Linking endoplasmic reticulum stress to cell death in hepatocytes: roles of C/EBP homologous protein and chemical chaperones in palmitate-mediated cell death

Abstract

Prolonged endoplasmic reticulum (ER) stress and activation of the unfolded protein response (UPR) have been linked to apoptosis via several mechanisms, including increased expression of C/EBP homologous protein (Chop). Increased long-chain fatty acids, in particular saturated fatty acids, induce ER stress, Chop expression, and apoptosis in liver cells. The first aim of the present study was to determine the role of Chop in lipid-induced hepatocyte cell death and liver injury induced by a methionine-choline-deficient diet. Albumin-bound palmitate increased Chop gene and protein expression in a dose-dependent fashion in H4IIE liver cells. siRNA-mediated silencing of Chop in H4IIE liver cells reduced thapsigargin-mediated cell death by approximately 40% and delayed palmitate-mediated cell death, but only at high concentrations of palmitate (400-500 microM). Similar results were observed in primary hepatocytes isolated from Chop-knockout mice. Indices of liver injury were also not reduced in Chop-knockout mice provided a methionine-choline-deficient diet. To ascertain whether ER stress was linked to palmitate-induced cell death, primary hepatocytes were incubated in the absence or presence of the chemical chaperones taurine-conjugated ursodeoxycholic acid or 4-phenylbutyric acid. The presence of either of these chemical chaperones protected liver cells from palmitate-mediated ER stress and cell death, in part, via inhibition of JNK activation. These data suggest that ER stress is linked to palmitate-mediated cell death via mechanisms that include JNK activation.

Fig. 1.

Fatty acid-mediated regulation of Chop gene and protein expression. H4IIE liver cells were incubated for varying periods of time in control medium (LG), control medium supplemented with albumin-bound oleate at 300 μM (O300), or palmitate at 100 (P100), 200 (P200), or 300 μM (P300). A: Chop mRNA. B: Chop protein. C: cell number and ELISA-based cell death as a function of palmitate concentration and duration of exposure. Data in graphs are reported as the mean ± SD of triplicate samples from 5 to 7 independent experiments. Representative gels are shown for Chop and actin protein expression. *Significantly (P < 0.05) different from LG and O300.

Fig. 2.

Small interfering RNA (siRNA)-mediated knockdown of Chop. H4IIE liver cells were transfected with control siRNA or Chop siRNA, as described in materials and methods, and cells were harvested for Western blot or RNA analysis following a 16-h incubation in LG or control medium supplemented with thapsigargin (Thap; 450 nM), 200 μM albumin-bound oleate (O200), or 200 μM albumin-bound palmitate (P200). A: Western blot analysis of Chop and actin protein in cells that were not transfected (control) and cells that were transfected with either control scrambled oligonucleotides (siRNA control) or Chop siRNA at the indicated concentrations. The gel shown is representative of 4 independent experiments. B: DNA fragmentation, ELISA-based cell death, caspase-3 activity, and cell survival based on MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] assay. Data in graphs are reported as the mean ± SD of triplicate samples from 5 independent experiments. Gel for DNA fragmentation is representative from a total of 5 independent experiments. *Significantly (P < 0.05) different from siRNA control.

Fig. 3.

Fatty acid and Thap-mediated cell death in primary hepatocytes from wild-type (WT) and Chop-knockout (KO) mice. Primary hepatocytes were incubated in LG or control medium supplemented with Thap (450 nM), albumin-bound oleate at 200 μM (O200), or palmitate at 200 μM (P200) for 16 h. Measurements of cell integrity include DNA fragmentation (representative gel), ELISA-based cell death, caspase-3 activity, and MTT assay. Data in graphs are reported as the mean ± SD of triplicate samples from a total of 6 independent experiments. *Significantly (P < 0.05) different from WT.

Fig. 4.

Liver injury in WT and Chop-KO mice. WT and Chop-KO mice were provided a purified high-starch diet (STD) or a methionine-choline-deficient (MCD) diet for 3 wk. A: hematoxylin and eosin staining. B: liver enzymes, alanine aminotransferase (ALT), and aspartate aminotransferase (AST). C: collagen-α1 (COL1a1), α-smooth muscle actin (SMA), and transforming growth factor-β1 (TGFb1) mRNA from liver samples. Data in graphs are reported as the mean ± SD; n = 6/group. *Significantly (P < 0.05) different from STD.

Fig. 5.

Cell integrity in primary hepatocytes isolated from WT mice. Primary hepatocytes were incubated in LG or control medium supplemented with Thap (450 nM), albumin-bound oleate at 300 μM (O300) or palmitate at 100 (P100), 200 (P200), or 300 μM (P300) in the absence or presence of chemical chaperones, taurine-conjugated ursodeoxycholic acid (TUDCA; 200 μM), or 4-phenylbutyric acid (PBA; 500 μM) for 16 h. Measurements of cell integrity include ELISA-based cell death (A), caspase-3 activity (B), and MTT assay (C). Data in graphs are reported as the mean ± SD of triplicate samples from a total of 6 independent experiments. *Significantly (P < 0.05) different from no addition.

Fig. 6.

JNK activity and cell integrity in primary hepatocytes. Primary hepatocytes were incubated in LG or control medium supplemented with Thap (450 nM), albumin-bound oleate at 300 μM (O300), or palmitate at 100 (P100), 200 (P200), or 300 μM (P300) in the absence or presence of chemical chaperones, TUDCA (200 μM), PBA (500 μM), or SP600125 (10 μM) for 16 h. A and B: ratio of phosphorylation of c-Jun to total JNK. C: ELISA-based cell death. D: caspase-3 activity. Data in graphs are reported as the mean ± SD of triplicate samples from a total of 6 independent experiments. *Significantly (P < 0.05) different from no addition or SP600125. Representative Western blots are provided in Supplemental Fig. S9.