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Saturated fatty acid induction of endoplasmic reticulum stress and apoptosis in human liver cells via the PERK/ATF4/CHOP signaling pathway

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Abstract

Accumulation of saturated fatty acids in the liver can cause nonalcoholic fatty liver disease (NAFLD). This study investigated saturated fatty acid induction of endoplasmic reticulum (ER) stress and apoptosis in human liver cells and the underlying causal mechanism. Human liver L02 and HepG2 cell lines were exposed to the saturated fatty acid sodium palmitate. MTT assay was used for cell viability, flow cytometry and Hoechst 33258 staining for apoptosis, RT-PCR for mRNA expression, and Western blot for protein expression. Silence of PRK-like ER kinase (PERK) expression in liver cells was through transient transfection of PERK shRNA. Treatment of L02 and HepG2 cells with sodium palmitate reduced cell viability through induction of apoptosis. Sodium palmitate also induced ER stress in the cells, indicated by upregulation of PERK phosphorylation and expression of BiP, ATF4, and CHOP proteins. Sodium palmitate had little effect on activating XBP-1, a common target of the other two canonical sensors of ER stress, ATF6, and IRE1. Knockdown of PERK gene expression suppressed the PERK/ATF4/CHOP signaling pathway during sodium palmitate-induced ER stress and significantly inhibited sodium palmitate-induced apoptosis in L02 and HepG2 cells. Saturated fatty acid-induced ER stress and apoptosis in these human liver cells were enacted through the PERK/ATF4/CHOP signaling pathway. Future study is warranted to investigate the role of these proteins in mediating saturated fatty acid-induced NAFLD in animal models.

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Abbreviations

7-AAD:

7-Amino-actinomycin D

ATF4:

Activated transcription factor 4

BCA:

Bicinchoninic acid

BiP:

Binding immunoglobulin protein

BSA:

Bovine serum albumin

ER:

Endoplasmic reticulum

GRP78:

Glucose-regulated protein 78

FFA:

Free fatty acid

JNK:

c-Jun NH2-terminal kinase

MTT:

Methyl-thiazolyl-tetrazolium

NAFLD:

Nonalcoholic fatty liver disease

NASH:

Nonalcoholic steatohepatitis

PBS:

Phosphate-buffered saline

PKR:

Protein kinase dependent on RNA

PERK:

PKR-like ER kinase

PVDF:

Polyvinylidene difluoride

RIPA:

Radioimmunoprecipitation

TBS:

Tris-buffered saline

TBST:

Triton X-100-TBS

UPR:

Unfolded protein response

XBP-1:

X-box-binding protein 1

CHOP:

C/EBP-homologous protein

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Acknowledgments

Our study was supported by grants from the Natural Science Foundation of China (#30871160), the Natural Science Foundation of Chongqing, China (#CSTC-2008BB5404) and the Project of Medical Science and Technology of Chongqing (#2008-2-401). We sincerely thank the staff of the Department of Gastroenterology and Hepatology at the Second Affiliated Hospital of Chongqing Medical University for their constant and unselfish help. We thank Ms. Yingxia Xiang and Mr. Jing Wang for their consistent and illuminating technical assistance. We also thank Medjaden Bioscience, Hong Kong, China, for assistance in the preparation of this manuscript.

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Authors and Affiliations

  1. Department of Gastroenterology and Hepatology, 2nd Affiliated Hospital of Chongqing Medical University, No. 74 Linjiang Road, Chongqing, 400010, China

    Jie Cao, Hui-Hong Yu, Bo Ning, Juan Chen, Wen-Hui Cheng, Wei Shen & Zhao-Xia Yang

  2. Department of Internal Medicine, The Children’s Hospital of Shenzhen, Shenzhen, China

    Dong-Ling Dai

  3. Department of Intensive-Care Medicine, The Central Hospital of Jiangjin, Chongqing, China

    Long Yao

  4. Department of Digestive Diseases, The First People’s Hospital of Liangshan, Liangshan, China

    Qin Zhang

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  1. Jie Cao
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  9. Wei Shen
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Correspondence to Wei Shen or Zhao-Xia Yang.

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Cao, J., Dai, DL., Yao, L. et al. Saturated fatty acid induction of endoplasmic reticulum stress and apoptosis in human liver cells via the PERK/ATF4/CHOP signaling pathway. Mol Cell Biochem 364, 115–129 (2012). https://doi.org/10.1007/s11010-011-1211-9

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