Effect of α-linolenic acid on endoplasmic reticulum stress-mediated apoptosis of palmitic acid lipotoxicity in primary rat hepatocytes

Abstract

Background: Hepatic inflammation and degeneration induced by lipid depositions may be the major cause of nonalcoholic fatty liver disease (NAFLD). In this study, we investigated the effects of saturated and unsaturated fatty acids (FA) on apoptosis in primary rat hepatocytes.

Methods: The primary rat hepatocytes were treated with palmitic acid and/or α-linolenic acid in vitro. The expression of proteins associated with endoplasmic reticulum (ER) stress, apoptosis, caspase-3 levels were detected after the treatment.

Results: The treatment with palmitic acid produced a significant increase in cell death. The unfolded protein response (UPR)-associated genes CHOP, GRP78, and GRP94 were induced to higher expression levels by palmitic acid. Co-treatment with α-linolenic acid reversed the apoptotic effect and levels of all three indicators of ER stress exerted by palmitic acid. Tunicamycin, which induces ER stress produced similar effects to those obtained using palmitic acid; its effects were also reversed by α-linolenic acid.

Conclusions: α-Linolenic acid may provide a useful strategy to avoid the lipotoxicity of dietary palmitic acid and nutrient overload accompanied with obesity and NAFLD.

Figure 1

Effects of PA on primary rat hepatocytes. (A) Apoptotic cell death elicited by increasing concentrations of palmitic acid (PA). (B) Treatment with PA for 16 h produced significant and concentration-dependent effects on MTT reduction and LDH release. Data represent mean ± S.E.M., n = 5,*P < 0.05 vs. control (0 μmol/l palmitic acid), **P < 0.05 vs. palmitic acid-only cells. (C) Treatment of primary rat hepatocytes with 250 μmol/L PA produced a significant increase in activity of caspase-3. For comparison, the effects of tunicamycin(Tn; 5 μg/ml)are also shown. Data represent mean ± S.E.M., n = 5,*P < 0.05 vs. control (0 μmol/l palmitic acid), **P < 0.05 vs. palmitic acid-only cells.

Figure 2

α-Linolenic acid protects primary rat hepatocytes against ER stress induced by palmitic acid. (A)Western blot and (B) densitometric analysis demonstrating the reduction of palmitic acid (PA)-induced GRP78 expression by 150 or 250 μmol/l α-linolenic acid (ALA) after 16 h. (A)Western blot and (C) densitometric analysis of GRP94 expression after 16 h incubation of cells with 250 μmol/l palmitic acid(PA) in presence of 150 or 250 μmol/l α-linolenic acid (ALA). (A)Western blot and (D) densitometric analysis demonstrating the reduction of palmitic acid (PA)-induced CHOP expression by 150 or 250 μmol/l α-linolenic acid (ALA) after 16 h. Data represent mean ± S.E.M., n = 5, *P < 0.05 vs. LG, low glucose control (0 μmol/l palmitic acid), **P < 0.05 vs. palmitic acid-only cells.

Figure 3

α-Linolenic acid protects against dysfunction and apoptosis of primary rat hepatocytes induced by tunicamycin. Relative cell death treated with 5 μg/ml tunicamycin (Tn) for 16 h in presence of 150 or 250 μmol/l α-linolenic acid (ALA). Data represent mean ± S.E.M., n = 5, *P < 0.05 vs. LG, low glucose control set to 1 (0 μmol/l palmitic acid), **P < 0.05 vs. tunicamycin-only cells.

Figure 4

α-Linolenic acid protects primary rat hepatocytes against ER stress induced by tunicamycin. (A)Western blot image and densitometric analysis of (B) CHOP and (C) GRP78 expression in cells treated with tunicamycin (Tn; 5 μg/ml) in presence of increasing concentrations of α-linolenic acid (ALA) for 16 h. Data represent mean ± S.E.M., n = 5, *P < 0.05 vs. LG, low glucose control (0 μmol/l palmitic acid), **P < 0.05 vs. tunicamycin-only cells.