Protective Effects of Oleic Acid Against Palmitic Acid-Induced Apoptosis in Pancreatic AR42J Cells and Its Mechanisms

Abstract

Palmitic acid (PAM), one of the most common saturated fatty acid (SFA) in animals and plants, has been shown to induce apoptosis in exocrine pancreatic AR42J cells. In this study, we investigated cellular mechanisms underlying protective effects of oleic acid (OLA) against the lipotoxic actions of PAM in AR42J cells. Exposure of cells to long-chain SFA induced apoptotic cell death determined by MTT cell viability assay and Hoechst staining. Co-treatment of OLA with PAM markedly protected cells against PAM-induced apoptosis. OLA significantly attenuated the PAM-induced increase in the levels of pro-apoptotic Bak protein, cleaved forms of apoptotic proteins (caspase-3, PARP). On the contrary, OLA restored the decreased levels of anti-apoptotic Bcl-2 family proteins (Bcl-2, Bcl-xL, and Mcl-1) in PAM-treated cells. OLA also induced up-regulation of the mRNA expression of Dgat2 and Cpt1 genes which are involved in triacylglycerol (TAG) synthesis and mitochondrial β-oxidation, respectively. Intracellular TAG accumulation was increased by OLA supplementation in accordance with enhanced expression of Dgat2 gene. These results indicate that restoration of anti-apoptotic/pro-apoptotic protein balance from apoptosis toward cell survival is involved in the cytoprotective effects of OLA against PAM-induced apoptosis in pancreatic AR42J cells. In addition, OLA-induced increase in TAG accumulation and up-regulation of Dgat2 and Cpt1 gene expressions may be possibly associated in part with the ability of OLA to protect cells from deleterious actions of PAM.

Keywords: Anti-apoptotic/pro-apoptotic proteins; Apoptosis; Oleic acid; Palmitic acid; Pancreatic AR42J cells.

Fig. 1

Co-treatment with oleic acid (OLA) inhibits palmitic acid (PAM)-induced apoptosis in AR42J cells. (A) Different saturated fatty acids induce cell death. Cells were incubated with 10~250 µM of myristic acid (C14:0), palmitic acid (C16:0), stearic acid (C18:0), and arachidic acid (C20:0) for 48 h and then, cell viability was estimated by MTT assay. (B) Different unsaturated fatty acids inhibit PAM-induced cell death. Cells were incubated with 10~250 µM of palmitoleic acid (C16:1), oleic acid (C18:1), linoleic acid (C18:2), and linolenic acid (C18:3) in the presence of 250 µM PAM for 48 h. (C) OLA inhibits PAM-induced apoptosis. Photographs shown on the left are the representative images of Hoechst 33342-stained cells exposed to PAM and OLA either alone or in combination for 48 h. The percentage of apoptotic cells with condensed and segmented chromatin (arrows) is calculated. The results are presented as means±SEM of 4~6 independent experiments. ^**p<0.01, ^***p<0.001, compared to untreated control and ^#p<0.05, ^##p<0.01, ^###p<0.001, compared to PAM-treated cells.

Fig. 2

Effects of oleic acid (OLA) on the levels of anti-apoptotic (Bcl-2, Bcl-xL, Mcl-1) and pro-apoptotic (Bak) Bcl-2 family proteins in palmitic acid (PAM)-treated AR42J cells. Cells were incubated with 250 µM OLA for 6~48 h in the presence or absence of 250 µM PAM. The levels of Bcl-2, Bcl-xL, Mcl-1, and Bak were detected by Western blotting. The results on the right are presented as means±SEM of 3~7 independent experiments. The expression level of each protein was normalized to that of a housekeeping protein, β-actin. ^*p<0.05, ^**p<0.01, ^***p<0.001, compared to untreated control and ^#p<0.05, ^##p<0.01, ^###p<0.001, compared to PAM-treated cells.

Fig. 3

Effects of oleic acid (OLA) on the levels of cleaved apoptotic proteins (caspase-3, PARP) in palmitic acid (PAM)-treated AR42J cells. Cells were incubated with 250 µM OLA for 6~48 h in the presence or absence of 250 µM PAM. The levels of cleaved caspase-3 (C-Casp3) and cleaved PARP (C-PARP) were detected by Western blotting. The results on the right are presented as means±SEM of 3 independent experiments. The expression level of each protein was normalized to that of a housekeeping protein, β-actin. ^*p<0.05, ^***p<0.001, compared to untreated control and ^##p<0.01, ^###p<0.001, compared to PAM-treated cells.

Fig. 4

Oleic acid (OLA) induces up-regulation of Dgat2 and Cpt1 gene expressions in AR42J cells. Cells were incubated with 250 µM OLA for 48 h in the presence or absence of 250 µM palmitic acid (PAM). The mRNA expressions of Dgat1, Dgat2, and Cpt1 genes were determined by RT-PCR. Density of each band was quantified using ImageQuant software. The results are presented as means±SEM of 3~6 independent experiments. The expression level of each gene was normalized to that of a housekeeping gene, β-actin. ^*p<0.05, ^***p<0.001, compared to untreated control and ^#p<0.05, ^###p<0.001, compared to PAM-treated cells.

Fig. 5

Oleic acid (OLA) increases the accumulation of intracellular triglyceride (TAG) in AR42J cells. Cells were incubated with 250 µM OLA for 48 h in the presence or absence of 250 µM palmitic acid (PAM). Photographs shown on the left are the representative images of Oil Red O-stained cells exposed to PAM and OLA either alone or in combination for 48 h. After staining cells with Oil Red O, stained dye was extracted with isopropanol and absorbance at 500 nm was measured to estimate the relative content of intracellular TAG. The results on the right are presented as means±SEM of 6 independent experiments. ^**p<0.01, compared to untreated control and ^#p<0.05, compared to PAM-treated cells.