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Role of lipid peroxidation and PPAR-δ in amplifying glucose-stimulated insulin secretion.

Cohen G, Riahi Y, Shamni O, Guichardant M, Chatgilialoglu C, Ferreri C, Kaiser N, Sasson S - Diabetes (2011)

Bottom Line: The latter mimicked the GSIS-amplifying effect of high glucose preexposure and of the PPAR-δ agonist GW501516 in INS-1E cells and isolated rat islets.Cytotoxic effects of 4-HNE were observed only above the physiologically effective concentration range.This molecule is an endogenous ligand for PPAR-δ, which amplifies insulin secretion in β-cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, School of Pharmacy, Faculty of Medicine, Institute for Drug Research, Hebrew University, Jerusalem, Israel.

ABSTRACT

Objective: Previous studies show that polyunsaturated fatty acids (PUFAs) increase the insulin secretory capacity of pancreatic β-cells. We aimed at identifying PUFA-derived mediators and their cellular targets that are involved in the amplification of insulin release from β-cells preexposed to high glucose levels.

Research design and methods: The content of fatty acids in phospholipids of INS-1E β-cells was determined by lipidomics analysis. High-performance liquid chromatography was used to identify peroxidation products in β-cell cultures. Static and dynamic glucose-stimulated insulin secretion (GSIS) assays were performed on isolated rat islets and/or INS-1E cells. The function of peroxisome proliferator-activated receptor-δ (PPAR-δ) in regulating insulin secretion was investigated using pharmacological agents and gene expression manipulations.

Results: High glucose activated cPLA(2) and, subsequently, the hydrolysis of arachidonic and linoleic acid (AA and LA, respectively) from phospholipids in INS-1E cells. Glucose also increased the level of reactive oxygen species, which promoted the peroxidation of these PUFAs to generate 4-hydroxy-2E-nonenal (4-HNE). The latter mimicked the GSIS-amplifying effect of high glucose preexposure and of the PPAR-δ agonist GW501516 in INS-1E cells and isolated rat islets. These effects were blocked with GSK0660, a selective PPAR-δ antagonist, and the antioxidant N-acetylcysteine or by silencing PPAR-δ expression. High glucose, 4-HNE, and GW501516 also induced luciferase expression in a PPAR-δ-mediated transactivation assay. Cytotoxic effects of 4-HNE were observed only above the physiologically effective concentration range.

Conclusions: Elevated glucose levels augment the release of AA and LA from phospholipids and their peroxidation to 4-HNE in β-cells. This molecule is an endogenous ligand for PPAR-δ, which amplifies insulin secretion in β-cells.

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NAC reduces ROS production, 4-HNE generation, and insulin secretion in β-cells. A: INS-1E cells were incubated for 16 h with the indicated glucose levels without or with 1 mmol/L NAC. ROS production was determined with the carboxy-DCF-fluorescence assay. Glc, glucose. *P < 0.05 for the difference from the 5 mmol/L glucose controls. #P < 0.05 for the difference from the cells incubated in the same glucose concentration without NAC. B: HPLC determination of 4-HNE in INS-1E cultures maintained at 25 mmol/L glucose, without or with 1 mmol/L NAC for 16 h, was performed as described in the legend to Fig. 2A. *P < 0.05 for the difference from the vehicle-treated controls. C: INS-1E cells were prepared for the PPRE-luciferase transactivation assay, as described in the legend to Fig. 4B, and incubated with the indicated glucose levels without or with 1 mmol/L NAC; luciferase activity was determined after 24 h of incubation. *P < 0.05 for the difference in luciferase activity in comparison with the control incubation at 5 mmol/L glucose in the absence of NAC. #P < 0.05 for the difference from cells incubated in the same glucose concentration without NAC. D: Rat islets and INS-1E cells were incubated with RPMI-1640 medium containing the indicated glucose concentrations in the absence or presence of 1 mmol/L NAC for 48 or 24 h, respectively. The islets and INS-1E cells were then washed and GSIS evaluated, as described in the Fig. 3A legend (white bars, 3.3 mmol/L glucose; dark bars, 16.7 mmol/L glucose). Insulin secretion is presented as percent of insulin content. *P < 0.05 for the difference in stimulated secretion compared with cells maintained at 5 mmol/L glucose. #P < 0.05 for differences from the respective NAC-free incubation. Results are mean ± SEM, n = 3.
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Figure 6: NAC reduces ROS production, 4-HNE generation, and insulin secretion in β-cells. A: INS-1E cells were incubated for 16 h with the indicated glucose levels without or with 1 mmol/L NAC. ROS production was determined with the carboxy-DCF-fluorescence assay. Glc, glucose. *P < 0.05 for the difference from the 5 mmol/L glucose controls. #P < 0.05 for the difference from the cells incubated in the same glucose concentration without NAC. B: HPLC determination of 4-HNE in INS-1E cultures maintained at 25 mmol/L glucose, without or with 1 mmol/L NAC for 16 h, was performed as described in the legend to Fig. 2A. *P < 0.05 for the difference from the vehicle-treated controls. C: INS-1E cells were prepared for the PPRE-luciferase transactivation assay, as described in the legend to Fig. 4B, and incubated with the indicated glucose levels without or with 1 mmol/L NAC; luciferase activity was determined after 24 h of incubation. *P < 0.05 for the difference in luciferase activity in comparison with the control incubation at 5 mmol/L glucose in the absence of NAC. #P < 0.05 for the difference from cells incubated in the same glucose concentration without NAC. D: Rat islets and INS-1E cells were incubated with RPMI-1640 medium containing the indicated glucose concentrations in the absence or presence of 1 mmol/L NAC for 48 or 24 h, respectively. The islets and INS-1E cells were then washed and GSIS evaluated, as described in the Fig. 3A legend (white bars, 3.3 mmol/L glucose; dark bars, 16.7 mmol/L glucose). Insulin secretion is presented as percent of insulin content. *P < 0.05 for the difference in stimulated secretion compared with cells maintained at 5 mmol/L glucose. #P < 0.05 for differences from the respective NAC-free incubation. Results are mean ± SEM, n = 3.

