- The Effect of Chronic High Glucose Concentration on Endoplasmic Reticulum Stress in INS-1 Cells.
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Mi Kyung Kim, Hye Young Seo, Tae Sung Yun, Nam Kyung Kim, Yu Jin Hah, Yun Jung Kim, Ho Chan Cho, Young Yun Jang, Hye Soon Kim, Seong Yeol Ryu, In Kyu Lee, Keun Gyu Park
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Korean Diabetes J. 2008;32(2):112-120. Published online April 1, 2008
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DOI: https://doi.org/10.4093/kdj.2008.32.2.112
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- BACKGROUND
The highly developed endoplasmic reticulum (ER) structure is one of the characteristic features of pancreatic beta-cells. Recent study showed that ER stress causes beta-cell dysfunction. However, little is known about the effects of high glucose concentration on induction of ER stress in pancreatic beta-cells. Therefore, this study was designed to evaluate whether exposure of high glucose concentration in rat insulinoma cell line, INS-1 cell induces ER stress and whether ER stress decreases insulin gene expression. METHODS: The effect of 30 mM glucose on insulin expression and secretion in INS-1 cells was evaluated by Northern blot analysis and glucose-stimulated insulin secretion (GSIS). Cell viability was evaluated by XTT assay. The effect of 30 mM glucose on phosphorylation of eIF2alpha and CHOP expression, which are markers of ER stress were evaluated by Western blot analysis. RT-PCR analysis was performed to determine whether high glucose concentration induces XBP-1 splicing. To investigate whether ER stress decreases insulin gene expression, the effect of tunicamycin on insulin mRNA expression was evaluated by Northern blot analysis. RESULTS: The prolonged exposure of INS-1 cells with the 30 mM glucose concentration decreased insulin mRNA expression in a time dependent manner and impaired GSIS while did not influence on cell viability. 30 mM glucose increased phosphorylation of eIF2alpha, XBP-1 splicing and CHOP expression in INS-1 cells. Tunicamycin-treated INS-1 increased XBP-1 splicing and decreased insulin mRNA expression in a dose dependent manner. CONCLUSION: This study showed that prolonged exposure of INS-1 with high glucose concentration induces ER stress and ER stress decreases insulin gene expression. Further studies about underlying molecular mechanism by which ER stress induces beta-cell dysfunction are needed.
- Transcriptional Regulation of Insulin and CXCL10 Gene by Peroxisome Proliferator Activated Receptor gamma Coactivator-1alpha.
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Won Gu Jang, In Kyu Lee, Eun Jung Kim, Seong Yeol Ryu, Bo Wan Kim, Jung Guk Kim
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Korean Diabetes J. 2007;31(4):326-335. Published online July 1, 2007
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DOI: https://doi.org/10.4093/jkda.2007.31.4.326
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- BACKGROUND
Peroxisome proliferator-activated receptor-gamma coactivator 1alpha (PGC-1alpha), which act as a coactivator of nuclear receptors and several other transcription factors. This study was performed to evaluate the expressional regulation of insulin and inflammatory response genes by PGC-1alpha. METHODS: Transient transfection assays were performed to measure the promoter activity of the insulin and CXCL10 gene. The insulin gene expression levels in INS-1 cells were determined by Northern blot analysis. Differentially expressed genes by PGC-1alpha overexpression in HASMCs were confirmed using DNA microarray, real-time PCR and Northen blot analysis. RESULTS: Insulin promoter activity and mRNA levels were suppressed by GR and Ad-PGC-1alpha. Northern blot analysis of the INS-1 cells revealed that infection with Ad-PGC-1alpha markedly reduced the amount of insulin mRNA and treatment of Dex enhanced this effect in an additive manner. The PGC-1alpha-specific siRNA decreased insulin expression that was induced by Dex in the GR-expressing INS-1 cells was nearly restored by this siRNA treatment. We found that when vascular smooth muscle cells (VSMCs) overexpressed PGC-1alpha, immune or inflammatory response genes were highly expressed. For example, promoter activity and mRNA level of CXCL10 gene were increased by PGC-1alpha. CONCLUSION: PGC-1alpha overexpression inhibited insulin promoter activity in INS-1 cells and enhanced expressions of inflammatory response genes (CXCL10, CXCL11, TNFLSF10) in VSMCs.
- The Effect of Alpha-lipoic Acid on the Cell Cycle Arrest and Apoptosis in Rat Vascular Smooth Muscle Cells.
