PKB autophosphorylation, MEK activity, or insulin receptor tyrosine phosphorylation. Insulin-stimulated glycogen synthesis and glycogen synthase were not affected by the MEK/MAPK inhibitor PD98059. These data suggest that the PI3K, and not the MAPK pathway plays an important role in the insulin-stimulated glycogen synthesis in the hepatocyte, partly mediated by FRAP and S6 kinase activation. However, the inhibition of FRAP and S6 kinase activation is not sufficient to block insulin-stimulated glycogen synthesis, suggesting an important role of a branching pathway upstream of S6 kinase and downstream of PI3K, which is probably mediated by PKB in the signaling of the insulin receptor in hepatoma HTC cells.     

Activated MEK Stimulates Expression of AP-1 Components Independently of Phosphatidylinositol 3-Kinase (PI3-Kinase) but Requires a PI3-Kinase Signal To Stimulate DNA Synthesis

To investigate the contribution that ERK/mitogen-activated protein kinase signalling makes to cell cycle progression and gene expression, we have constructed cell lines to express an inducible version of activated MEK1. Using these cells, we show that activation of MEK leads to the expression of Fra-1 and Fra-2 but not c-Fos. Treatment of Ras-transformed cells with the MEK inhibitor PD098059 blocks expression of Fra-1 and Fra-2, showing that in Ras transformation ERK signalling is responsible for Fra-1 and Fra-2 expression. Activation of MEK1 in growth-arrested cells leads to DNA synthesis; however, ERK activation alone is insufficient because the induction of DNA synthesis is blocked by inhibition of phosphatidylinositol 3-kinase (PI3-kinase). Activation of PI3-kinase is indirect, perhaps through autocrine growth factors, and is required for the induction of cyclin D1.     

Use of lithium and SB-415286 to explore the role of glycogen synthase kinase-3 in the regulation of glucose transport and glycogen synthase.

Glycogen synthase kinase 3 (GSK3) is inactivated by insulin and lithium and, like insulin, Li also activates glycogen synthase (GS) via inhibition of GSK3. Li also mimics insulin's ability to stimulate glucose transport (GT), an observation that has led to the suggestion that GSK3 may coordinate hormonal increases in GT and glycogen synthesis. Here we have used Li and SB-415286, a selective GSK3 inhibitor, to establish the importance of GSK3 in the hormonal activation of GT in terms of its effect on GS in L6 myotubes and 3T3-L1 adipocytes. Insulin, Li and SB-415286 all induced a significant inhibition of GSK3, which was associated with a marked dephosphorylation and activation of GS. In L6 myotubes, SB-415286 induced a much greater activation of GS (6.8-fold) compared to that elicited by insulin (4.2-fold) or Li (4-fold). In adipocytes, insulin, Li and SB-415286 all caused a comparable activation of GS despite a substantial differentiation-linked reduction in GSK3 expression ( approximately 85%) indicating that GSK3 remains an important determinant of GS activation in fat cells. Whilst Li and SB-415286 both inhibit GSK3 in muscle and fat cells, only Li stimulated GT. This increase in GT was not sensitive to inhibitors of PI3-kinase, MAP kinase or mTOR, but was suppressed by the p38 MAP kinase inhibitor, SB-203580. Consistent with this, phosphorylation of p38 MAP kinase induced by Li correlated with its stimulatory effect on GT. Our findings support a crucial role for GSK3 in the regulation of GS, but based on the differential effects of Li and SB-415286, it is unlikely that acute inhibition of GSK3 contributes towards the rapid stimulation of GT by insulin in muscle and fat cells.     

Dissociation of PTPase levels from their modulation of insulin receptor signal transduction

Protein tyrosine phosphatases have been implicated in the regulation of receptor tyrosine kinase signalling pathways, including that of the insulin receptor. Here, cell density-dependent changes in PTPase expression have been exploited to investigate the relationship between cellular PTPase levels and the insulin receptor signal transduction pathway. Increasing cell density (20%, 50%, and >90%) in the rat McA-RH7777 hepatoma cell line resulted in increased protein expression of the receptor-like PTPase LAR (14-fold), and the nonreceptor PTPases PTP1B (11-fold) and SHP2 (10-fold). Each of these PTPases has previously been implicated in regulating insulin receptor signal transduction. Despite these marked increases, maximum insulin receptor autophosphorylation as well as receptor expression actually increased 2-fold. MAP kinase also increased approximately 2-fold as a function of cell density and paralleled increases in expression levels. Neither sensitivity nor maximum responsiveness to insulin were decreased at increasing cell densities as assessed by activation of PI 3-kinase. Duration of response was also unimpaired. These results suggest that expression levels of relevant PTPases are not the primary determinant in their modulation of insulin receptor kinase activity. Restricted accessibility at the molecular level or involvement of accessory proteins may be more critical parameters.     

