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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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Effect of high glucose on PUFA peroxidation in β-cells. A: INS-1E cells were exposed to the indicated d-glucose (D-Glc) and l-glucose (L-Glc) concentrations for 48 h; during the last 16 h, the cells were incubated with serum-free culture medium with the same additions. The media (10 mL) were then collected, extracted, and analyzed by HPLC. Data are given as nanogram 4-HNE per milligram cellular protein. Representative HPLC tracings are depicted (inset) and the arrows point to 4-HNE peaks. Results are mean ± SEM, n = 3–4. *P < 0.05 for the difference from the 5 mmol/L glucose controls. B: P. obesus gerbils fed LE or HE diet were killed, and sera were collected for glucose and 4-HNE determinations, as described in research design and methods. Serum glucose levels were 3.9 ± 0.1 (LE group) and 14.3 ± 0.7* mmol/L (HE group). Results are mean ± SEM, n = 5–12 animals. *P < 0.05 for the differences from the LE-diet control group. C: ROS production in INS-1E cells incubated for 16 h with the indicated glucose levels was determined by the carboxy-DCF-fluorescence method. Results are mean ± SEM, n = 3–4. *P < 0.05 for the difference from the 5 mmol/L glucose controls.
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Figure 2: Effect of high glucose on PUFA peroxidation in β-cells. A: INS-1E cells were exposed to the indicated d-glucose (D-Glc) and l-glucose (L-Glc) concentrations for 48 h; during the last 16 h, the cells were incubated with serum-free culture medium with the same additions. The media (10 mL) were then collected, extracted, and analyzed by HPLC. Data are given as nanogram 4-HNE per milligram cellular protein. Representative HPLC tracings are depicted (inset) and the arrows point to 4-HNE peaks. Results are mean ± SEM, n = 3–4. *P < 0.05 for the difference from the 5 mmol/L glucose controls. B: P. obesus gerbils fed LE or HE diet were killed, and sera were collected for glucose and 4-HNE determinations, as described in research design and methods. Serum glucose levels were 3.9 ± 0.1 (LE group) and 14.3 ± 0.7* mmol/L (HE group). Results are mean ± SEM, n = 5–12 animals. *P < 0.05 for the differences from the LE-diet control group. C: ROS production in INS-1E cells incubated for 16 h with the indicated glucose levels was determined by the carboxy-DCF-fluorescence method. Results are mean ± SEM, n = 3–4. *P < 0.05 for the difference from the 5 mmol/L glucose controls.

Mentions: The major peroxidation products of AA and LA are 4-HNE and 4-HDDE (15). We measured their levels in culture media of INS-1E cells exposed to increasing glucose concentrations in a serum-free medium during the last 16 h of incubation. This procedure eliminates the formation of 4-HNE adducts with serum proteins. Figure 2A shows a clear glucose-dependent increase in 4-HNE generation, up to ninefold at 25 mmol/L glucose. In contrast, 4-HDDE was not detectable in INS-1E culture medium extracts at all glucose concentrations used (data not shown). The gerbil P. obesus is an established animal model of diet-induced diabetes (5). Figure 2B shows that hyperglycemia induced by HE diet was accompanied by a marked increase in serum 4-HNE. The plasma insulin levels in the normo- and hyperglycemic animal were 1.41 ± 0.16 and 6.38 ± 0.59 nmol/L, respectively. ROS mediate the initiation step in the peroxidation cascade of AA and LA (15). Figure 2C shows increased ROS production in INS-1E cells incubated at 11 and 25 mmol/L glucose.


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)

Effect of high glucose on PUFA peroxidation in β-cells. A: INS-1E cells were exposed to the indicated d-glucose (D-Glc) and l-glucose (L-Glc) concentrations for 48 h; during the last 16 h, the cells were incubated with serum-free culture medium with the same additions. The media (10 mL) were then collected, extracted, and analyzed by HPLC. Data are given as nanogram 4-HNE per milligram cellular protein. Representative HPLC tracings are depicted (inset) and the arrows point to 4-HNE peaks. Results are mean ± SEM, n = 3–4. *P < 0.05 for the difference from the 5 mmol/L glucose controls. B: P. obesus gerbils fed LE or HE diet were killed, and sera were collected for glucose and 4-HNE determinations, as described in research design and methods. Serum glucose levels were 3.9 ± 0.1 (LE group) and 14.3 ± 0.7* mmol/L (HE group). Results are mean ± SEM, n = 5–12 animals. *P < 0.05 for the differences from the LE-diet control group. C: ROS production in INS-1E cells incubated for 16 h with the indicated glucose levels was determined by the carboxy-DCF-fluorescence method. Results are mean ± SEM, n = 3–4. *P < 0.05 for the difference from the 5 mmol/L glucose controls.
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Figure 2: Effect of high glucose on PUFA peroxidation in β-cells. A: INS-1E cells were exposed to the indicated d-glucose (D-Glc) and l-glucose (L-Glc) concentrations for 48 h; during the last 16 h, the cells were incubated with serum-free culture medium with the same additions. The media (10 mL) were then collected, extracted, and analyzed by HPLC. Data are given as nanogram 4-HNE per milligram cellular protein. Representative HPLC tracings are depicted (inset) and the arrows point to 4-HNE peaks. Results are mean ± SEM, n = 3–4. *P < 0.05 for the difference from the 5 mmol/L glucose controls. B: P. obesus gerbils fed LE or HE diet were killed, and sera were collected for glucose and 4-HNE determinations, as described in research design and methods. Serum glucose levels were 3.9 ± 0.1 (LE group) and 14.3 ± 0.7* mmol/L (HE group). Results are mean ± SEM, n = 5–12 animals. *P < 0.05 for the differences from the LE-diet control group. C: ROS production in INS-1E cells incubated for 16 h with the indicated glucose levels was determined by the carboxy-DCF-fluorescence method. Results are mean ± SEM, n = 3–4. *P < 0.05 for the difference from the 5 mmol/L glucose controls.
Mentions: The major peroxidation products of AA and LA are 4-HNE and 4-HDDE (15). We measured their levels in culture media of INS-1E cells exposed to increasing glucose concentrations in a serum-free medium during the last 16 h of incubation. This procedure eliminates the formation of 4-HNE adducts with serum proteins. Figure 2A shows a clear glucose-dependent increase in 4-HNE generation, up to ninefold at 25 mmol/L glucose. In contrast, 4-HDDE was not detectable in INS-1E culture medium extracts at all glucose concentrations used (data not shown). The gerbil P. obesus is an established animal model of diet-induced diabetes (5). Figure 2B shows that hyperglycemia induced by HE diet was accompanied by a marked increase in serum 4-HNE. The plasma insulin levels in the normo- and hyperglycemic animal were 1.41 ± 0.16 and 6.38 ± 0.59 nmol/L, respectively. ROS mediate the initiation step in the peroxidation cascade of AA and LA (15). Figure 2C shows increased ROS production in INS-1E cells incubated at 11 and 25 mmol/L glucose.

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