Mentions: The role of endogenous 4-HNE in regulating insulin secretion was further substantiated by measuring insulin secretion after the blocking of 4-HNE production in β-cells by the antioxidant N-acetylcysteine (NAC), as described previously (18). Figure 6A and B shows that glucose-induced generation of ROS and 4-HNE in INS-1E cells was nearly abolished in the presence of 1 mmol/L NAC. This treatment also prevented high glucose-induced expression of luciferase in the PPRE-dependent transactivation assay in cells overexpressing PPAR-δ. Finally, glucose-amplified insulin secretion was markedly reduced in NAC-treated rat islets and in INS-1E cells (Fig. 6D).


Role of lipid peroxidation and PPAR-δ in amplifying glucose-stimulated insulin secretion.

Cohen G, Riahi Y, Shamni O, Guichardant M, Chatgilialoglu C, Ferreri C, Kaiser N, Sasson S - Diabetes (2011)

NAC reduces ROS production, 4-HNE generation, and insulin secretion in β-cells. A: INS-1E cells were incubated for 16 h with the indicated glucose levels without or with 1 mmol/L NAC. ROS production was determined with the carboxy-DCF-fluorescence assay. Glc, glucose. *P < 0.05 for the difference from the 5 mmol/L glucose controls. #P < 0.05 for the difference from the cells incubated in the same glucose concentration without NAC. B: HPLC determination of 4-HNE in INS-1E cultures maintained at 25 mmol/L glucose, without or with 1 mmol/L NAC for 16 h, was performed as described in the legend to Fig. 2A. *P < 0.05 for the difference from the vehicle-treated controls. C: INS-1E cells were prepared for the PPRE-luciferase transactivation assay, as described in the legend to Fig. 4B, and incubated with the indicated glucose levels without or with 1 mmol/L NAC; luciferase activity was determined after 24 h of incubation. *P < 0.05 for the difference in luciferase activity in comparison with the control incubation at 5 mmol/L glucose in the absence of NAC. #P < 0.05 for the difference from cells incubated in the same glucose concentration without NAC. D: Rat islets and INS-1E cells were incubated with RPMI-1640 medium containing the indicated glucose concentrations in the absence or presence of 1 mmol/L NAC for 48 or 24 h, respectively. The islets and INS-1E cells were then washed and GSIS evaluated, as described in the Fig. 3A legend (white bars, 3.3 mmol/L glucose; dark bars, 16.7 mmol/L glucose). Insulin secretion is presented as percent of insulin content. *P < 0.05 for the difference in stimulated secretion compared with cells maintained at 5 mmol/L glucose. #P < 0.05 for differences from the respective NAC-free incubation. Results are mean ± SEM, n = 3.
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Figure 6: NAC reduces ROS production, 4-HNE generation, and insulin secretion in β-cells. A: INS-1E cells were incubated for 16 h with the indicated glucose levels without or with 1 mmol/L NAC. ROS production was determined with the carboxy-DCF-fluorescence assay. Glc, glucose. *P < 0.05 for the difference from the 5 mmol/L glucose controls. #P < 0.05 for the difference from the cells incubated in the same glucose concentration without NAC. B: HPLC determination of 4-HNE in INS-1E cultures maintained at 25 mmol/L glucose, without or with 1 mmol/L NAC for 16 h, was performed as described in the legend to Fig. 2A. *P < 0.05 for the difference from the vehicle-treated controls. C: INS-1E cells were prepared for the PPRE-luciferase transactivation assay, as described in the legend to Fig. 4B, and incubated with the indicated glucose levels without or with 1 mmol/L NAC; luciferase activity was determined after 24 h of incubation. *P < 0.05 for the difference in luciferase activity in comparison with the control incubation at 5 mmol/L glucose in the absence of NAC. #P < 0.05 for the difference from cells