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Inhibition of Calcium Influx Reduces Dysfunction and Apoptosis in Lipotoxic Pancreatic β-Cells via Regulation of Endoplasmic Reticulum Stress.

Zhou Y, Sun P, Wang T, Chen K, Zhu W, Wang H - PLoS ONE (2015)

Bottom Line: And whether the compounds could reduce palmitic acid-induced β-cell failure and the underlying mechanism were also investigated.It was found that both nifedipine and diazoxide protected MIN6 pancreatic β-cells and primary cultured murine islets from palmitic acid-induced apoptosis.Our results verified that nifedipine and diazoxide could reduce palmitic acid-induced endoplasmic reticulum stress to generate protective effects on pancreatic β-cells.

View Article: PubMed Central - PubMed

Affiliation: Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 555 Zuchongzhi Road, Shanghai, 201203, China.

ABSTRACT
Lipotoxicity plays an important role in pancreatic β-cell failure during the development of type 2 diabetes. Prolonged exposure of β-cells to elevated free fatty acids level could cause deterioration of β-cell function and induce cell apoptosis. Therefore, inhibition of fatty acids-induced β-cell dysfunction and apoptosis might provide benefit for the therapy of type 2 diabetes. The present study examined whether regulation of fatty acids-triggered calcium influx could protect pancreatic β-cells from lipotoxicity. Two small molecule compounds, L-type calcium channel blocker nifedipine and potassium channel activator diazoxide were used to inhibit palmitic acid-induced calcium influx. And whether the compounds could reduce palmitic acid-induced β-cell failure and the underlying mechanism were also investigated. It was found that both nifedipine and diazoxide protected MIN6 pancreatic β-cells and primary cultured murine islets from palmitic acid-induced apoptosis. Meanwhile, the impaired insulin secretion was also recovered to varying degrees by these two compounds. Our results verified that nifedipine and diazoxide could reduce palmitic acid-induced endoplasmic reticulum stress to generate protective effects on pancreatic β-cells. More importantly, it suggested that regulation of calcium influx by small molecule compounds might provide benefits for the prevention and therapy of type 2 diabetes.

No MeSH data available.


Related in: MedlinePlus

Nifedipine and diazoxide attenuate PA-activated ER-stress.(A) After MIN6 cells were co-treated with nifedipine and 0.5 mM PA for 48 h, the phosphorylation of eIF2α and the expression of CHOP were detected by western blot. β-actin was used for normalization. (B) The phosphorylation rate of eIF2α was calculated as optical density of phosphorylated-eIF2α (p-eIF2α) divide by total eIF2α (t-eIF2α). * p < 0.05 denote significant difference versus the PA-treated alone group, n = 3. (C) The expression of CHOP was calculated by optical density. * p<0.05 denote significant difference versus the PA-treated alone group, n = 3. (D) After MIN6 cells were co-treated with diazoxide and 0.5 mM PA for 48 h, the phosphorylation of eIF2α, the expression of CHOP were detected by western blot. (E) The phosphorylation rate of eIF2α was calculated by optical density. * p < 0.05 denote significant difference versus the PA-treated alone group, n = 3. (F) The expression of CHOP was calculated by optical density. * p < 0.05 denote significant difference versus the PA-treated alone group, n = 3. (G) After treatment, the cells were fixed and stained with CHOP and insulin antibodies. Red fluorescence indicated CHOP expression while green marked insulin. The nuclei were stained with DAPI dye. Scale bar = 50 μm and referred to all panels.
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pone.0132411.g003: Nifedipine and diazoxide attenuate PA-activated ER-stress.(A) After MIN6 cells were co-treated with nifedipine and 0.5 mM PA for 48 h, the phosphorylation of eIF2α and the expression of CHOP were detected by western blot. β-actin was used for normalization. (B) The phosphorylation rate of eIF2α was calculated as optical density of phosphorylated-eIF2α (p-eIF2α) divide by total eIF2α (t-eIF2α). * p < 0.05 denote significant difference versus the PA-treated alone group, n = 3. (C) The expression of CHOP was calculated by optical density. * p<0.05 denote significant difference versus the PA-treated alone group, n = 3. (D) After MIN6 cells were co-treated with diazoxide and 0.5 mM PA for 48 h, the phosphorylation of eIF2α, the expression of CHOP were detected by western blot. (E) The phosphorylation rate of eIF2α was calculated by optical density. * p < 0.05 denote significant difference versus the PA-treated alone group, n = 3. (F) The expression of CHOP was calculated by optical density. * p < 0.05 denote significant difference versus the PA-treated alone group, n = 3. (G) After treatment, the cells were fixed and stained with CHOP and insulin antibodies. Red fluorescence indicated CHOP expression while green marked insulin. The nuclei were stained with DAPI dye. Scale bar = 50 μm and referred to all panels.

Mentions: We than investigated the effects of nifedipine and diazoxide on the expressions of some ER-stress markers in PA-induced β-cell apoptosis. Fig 3 showed that after 48 h PA incubation, the expression of CHOP and phosphorylation rate of eIF2α were significantly increased in MIN6 cells. Nifedipine and diazoxide dose-dependently suppressed PA-induced phosphorylation of eIF2α. Meanwhile, both western blot (Fig 3A, 3C, 3D and 3F) and immunofluorescence (Fig 3G) experiments showed that the expression of CHOP in nifedipine or diazoxide co-treated with PA cells was obviously decreased in comparison with PA-treated alone group.


