Saturated fatty acids induce endoplasmic reticulum stress and apoptosis independently of ceramide in liver cells.

Accumulation of lipids in nonadipose tissues can lead to cell dysfunction and cell death, a phenomenon known as lipotoxicity. However, the signaling pathways and mechanisms linking lipid accumulation to cell death are poorly understood. The present study examined the hypothesis that saturated fatty acids disrupt endoplasmic reticulum (ER) homeostasis and promote apoptosis in liver cells via accumulation of ceramide. H4IIE liver cells were exposed to varying concentrations of saturated (palmitate or stearate) or unsaturated (oleate or linoleate) fatty acids. ER homeostasis was monitored using markers of the ER stress response pathway, including phosphorylation of IRE1alpha and eIF2alpha, splicing of XBP1 mRNA, and expression of molecular chaperone (e.g., GRP78) and proapoptotic (CCAAT/enhancer-binding protein homologous protein) genes. Apoptosis was monitored using caspase activity and DNA laddering. Palmitate and stearate induced ER stress, caspase activity, and DNA laddering. Inhibition of caspase activation prevented DNA laddering. Unsaturated fatty acids did not induce ER stress or apoptosis. Saturated fatty acids increased ceramide concentration; however, inhibition of de novo ceramide synthesis did not prevent saturated fatty acid-induced ER stress and apoptosis. Unsaturated fatty acids rescued palmitate-induced ER stress and apoptosis. These data demonstrate that saturated fatty acids disrupt ER homeostasis and induce apoptosis in liver cells via mechanisms that do not involve ceramide accumulation.

[1]  J. D. de Vries,et al.  Saturated but not mono-unsaturated fatty acids induce apoptotic cell death in neonatal rat ventricular myocytes. , 1997, Journal of lipid research.

[2]  Zuzana Dobbie,et al.  Processing of gene expression data generated by quantitative real-time RT-PCR. , 2002, BioTechniques.

[3]  D. Eizirik,et al.  Inverse relationship between cytotoxicity of free fatty acids in pancreatic islet cells and cellular triglyceride accumulation. , 2001, Diabetes.

[4]  G. Shulman,et al.  Mechanism of Insulin Resistance in A-ZIP/F-1 Fatless Mice* , 2000, The Journal of Biological Chemistry.

[5]  Masataka Mori,et al.  Targeted disruption of the Chop gene delays endoplasmic reticulum stress-mediated diabetes. , 2002, The Journal of clinical investigation.

[6]  L. Berglund,et al.  The Risk to Develop NIDDM Is Related to the Fatty Acid Composition of the Serum Cholesterol Esters , 1994, Diabetes.

[7]  M. Czaja.  The future of GI and liver research: editorial perspectives. III. JNK/AP-1 regulation of hepatocyte death. , 2003, American journal of physiology. Gastrointestinal and liver physiology.

[8]  Robert V Farese,et al.  Triglyceride accumulation protects against fatty acid-induced lipotoxicity , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[9]  D. Ron,et al.  Lipid phase perturbations and the unfolded protein response. , 2004, Developmental cell.

[10]  M. Pagliassotti,et al.  Saturated fatty acids promote endoplasmic reticulum stress and liver injury in rats with hepatic steatosis. , 2006, Endocrinology.

[11]  George Kuriakose,et al.  The endoplasmic reticulum is the site of cholesterol-induced cytotoxicity in macrophages , 2003, Nature Cell Biology.

[12]  G. Gores,et al.  Hepatocyte apoptosis and fas expression are prominent features of human nonalcoholic steatohepatitis. , 2003, Gastroenterology.

[13]  R. Kaufman,et al.  The mammalian unfolded protein response. , 2003, Annual review of biochemistry.

[14]  G. Marchesini,et al.  Association of nonalcoholic fatty liver disease with insulin resistance. , 1999, The American journal of medicine.

[15]  R. Kitsis,et al.  Myocyte apoptosis during acute myocardial infarction in the mouse localizes to hypoxic regions but occurs independently of p53. , 1997, The Journal of clinical investigation.

[16]  P. Cullen,et al.  Thapsigargin, a tumor promoter, discharges intracellular Ca2+ stores by specific inhibition of the endoplasmic reticulum Ca2(+)-ATPase. , 1990, Proceedings of the National Academy of Sciences of the United States of America.

[17]  Jeanne M Clark,et al.  Nonalcoholic fatty liver disease: an underrecognized cause of cryptogenic cirrhosis. , 2003, JAMA.

