No abstract

The highly developed endoplasmic reticulum (ER) structure of pancreatic beta-cells is a key factor in beta-cell function. Here we examined whether ER stress-induced activation of activating transcription factor (ATF)-6 impairs insulin gene expression via up-regulation of the orphan nuclear receptor small heterodimer partner (SHP; NR0B2), which has been shown to play a role in beta-cell dysfunction. We examined whether ER stress decreases insulin gene expression, and this process is mediated by ATF6. A small interfering RNA that targeted SHP was used to determine whether the effect of ATF6 on insulin gene expression is mediated by SHP. We also measured the expression level of ATF6 in pancreatic islets in Otsuka Long Evans Tokushima Fatty rats, a rodent model of type 2 diabetes. High glucose concentration (30 mmol/liter glucose) increased ER stress in INS-1 cells. ER stress induced by tunicamycin, thapsigargin, or dithiotreitol decreased insulin gene transcription. ATF6 inhibited insulin promoter activity, whereas X-box binding protein-1 and ATF4 did not. Adenovirus-mediated overexpression of active form of ATF6 in INS-1 cells impaired insulin gene expression and secretion. ATF6 also down-regulated pancreatic duodenal homeobox factor-1 and RIPE3b1/MafA gene expression and repressed the cooperative action of pancreatic duodenal homeobox factor-1, RIPE3b1/MafA, and beta-cell E box transactivator 2 in stimulating insulin transcription. The ATF6-induced suppression of insulin gene expression was associated with up-regulation of SHP gene expression. Finally, we found that expression of ATF6 was increased in the pancreatic islets of diabetic Otsuka Long Evans Tokushima Fatty rats, compared with their lean, nondiabetic counterparts, Long-Evans Tokushima Otsuka rats. Collectively, this study shows that ER stress-induced activation of ATF6 plays an important role in the development of beta-cell dysfunction.

Increased oxidative stress in vascular cells is implicated in the pathogenesis of atherosclerosis. Reactive oxygen species (ROS) induce vascular inflammation via the proinflammatory cytokine/NF-kappaB pathway. Several lines of evidence suggest that peroxisome proliferator-activated receptor-gamma coactivator 1-alpha (PGC-1alpha) is an important regulator of intracellular ROS levels. However, no studies have examined the effects of PGC-1alpha on this process. We investigated the effects of PGC-1alpha on inflammatory molecule expression and activity of the redox-sensitive transcription factor, NF-kappaB, in vascular cells. PGC-1alpha expressed in human aortic smooth (HASMCs) and endothelial cells (HAECs) is upregulated by AMP-activated protein kinase activators, including metformin, rosiglitazone and alpha-lipoic acid. Tumor necrosis factor-alpha (TNF-alpha), a major proinflammatory factor in the development of vascular inflammation, stimulates intracellular ROS production through an increase in both mitochondrial ROS and NAD(P)H oxidase activity. Adenovirus-mediated overexpression of the PGC-1alpha gene in HASMCs and HAECs leads to a significant reduction in intracellular and mitochondrial ROS production as well as NAD(P)H oxidase activity. Consequently, NF-kappaB activity and MCP-1 and VCAM-1 induced by TNF-alpha are suppressed. Our data support the possibility that agents stimulating PGC-1alpha expression in the vasculature aid in preventing the development of atherosclerosis.

Abbreviations: ALA, α-lipoic acid; AP-1, activator protein-1; MMP-9, matrix metalloproteinase-9; VSMC, vascular smooth muscle cell AbstractThe migration of vascular smooth muscle cells (VSMCs) into the intima, an important step in injury-induced neointimal hyperplasia, requires the activation of nuclear factor-κB (NF-κB) and the consequent up-regulation of matrix metalloproteinase-9 (MMP-9). This study was undertaken to test for a possible effect of α-lipoic acid (ALA), a potent inhibitor of NF-κB, on MMP-9 expression. ALA inhibited high-glucose-and TNF-α-stimulated VSMC migrations in vitro. It also inhibited high-glucoseand TNF-α-induced increases in MMP-9 expression. The activity of MMP-9-promoter constructs with mutations in the NF-κB binding site was not inhibited by ALA, indicating an involvement of the NF-κB signaling pathway in the ALA-specific inhibition of MMP-9. These data suggest the possibility that ALA may be useful for the prevention of neointimal hyperplasia after angioplasty, by inhibiting the NF-κB/ MMP-9 pathway, especially with hyperglycemia.

