Early growth response gene-1 expression in vascular smooth muscle cells effects of insulin and oxidant stress

BACKGROUND: Cardiovascular mortality is increased in individuals with insulin resistance, and increased oxidant stress is strongly implicated in atherogenesis. Early growth response gene-1 (Egr-1) may be an important link between insulin resistance and oxidant stress. In this study we examined the effects of insulin and oxidant stress on Egr-1 expression in vascular smooth muscle cells (VSMC), and identified mechanisms for these effects on Egr-1. METHODS: Rat VSMC were used to obtain time course and dose-response curves for insulin and oxidant stress on Egr-1 protein expression. Intracellular signaling pathway inhibitors and adenoviral vectors with dominant negative effects on specific signaling pathways were used to determine mechanisms for these effects. RESULTS: Insulin and oxidant stress each significantly stimulate Egr-1 protein expression. Insulin and oxidant stress combined have a greater effect on Egr-1 than either alone. Insulin effects are mediated via the ERK1/2 MAP kinase pathway, whereas oxidant stress effects may be mediated via the ERK5 and p38 MAP kinase pathways. CONCLUSIONS: We demonstrated that insulin and oxidant stress stimulate Egr-1 expression in VSMC. Insulin effects are mediated via the ERK1/2 MAP kinase pathway, whereas oxidant stress effects may be mediated via the ERK5 and p38 MAP kinase pathways. As insulin resistance is characterized by compensatory hyperinsulinemia and selective impairment of the PI 3-kinase pathway with intact signaling along the ERK1/2 MAP kinase pathway, this may have implications for accelerated atherosclerosis in insulin resistance.     

Hypothalamic preproghrelin gene expression is repressed by insulin via both PI3-K/Akt and ERK1/2 MAPK pathways in immortalized, hypothalamic neurons

The gut peptide ghrelin is expressed within neurons of the hypothalamus. Using a hypothalamic cell line, mHypoE-38 neurons, the effect of insulin on preproghrelin gene expression was assayed. These cells contain neuron-specific markers, preproghrelin and the insulin receptor. We determined that insulin has direct effects on preproghrelin gene expression. Insulin (10 nM) stimulated protein kinase B (Akt) and extracellular signal-regulated kinase 1 and 2 (ERK1/2) phosphorylation from 5 to 60 min and 5 min, respectively, and led to repression of preproghrelin gene expression at 2 h. Pharmacological inhibitors to phosphoinositide-3-kinase (PI3-K; LY294002) and MEK (PD98059) demonstrated that basal ghrelin gene expression is regulated by the PI3-K pathway and requires the mitogen-activated protein kinase pathway for insulin-stimulated preproghrelin repression. These results demonstrate that insulin has a direct effect on hypothalamic neurons to decrease preproghrelin gene expression through classic insulin pathways.     

Differentiation state-selective roles of p38 isoforms in human intestinal epithelial cell anoikis

BACKGROUND & AIMS: Little is known of the signaling events implicated in the induction of human enterocytic anoikis. In the present study, we analyzed the role of the stress-activated protein kinase p38 in this process. METHODS: Anoikis was induced in undifferentiated and differentiated enterocytes by inhibition of focal adhesion kinase (Fak; pharmacologic inhibition or overexpression of a dominant negative form) or beta1 integrins (antibody blocking), or by maintaining cells in suspension. Expression/activation parameters of p38 (isoforms alpha, beta, gamma, delta) and of the Fak/phosphatidylinositol-3-kinase (PI3-K)/Akt anoikis-suppressing pathways were analyzed. Kinase activities of p38 isoforms also were blocked by pharmacologic inhibitors or by overexpression of dominant-negative forms. RESULTS: (1) p38 activation is sustained transiently after induction of anoikis in both undifferentiated and differentiated enterocytes; (2) such sustenance of p38 activation is associated with a down-regulation of the Fak/PI3-K/Akt pathway; (3) distinct profiles of p38 isoform expression are exhibited by undifferentiated (alpha, beta, gamma) and differentiated (alpha, gamma, delta) enterocytes; (4) none of the 4 known p38 isoforms was found to promote cell survival in either differentiation state; and (5) only p38beta and p38delta are required specifically for anoikis in undifferentiated and differentiated cells, respectively. CONCLUSIONS: Distinct p38 isoforms play a major role in the induction of enterocytic anoikis and the regulation of such selective p38 isoform-mediated anoikis is linked with the state of cell differentiation. These data provide novel insights into the synchronized regulation of cell survival/death required in the epithelial renewal process along the human intestinal crypt-villus axis.     