Inhibition of Calcium Influx Reduces Dysfunction and Apoptosis in Lipotoxic Pancreatic β-Cells via Regulation of Endoplasmic Reticulum Stress.

Zhou Y, Sun P, Wang T, Chen K, Zhu W, Wang H - PLoS ONE (2015)

Nifedipine and diazoxide attenuate PA-activated ER-stress.(A) After MIN6 cells were co-treated with nifedipine and 0.5 mM PA for 48 h, the phosphorylation of eIF2α and the expression of CHOP were detected by western blot. β-actin was used for normalization. (B) The phosphorylation rate of eIF2α was calculated as optical density of phosphorylated-eIF2α (p-eIF2α) divide by total eIF2α (t-eIF2α). * p < 0.05 denote significant difference versus the PA-treated alone group, n = 3. (C) The expression of CHOP was calculated by optical density. * p<0.05 denote significant difference versus the PA-treated alone group, n = 3. (D) After MIN6 cells were co-treated with diazoxide and 0.5 mM PA for 48 h, the phosphorylation of eIF2α, the expression of CHOP were detected by western blot. (E) The phosphorylation rate of eIF2α was calculated by optical density. * p < 0.05 denote significant difference versus the PA-treated alone group, n = 3. (F) The expression of CHOP was calculated by optical density. * p < 0.05 denote significant difference versus the PA-treated alone group, n = 3. (G) After treatment, the cells were fixed and stained with CHOP and insulin antibodies. Red fluorescence indicated CHOP expression while green marked insulin. The nuclei were stained with DAPI dye. Scale bar = 50 μm and referred to all panels.
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Related In: Results  -  Collection

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pone.0132411.g003: Nifedipine and diazoxide attenuate PA-activated ER-stress.(A) After MIN6 cells were co-treated with nifedipine and 0.5 mM PA for 48 h, the phosphorylation of eIF2α and the expression of CHOP were detected by western blot. β-actin was used for normalization. (B) The phosphorylation rate of eIF2α was calculated as optical density of phosphorylated-eIF2α (p-eIF2α) divide by total eIF2α (t-eIF2α). * p < 0.05 denote significant difference versus the PA-treated alone group, n = 3. (C) The expression of CHOP was calculated by optical density. * p<0.05 denote significant difference versus the PA-treated alone group, n = 3. (D) After MIN6 cells were co-treated with diazoxide and 0.5 mM PA for 48 h, the phosphorylation of eIF2α, the expression of CHOP were detected by western blot. (E) The phosphorylation rate of eIF2α was calculated by optical density. * p < 0.05 denote significant difference versus the PA-treated alone group, n = 3. (F) The expression of CHOP was calculated by optical density. * p < 0.05 denote significant difference versus the PA-treated alone group, n = 3. (G) After treatment, the cells were fixed and stained with CHOP and insulin antibodies. Red fluorescence indicated CHOP expression while green marked insulin. The nuclei were stained with DAPI dye. Scale bar = 50 μm and referred to all panels.
Mentions: We than investigated the effects of nifedipine and diazoxide on the expressions of some ER-stress markers in PA-induced β-cell apoptosis. Fig 3 showed that after 48 h PA incubation, the expression of CHOP and phosphorylation rate of eIF2α were significantly increased in MIN6 cells. Nifedipine and diazoxide dose-dependently suppressed PA-induced phosphorylation of eIF2α. Meanwhile, both western blot (Fig 3A, 3C, 3D and 3F) and immunofluorescence (Fig 3G) experiments showed that the expression of CHOP in nifedipine or diazoxide co-treated with PA cells was obviously decreased in comparison with PA-treated alone group.

Bottom Line: And whether the compounds could reduce palmitic acid-induced β-cell failure and the underlying mechanism were also investigated.It was found that both nifedipine and diazoxide protected MIN6 pancreatic β-cells and primary cultured murine islets from palmitic acid-induced apoptosis.Our results verified that nifedipine and diazoxide could reduce palmitic acid-induced endoplasmic reticulum stress to generate protective effects on pancreatic β-cells.

View Article: PubMed Central - PubMed

Affiliation: Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 555 Zuchongzhi Road, Shanghai, 201203, China.

ABSTRACT
Lipotoxicity plays an important role in pancreatic β-cell failure during the development of type 2 diabetes. Prolonged exposure of β-cells to elevated free fatty acids level could cause deterioration of β-cell function and induce cell apoptosis. Therefore, inhibition of fatty acids-induced β-cell dysfunction and apoptosis might provide benefit for the therapy of type 2 diabetes. The present study examined whether regulation of fatty acids-triggered calcium influx could protect pancreatic β-cells from lipotoxicity. Two small molecule compounds, L-type calcium channel blocker nifedipine and potassium channel activator diazoxide were used to inhibit palmitic acid-induced calcium influx. And whether the compounds could reduce palmitic acid-induced β-cell failure and the underlying mechanism were also investigated. It was found that both nifedipine and diazoxide protected MIN6 pancreatic β-cells and primary cultured murine islets from palmitic acid-induced apoptosis. Meanwhile, the impaired insulin secretion was also recovered to varying degrees by these two compounds. Our results verified that nifedipine and diazoxide could reduce palmitic acid-induced endoplasmic reticulum stress to generate protective effects on pancreatic β-cells. More importantly, it suggested that regulation of calcium influx by small molecule compounds might provide benefits for the prevention and therapy of type 2 diabetes.

No MeSH data available.


Related in: MedlinePlus