Prolonged elevations of glucose concentration have deleterious effects on ␤-cell function. One of the hallmarks of such glucotoxicity is a reduction in insulin gene expression, resulting from decreased insulin promoter activity. Small heterodimer partner (SHP; NR0B2) is an atypical orphan nuclear receptor that inhibits nuclear receptor signaling in diverse metabolic pathways. In this study, we found that sustained culture of INS-1 cells at high glucose concentrations leads to an increase in SHP mRNA expression, followed by a decrease in insulin gene expression. Inhibition of endogenous SHP gene expression by small interfering RNA partially restored high-glucose-induced suppression of the insulin gene. Adenovirus-mediated overexpression of SHP in INS-1 cells impaired glucose-stimulated insulin secretion as well as insulin gene expression. SHP downregulates insulin gene expression via two mechanisms: by downregulating PDX-1 and MafA gene expression and by inhibiting p300-mediated pancreatic duodenal homeobox factor 1-and BETA2-dependent transcriptional activity from the insulin promoter. Finally, the pancreatic islets of diabetic OLETF rats express SHP mRNA at higher levels than the islets from LETO rats. These results collectively suggest that SHP plays an important role in the development of ␤-cell dysfunction induced by glucotoxicity.

No abstract

Abstract-Neointimal formation, the leading cause of restenosis, is caused by proliferation of vascular smooth muscle cells (VSMCs). Patients with diabetes mellitus have higher restenosis rates after coronary angioplasty than nondiabetic patients. Cilostazol, a selective type 3 phosphodiesterase inhibitor, is currently used to treat patients with diabetic vascular complications. Cilostazol is a potent antiplatelet agent that inhibits VSMC proliferation. In the present study, we examine whether the antiproliferative effect of cilostazol on VSMCs is mediated by inhibition of an important cell cycle transcription factor, E2F. Cilostazol inhibited the proliferation of human VSMCs in response to high glucose in vitro and virtually abolished neointimal formation in rats subjected to carotid artery injury in vivo. Moreover, the compound suppressed high-glucose-induced E2F-DNA binding activity, and the expression of E2F1, E2F2, cyclin A, and PCNA proteins. These data suggest that the beneficial effects of cilostazol on high-glucose-stimulated proliferation of VSMCs are mediated by the downregulation of E2F activity and expression of its downstream target genes, including E2F1, E2F2, cyclin A, and PCNA. The transcription factor, E2F, has been implicated in the periodic regulation of cellular genes required for transition through G1 and entry into the S phase, including dihydrofolate reductase, c-myc, DNA polymerase, cdc2, and proliferating cell nuclear antigen (PCNA). 5-7 E2F activity is regulated by interactions with RB family members. As cells progress toward S phase, RB family proteins are phosphorylated by G1 cyclin-complexes, resulting in the release of transcriptionally active E2F, which then leads to the activation of genes required for cell cycle progression. 8 -10 We recently showed that high glucose activates the DNA-binding activity of E2F, and decoy oligodeoxynucleotides against E2F inhibit the proliferation of VSMCs. 11 These data suggest that downregulation of E2F could constitute a therapeutic target to prevent restenosis after angioplasty in patients with diabetes.Cilostazol increases intracellular cAMP concentrations by selectively blocking phosphodiesterase type III. The clinical implications and pharmacokinetics with respect to the effects and safety of this drug have been well-established, especially in peripheral vascular disease. 12 Cilostazol is a potent antiplatelet agent currently used in clinical practice to treat patients with diabetic vascular complications. [13][14][15] Several lines of evidence indicate that cilostazol additionally inhibits the proliferation of VSMCs, reduces neointimal formation in balloon-injured rat carotid arteries, 16 -18 and inhibits restenosis after percutaneous transluminal coronary angioplasty. 19,20 One mechanism by which cilostazol may inhibit VSMC proliferation is via an increase in intracellular cAMP, because cAMP inhibits the proliferation of VSMCs by induction of p53-mediated and p21-mediated apoptosis. 21 However, Nadri et al demonstrated that increased cAMP...

No abstract