Effect of TNF-α on the expression of ABCA1 in pancreatic β-cells

in Journal of Molecular Endocrinology
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ATP-binding cassette transporter A1 (ABCA1), a 254-kD membrane protein, is a key regulator of lipid efflux from cells to apolipoproteins. ABCA1 in pancreatic β-cells influences insulin secretion and cholesterol homeostasis. Tumor necrosis factor (TNF)-α is a pleiotropic cytokine that elicits a wide spectrum of physiological events, including cell proliferation, differentiation and apoptosis and is also known to decrease glucose-dependent insulin secretion in pancreatic islets. In the present study, we examined the role of TNF-α on ABCA1 expression in rat pancreatic islets and INS-1 cells. ABCA1 protein levels decreased in response to rising concentrations of TNF-α in pancreatic islets. Real-time polymerase chain reaction analysis showed a significant decrease in ABCA1 mRNA expression. In parallel with its effect on endogenous ABCA1 mRNA levels, TNF-α suppressed the activity of a reporter construct containing the ABCA1 promoter. This effect was abrogated by BIRB796, but not by SB203580 or LY-294002. The constitutively active form of p38 mitogen-activated protein kinase (MAPK) γ suppressed ABCA1 promoter activity but not p38-MAPK (α, β), while a dominant-negative mutant of p38-MAPK γ blocked the effect of TNF-α on ABCA1 promoter activity. BIRB796 inhibited the increased cholesterol ester content induced by TNF-α. However, BIRB796 had no effect on either the decreased insulin content or the ABCA1 suppression caused by TNF-α in INS-1 cells. We checked the influence of TNF-α of insulin secretion and glucose-stimulated insulin secretion in rat pancreatic islet and INS-1 cell. TNF-α suppressed the insulin secretion and glucose-stimulated insulin secretion in both rat pancreatic islet and INS-1 cell. In summary, TNF-α suppressed the expression of endogenous ABCA1 and suppress the insulin secretion in pancreatic islets and INS-1 cells. These findings raise the possibility that TNF-α may affect insulin secretion by controlling ABCA1 expression.

 

      Society for Endocrinology

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    Effect of tumor necrosis factor (TNF)-α on insulin secretion and glucose stimulated insulin secretion in INS-1 cells and pancreatic islet. (A) INS-1 cells were incubated with 10 ng/mL TNF-α for 24 h. Fold of insulin secretion has significantly decreased in treated with TNF-α (P < 0.05). (B) To compare with 3.3 mM glucose group, 16.7 mM glucose group has significantly increased of insulin secretion. But treated with TNF-α, the increased level of insulin secretion in 16.7 mM glucose has canceled. (C) Rat pancreatic islet was incubated with 10 ng/mL TNF-α for 24 h. Fold of insulin secretion has significantly decreased in treated with TNF-α (P < 0.05). (D) To compare with 3.3 mM glucose group, 16.7 mM glucose group has significantly increased of insulin secretion. But treated with TNF-α, the increased level of insulin secretion in 16.7 mM glucose has canceled.

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    Effect of tumor necrosis factor (TNF)-α on the expression of ATP-binding cassette transporter A1 (ABCA1) in rat pancreatic islets. The effect of TNF-α on the expression of ABCA1 protein (A) and mRNA (B) in rat pancreatic islets. The ABCA1/GAPDH ratios are shown as percentages of control. Each data point shows the mean ± s.e.m. of three separate experiments for each treatment group. *Significantly different compared to the control (P < 0.05).

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    Effect of tumor necrosis factor (TNF)-α on the expression of ATP-binding cassette transporter A1 (ABCA1) in INS-1 cells. (A) Dose-dependent suppression of ABCA1 protein by TNF-α. INS-1 cells were seeded in six-well plates and exposed to the indicated amounts of TNF-α for 24 h. ABCA1 in the total cell lysate was detected by Western blot analysis. GAPDH served as the control and is shown at the bottom of each lane. The ABCA1/GAPDH ratios are shown as percentages of control in the lower panel. Each data point shows the mean ± s.e. of three separate experiments. *Significantly different from control (P < 0.05). (B) Time-dependent suppression of ABCA1 protein by TNF-α. INS-1 cells were incubated for the indicated periods of time in the presence of 10 ng/mL TNF-α. GAPDH served as the control and is shown at the bottom of each lane. The ABCA1/GAPDH ratios are shown as percentages of control in the lower panel. Each data point shows the mean ± s.e. of three separate experiments. *Significantly different from control (P < 0.05). (C) Abundance of ABCA1 mRNA in INS-1 cells treated with 10 ng/mL TNF-α. The ABCA1/GAPDH ratios are shown as percentages of control. Each data point shows the mean ± s.e.m. of three separate experiments for each treatment group. *Significantly different from control (P < 0.05).