Insulin-mediated cell proliferation and survival involve inhibition of c-Jun N-terminal kinases through a phosphatidylinositol 3-kinase- and mitogen-activated protein kinase phosphatase-1-dependent pathway

We previously reported that long term treatment with insulin led to sustained inhibition of c-Jun N-terminal kinases (JNKs) in CHO cells overexpressing insulin receptors. Here we investigated the signaling molecules involved in insulin inhibition of JNKs, focusing on phosphatidylinositol 3-kinase (PI 3-K) and mitogen-activated protein kinase phosphatase-1 (MKP-1). In addition, we examined the relevance of JNK inhibition for insulin-mediated proliferation and survival. Insulin inhibition of JNKs was mediated by PI 3-K, as it was blocked by wortmannin and LY294002 and required the de novo synthesis of a phosphatase(s), as it was abolished by orthovanadate and actinomycin D. MKP-1 was a good candidate because 1) insulin stimulation of MKP-1 expression correlated with insulin inhibition of JNKs; 2) insulin stimulation of MKP-1 expression, like insulin inhibition of JNKs, was mediated by PI 3-K; and 3) the transient expression of an antisense MKP-1 RNA reduced the insulin inhibitory effect on JNKs. The overexpression of a dominant negative JNK1 mutant increased insulin stimulation of DNA synthesis and mimicked the protective effect of insulin against serum withdrawal-induced apoptosis. The overexpression of wild-type JNK1 or antisense MKP-1 RNA reduced the proliferative and/or antiapoptotic responses to insulin. Altogether, these results demonstrate that insulin inhibits JNKs through a PI 3-K- and MKP-1-dependent pathway and provide evidence for a key role for JNK inhibition in insulin regulation of proliferation and survival.     

Long-term denervation impairs insulin receptor substrate-1-mediated insulin signaling in skeletal muscle

Long-term denervation is associated with insulin resistance. To investigate the molecular bases of insulin resistance, the downstream signaling molecules of insulin receptor including insulin receptor substrate-1 (IRS-1) and phosphatidylinositol 3-kinase (PI 3-K) were examined in skeletal muscle of rats after 7 days of denervation. Long-term denervation attenuated insulin-stimulated activation of the initial steps of the intracellular signaling pathway. Insulin-stimulated tyrosine phosphorylation of insulin receptor was reduced to 36% (P < .005), as was the phosphorylation of IRS-1 to 34% (P < .0001) of control. While insulin receptor protein level was unchanged, the protein expression of IRS-1 was significantly decreased in denervated muscles. Insulin-stimulated percent tyrosine phosphorylation of IRS-1, normalized to the IRS-1 protein expression, was also reduced to 55% (P < .01) of control in denervated muscle. Denervation caused a decline in the insulin-induced binding of p85 regulatory subunit of PI 3-K to IRS-1 to 61% (P < .001) and IRS-1-associated PI 3-K activity to 57% (P < .01). These results provide evidence that long-term denervation results in insulin resistance because of derangements at multiple points, including tyrosine phosphorylation of insulin receptor and its downstream signaling molecule, IRS-1, protein expression of IRS-1, and activation of PI 3-K.     

Involvement of both phosphatidylinositol 3-kinase and p44/p42 mitogen-activated protein kinase pathways in the short-term regulation of pyruvate kinase L by insulin

Pyruvate kinase L (PK-L) is a key regulatory enzyme of the hepatic glycolytic/gluconeogenic pathway that can be dephosphorylated and activated in response to insulin. However, the signaling cascades involved in this insulin effect have not been established. In this work we have investigated the potential involvement of phosphatidylinositol 3-kinase (PI 3-K) and p44/p42 mitogen-activated protein kinase (MAPK) pathways in the short-term modulation of PK-L by insulin in primary cultures of rat hepatocytes. Wortmannin, at a concentration of 100 nM, caused a marked inhibition of the PI 3-K/protein kinase B pathway, which became complete at 500 nM wortmannin. Likewise, wortmannin at 100 and 500 nM, elicited partial and total inhibitions of insulin-mediated activation of PK-L, respectively. However, this PI 3-K inhibitor also reduced insulin-mediated phosphorylation of p44/p42 MAPK in cultured rat hepatocytes, indicating that both the PI 3-K and MAPK pathways could be involved in PK-L activation by insulin. Three facts appear to reinforce this hypothesis: 1) the selective and complete inhibition of the PI 3-K/protein kinase B pathway by LY294002 (50 microM) was accompanied by a partial blockade of insulin-induced PK-L activation; 2) when signaling through the MAPK cascade was selectively suppressed by the presence of PD98059 (50 microM), a 50% reduction of insulin-induced activation of PK-L was observed; and 3) the effect of PD98059 (50 microM) on PK-L activation was reinforced by the additional presence of 100 nM wortmannin. We also observed that the blockade of p70 S6-kinase by rapamycin did not affect the activation of PK-L by insulin. From these findings it can be concluded that both PI 3-K and MAPK pathways, but not p70 S6-kinase, are involved in the short-term activation of PK-L by insulin in rat hepatocytes.