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

High glucose and 4-HNE activate PPAR-δ in INS-1E cells. A: INS-1E cells were transfected with the hPPAR-α, hPPAR-γ1, hPPAR-γ2, or hPPAR-δ expression vectors; the hRXR expression vector; and the 3XPPRE-TK-luciferase reporter plasmid. Renilla luciferase plasmid was transfected for normalizing the luciferase activity data. The transfected cells were treated with 60 μmol/L WY14643, 30 μmol/L troglitazone, 0.1 μmol/L GW501516 (GW), or 1.0 μmol/L 4-HNE for 24 h and luciferase activity was then measured. The 100% values were assigned to the respective untreated control groups. *P < 0.05 for differences from the respective controls. B: INS-1E cells were transfected as described in A using the hPPAR-δ expression vector. The cells were then incubated for 24 h with increasing concentrations of 4-HNE without (●) or with (○) 1 μmol/L GSK0660. Luciferase activity was then measured. The value of light units measured in lysates of vehicle-treated cells was taken as 100%. *P < 0.05 for differences from untreated controls. #P < 0.05 for differences from the respective 4-HNE–treated cells. C: INS-1E cells were transfected as described in B and incubated for 48 h in RPMI-1640 medium containing the indicated glucose levels. GSK0660 (1 μmol/L) was present during the last 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 GSK0660. #P < 0.05 for difference from the cells incubated in the same glucose concentration without GSK0660. The 100% value was taken as the light units of control cells at 5 mmol/L glucose. Results are mean ± SEM, n = 3. The vehicle DMSO, used at a 1:1,000 dilution in the incubation medium, had no significant effect on GSIS. Glc, glucose.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 4: High glucose and 4-HNE activate PPAR-δ in INS-1E cells. A: INS-1E cells were transfected with the hPPAR-α, hPPAR-γ1, hPPAR-γ2, or hPPAR-δ expression vectors; the hRXR expression vector; and the 3XPPRE-TK-luciferase reporter plasmid. Renilla luciferase plasmid was transfected for normalizing the luciferase activity data. The transfected cells were treated with 60 μmol/L WY14643, 30 μmol/L troglitazone, 0.1 μmol/L GW501516 (GW), or 1.0 μmol/L 4-HNE for 24 h and luciferase activity was then measured. The 100% values were assigned to the respective untreated control groups. *P < 0.05 for differences from the respective controls. B: INS-1E cells were transfected as described in A using the hPPAR-δ expression vector. The cells were then incubated for 24 h with increasing concentrations of 4-HNE without (●) or with (○) 1 μmol/L GSK0660. Luciferase activity was then measured. The value of light units measured in lysates of vehicle-treated cells was taken as 100%. *P < 0.05 for differences from untreated controls. #P < 0.05 for differences from the respective 4-HNE–treated cells. C: INS-1E cells were transfected as described in B and incubated for 48 h in RPMI-1640 medium containing the indicated glucose levels. GSK0660 (1 μmol/L) was present during the last 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 GSK0660. #P < 0.05 for difference from the cells incubated in the same glucose concentration without GSK0660. The 100% value was taken as the light units of control cells at 5 mmol/L glucose. Results are mean ± SEM, n = 3. The vehicle DMSO, used at a 1:1,000 dilution in the incubation medium, had no significant effect on GSIS. Glc, glucose.

Mentions: Recent reports show that 4-HNE is an endogenous ligand of PPAR-δ (21); therefore, we tested its ability to activate PPAR-δ in INS-1E cells. The cells were transfected with hPPAR-α, hPPAR-γ1, hPPAR-γ2, or hPPAR-δ expression vectors along with hRXR- and 3XPPRE (PPAR response element)-luciferase expression vectors, as described before (18). Each hPPAR isotype was successfully expressed in INS-1E cells (Supplementary Fig. 2). Furthermore, WY14643, troglitazone, and GW501516, the pharmacological agonists of PPAR-α, PPAR-γ, and PPAR-δ, respectively (18), transactivated luciferase expression in an hPPAR isotype–specific manner (Fig. 4A). Exogenously added 4-HNE mimicked GW501516 and induced luciferase activity only in cells ectopically expressing hPPAR-δ. The finding that 4-HNE activated PPAR-δ in β-cells was further confirmed by the use of the selective PPAR-δ antagonist GSK0660; it abolished 4-HNE–induced luciferase activity in hPPAR-δ–expressing cells (Fig. 4B). The corresponding analysis of GSK0660 inhibitory effects on GW501516-treated INS-1E cells is shown in Supplementary Fig. 3. Moreover, 4-HNA, the acidic metabolite of 4-HNE, lacked any PPAR-δ stimulating activity (Supplementary Fig. 1B). Figure 4C shows a similar transactivation assay with increasing glucose concentrations in the absence or presence of GSK0660. Glucose stimulated luciferase activity in a concentration-dependent manner, which was abolished by the PPAR-δ antagonist.