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Hye Jin Kim, In Kyu Lee, Young Ho Kim, Soon Young Shin, Young Han Lee, Jung Guk Kim, Bo Wan Kim, Hye Soon Kim, Mi Kyoung Kim, Keun Gyu Park, Seong Yeol Ryu
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Korean Diabetes J. 2007;31(3):200-207. Published online May 1, 2007
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DOI: https://doi.org/10.4093/jkda.2007.31.3.200
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- BACKGROUND
The proliferation of vascular smooth muscle cells (VSMCs) is a hallmark of atheroscelrosis and post-angioplasty restenosis. We previously showed that alpha-lipoic acid (ALA) inhibited neointimal hyperplasia and has potential anti-atherosclerosis effect in rat carotid artery balloon injured model. Here, we investigated whether alpha-lipoic acid inhibited proliferation of cells and induced apoptosis in rat vascular smooth muscle cells. METHODS: VSMCs were treated with ALA under each condition, harvested and protein was extracted. Same amount of protein was loaded into SDS-PAGE and western blot analysis was performed with various cell cycle regulation protein. To examine ALA induce apoptosis in VSMCs, FACS and DNA fragmentation assay were performed. Antioxidant effect of ALA was determined by DCF-DA staining. RESULTS: ALA induced VSMCs cell cycle arrest and induced p21, p27 and p53 proteins. Also ALA induced PTEN expression and AMPK phosphorylation. Increased AMPK phosphorylation reduced Erk-2 phosphorylation and finally arrested cell cycle promotion. The apoptotic effect was also shown by ALA treatment. Also we confirmed that ALA reduced ROS generation in VSMCs. CONCLUSION: The present data suggest that ALA has anti-proliferative effect and arrests cell proliferation. Therefore, ALA may provide new strategies for the prevention of neointimal hyperplasia after angioplasty.
- Alpha-Lipoic acid Inhibits TNF-alpha-Induced Fractalkine Expression in Rat aortic Smooth Muscle Cells.
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Keun Gyu Park, Hye Soon Kim, Seong Yeol Ryu, Chang Wook Nam, Byung Kyu Chae, Eui Dal Jung, Jung Guk Kim, Bo Wan Kim, In Kyu Lee
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Korean Diabetes J. 2005;29(5):409-417. Published online September 1, 2005
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- BACKGOUND: The induction of vascular inflammation via the proinflammatory cytokine/ nuclear factor (NF)-kappaB pathway is one of the key mechanisms in the development and progression of atherosclerosis. Accumulating evidence suggests a recently identified chemokine, fractalkine, is involved in arterial inflammation and atherogenesis; however, few studies have examined the effects of pharmacological agents on this process. The purposes of this study were to determine if alpha-lipoic acid (ALA) inhibits the expression of tumor necrosis factor (TNF)-alpha-stimulated fractalkine in vascular smooth muscle cells(VSMCs). METHODS: Rat VSMCs were isolated and cultured. Northern and Western blot analyses were performed to evaluate the effects of ALA on the expression of TNF-alpha-stimulated fractalkine in VSMCs. A gel shift assay was performed to examine the mechanism by which ALA inhibits the expression of fractalkine. RESULTS: TNF-alpha markedly induced the expression of fractalkine in primary cultured VSMCs. ALA inhibited the expression of TNF-alpha-stimulated fractalkine in cultured VSMCs. The result of the gel shift assay suggested the inhibitory effects of AS-6 on the expression of TNF-alpha-stimulated fractalkine were mediated via the NF-kappaB pathway. CONCLUSION: This study has shown that ALA has anti-inflammatory effects on VSMCs, which are mediated by the inhibitoin, at least in part, of the NF-kappaB dependent inflammatory signal-stimulated expression of fractalkine. Our data suggest the possibility that antioxidants, such as ALA, inhibit the NF-kappaB pathway, which may be used to prevent the development and progression of atherosclerosis.
- Ascochlorin Derivative, AS-6, Inhibits TNF-alpha-Induced fractalkine, MCP-1 and VCAM-1 Expression in Rat Aortic Smooth Muscle Cells.
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Young Yun Jang, Sang Yoon Kim, Nam Keong Kim, Mi Kyung Kim, Hee Kyoung Kim, Hye Soon Kim, Chang Wook Nam, Seong Yeol Ryu, Sung Il Nam, Keun Gyu Park
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Korean Diabetes J. 2005;29(5):401-408. Published online September 1, 2005
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- BACKGOUND: Inflammation is one of the key mechanisms in the development and progression of atherosclerosis. Accumulating evidence suggests that peroxisome proliferators- activated receptorgamma(PPARgamma) plays an important role in the prevention of arterial inflammation and the formation of atherogenesis. This study was designed to evaluate whether the new synthetic PPARgamma, ascochlorin-6(AS-6) has anti-inflammatory and anti-atherogenic effects in primary cultured rat vascular smooth muscle cells(VSMCs). METHODS: Rat VSMCs were isolated and cultured. Northern and Western blot analyses were performed to evaluate the effects of AS-6 on the expressions of tumor necrosis factor (TNF)-alpha-stimulated fractalkine, monocyte chemoattractant protein(MCP)-1 and vascular cell adhesion molecule (VCAM)-1 in VSMCs. A gel shift assay was performed to examine the mechanism by which AS-6 inhibits the expressions of fractalkine, MCP-1 and VCAM-1. RESULTS: TNF-alpha markedly induced the expressions of fractalkine, MCP-1 and VCAM-1 in primary cultured VSMCs. AS-6 inhibited the expressions of TNF-alpha-stimulated fractalkine, MCP-1 and VCAM-1 in primary cultured VSMCs. The result of the gel shift assay suggested the inhibitory effects of AS-6 on the expressions of TNF-alpha-stimulated fractalkine, MCP-1 and VCAM-1 were mediated through a nuclear factor kappaB associated pathway. CONCLUSION: The present study shows that AS-6 has anti-inflammatory effects on VSMCs, suggesting the possibility for the use of AS-6 for prevention of the development and progression of atherosclerosis.
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