Functional characterisation of the regulation of CAAT enhancer binding protein alpha by GSK-3 phosphorylation of Threonines 222/226

Background  

Glycogen Synthase Kinase-3 (GSK3) activity is repressed following insulin treatment of cells. Pharmacological inhibition of GSK3 mimics the effect of insulin on Phosphoenolpyruvate Carboxykinase (PEPCK), Glucose-6 Phosphatase (G6Pase) and IGF binding protein-1 (IGFBP1) gene expression. CAAT/enhancer binding protein alpha (C/EBPα) regulates these gene promoters in liver and is phosphorylated on two residues (T222/T226) by GSK3, although the functional outcome of the phosphorylation has not been established. We aimed to establish whether CEBPα is a link between GSK3 and these gene promoters.     

Different mechanisms are used by insulin to repress three genes that contain a homologous thymine-rich insulin response element

Insulin rapidly and completely inhibits expression of the hepatic insulin-like growth factor binding protein-1 (IGFBP-1), phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6Pase) genes. This inhibition is mediated through a phosphatidyl inositol 3-kinase-dependent regulation of a DNA element, termed the thymine-rich insulin response element, found within the promoters of each of these genes. This has led to the conclusion that these three promoters are regulated by insulin using the same molecular mechanism. However, we recently found that the regulation of the IGFBP1 but not the PEPCK or G6Pase genes by insulin was sensitive to rapamycin, an inhibitor of mTOR. Here, we present further evidence that different regulatory pathways mediate the insulin regulation of these promoters. Importantly, we identify a protein phosphatase activity in the pathway connecting mTOR to the IGFBP-1 promoter. These data have major implications for the development of molecular therapeutics for the treatment of insulin-resistant states such as diabetes and hypertension.     

Activation of translation initiation factor eIF2B by insulin requires phosphatidyl inositol 3-kinase

Eukaryotic initiation factor eIF2B mediates a key regulatory step in peptide-chain initiation and is acutely activated by insulin, although it is not clear how. Inhibitors of phosphatidylinositide 3-kinase blocked activation of eIF2B, although rapamycin, which inhibits the p70 S6 kinase pathway, did not. Furthermore, a dominant negative mutant of PI 3-kinase also prevented activation of eIF2B, while a Sos-mutant, which blocks MAP kinase activation, did not. The data demonstrate that a pathway distinct from MAP and p70 S6 kinases regulates eIF2B. Glycogen synthase kinase-3 (GSK-3) phosphorylates and inactivates eIF2B. In all cases, eIF2B and GSK-3 were regulated reciprocally. Dominant negative PI 3-kinase abolished the insulin-induced inhibition of GSK-3. These data strongly support the hypothesis that insulin activates eIF2B through a signalling pathway involving PI 3-kinase and inhibition of GSK-3.     

Erythropoietin Inhibits Gluconeogenesis and Inflammation in the Liver and Improves Glucose Intolerance in High-Fat Diet-Fed Mice

Erythropoietin (EPO) has multiple biological functions, including the modulation of glucose metabolism. However, the mechanisms underlying the action of EPO are still obscure. This study is aimed at investigating the potential mechanisms by which EPO improves glucose tolerance in an animal model of type 2 diabetes. Male C57BL/6 mice were fed with high-fat diet (HFD) for 12 weeks and then treated with EPO (HFD-EPO) or vehicle saline (HFD-Con) for two week. The levels of fasting blood glucose, serum insulin and glucose tolerance were measured and the relative levels of insulin-related phosphatidylinositol 3-kinase (PI3K)/Akt, insulin receptor (IR) and IR substrate 1 (IRS1) phosphorylation were determined. The levels of phosphoenolpyruvate carboxykinase (PEPCK), glucose-6- phosphatase (G6Pase), toll like receptor 4 (TLR4), tumor necrosis factor (TNF)-α and IL-6 expression and nuclear factor-κB (NF-κB) and c-Jun N-terminal kinase (JNK), extracellular-signal-regulated kinase (ERK) and p38 MAPK activation in the liver were examined. EPO treatment significantly reduced the body weights and the levels of fasting blood glucose and serum insulin and improved the HFD-induced glucose intolerance in mice. EPO treatment significantly enhanced the levels of Akt, but not IR and IRS1, phosphorylation, accompanied by inhibiting the PEPCK and G6Pase expression in the liver. Furthermore, EPO treatment mitigated the HFD-induced inflammatory TNF-α and IL-6 production, TLR4 expression, NF-κB and JNK, but not ERK and p38 MAPK, phosphorylation in the liver. Therefore, our data indicated that EPO treatment improved glucose intolerance by inhibiting gluconeogenesis and inflammation in the livers of HFD-fed mice.     