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    Effect of tumor necrosis factor (TNF)-α on ATP-binding cassette transporter A1 (ABCA1) promoter activity in INS-1 cells. (A) TNF-α decreases ABCA1 gene transcription. INS-1 cells were transfected with 1 μg of pABCA1-LUC and treated with TNF-α for 24 h prior to harvesting. All assays were corrected for β-galactosidase activity. The total amount of protein in every reaction was identical. The results are expressed as relative luciferase activity compared with control cells arbitrarily set at 100. Each data point shows the mean ± s.e. of three separate transfections that were performed. *Significantly different from control (P < 0.05). (B) Effects of the phosphatidylinositol 3-kinase inhibitor, LY-294002 (LY), and the p38-mitogen-activated protein kinase (MAPK) inhibitors, SB-203580 (SB) and BIRB796 (BIRB), on ABCA1 transcriptional activity in INS-1 cells treated with TNF-α. The p38-MAPK inhibitors block the actions of TNF-α. Vehicle was 0.1% dimethyl sulfoxide. Each data point shows the mean ± s.e. of three independent transfections that were performed . *Significantly different from only TNF-α (P < 0.05).

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    Role of the p38-mitogen-activated protein kinase (MAPK) signal transduction pathway on ABCA1 promoter activity inhibited by tumor necrosis factor (TNF)-α. (A) Effects of p38-MAPK isoforms on ABCA1 promoter activity. INS-1 cells were transfected with pABCA1-LUC and either an empty vector (control) or expression vectors of constitutively active forms of p38-MAPK (α, -β, -γ) for 24 h prior to cell harvesting. All assays were corrected for β-galactosidase activity. The total amount of protein in each reaction was identical. The results are expressed as relative luciferase activity compared with control cells arbitrarily set at 100. Each data point shows the mean ± s.e. of four separate transfections that were performed on different days. *Significantly different from control (*P < 0.05). N.S., no significant difference. (B) Dominant-negative p38-MAPKγ (p38γ-DN) blocks the TNF-α-induced inhibition of ABCA1 transcription. INS-1 cells were transfected with pABCA1-LUC and either the empty vector or p38γ-DN, then treated with TNF-α for 24 h prior to cell harvesting. The results are expressed as relative luciferase activity compared with control cells arbitrarily set at 100. Each data point shows the mean ± s.e. of four separate transfections that were performed on different days. *Significantly different from control (P < 0.05). N.S., no significant difference.

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    Effect of tumor necrosis factor (TNF)-α on cholesterol accumulation in INS-1 cells. INS-1 cells were pretreated with dimethyl sulfoxide or BIRB796 (BIRB) for 30 min, then incubated with 10 ng/mL TNF-α for 24 h. Cells were then stained with Oil Red O (A) or lysed to measure intracellular cholesterol content (B). Percentages of cholesterol content per cell relative to control are shown as means ± s.e.m. of three separate experiments. *Significantly different compared to control (P < 0.05). #Significantly different compared to dimethyl sulfoxide plus TNF-α (P < 0.05). A full colour version of this figure is available at https://doi.org/10.1530/JME-18-0167.

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    Effect of tumor necrosis factor (TNF)-α on ABCA1 expression and insulin synthesis in INS-1 cells. (A) INS-1 cells were pretreated with dimethyl sulfoxide or BIRB796 (BIRB) for 30 min and then incubated with 10 ng/mL TNF-α for 24 h. Protein extracts were subjected to Western blot analysis to determine ABCA1 expression. The ABCA1/GAPDH ratios are shown as mean ± s.e.m. percentages of control from three separate experiments for each treatment group. (B) Abundance of insulin in INS-1 cells treated with TNF-α. INS-1 cells were pretreated with dimethyl sulfoxide or BIRB for 30 min, and then incubated with 10 ng/mL TNF-α for 24 h. Protein extracts were subjected to Western blot analysis to determine insulin expression. The insulin/GAPDH ratios are shown as mean ± s.e.m. percentages of control from three separate experiments for each treatment group. *Significantly different compared to DMSO (P < 0.05). #Significantly different compared to DMSO + TNF-α (P < 0.05).

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