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

Zhang Y, Xue R, Zhang Z, Yang X, Shi H - Lipids Health Dis (2012)

Bottom Line: 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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Anesthesia Department, the Second Affiliated Hospital of Xi'an Jiaotong University, No, 157, West 5th Road, Xi'an, Shaanxi Province-710004, China.

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.

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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.
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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.

Mentions: The unfolded protein response (UPR) is the main pathway related to ER stress [20]. Therefore, the expression of genes involved in the UPR, ATF4, CHOP, GADD34, GRP78, and GRP94, was assessed after fatty acid treatment for 16 h (Figure 2 and 3). Significantly increased ATF4, CHOP, GADD34, and GRP78 mRNA expression was observed upon treatment with thapsigargin (2 μM); no changes in GRP94 expression were observed (Figure 2E). Changes in the H4IIE cells were observed upon cotreatment with palmitic and linoleic acid. Significantly increased CHOP mRNA expression was observed upon cotreatment (125 μM) whereas GRP78 mRNA expression was significantly decreased with cotreatment at both concentrations (Figure 2B and 2D). However, these changes in the H4IIE cells were not observed at the protein level for either of these genes (Figure 3A and 3B). Increased GRP78 and GRP94 protein expression (Figure 3B and 3C, respectively) were also observed upon thapsigargin treatment; however, no changes in CHOP protein expression were observed (Figure 3A). In contrast, treatment of H4IIE cells with 250 μM palmitic or linoleic acid alone failed to alter the mRNA or protein expression of any of these genes. A representative Western blot for each protein after the indicated treatments is shown in Figure 3D.


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

Zhang Y, Xue R, Zhang Z, Yang X, Shi H - Lipids Health Dis (2012)

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.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3306830&req=5

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.
Mentions: The unfolded protein response (UPR) is the main pathway related to ER stress [20]. Therefore, the expression of genes involved in the UPR, ATF4, CHOP, GADD34, GRP78, and GRP94, was assessed after fatty acid treatment for 16 h (Figure 2 and 3). Significantly increased ATF4, CHOP, GADD34, and GRP78 mRNA expression was observed upon treatment with thapsigargin (2 μM); no changes in GRP94 expression were observed (Figure 2E). Changes in the H4IIE cells were observed upon cotreatment with palmitic and linoleic acid. Significantly increased CHOP mRNA expression was observed upon cotreatment (125 μM) whereas GRP78 mRNA expression was significantly decreased with cotreatment at both concentrations (Figure 2B and 2D). However, these changes in the H4IIE cells were not observed at the protein level for either of these genes (Figure 3A and 3B). Increased GRP78 and GRP94 protein expression (Figure 3B and 3C, respectively) were also observed upon thapsigargin treatment; however, no changes in CHOP protein expression were observed (Figure 3A). In contrast, treatment of H4IIE cells with 250 μM palmitic or linoleic acid alone failed to alter the mRNA or protein expression of any of these genes. A representative Western blot for each protein after the indicated treatments is shown in Figure 3D.

Bottom Line: 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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Anesthesia Department, the Second Affiliated Hospital of Xi'an Jiaotong University, No, 157, West 5th Road, Xi'an, Shaanxi Province-710004, China.

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.

Show MeSH
Related in: MedlinePlus