Limits...
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.

Show MeSH

Related in: MedlinePlus

PPAR-δ mediates 4-HNE effects in β-cells. A: PPAR-δ expression was silenced in INS-1E cells with targeted siRNA sequences (Si), as described in research design and methods. The cells were then incubated for 24 h at the indicated glucose concentrations, washed, processed, and taken for the standard GSIS analysis, as described in the legend to Fig. 3A. Cells transfected with scrambled RNA (Sc) served as controls. C, nontransfected cells; Glc, glucose. *P < 0.05 for differences from the respective controls. B: Rat islets and INS-1E cells were incubated in RPMI-1640 medium containing 11 mmol/L glucose with increasing GW501516 concentrations for 48 and 24 h, respectively, and taken for GSIS analysis. *P < 0.05 for difference from untreated cells. C: Similar β-cell preparations were incubated as described in A for 48 and 24 h, respectively, without or with 0.1 μmol/L GW501516 and increasing concentrations of GSK0660, and taken for GSIS analysis. *P < 0.05 for the difference from untreated cells. #P < 0.05 for the difference from GW501516-treated cells in the absence of GSK0660. D: The β-cell preparations were incubated at 11 mmol/L glucose without or with 15 (rat islets) or 1 μmol/L 4-HNE (INS-1E cells) with increasing concentrations of GSK0660. At the end of incubations, the islets and INS-1E cells were taken for GSIS analysis. *P < 0.05 for difference from untreated cells. #P < 0.05 for the differences from 4-HNE–treated cells in the absence of GSK0660. Results (A–D) are mean ± SEM, n = 3. E: Isolated rat islets were preincubated for 48 h in RPMI-1640 medium (11 mmol/L glucose) with 15 μmol/L 4-HNE (○), 0.1 μmol/L GW501516 (●), or the vehicle (△) and taken for perfusion experiments. AUC of the first and second phases of insulin release of each treatment are shown (inset). P < 0.05 for differences of the AUC values of the first (*) and second phase (**) insulin secretion from 4-HNE– and GW501516-treated islets in comparison with control islets. Results are mean ± SEM, n = 3.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3198069&req=5

Figure 5: PPAR-δ mediates 4-HNE effects in β-cells. A: PPAR-δ expression was silenced in INS-1E cells with targeted siRNA sequences (Si), as described in research design and methods. The cells were then incubated for 24 h at the indicated glucose concentrations, washed, processed, and taken for the standard GSIS analysis, as described in the legend to Fig. 3A. Cells transfected with scrambled RNA (Sc) served as controls. C, nontransfected cells; Glc, glucose. *P < 0.05 for differences from the respective controls. B: Rat islets and INS-1E cells were incubated in RPMI-1640 medium containing 11 mmol/L glucose with increasing GW501516 concentrations for 48 and 24 h, respectively, and taken for GSIS analysis. *P < 0.05 for difference from untreated cells. C: Similar β-cell preparations were incubated as described in A for 48 and 24 h, respectively, without or with 0.1 μmol/L GW501516 and increasing concentrations of GSK0660, and taken for GSIS analysis. *P < 0.05 for the difference from untreated cells. #P < 0.05 for the difference from GW501516-treated cells in the absence of GSK0660. D: The β-cell preparations were incubated at 11 mmol/L glucose without or with 15 (rat islets) or 1 μmol/L 4-HNE (INS-1E cells) with increasing concentrations of GSK0660. At the end of incubations, the islets and INS-1E cells were taken for GSIS analysis. *P < 0.05 for difference from untreated cells. #P < 0.05 for the differences from 4-HNE–treated cells in the absence of GSK0660. Results (A–D) are mean ± SEM, n = 3. E: Isolated rat islets were preincubated for 48 h in RPMI-1640 medium (11 mmol/L glucose) with 15 μmol/L 4-HNE (○), 0.1 μmol/L GW501516 (●), or the vehicle (△) and taken for perfusion experiments. AUC of the first and second phases of insulin release of each treatment are shown (inset). P < 0.05 for differences of the AUC values of the first (*) and second phase (**) insulin secretion from 4-HNE– and GW501516-treated islets in comparison with control islets. Results are mean ± SEM, n = 3.

Mentions: To establish the role of PPAR-δ in mediating high glucose-induced amplification of insulin secretion, we silenced its expression. Supplementary Fig. 4A and B shows that siRNA targeted to PPAR-δ mRNA reduced the mRNA and protein levels by 65–75 and 45–60%, respectively. Figure 5A shows that this treatment resulted in loss of the cells’ ability to amplify GSIS after preexposure to high glucose levels. Noteworthy, the activation of PPAR-δ by increasing glucose concentrations was not associated with increased expression of this nuclear receptor (Supplementary Fig. 4A and B).


