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Arachidonic acid actions on functional integrity and attenuation of the negative effects of palmitic acid in a clonal pancreatic β-cell line.

Keane DC, Takahashi HK, Dhayal S, Morgan NG, Curi R, Newsholme P - Clin. Sci. (2011)

Bottom Line: Experiments using specific COX and LOX (lipoxygenase) inhibitors demonstrated the importance of COX-1 activity for acute (20 min) stimulation of insulin secretion, suggesting that AA metabolites may be responsible for the insulinotropic effects.AA decreased the protein expression of iNOS (inducible NO synthase), the p65 subunit of NF-κB (nuclear factor κB) and the p47 subunit of NADPH oxidase in PA-treated cells.These findings indicate that AA has an important regulatory and protective β-cell action, which may be beneficial to function and survival in the 'lipotoxic' environment commonly associated with Type 2 diabetes mellitus.

View Article: PubMed Central - PubMed

Affiliation: UCD School of Biomolecular and Biomedical Science, UCD Conway Institute and UCD Institute of Sport and Health, UCD Dublin, Belfield, Dublin 4, Ireland.

ABSTRACT
Chronic exposure of pancreatic β-cells to saturated non-esterified fatty acids can lead to inhibition of insulin secretion and apoptosis. Several previous studies have demonstrated that saturated fatty acids such as PA (palmitic acid) are detrimental to β-cell function compared with unsaturated fatty acids. In the present study, we describe the effect of the polyunsaturated AA (arachidonic acid) on the function of the clonal pancreatic β-cell line BRIN-BD11 and demonstrate AA-dependent attenuation of PA effects. When added to β-cell incubations at 100 μM, AA can stimulate cell proliferation and chronic (24 h) basal insulin secretion. Microarray analysis and/or real-time PCR indicated significant AA-dependent up-regulation of genes involved in proliferation and fatty acid metabolism [e.g. Angptl (angiopoietin-like protein 4), Ech1 (peroxisomal Δ3,5,Δ2,4-dienoyl-CoA isomerase), Cox-1 (cyclo-oxygenase-1) and Cox-2, P<0.05]. Experiments using specific COX and LOX (lipoxygenase) inhibitors demonstrated the importance of COX-1 activity for acute (20 min) stimulation of insulin secretion, suggesting that AA metabolites may be responsible for the insulinotropic effects. Moreover, concomitant incubation of AA with PA dose-dependently attenuated the detrimental effects of the saturated fatty acid, so reducing apoptosis and decreasing parameters of oxidative stress [ROS (reactive oxygen species) and NO levels] while improving the GSH/GSSG ratio. AA decreased the protein expression of iNOS (inducible NO synthase), the p65 subunit of NF-κB (nuclear factor κB) and the p47 subunit of NADPH oxidase in PA-treated cells. These findings indicate that AA has an important regulatory and protective β-cell action, which may be beneficial to function and survival in the 'lipotoxic' environment commonly associated with Type 2 diabetes mellitus.

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Rescue of PA-incubated cells by AA determined by flow cytometry (A), and effect of PA and AA on chronic (24 h) insulin secretion (B)(A) BRIN-BD11 cells were incubated in the presence of 250 μM PA complexed to BSA in the absence or increasing concentrations of AA as indicated for 24 h. After this time, the cells were harvested and their viability assessed by flow cytometry after staining with PI. A significant improvement in cell viability (P<0.01) was observed at all concentrations of AA tested. (B) BRIN-BD11 β-cells were incubated in the presence of various concentrations of PA (50–150 μM non-complexed) in the absence or presence of 100 μM AA for 24 h. After the 24-h incubation period, an aliquot of the medium was removed, and the concentration of insulin was determined. The results are expressed as means±S.E.M., for three separate determinations each measured in duplicate, relative to cells incubated in the absence of fatty acid (#P<0.05) or compared with the equivalent PA-only-treated cells (*P<0.05).
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Figure 6: Rescue of PA-incubated cells by AA determined by flow cytometry (A), and effect of PA and AA on chronic (24 h) insulin secretion (B)(A) BRIN-BD11 cells were incubated in the presence of 250 μM PA complexed to BSA in the absence or increasing concentrations of AA as indicated for 24 h. After this time, the cells were harvested and their viability assessed by flow cytometry after staining with PI. A significant improvement in cell viability (P<0.01) was observed at all concentrations of AA tested. (B) BRIN-BD11 β-cells were incubated in the presence of various concentrations of PA (50–150 μM non-complexed) in the absence or presence of 100 μM AA for 24 h. After the 24-h incubation period, an aliquot of the medium was removed, and the concentration of insulin was determined. The results are expressed as means±S.E.M., for three separate determinations each measured in duplicate, relative to cells incubated in the absence of fatty acid (#P<0.05) or compared with the equivalent PA-only-treated cells (*P<0.05).

Mentions: Incubation of BRIN-BD11 cells with PA for 24 h decreased relative cell number in a dose-dependent manner as determined by WST-1 analysis (P<0.001; Table 2). However, concomitant incubation with AA protected BRIN-BD11 cells from the toxic effect of PA in a dose-dependent manner as determined either by WST-1 analysis (Table 2) or by flow cytometry (Figure 6A). The two experiments described in Table 2 and Figure 6(A) were performed using different methods of fatty acid delivery. The fatty acids were added to the incubation medium in ethanol only in the experiment described in Table 2, while the fatty acids were delivered complexed to BSA in the experiment described in Figure 6(A). The latter revealed that the cytoprotective response was extremely potent with an EC50 ~2 μM (total AA concentration) under the experimental conditions used and that AA caused complete protection against the cytotoxicity induced by 250 μM PA.