[18]  M. Prentki,et al.  Oleate activates phosphatidylinositol 3-kinase and promotes proliferation and reduces apoptosis of MDA-MB-231 breast cancer cells, whereas palmitate has opposite effects. , 2000, Cancer Research.

[19]  S. Kurinna,et al.  Ceramide Promotes Apoptosis in Lung Cancer-Derived A549 Cells by a Mechanism Involving c-Jun NH2-Terminal Kinase , 2004, Cancer Research.

[20]  S. Linse,et al.  Lipids as cofactors in protein folding: Stereo‐specific lipid–protein interactions are required to form HAMLET (human α‐lactalbumin made lethal to tumor cells) , 2003, Protein science : a publication of the Protein Society.

[21]  D. Schmoll,et al.  Endoplasmic reticulum stress increases glucose-6-phosphatase and glucose cycling in liver cells. , 2006, Endocrinology.

[22]  J. Jessurun,et al.  Sources of fatty acids stored in liver and secreted via lipoproteins in patients with nonalcoholic fatty liver disease. , 2005, The Journal of clinical investigation.

[23]  R. Evershed,et al.  Adverse physicochemical properties of tripalmitin in beta cells lead to morphological changes and lipotoxicity in vitro , 2005, Diabetologia.

[24]  R. Kaufman,et al.  Stress signaling from the lumen of the endoplasmic reticulum: coordination of gene transcriptional and translational controls. , 1999, Genes & development.

[25]  R. Unger,et al.  Fatty acid-induced β cell apoptosis: A link between obesity and diabetes , 1998 .

[26]  R. Kolesnick,et al.  Regulation of ceramide production and apoptosis. , 1998, Annual review of physiology.

[27]  D. Ron,et al.  Diabetes mellitus and exocrine pancreatic dysfunction in perk-/- mice reveals a role for translational control in secretory cell survival. , 2001, Molecular cell.

[28]  R. Kaufman.  Orchestrating the unfolded protein response in health and disease. , 2002, The Journal of clinical investigation.

[29]  Daniel S. Ory,et al.  Palmitate-induced Apoptosis Can Occur through a Ceramide-independent Pathway* , 2001, The Journal of Biological Chemistry.

[30]  M. Rider,et al.  Sustained activation of AMP‐activated protein kinase induces c‐Jun N‐terminal kinase activation and apoptosis in liver cells , 2002, FEBS letters.

[31]  L. Kuller,et al.  Fatty liver in type 2 diabetes mellitus: relation to regional adiposity, fatty acids, and insulin resistance. , 2003, American journal of physiology. Endocrinology and metabolism.

[32]  L. Glimcher,et al.  Endoplasmic Reticulum Stress Links Obesity, Insulin Action, and Type 2 Diabetes , 2004, Science.

[33]  E. Zafrani.  Non-alcoholic fatty liver disease: an emerging pathological spectrum , 2003, Virchows Archiv.

[34]  C. Day,et al.  Steatohepatitis: a tale of two "hits"? , 1998, Gastroenterology.

[35]  Hiderou Yoshida,et al.  IRE1-mediated unconventional mRNA splicing and S2P-mediated ATF6 cleavage merge to regulate XBP1 in signaling the unfolded protein response. , 2002, Genes & development.

[36]  J. Finnie.  Effect of tunicamycin on hepatocytes in vitro. , 2001, Journal of comparative pathology.

[37]  F. Schaffner,et al.  Nonalcoholic fatty liver disease. , 1986, Progress in liver diseases.

[38]  K. Maedler,et al.  Distinct effects of saturated and monounsaturated fatty acids on beta-cell turnover and function. , 2001, Diabetes.

[39]  H. Kaneto,et al.  Involvement of Endoplasmic Reticulum Stress in Insulin Resistance and Diabetes* , 2005, Journal of Biological Chemistry.

[40]  E McEwen,et al.  Translational control is required for the unfolded protein response and in vivo glucose homeostasis. , 2001, Molecular cell.

[41]  M. Donath,et al.  Monounsaturated fatty acids prevent the deleterious effects of palmitate and high glucose on human pancreatic beta-cell turnover and function. , 2003, Diabetes.

[42]  D. Steindler,et al.  GM1-ganglioside-mediated activation of the unfolded protein response causes neuronal death in a neurodegenerative gangliosidosis. , 2004, Molecular cell.

[43]  T. Honjo,et al.  Inhibition of Carnitine Palmitoyltransferase I Augments Sphingolipid Synthesis and Palmitate-induced Apoptosis* , 1997, The Journal of Biological Chemistry.

[44]  R. Kaufman,et al.  A trip to the ER: coping with stress. , 2004, Trends in cell biology.