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Effect of Saturated Stearic Acid on MAP Kinase and ER Stress Signaling Pathways During Apoptosis Induction in Human Pancreatic β-Cells Is Inhibited by Unsaturated Oleic Acid

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Effect of Saturated Stearic Acid on MAP Kinase and ER Stress Signaling Pathways During Apoptosis Induction in Human Pancreatic β-Cells Is Inhibited by Unsaturated Oleic Acid

Jan Šrámek et al. Int J Mol Sci.

Abstract

It has been shown that saturated fatty acids (FAs) have a detrimental effect on pancreatic β-cells function and survival, leading to apoptosis, whereas unsaturated FAs are well tolerated and are even capable of inhibiting the pro-apoptotic effect of saturated FAs. Molecular mechanisms of apoptosis induction and regulation by FAs in β-cells remain unclear; however, mitogen-activated protein (MAP) kinase and endoplasmic reticulum (ER) stress signaling pathways may be involved. In this study, we tested how unsaturated oleic acid (OA) affects the effect of saturated stearic acid (SA) on the p38 mitogen-activated protein kinase (MAPK) and extracellular signal-regulated kinase (ERK) pathways as well as the ER stress signaling pathways during apoptosis induction in the human pancreatic β-cells NES2Y. We demonstrated that OA is able to inhibit all effects of SA. OA alone has only minimal or no effects on tested signaling in NES2Y cells. The point of OA inhibitory intervention in SA-induced apoptotic signaling thus seems to be located upstream of the discussed signaling pathways.

Keywords: NES2Y; apoptosis; endoplasmic reticulum (ER) stress; extracellular signal-regulated kinase (ERK); fatty acids; p38 mitogen-activated protein kinase (MAPK); pancreatic β-cells.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Effect of 1 mM stearic acid (SA), 1 mM stearic acid applied simultaneously with 0.2 mM oleic acid (OA), and 0.2 mM oleic acid (see Section 4) on (A) cell growth and viability, (B) the level of cleaved caspase-9 (C9), caspase-8 (C8), caspase-7 (C7), and PARP in NES2Y cells. Cells incubated without fatty acids represented control cells. (A) Cells were seeded at a concentration of 5 × 103 cells/100 µL of culture medium per well of 96-well plate (see Section 4). The number of living cells was determined after 96 h of incubation. The number of cells of the inoculum is shown as a dashed line. Each column represents the mean of four separate cultures ± standard error of the mean (SEM). ** p < 0.01 when comparing the effect of 1 mM stearic acid applied together with 0.2 mM oleic acid and the effect of 1 mM stearic acid alone. (B) After 18 h of incubation (see Section 4), the levels of individual proteins were assessed using Western blot analysis and relevant antibodies (see Section 4). Actin was included to confirm equal protein loading. The data presented were obtained in one representative experiment from at least three independent experiments. (C) Densitometric analysis of data from Western blotting are also shown. Each column represents the mean of three experimental values ± SEM. ** p < 0.01 when comparing the effect of SA with control cells, ++ p < 0.05 when comparing the effect of SA plus OA with the effect of SA alone.
Figure 2
Figure 2
Effect of 1 mM stearic acid (SA), 1 mM stearic acid applied simultaneously with 0.2 mM oleic acid (OA), and 0.2 mM oleic acid (see Section 4) on (A) the levels of phospho-MKK3/6, p38 MAPK, phospho-p38 MAPK, and phospho-MAPKAPK-2 (the p38 MAPK signaling pathway); and (B) the levels of phospho-c-Raf, phospho-MEK1/2, ERK1/2, and phospho-ERK1/2 (the ERK signaling pathway) in NES2Y cells. Cells incubated without fatty acids represented control cells. After 3, 6, 12, and 24 h of incubation (see Section 4), the levels of individual proteins were assessed using Western blot analysis and relevant antibodies (see Section 4). Actin was included to confirm equal protein loading. The data presented were obtained in one representative experiment from at least three independent experiments. Densitometric analysis of data from Western blotting is also shown. The analysis was carried out for 12 h after fatty acids application in the case of the p38 MAPK pathway and for 24 h in the case of the ERK pathway. Each column represents the mean of three experimental values ± SEM. ** p < 0.01 when comparing the effect of SA with control cells, + p < 0.05, ++ p < 0.01 when comparing the effect of SA plus OA with the effect of SA alone.
Figure 3
Figure 3
Effect of 1 mM stearic acid (SA), 1 mM stearic acid applied simultaneously with 0.2 mM oleic acid (OA), and 0.2 mM oleic acid (see Section 4) on the level of ER stress markers BiP and CHOP in NES2Y cells. Cells incubated without fatty acids represented control cells. After 3, 6, 12, and 24 h of incubation (see Section 4), the levels of individual proteins were assessed using Western blot analysis and relevant antibodies (see Section 4). Actin was included to confirm equal protein loading. The data presented were obtained in one representative experiment from at least three independent experiments. Densitometric analysis of data from Western blotting is also shown. The analysis was carried out for 24 h after fatty acids application. Each column represents the mean of three experimental values ± SEM. ** p < 0.01 when comparing the effect of SA with control cells, + p < 0.05, ++ p < 0.01 when comparing the effect of SA plus OA with the effect of SA alone.
Figure 4
Figure 4
Effect of 1 mM stearic acid (SA), 1 mM stearic acid applied simultaneously with 0.2 mM oleic acid (OA), and 0.2 mM oleic acid (see Section 4) on the levels of (A) eIF2α and phospho-eIF2α, and (B) IRE1α, phospho-IRE1α, JNK, phospho-JNK, c-Jun, and phospho-c-Jun in NES2Y cells. Cells incubated without fatty acids represented control cells. After 3, 6, 12, and 24 h of incubation (see Section 4) (A,B), the levels of individual proteins were assessed using Western blot analysis and relevant antibodies (see Section 4). Actin was included to confirm equal protein loading. (C) Densitometric analysis of data from Western blotting is also shown. The analysis was carried out for 6 h after fatty acids application in the case of phospho-eIF2α, for 12 h in the case of phospho-JNK, c-Jun and phospho-c-Jun, and for 24 h in the case of IRE1α and phospho-IRE1α. Each column represents the mean of three experimental values ± SEM. * p < 0.05, ** p < 0.01 when comparing the effect of SA with control cells. + p < 0.05, ++ p < 0.01 when comparing the effect of SA plus OA with the effect of SA alone.
Figure 5
Figure 5
Effect of 1 mM stearic acid (SA), 1 mM stearic acid applied simultaneously with 0.2 mM oleic acid (OA), and 0.2 mM oleic acid (see Section 4) on XBP1 splicing in NES2Y cells. Cells incubated without fatty acids represented control cells. After 3, 6, 12, and 24 h of incubation (see Section 4), the XBP1 splicing was assessed by RT-PCR using relevant primers (see Section 4). NES2Y cells were treated with 1 μM thapsigargin as a positive control of XBP1 splicing. GAPDH (glyceraldehyde-3-phosphate dehydrogenase) was used as a control gene for RT-PCR. The data presented were obtained in one representative experiment from at least three independent experiments. Densitometric analysis of data from RT-PCR is also shown. The analysis was carried out for 24 h after fatty acids application. Each column represents the mean of three experimental values ± SEM. ** p < 0.01 when comparing the effect of SA with control cells, ++ p < 0.01 when comparing the effect of SA plus OA with the effect of SA alone.

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