Glycogen synthase kinase-3 beta activity is critical for neuronal death caused by inhibiting phosphatidylinositol 3-kinase or Akt but not for death caused by nerve growth factor withdrawal

Numerous studies reveal that phosphatidylinositol (PI) 3-kinase and Akt protein kinase are important mediators of cell survival. However, the survival-promoting mechanisms downstream of these enzymes remain uncharacterized. Glycogen synthase kinase-3 beta (GSK-3 beta), which is inhibited upon phosphorylation by Akt, was recently shown to function during cell death induced by PI 3-kinase inhibitors. In this study, we tested whether GSK-3 beta is critical for the death of sympathetic neurons caused by the withdrawal of their physiological survival factor, the nerve growth factor (NGF). Stimulation with NGF resulted in PI 3-kinase-dependent phosphorylation of GSK-3 beta and inhibition of its protein kinase activity, indicating that GSK-3 beta is targeted by PI 3-kinase/Akt in these neurons. Expression of the GSK-3 beta inhibitor Frat1, but not a mutant Frat1 protein that does not bind GSK-3 beta, rescued neurons from death caused by inhibiting PI 3-kinase. Similarly, expression of Frat1 or kinase-deficient GSK-3 beta reduced death caused by inhibiting Akt. In NGF-maintained neurons, overexpression of GSK-3 beta caused a small but significant decrease in survival. However, expression of neither Frat1, kinase-deficient GSK-3 beta, nor GSK-3-binding protein inhibited NGF withdrawal-induced death. Thus, although GSK-3 beta function is required for death caused by inactivation of PI 3-kinase and Akt, neuronal death caused by NGF withdrawal can proceed through GSK-3 beta-independent pathways.     

Selective small-molecule inhibitors of glycogen synthase kinase-3 activity protect primary neurones from death

The phosphatidylinositol 3-kinase (PI 3-kinase)/protein kinase B (PKB; also known as Akt) signalling pathway is recognized as playing a central role in the survival of diverse cell types. Glycogen synthase kinase-3 (GSK-3) is a ubiquitously expressed serine/threonine protein kinase that is one of several known substrates of PKB. PKB phosphorylates GSK-3 in response to insulin and growth factors, which inhibits GSK-3 activity and leads to the modulation of multiple GSK-3 regulated cellular processes. We show that the novel potent and selective small-molecule inhibitors of GSK-3; SB-415286 and SB-216763, protect both central and peripheral nervous system neurones in culture from death induced by reduced PI 3-kinase pathway activity. The inhibition of neuronal death mediated by these compounds correlated with inhibition of GSK-3 activity and modulation of GSK-3 substrates tau and beta-catenin. Thus, in addition to the previously assigned roles of GSK-3, our data provide clear pharmacological and biochemical evidence that selective inhibition of the endogenous pool of GSK-3 activity in primary neurones is sufficient to prevent death, implicating GSK-3 as a physiologically relevant principal regulatory target of the PI 3-kinase/PKB neuronal survival pathway.     

