Palmitic and linoleic acids induce ER stress and apoptosis in hepatoma cells

Abstract

Objectives: Hepatic inflammation and degeneration induced by lipid depositions may be the major cause of nonalcoholic fatty liver disease. In this study, we tried to investigate the effects of saturated and unsaturated fatty acids on hepatoma cell apoptosis.

Methods: H4IIE liver cells were treated with palmitic acid, linoleic acid, or both with or without the calcium-specific chelator BAPTA-AM after which the expression of proteins associated with endoplasmic reticulum (ER) stress, apoptosis, caspase-3 levels, and calcium flux were measured.

Results: Palmitic or linoleic acid (250 μM) induced H4IIE cell apoptosis, which required calcium flux but not caspase-3. Apoptosis was not observed when cells were co-treated with linoleic acid (125 μM) and palmitic acid (250 μM). Importantly, the release of cytochrome C from mitochondria into cytoplasm during cell apoptosis was specifically detected only when linoleic acid (125 μM), but not palmitic acid (250 μM), was added to the cells. Depletion of intracellular calcium flux by the calcium-specific chelator, BAPTA-AM, abolished linoleic acid-induced apoptosis. Moreover, in the presence of BAPTA-AM, expression of the unfolded protein response (UPR)-associated genes, CHOP, GRP78, and GRP94, was induced by linoleic acid, but not palmitic acid.

Conclusions: The results suggest that linoleic acid promotes cell apoptosis through the release of cytochrome C, only if the intracellular calcium flux is unperturbed and intact. These results confirm that ER stress contributes to fatty acid-induced liver cell apoptosis.

Figure 1

Effects of palmitic and linoleic acids on apoptosis, caspase-3 activity and calcium flux in H4IIE liver cells. Rat liver cells were treated with thapsigargin (TG), palmitic acid (PA, 250 μM), linoleic acid (LA, 250 μM) alone or the combination of PA (250 μM) and LA (125 or 250 μM) for 16 hours. The cells were treated with phosphate buffered saline (PBS) as Control group. (A) Apoptotic index was measured by determining the cytoplasmic histone-associated DNA fragments with an ELISA assay. (B) The caspase-3 level was detected according to a colorimetric assay that measures the extent of cleavage of a caspase-specific peptide derivatized with p-nitroalanine. (C) The calcium level was determined with the Fluo-4 NW calcium assay kit. a: P < 0.05 compared to Control; b: P < 0.05 compared to PA; c: P < 0.05 compared to TG; d: P < 0.05 compared to LA; e: P < 0.05 compared to PA+LA (125uM) after Bonferroni adjustment. n = 6 in each group.

Figure 2

Effects of palmitic and linoleic acids on the expression of UPR-associated genes in mRNA level. The tumor cells were treated with TG, PA(250 μM), LA (250 μM) alone or PA+LA (125 μM or 250 μM) for 16 hr. The cells were treated with PBS as Control group. The levels of the respective mRNAs for (A) ATF4, (B) CHOP, (C) GADD34, (D) GRP78, and (E) GRP94 were determined by real-time RT-PCR relative to β2-microglobulin. a: P < 0.05 compared to Control; b: P < 0.05 compared to PA; c: P < 0.05 compared to TG; d: P < 0.05 compared to LA after Bonferroni adjustment. n = 3 in each group.

Figure 3

Effects of palmitic and linoleic acids on the expression of CHOP, GRP78, and GRP94 in protein level. The Rat liver tumor cells were treated with TG, PA(250 μM), LA (250 μM) alone or PA+LA (125 μM or 250 μM) for 16 hr. The cells were treated with PBS as Control group. The levels of the respective proteins were determined by Western blot and normalized to the level of β-actin (n = 3 in each group). The relative expression of protein (A) CHOP, (B) GRP78 and (C) GRP94 were summarized and representative western blot photos were presented in (D). a: P < 0.05 compared to Control; b: P < 0.05 compared to PA; c: P < 0.05 compared to TG after Bonferroni adjustment. In (D), lane 1 and 5 were loaded with Control, lane 2 for TG, lane 3 and 6 for PA, lane 4 for LA, lane 7 for PA+LA (125 μM) and lane 8 for PA+LA (250 μM).

Figure 4

Impact of calcium flux on cell death, cytochrome C release, and expression of CHOP, GRP78, and GRP94 genes in cells treated with palmitic acid or linoleic acid in the absence and presence of 10 μM calcium-specific chelator BAPTA-AM. H4IIE liver cells were treated with PA or LA (125 μM) in the absence or presence of 10 μM calcium-specific chelator BAPTA-AM for 16 hours (n = 3 in each group).