Activation of p70 ribosomal protein S6 kinase is an essential step in the DNA damage-dependent signaling pathway responsible for the ultraviolet B-mediated increase in interstitial collagenase (MMP-1) and stromelysin-1 (MMP-3) protein levels in human dermal fibroblasts

Ultraviolet B (UVB) irradiation has been shown to stimulate the expression of matrix-degrading metalloproteinases via generation of DNA damage and/or reactive oxygen species. Matrix-degrading metalloproteinases promote UVB-triggered detrimental long term effects like cancer formation and premature skin aging. Here, we were interested in identifying components of the signal transduction pathway that causally link UVB-mediated DNA damage and induction of matrix-degrading metalloproteinase (MMP)-1/interstitial collagenase and MMP-3/stromelysin-1 in human dermal fibroblasts in vitro. The activity of p70 ribosomal S6 kinase, a downstream target of the FK506-binding protein-12/rapamycin-associated protein kinase (FRAP) kinase (RAFT1, mTOR), was identified to be 4.8 +/- 0.8-fold, and MMP-1 and MMP-3 protein levels 2.4- and 11.5-fold increased upon UVB irradiation compared with mock-irradiated controls. The FRAP kinase inhibitor rapamycin and the DNA repair inhibitor aphidicolin significantly suppressed the UVB-mediated increase in p70 ribosomal S6 kinase activity by 50-65% and MMP-1 and MMP-3 protein levels by 34-68% and 42-88% compared with UVB-irradiated fibroblasts. By contrast, the interleukin-1beta-mediated increase in MMP-1 and MMP-3 protein levels could not be suppressed by rapamycin. Collectively, our data suggest that the FRAP-controlled p70 ribosomal S6 kinase is an essential component of a DNA damage-dependent, but not of the interleukin-1/cell membrane receptor-dependent signaling.     

Zinc stimulates the activity of the insulin- and nutrient-regulated protein kinase mTOR

Recent studies indicate that zinc activates p70 S6 kinase (p70(S6k)) by a mechanism involving phosphatidylinositol 3-kinase (PI 3-kinase) and Akt (protein kinase B). Here it is shown that phenanthroline, a zinc and heavy metal chelator, inhibited both amino acid- and insulin-stimulated phosphorylation of p70(S6k). Both amino acid and insulin activations of p70(S6k) involve a rapamycin-sensitive step that involves the mammalian target of rapamycin (mTOR, also known as FRAP and RAFT). However, in contrast to insulin, amino acids activate p70(S6k) by an unknown PI 3-kinase- and Akt-independent mechanism. Thus the effects of chelator on amino acid activation of p70(S6k) were surprising. For this reason, we tested the hypothesis that zinc directly regulates mTOR activity, independently of PI 3-kinase activation. In support of this, basal and amino acid stimulation of p70(S6k) phosphorylation was increased by zinc addition to the incubation media. Furthermore, the protein kinase activities of mTOR immunoprecipitated from rat brain lysates were stimulated two- to fivefold by 10-300 microM Zn2+ in the presence of an excess of either Mn2+ or Mg2+, whereas incubation with 1,10-phenanthroline had no effect. These findings indicate that Zn2+ regulates, but is not absolutely required for, mTOR protein kinase activity. Zinc also stimulated a recombinant human form of mTOR. The stimulatory effects of Zn2+ were maximal at approximately 100 microM but decreased and became inhibitory at higher physiologically irrelevant concentrations. Micromolar concentrations of other divalent cations, Ca2+, Fe2+, and Mn2+, had no effect on the protein kinase activity of mTOR in the presence of excess Mg2+. Our results and the results of others suggest that zinc acts at multiple steps in amino acid- and insulin cell-signaling pathways, including mTOR, and that the additive effects of Zn2+ on these steps may thereby promote insulin and nutritional signaling.     