Interleukin-6 impairs the insulin signaling pathway, promoting production of nitric oxide in human umbilical vein endothelial cells

Interleukin 6 (IL-6) is an independent predictor of type 2 diabetes and cardiovascular disease and is correlated with insulin resistance. Insulin stimulates nitric oxide (NO) production through the IRS-1/PI3-kinase/Akt/eNOS pathway (where IRS-1 is insulin receptor substrate 1, PI3-kinase is phosphatidylinositol 3-kinase, and eNOS is endothelial NO synthase). We asked if IL-6 affects insulin vasodilator action both in human umbilical vein endothelial cells (HUVEC) and in the aortas of C57BL/6J mice and whether this inhibitory effect was caused by increased Ser phosphorylation of IRS-1. We observed that IL-6 increased IRS-1 phosphorylation at Ser(312) and Ser(616); these effects were paralleled by increased Jun N-terminal protein kinase (JNK) and extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation and reversed by JNK and ERK1/2 inhibition. In addition, IL-6 treatment resulted in impaired IRS-1 phosphorylation at Tyr(612), a site essential for engaging PI3-kinase. Furthermore, IL-6 treatment reduced insulin-stimulated phosphorylation of eNOS at the stimulatory Ser(1177) site and impaired insulin-stimulated eNOS dephosphorylation at the inhibitory Thr(495) site. Insulin-stimulated eNOS activation and NO production were also inhibited by IL-6; these effects were reversed by inhibition of JNK and ERK1/2. Treatment of C57BL/6J mice with IL-6 resulted in impaired insulin-dependent activation of the Akt/eNOS pathway in the aorta as a result of JNK and ERK1/2 activation. Our data suggest that IL-6 impairs the vasodilator effects of insulin that are mediated by the IRS-1/PI3-kinase/Akt/eNOS pathway through activation of JNK and ERK1/2.     

Insulin activates caspase-3 by a phosphatidylinositol 3'-kinase-dependent pathway

Activation of the caspase proteases by c-Jun N-terminal kinase 1 (JNK1) has been proposed as a mechanism of apoptotic cell death. Here we report that insulin activates caspase-3 by a pathway requiring phosphatidylinositol 3'-kinase (PI3-kinase). JNK1 assays demonstrated that insulin treatment of myeloma cells induced 3-fold activation of JNK1. Inhibition of PI3-kinase with wortmannin and LY294002 blocked insulin-dependent activation of JNK1. Caspase assays demonstrated that insulin increased caspase-3 activity 3-fold and that inhibition of PI3-kinase blocked this effect. Cell death was doubled by insulin and was due to a 3-fold increase in apoptosis of cells in the G1/G0 phase of the cell cycle. Inhibition of PI3-kinase completely blocked this effect. Finally, inhibition of caspase-3 with benzyloxycarbonyl-Asp-2,6-dichlorobenzoyloxymethylketone blocked cell death due to insulin. Taken together, these findings indicate that insulin activates caspase-3 by a PI3-kinase-dependent pathway resulting in increased apoptosis and cell death.     

Translocation and activation of AKT2 in response to stimulation by insulin

The AKT2 oncogene encodes a protein-serine/threonine kinase that was recently shown to be activated by a variety of growth factors. In addition, we previously showed that AKT2 is abundant in brown fat and skeletal muscle, tissues that are highly insulin responsive and that play a role in glucose metabolism. In this study, we demonstrate that AKT2 is activated in response to stimulation by insulin in a dose- and time-dependent manner in human ovarian carcinoma cells and that activation of AKT2 is abolished in cells pretreated with wortmannin, an inhibitor of phosphatidylinositol 3-kinase (PI 3-kinase). Activation of AKT2 is manifested by changes in its phosphorylation state. Immunofluorescence experiments demonstrate that AKT2 is translocated to the plasma membrane after insulin stimulation, and this translocation is abolished by wortmannin. Both wild-type AKT2 activated by insulin and constitutively active AKT2, which has been targeted to the membrane by the addition of a myristoylation signal, were found to inactivate glycogen synthase kinase-3 (GSK-3) in vitro. GSK-3 was not inactivated by a catalytically inactive AKT2 mutant. Collectively, these data indicate that activation of AKT2 by insulin is mediated by PI 3-kinase and that GSK-3 is a downstream target of AKT2, suggesting a potentially important role of AKT2 in glycogen synthesis and other GSK-3 signaling pathways. J. Cell. Biochem. 70:433–441, 1998. © 1998 Wiley-Liss, Inc.