Abstract-Vascular smooth muscle cell (VSMC) proliferation is a critical event in the development and progression of vascular diseases, including atherosclerosis. We investigated whether the activation of adenosine monophosphate-activated protein kinase (AMPK) could suppress VSMC proliferation and inhibit cell cycle progression. Treatment of human aortic smooth muscle cells (HASMCs) or isolated rabbit aortas with the AMPK activator 5-Aminoimidazole-4-carboxamide ribonucleoside (AICAR) induced phosphorylation of AMPK and acetyl Co-A carboxylase. AICAR significantly inhibited HASMC proliferation induced by both platelet-derived growth factor-BB (PDGF-BB) and fetal calf serum (FCS). Treatment with AICAR inhibited the phosphorylation of retinoblastoma gene product (Rb) induced by PDGF-BB or FCS, and increased the expression of cyclin-dependent kinase inhibitor p21 CIP but not that of p27 KIP . Pharmacological inhibition of AMPK or overexpression of dominant negative-AMPK inhibited both the suppressive effect of AICAR on cell proliferation and the phosphorylation of Rb, suggesting that the effect of AICAR is mediated through the activation of AMPK. Cell cycle analysis in HASMCs showed that AICAR significantly increased cell population in G0/G1-phase and reduced that in S-and G2/M-phase, suggesting AICAR induced cell cycle arrest. AICAR increased both p53 protein and Ser-15 phosphorylated p53 in HASMCs, which were blocked by inhibition of AMPK. In isolated rabbit aortas, AICAR also increased Ser-15 phosphorylation and protein expression of p53 and inhibited Rb phosphorylation induced by FCS. These data suggest for the first time that AMPK suppresses VSMC proliferation via cell cycle regulation by p53 upregulation. Therefore, AMPK activation in VSMCs may be a therapoietic target for the prevention of vascular diseases.

Tumor necrosis factor (TNF)-␣ inhibits insulin action;however, the precise mechanisms are unknown. It was reported that TNF-␣ could increase mitochondrial reactive oxygen species (ROS) production, and apoptosis signalregulating kinase 1 (ASK1) was reported to be required for TNF-␣-induced apoptosis. Here, we examined roles of mitochondrial ROS and ASK1 in TNF-␣-induced impaired insulin signaling in cultured human hepatoma (Huh7) cells. Using reduced MitoTracker Red probe, we confirmed that TNF-␣ increased mitochondrial ROS production, which was suppressed by overexpression of either uncoupling protein-1 (UCP)-1 or manganese superoxide dismutase (Mn-SOD). TNF-␣ significantly activated ASK1, increased serine phosphorylation of insulin receptor substrate (IRS)-1, and decreased insulin-stimulated tyrosine phosphorylation of IRS-1 and serine phosphorylation of Akt, and all of these effects were inhibited by overexpression of either UCP-1 or MnSOD. Similar to TNF-␣, overexpression of wild-type ASK1 increased serine phosphorylation of IRS-1 and decreased insulin-stimulated tyrosine phosphorylation of IRS-1, whereas overexpression of dominant-negative ASK1 ameliorated these TNF-␣-induced events. In addition, TNF-␣ activated c-jun NH 2 -terminal kinases (JNKs), and this observation was partially inhibited by overexpression of UCP-1, MnSOD, or dominant-negative ASK1. These results suggest that TNF-␣ increases mitochondrial ROS and activates ASK1 in Huh7 cells and that these TNF-␣-induced phenomena contribute, at least in part, to impaired insulin signaling.

To investigate the roles of insulin receptor substrate 3 (IRS-3) and IRS-4 in the insulin-like growth factor 1 (IGF-1) signaling cascade, we introduced these proteins into 3T3 embryonic fibroblast cell lines prepared from wild-type (WT) and IRS-1 knockout (KO) mice by using a retroviral system. Following transduction of IRS-3 or IRS-4, the cells showed a significant decrease in IRS-2 mRNA and protein levels without any change in the IRS-1 protein level. In these cell lines, IGF-1 caused the rapid tyrosine phosphorylation of all four IRS proteins. However, IRS-3-or IRS-4-expressing cells also showed a marked decrease in IRS-1 and IRS-2 phosphorylation compared to the host cells. This decrease was accounted for in part by a decrease in the level of IRS-2 protein but occurred with no significant change in the IRS-1 protein level. IRS-3-or IRS-4-overexpressing cells showed an increase in basal phosphatidylinositol 3-kinase activity and basal Akt phosphorylation, while the IGF-1-stimulated levels correlated well with total tyrosine phosphorylation level of all IRS proteins in each cell line. IRS-3 expression in WT cells also caused an increase in IGF-1-induced mitogenactivated protein kinase phosphorylation and egr-1 expression (ϳ1.8-and ϳ2.4-fold with respect to WT). In the IRS-1 KO cells, the impaired mitogenic response to IGF-1 was reconstituted with IRS-1 to supranormal levels and was returned to almost normal by IRS-2 or IRS-3 but was not improved by overexpression of IRS-4. These data suggest that IRS-3 and IRS-4 may act as negative regulators of the IGF-1 signaling pathway by suppressing the function of other IRS proteins at several steps.Insulin and insulin-like growth factor 1 (IGF-1) initiate their diverse biological effects by binding to and activating their endogenous tyrosine kinase receptors (22,44). The insulin receptor substrate (IRS) proteins are major substrates of both insulin receptor and IGF-1 receptor tyrosine kinases and are rapidly phosphorylated on their tyrosine residues following ligand stimulation (21). The resulting phosphotyrosine motifs in these substrates then bind proteins containing Src homology 2 (SH2) domains, notably phosphatidylinositol 3-kinase (PI 3-kinase) (5), growth factor receptor binding protein 2 (Grb-2) (36), and the protein tyrosine phosphatase SHP-2/Syp (38), thereby activating specific signaling cascades. In addition, depending on the cell type, IGF-1 and insulin receptor can phosphorylate other substrates, such as Shc (16, 28), and Gab1 (18), which link to one or another of these pathways. Together, these intermediate signals stimulate a variety of different downstream biological effects including mitogenesis, gene expression, glucose transport, and glycogen synthesis.To date, four members of the IRS family (IRS-1, IRS-2, IRS-3, and IRS-4) have been identified (23,24,33,40,41). IRS-1 and IRS-2 are the best-characterized members and are very similar in their overall structure. Both are high-molecularweight proteins consisting of a pleckstrin homology domain at the N t...

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