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)

High glucose and 4-HNE activate PPAR-δ in INS-1E cells. A: INS-1E cells were transfected with the hPPAR-α, hPPAR-γ1, hPPAR-γ2, or hPPAR-δ expression vectors; the hRXR expression vector; and the 3XPPRE-TK-luciferase reporter plasmid. Renilla luciferase plasmid was transfected for normalizing the luciferase activity data. The transfected cells were treated with 60 μmol/L WY14643, 30 μmol/L troglitazone, 0.1 μmol/L GW501516 (GW), or 1.0 μmol/L 4-HNE for 24 h and luciferase activity was then measured. The 100% values were assigned to the respective untreated control groups. *P < 0.05 for differences from the respective controls. B: INS-1E cells were transfected as described in A using the hPPAR-δ expression vector. The cells were then incubated for 24 h with increasing concentrations of 4-HNE without (●) or with (○) 1 μmol/L GSK0660. Luciferase activity was then measured. The value of light units measured in lysates of vehicle-treated cells was taken as 100%. *P < 0.05 for differences from untreated controls. #P < 0.05 for differences from the respective 4-HNE–treated cells. C: INS-1E cells were transfected as described in B and incubated for 48 h in RPMI-1640 medium containing the indicated glucose levels. GSK0660 (1 μmol/L) was present during the last 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 GSK0660. #P < 0.05 for difference from the cells incubated in the same glucose concentration without GSK0660. The 100% value was taken as the light units of control cells at 5 mmol/L glucose. Results are mean ± SEM, n = 3. The vehicle DMSO, used at a 1:1,000 dilution in the incubation medium, had no significant effect on GSIS. Glc, glucose.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 4: High glucose and 4-HNE activate PPAR-δ in INS-1E cells. A: INS-1E cells were transfected with the hPPAR-α, hPPAR-γ1, hPPAR-γ2, or hPPAR-δ expression vectors; the hRXR expression vector; and the 3XPPRE-TK-luciferase reporter plasmid. Renilla luciferase plasmid was transfected for normalizing the luciferase activity data. The transfected cells were treated with 60 μmol/L WY14643, 30 μmol/L troglitazone, 0.1 μmol/L GW501516 (GW), or 1.0 μmol/L 4-HNE for 24 h and luciferase activity was then measured. The 100% values were assigned to the respective untreated control groups. *P < 0.05 for differences from the respective controls. B: INS-1E cells were transfected as described in A using the hPPAR-δ expression vector. The cells were then incubated for 24 h with increasing concentrations of 4-HNE without (●) or with (○) 1 μmol/L GSK0660. Luciferase activity was then measured. The value of light units measured in lysates of vehicle-treated cells was taken as 100%. *P < 0.05 for differences from untreated controls. #P < 0.05 for differences from the respective 4-HNE–treated cells. C: INS-1E cells were transfected as described in B and incubated for 48 h in RPMI-1640 medium containing the indicated glucose levels. GSK0660 (1 μmol/L) was present during the last 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 GSK0660. #P < 0.05 for difference from the cells incubated in the same glucose concentration without GSK0660. The 100% value was taken as the light units of control cells at 5 mmol/L glucose. Results are mean ± SEM, n = 3. The vehicle DMSO, used at a 1:1,000 dilution in the incubation medium, had no significant effect on GSIS. Glc, glucose.
Mentions: Recent reports show that 4-HNE is an endogenous ligand of PPAR-δ (21); therefore, we tested its ability to activate PPAR-δ in INS-1E cells. The cells were transfected with hPPAR-α, hPPAR-γ1, hPPAR-γ2, or hPPAR-δ expression vectors along with hRXR- and 3XPPRE (PPAR response element)-luciferase expression vectors, as described before (18). Each hPPAR isotype was successfully expressed in INS-1E cells (Supplementary Fig. 2). Furthermore, WY14643, troglitazone, and GW501516, the pharmacological agonists of PPAR-α, PPAR-γ, and PPAR-δ, respectively (18), transactivated luciferase expression in an hPPAR isotype–specific manner (Fig. 4A). Exogenously added 4-HNE mimicked GW501516 and induced luciferase activity only in cells ectopically expressing hPPAR-δ. The finding that 4-HNE activated PPAR-δ in β-cells was further confirmed by the use of the selective PPAR-δ antagonist GSK0660; it abolished 4-HNE–induced luciferase activity in hPPAR-δ–expressing cells (Fig. 4B). The corresponding analysis of GSK0660 inhibitory effects on GW501516-treated INS-1E cells is shown in Supplementary Fig. 3. Moreover, 4-HNA, the acidic metabolite of 4-HNE, lacked any PPAR-δ stimulating activity (Supplementary Fig. 1B). Figure 4C shows a similar transactivation assay with increasing glucose concentrations in the absence or presence of GSK0660. Glucose stimulated luciferase activity in a concentration-dependent manner, which was abolished by the PPAR-δ antagonist.

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