Nerve growth factor inhibits Na+/H+ exchange and formula absorption through parallel phosphatidylinositol 3-kinase-mTOR and ERK pathways in thick ascending limb

In the medullary thick ascending limb, inhibiting the basolateral NHE1 Na(+)/H(+) exchanger with nerve growth factor (NGF) induces actin cytoskeleton remodeling that secondarily inhibits apical NHE3 and transepithelial HCO(3)(-) absorption. The inhibition by NGF is mediated 50% through activation of extracellular signal-regulated kinase (ERK). Here we examined the signaling pathway responsible for the remainder of the NGF-induced inhibition. Inhibition of HCO(3)(-) absorption was reduced 45% by the phosphatidylinositol 3-kinase (PI3K) inhibitors wortmannin or LY294002 and 50% by rapamycin, a specific inhibitor of mammalian target of rapamycin (mTOR), a downstream effector of PI3K. The combination of a PI3K inhibitor plus rapamycin did not cause a further reduction in the inhibition by NGF. In contrast, the combination of a PI3K inhibitor plus the MEK/ERK inhibitor U0126 completely eliminated inhibition by NGF. Rapamycin decreased NGF-induced inhibition of basolateral NHE1 by 45%. NGF induced a 2-fold increase in phosphorylation of Akt, a PI3K target linked to mTOR activation, and a 2.2-fold increase in the activity of p70 S6 kinase, a downstream effector of mTOR. p70 S6 kinase activation was blocked by wortmannin and rapamycin, consistent with PI3K, mTOR, and p70 S6 kinase in a linear pathway. Rapamycin-sensitive inhibition of NHE1 by NGF was associated with an increased level of phosphorylated mTOR in the basolateral membrane domain. These findings indicate that NGF inhibits HCO(3)(-) absorption in the medullary thick ascending limb through the parallel activation of PI3K-mTOR and ERK signaling pathways, which converge to inhibit NHE1. The results identify a role for mTOR in the regulation of Na(+)/H(+) exchange activity and implicate NHE1 as a possible downstream effector contributing to mTOR's effects on cell growth, proliferation, survival, and tumorigenesis.     

Insulin regulates MAP kinase phosphatase-1 induction in Hirc B cells via activation of both extracellular signal-regulated kinase (ERK) and c-Jun-N-terminal kinase (JNK)

Previously, we have reported that insulin induces the expression of the dual-specificity tyrosine phosphatase Mitogen-activated protein (MAP) kinase phosphatase-1 (MKP-1) and that this may represent a negative feedback mechanism to regulate insulin-stimulated MAP kinase activity. In this work, the mechanism of regulation of MKP-1 expression by insulin was examined, particularly the role of the MAP kinase superfamily. Inhibition of the ERK pathway attenuated insulin-stimulated MKP-1 mRNA expression. Expression of dominant negative molecules of the JNK pathway also abolished insulin-stimulated MKP-1 expression. However, inhibition of p38^MAPK activity by SB202190 had no effect on insulin-stimulated MKP-1 induction. Simultaneous inhibition of the ERK and JNK pathways abolished the ability of insulin to stimulate MKP-1 expression, however, this combined inhibition was neither additive nor synergistic, suggesting these pathways converge to act on a common final effector. In conclusion, induction of MKP-1 mRNA expression in Hirc B cells by insulin requires activation of both the ERK and JNK pathways, but not p38^MAPK.     

Fatty acid infusion selectively impairs insulin action on Akt1 and protein kinase C lambda /zeta but not on glycogen synthase kinase-3

To determine the mechanism(s) for insulin resistance induced by fatty acids, we measured the ability of insulin to activate phosphoinositide 3-kinase (PI3K) and multiple distal pathways in rats. Following a 5-h infusion of lipid or glycerol (control), rats underwent a euglycemic hyperinsulinemic clamp. Insulin stimulated IRS-1-associated PI3K activity in muscle of glycerol-infused rats 2.4-fold but had no effect in lipid-infused rats. IRS-2- and phosphotyrosine-associated PI3K activity were increased 3.5- and 4.8-fold, respectively, by insulin in glycerol-infused rats but only 1.6- and 2.3-fold in lipid-infused rats. Insulin increased Akt1 activity 3.9-fold in glycerol-infused rats, and this was impaired 41% in lipid-infused rats. Insulin action on Akt2 and p70S6K were not impaired, whereas activation of protein kinase C lambda/zeta activity was reduced 47%. Insulin inhibited glycogen synthase kinase 3alpha (GSK-3alpha) activity by 30% and GSK-3beta activity by approximately 65% and increased protein phosphatase-1 activity by 40-47% in both glycerol- and lipid-infused rats. Insulin stimulated glycogen synthase activity 2.0-fold in glycerol-infused rats but only 1.4-fold in lipid-infused rats. Thus, 1) elevation of fatty acids differentially affects insulin action on pathways distal to PI3K, impairing activation of Akt1 and protein kinase C lambda/zeta and 2) insulin action on glycogen synthase can be regulated independent of effects on GSK-3 and protein phosphatase-1 activity in vivo.     