incubated in the same glucose concentration without NAC. D: Rat islets and INS-1E cells were incubated with RPMI-1640 medium containing the indicated glucose concentrations in the absence or presence of 1 mmol/L NAC for 48 or 24 h, respectively. The islets and INS-1E cells were then washed and GSIS evaluated, as described in the Fig. 3A legend (white bars, 3.3 mmol/L glucose; dark bars, 16.7 mmol/L glucose). Insulin secretion is presented as percent of insulin content. *P < 0.05 for the difference in stimulated secretion compared with cells maintained at 5 mmol/L glucose. #P < 0.05 for differences from the respective NAC-free incubation. Results are mean ± SEM, n = 3.
Mentions: The role of endogenous 4-HNE in regulating insulin secretion was further substantiated by measuring insulin secretion after the blocking of 4-HNE production in β-cells by the antioxidant N-acetylcysteine (NAC), as described previously (18). Figure 6A and B shows that glucose-induced generation of ROS and 4-HNE in INS-1E cells was nearly abolished in the presence of 1 mmol/L NAC. This treatment also prevented high glucose-induced expression of luciferase in the PPRE-dependent transactivation assay in cells overexpressing PPAR-δ. Finally, glucose-amplified insulin secretion was markedly reduced in NAC-treated rat islets and in INS-1E cells (Fig. 6D).

Bottom Line: The latter mimicked the GSIS-amplifying effect of high glucose preexposure and of the PPAR-δ agonist GW501516 in INS-1E cells and isolated rat islets.Cytotoxic effects of 4-HNE were observed only above the physiologically effective concentration range.This molecule is an endogenous ligand for PPAR-δ, which amplifies insulin secretion in β-cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, School of Pharmacy, Faculty of Medicine, Institute for Drug Research, Hebrew University, Jerusalem, Israel.

ABSTRACT

Objective: Previous studies show that polyunsaturated fatty acids (PUFAs) increase the insulin secretory capacity of pancreatic β-cells. We aimed at identifying PUFA-derived mediators and their cellular targets that are involved in the amplification of insulin release from β-cells preexposed to high glucose levels.

Research design and methods: The content of fatty acids in phospholipids of INS-1E β-cells was determined by lipidomics analysis. High-performance liquid chromatography was used to identify peroxidation products in β-cell cultures. Static and dynamic glucose-stimulated insulin secretion (GSIS) assays were performed on isolated rat islets and/or INS-1E cells. The function of peroxisome proliferator-activated receptor-δ (PPAR-δ) in regulating insulin secretion was investigated using pharmacological agents and gene expression manipulations.

Results: High glucose activated cPLA(2) and, subsequently, the hydrolysis of arachidonic and linoleic acid (AA and LA, respectively) from phospholipids in INS-1E cells. Glucose also increased the level of reactive oxygen species, which promoted the peroxidation of these PUFAs to generate 4-hydroxy-2E-nonenal (4-HNE). The latter mimicked the GSIS-amplifying effect of high glucose preexposure and of the PPAR-δ agonist GW501516 in INS-1E cells and isolated rat islets. These effects were blocked with GSK0660, a selective PPAR-δ antagonist, and the antioxidant N-acetylcysteine or by silencing PPAR-δ expression. High glucose, 4-HNE, and GW501516 also induced luciferase expression in a PPAR-δ-mediated transactivation assay. Cytotoxic effects of 4-HNE were observed only above the physiologically effective concentration range.

Conclusions: Elevated glucose levels augment the release of AA and LA from phospholipids and their peroxidation to 4-HNE in β-cells. This molecule is an endogenous ligand for PPAR-δ, which amplifies insulin secretion in β-cells.

Show MeSH
Related in: MedlinePlus