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)

PPAR-δ mediates 4-HNE effects in β-cells. A: PPAR-δ expression was silenced in INS-1E cells with targeted siRNA sequences (Si), as described in research design and methods. The cells were then incubated for 24 h at the indicated glucose concentrations, washed, processed, and taken for the standard GSIS analysis, as described in the legend to Fig. 3A. Cells transfected with scrambled RNA (Sc) served as controls. C, nontransfected cells; Glc, glucose. *P < 0.05 for differences from the respective controls. B: Rat islets and INS-1E cells were incubated in RPMI-1640 medium containing 11 mmol/L glucose with increasing GW501516 concentrations for 48 and 24 h, respectively, and taken for GSIS analysis. *P < 0.05 for difference from untreated cells. C: Similar β-cell preparations were incubated as described in A for 48 and 24 h, respectively, without or with 0.1 μmol/L GW501516 and increasing concentrations of GSK0660, and taken for GSIS analysis. *P < 0.05 for the difference from untreated cells. #P < 0.05 for the difference from GW501516-treated cells in the absence of GSK0660. D: The β-cell preparations were incubated at 11 mmol/L glucose without or with 15 (rat islets) or 1 μmol/L 4-HNE (INS-1E cells) with increasing concentrations of GSK0660. At the end of incubations, the islets and INS-1E cells were taken for GSIS analysis. *P < 0.05 for difference from untreated cells. #P < 0.05 for the differences from 4-HNE–treated cells in the absence of GSK0660. Results (A–D) are mean ± SEM, n = 3. E: Isolated rat islets were preincubated for 48 h in RPMI-1640 medium (11 mmol/L glucose) with 15 μmol/L 4-HNE (○), 0.1 μmol/L GW501516 (●), or the vehicle (△) and taken for perfusion experiments. AUC of the first and second phases of insulin release of each treatment are shown (inset). P < 0.05 for differences of the AUC values of the first (*) and second phase (**) insulin secretion from 4-HNE– and GW501516-treated islets in comparison with control islets. Results are mean ± SEM, n = 3.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3198069&req=5

Figure 5: PPAR-δ mediates 4-HNE effects in β-cells. A: PPAR-δ expression was silenced in INS-1E cells with targeted siRNA sequences (Si), as described in research design and methods. The cells were then incubated for 24 h at the indicated glucose concentrations, washed, processed, and taken for the standard GSIS analysis, as described in the legend to Fig. 3A. Cells transfected with scrambled RNA (Sc) served as controls. C, nontransfected cells; Glc, glucose. *P < 0.05 for differences from the respective controls. B: Rat islets and INS-1E cells were incubated in RPMI-1640 medium containing 11 mmol/L glucose with increasing GW501516 concentrations for 48 and 24 h, respectively, and taken for GSIS analysis. *P < 0.05 for difference from untreated cells. C: Similar β-cell preparations were incubated as described in A for 48 and 24 h, respectively, without or with 0.1 μmol/L GW501516 and increasing concentrations of GSK0660, and taken for GSIS analysis. *P < 0.05 for the difference from untreated cells. #P < 0.05 for the difference from GW501516-treated cells in the absence of GSK0660. D: The β-cell preparations were incubated at 11 mmol/L glucose without or with 15 (rat islets) or 1 μmol/L 4-HNE (INS-1E cells) with increasing concentrations of GSK0660. At the end of incubations, the islets and INS-1E cells were taken for GSIS analysis. *P < 0.05 for difference from untreated cells. #P < 0.05 for the differences from 4-HNE–treated cells in the absence of GSK0660. Results (A–D) are mean ± SEM, n = 3. E: Isolated rat islets were preincubated for 48 h in RPMI-1640 medium (11 mmol/L glucose) with 15 μmol/L 4-HNE (○), 0.1 μmol/L GW501516 (●), or the vehicle (△) and taken for perfusion experiments. AUC of the first and second phases of insulin release of each treatment are shown (inset). P < 0.05 for differences of the AUC values of the first (*) and second phase (**) insulin secretion from 4-HNE– and GW501516-treated islets in comparison with control islets. Results are mean ± SEM, n = 3.
Mentions: To establish the role of PPAR-δ in mediating high glucose-induced amplification of insulin secretion, we silenced its expression. Supplementary Fig. 4A and B shows that siRNA targeted to PPAR-δ mRNA reduced the mRNA and protein levels by 65–75 and 45–60%, respectively. Figure 5A shows that this treatment resulted in loss of the cells’ ability to amplify GSIS after preexposure to high glucose levels. Noteworthy, the activation of PPAR-δ by increasing glucose concentrations was not associated with increased expression of this nuclear receptor (Supplementary Fig. 4A and B).

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