Arachidonic acid actions on functional integrity and attenuation of the negative effects of palmitic acid in a clonal pancreatic β-cell line.

Keane DC, Takahashi HK, Dhayal S, Morgan NG, Curi R, Newsholme P - Clin. Sci. (2011)

Rescue of PA-incubated cells by AA determined by flow cytometry (A), and effect of PA and AA on chronic (24 h) insulin secretion (B)(A) BRIN-BD11 cells were incubated in the presence of 250 μM PA complexed to BSA in the absence or increasing concentrations of AA as indicated for 24 h. After this time, the cells were harvested and their viability assessed by flow cytometry after staining with PI. A significant improvement in cell viability (P<0.01) was observed at all concentrations of AA tested. (B) BRIN-BD11 β-cells were incubated in the presence of various concentrations of PA (50–150 μM non-complexed) in the absence or presence of 100 μM AA for 24 h. After the 24-h incubation period, an aliquot of the medium was removed, and the concentration of insulin was determined. The results are expressed as means±S.E.M., for three separate determinations each measured in duplicate, relative to cells incubated in the absence of fatty acid (#P<0.05) or compared with the equivalent PA-only-treated cells (*P<0.05).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2990202&req=5

Figure 6: Rescue of PA-incubated cells by AA determined by flow cytometry (A), and effect of PA and AA on chronic (24 h) insulin secretion (B)(A) BRIN-BD11 cells were incubated in the presence of 250 μM PA complexed to BSA in the absence or increasing concentrations of AA as indicated for 24 h. After this time, the cells were harvested and their viability assessed by flow cytometry after staining with PI. A significant improvement in cell viability (P<0.01) was observed at all concentrations of AA tested. (B) BRIN-BD11 β-cells were incubated in the presence of various concentrations of PA (50–150 μM non-complexed) in the absence or presence of 100 μM AA for 24 h. After the 24-h incubation period, an aliquot of the medium was removed, and the concentration of insulin was determined. The results are expressed as means±S.E.M., for three separate determinations each measured in duplicate, relative to cells incubated in the absence of fatty acid (#P<0.05) or compared with the equivalent PA-only-treated cells (*P<0.05).
Mentions: Incubation of BRIN-BD11 cells with PA for 24 h decreased relative cell number in a dose-dependent manner as determined by WST-1 analysis (P<0.001; Table 2). However, concomitant incubation with AA protected BRIN-BD11 cells from the toxic effect of PA in a dose-dependent manner as determined either by WST-1 analysis (Table 2) or by flow cytometry (Figure 6A). The two experiments described in Table 2 and Figure 6(A) were performed using different methods of fatty acid delivery. The fatty acids were added to the incubation medium in ethanol only in the experiment described in Table 2, while the fatty acids were delivered complexed to BSA in the experiment described in Figure 6(A). The latter revealed that the cytoprotective response was extremely potent with an EC50 ~2 μM (total AA concentration) under the experimental conditions used and that AA caused complete protection against the cytotoxicity induced by 250 μM PA.

Bottom Line: Experiments using specific COX and LOX (lipoxygenase) inhibitors demonstrated the importance of COX-1 activity for acute (20 min) stimulation of insulin secretion, suggesting that AA metabolites may be responsible for the insulinotropic effects.AA decreased the protein expression of iNOS (inducible NO synthase), the p65 subunit of NF-κB (nuclear factor κB) and the p47 subunit of NADPH oxidase in PA-treated cells.These findings indicate that AA has an important regulatory and protective β-cell action, which may be beneficial to function and survival in the 'lipotoxic' environment commonly associated with Type 2 diabetes mellitus.

View Article: PubMed Central - PubMed

Affiliation: UCD School of Biomolecular and Biomedical Science, UCD Conway Institute and UCD Institute of Sport and Health, UCD Dublin, Belfield, Dublin 4, Ireland.

ABSTRACT
Chronic exposure of pancreatic β-cells to saturated non-esterified fatty acids can lead to inhibition of insulin secretion and apoptosis. Several previous studies have demonstrated that saturated fatty acids such as PA (palmitic acid) are detrimental to β-cell function compared with unsaturated fatty acids. In the present study, we describe the effect of the polyunsaturated AA (arachidonic acid) on the function of the clonal pancreatic β-cell line BRIN-BD11 and demonstrate AA-dependent attenuation of PA effects. When added to β-cell incubations at 100 μM, AA can stimulate cell proliferation and chronic (24 h) basal insulin secretion. Microarray analysis and/or real-time PCR indicated significant AA-dependent up-regulation of genes involved in proliferation and fatty acid metabolism [e.g. Angptl (angiopoietin-like protein 4), Ech1 (peroxisomal Δ3,5,Δ2,4-dienoyl-CoA isomerase), Cox-1 (cyclo-oxygenase-1) and Cox-2, P<0.05]. Experiments using specific COX and LOX (lipoxygenase) inhibitors demonstrated the importance of COX-1 activity for acute (20 min) stimulation of insulin secretion, suggesting that AA metabolites may be responsible for the insulinotropic effects. Moreover, concomitant incubation of AA with PA dose-dependently attenuated the detrimental effects of the saturated fatty acid, so reducing apoptosis and decreasing parameters of oxidative stress [ROS (reactive oxygen species) and NO levels] while improving the GSH/GSSG ratio. AA decreased the protein expression of iNOS (inducible NO synthase), the p65 subunit of NF-κB (nuclear factor κB) and the p47 subunit of NADPH oxidase in PA-treated cells. These findings indicate that AA has an important regulatory and protective β-cell action, which may be beneficial to function and survival in the 'lipotoxic' environment commonly associated with Type 2 diabetes mellitus.

Show MeSH
Related in: MedlinePlus