Abscisic Acid Induces Mitogen-Activated Protein Kinase Activation in Barley Aleurone Protoplasts

Abscisic acid (ABA) induces a rapid and transient mitogen-activated protein (MAP) kinase activation in barley aleurone protoplasts. MAP kinase activity, measured as myelin basic protein phosphorylation by MAP kinase immunoprecipitates, increased after 1 min, peaked after 3 min, and decreased to basal levels after ~5 min of ABA treatment in vivo. Antibodies recognizing phosphorylated tyrosine residues precipitate with myelin basic protein kinase activity that has identical ABA activation characteristics and demonstrate that tyrosine phosphorylation of MAP kinase occurs during activation. The half-maximal concentration of ABA required for MAP kinase activation, 3 x 10-7 M, is very similar to that required for ABA-induced rab16 gene expression. The tyrosine phosphatase inhibitor phenylarsine oxide can completely block ABA-induced MAP kinase activation and rab16 gene expression. These results lead us to conclude that ABA activates MAP kinase via a tyrosine phosphatase and that these steps are a prerequisite for ABA induction of rab16 gene expression.     

Involvement of mitogen-activated protein kinase homologues in the regulation of lipopolysaccharide-mediated induction of cyclo-oxygenase-2 but not nitric oxide synthase in RAW 264.7 macrophages.

In RAW 264.7 macrophages lipopolysaccharide (LPS) stimulated the activation of p42 and p44 MAP kinases and their upstream activator mitogen-activated protein (MAP) kinase kinase (MAPKK), and induced the 69-kDa isoform of cyclo-oxygenase-2 (COX-2) and the 130-kDa isoform of nitric oxide synthase (iNOS). PD 098059, a specific inhibitor of the activation of MAPKK, prevented LPS-mediated activation of MAPKK (IC50 = 3.0 +/- 0.1 microM, n = 3) and p42/44 MAP kinases and substantially reduced the induction of COX-2 by approximately 40%-70%, but was without effect upon the induction of iNOS. In parallel, LPS also stimulated the activation of p38 MAP kinase and the MAPKAP kinase-2, a downstream target of p38 MAP kinase. SB 203580, a specific inhibitor of p38 MAP kinase prevented the activation of p38 MAP kinase (IC50 = 3.3 +/- 1.4 microM, n = 3) and MAPKAP kinase-2 by LPS and reduced the induction of COX-2 by approximately 50-90%, with no significant effect upon iNOS expression. These studies indicate the involvement of both the classical p42/44 MAP kinases and p38 MAP kinase in the regulation of COX-2 but not iNOS induction following exposure to LPS.     

The role of phosphatidylinositol 3-kinase, Src kinase, and protein kinase A signaling pathways in insulin and glucagon regulation of CYP2E1 expression

The signaling pathways involved in insulin and glucagon regulation of CYP2E1 expression were examined in primary cultured rat hepatocytes. Insulin addition to primary cultured rat hepatocytes for 24 h resulted in an approximately 80% and >90% decrease in CYP2E1 mRNA levels at 1 and 10 nM insulin, respectively, relative to untreated cells. Addition of the phosphatidylinositol 3-kinase inhibitor wortmannin, or the Src kinase inhibitor geldanamycin, prior to insulin addition, inhibited the insulin-mediated decline in CYP2E1 mRNA. In contrast, treatment of cells with glucagon (100 nM), or the cAMP analogue dibutyryl-cAMP (50 microM), for 24 h increased CYP2E1 mRNA levels by approximately 7-fold. Addition of the protein kinase A inhibitor H89 prior to glucagon treatment attenuated the glucagon-mediated increase in CYP2E1 mRNA by approximately 70%. Glucagon (100 nM) opposed the effects of insulin (1 nM) on CYP2E1 mRNA expression and conversely, insulin blocked the effects of glucagon. These data provide compelling evidence for the regulation of CYP2E1 expression via mutually antagonistic signaling pathways involving insulin and glucagon.