Induction of liver steatosis and lipid droplet formation in ATF6alpha-knockout mice burdened with pharmacological endoplasmic reticulum stress

Abstract

Accumulation of unfolded proteins in the endoplasmic reticulum (ER) activates homeostatic responses collectively termed the unfolded protein response. Among the three principal signaling pathways operating in mammals, activating transcription factor (ATF)6alpha plays a pivotal role in transcriptional induction of ER-localized molecular chaperones and folding enzymes as well as components of ER-associated degradation, and thereby mouse embryonic fibroblasts deficient in ATF6alpha are sensitive to ER stress. However, ATF6alpha-knockout mice show no apparent phenotype under normal growing conditions. In this report, we burdened mice with intraperitoneal injection of the ER stress-inducing reagent tunicamycin and found that wild-type mice were able to recover from the insult, whereas ATF6alpha-knockout mice exhibited liver dysfunction and steatosis. Thus, ATF6alpha-knockout mice accumulated neutral lipids in the liver such as triacylglycerol and cholesterol, which was ascribable to blockage of beta-oxidation of fatty acids caused by decreased mRNA levels of the enzymes involved in the process, suppression of very-low-density lipoprotein formation due to destabilized apolipoprotein B-100, and stimulation of lipid droplet formation resulting from transcriptional induction of adipose differentiation-related protein. Accordingly, the hepatocytes of tunicamycin-injected knockout mice were filled with many lipid droplets. These results establish links among ER stress, lipid metabolism, and steatosis.

Figure 1.

Effect of tunicamycin injection on body weight and liver appearance of ATF6α +/+ and −/− mice. (A) Tunicamycin (Tm) or LPS was intraperitoneally injected into ATF6α +/+ and −/− mice each at a dose of 1 or 5 μg/g body weight, respectively. Body weights of injected mice were determined daily for 7 d and are presented after normalization to that at day 0 of injection as the means ± SEM (n = 4 for Tm-injected ATF6α −/− mice and n = 5 for others). All ATF6α −/− mice injected with tunicamycin died at day 3. (B) Tm or LPS was injected into ATF6α +/+ and −/− mice as well as ATF6β −/− mice as described in A. The abdomen of injected mice was dissected and photographed after 48 h.

Figure 2.

Effect of tunicamycin injection on liver function of ATF6α +/+ and −/− mice. (A) Tunicamycin (Tm) was injected into ATF6α +/+ and −/− mice as described in Figure 1A. Liver tissue sections were prepared at the indicated time points after injection and stained with hematoxylin and eosin. Bar, 100 μm (a–h) and 50 μm (i and j). (B) Tm was injected into ATF6α +/+ and −/− mice as described in Figure 1A. Levels of ALT and total protein in the serum of injected mice were determined at the indicated time points and are presented as the means ± SEM (n = 3). *p < 0.05. (C) Tm was injected into ATF6α +/+ and −/− mice as described in Figure 1A. Liver lysates were prepared at the indicated time points after injection and analyzed by immunoblotting using antibody to BiP, GRP94, ERp72, CHOP, PDI, ADRP, or actin. (D) Tm was injected into ATF6α +/+ and −/− mice as described in Figure 1A. TUNEL-positive cells were determined in liver tissue sections prepared 12 h after injection and are presented as the means ± SEM (n = 3). *p < 0.05. (E) HEK293T cells were untreated (−) or treated for 12 h with 2 μg/ml Tm, 300 nM thapsigargin (Tg), or 2 mM dithiothreitol (DTT). The cell lysates were prepared and analyzed by immunoblotting using antibody to ADRP, BiP, or actin.

Figure 3.

Effect of tunicamycin injection on lipid contents in the liver of ATF6α +/+ and −/− mice. (A) Tunicamycin (Tm) was injected into ATF6α +/+ and −/− mice as described in Figure 1A. Liver tissue sections were prepared at the indicated time points after injection and stained with oil red O. Bar, 50 μm. (B) Tm was injected into ATF6α +/+ and −/− mice as described in Figure 1A. Levels of triacylglycerol (triglyceride) in the liver were determined at the indicated time points after injection and are presented as the means ± SEM (n = 3). *p < 0.05. (C) Tm was injected into ATF6α +/+ and −/− mice as described in Figure 1A. Levels of total cholesterol in the liver were determined at the indicated time points after injection and are presented as the means ± SEM (n = 3). *p < 0.05. (D) Tm was injected into ATF6α +/+ and −/− mice as in Figure 1A. Levels of triacylglycerol (triglyceride) in plasma were determined at the indicated time points after injection and are presented as the means ± SEM (n = 4). *p < 0.05. (E) Tm was injected into ATF6α +/+ and −/− mice as described in Figure 1A. Levels of total cholesterol in plasma were determined at the indicated time points after injection and are presented as the means ± SEM (n = 4). *p < 0.05. (F) Schematic presentation of metabolism of fatty acids and cholesterol.

Figure 4.

Effect of tunicamycin injection on expression levels of various genes involved in fatty acid metabolism in the liver of ATF6α +/+ and −/− mice. (A) Tunicamycin (Tm) was injected into ATF6α +/+ and −/− mice as described in Figure 1A. Total RNA was prepared from livers at the indicated time points after injection and analyzed by Northern blot hybridization by using a digoxigenin-labeled probe specific to SREBP-1, ChREBP, or PPARγ. (B) Total RNA was prepared and analyzed as described in A by using a probe specific to PPARα, CPT-II, or Acox1. (C) Total RNA was prepared and analyzed as described in A by using a probe specific to MTP, ApoB, or ADRP. (D) Intensities of each band obtained in A–C were quantified and are presented as the means ± SEM (n = 3) after normalization to the value obtained for uninjected ATF6α +/+ mice. *p < 0.05.

Figure 5.

Effect of tunicamycin injection on the level of apoB-100 in the liver of ATF6α +/+ and −/− mice. (A) Tunicamycin (Tm) was injected into ATF6α +/+ and −/− mice as described in Figure 1A. Microsomes were prepared from livers at the indicated time points after injection. Aliquots (100 μg) of microsomal lysates were analyzed by immunoblotting using anti-apoB-100 antibody. The results of four independent experiments are shown. Bottom right, intensity of each band was quantified, normalized with the respective value at 0 h, and plotted against time after tunicamycin injection. (B) Top, 100-μg aliquots of microsomal lysates prepared as described in A were digested with (+) or without (−) endo H and then analyzed by immunoblotting using anti-ATF6β antibody. The migration positions of pATF6β(P) and pATF6β(P*), the unglycosylated form of pATF6β(P), are indicated. Bottom, 100-μg aliquots of microsomal lysates prepared as described in A were analyzed by immunoblotting using anti-HSP47 antibody. (C) HepG2 cells transfected with vector alone (Mock) or vector to express a dominant-negative form of ATF6α (ATF6αDN) were treated with cycloheximide (CHX) for the indicated periods. Cell lysates were prepared and analyzed by immunoblotting using antibody to ApoB-100 or actin. Intensity of each band was quantified, normalized with the respective value at 0 h, and plotted against time of cycloheximide treatment. Values are presented as the mean ± SEM (n = 3).

Figure 6.

Induction of lipid droplet formation in ATF6α −/− mice in response to tunicamycin injection. (A) Tunicamycin (Tm) was injected into ATF6α +/+ and −/− mice as described in Figure 1A. Liver tissue sections were prepared from untreated or tunicamycin-injected mice after 48 h and analyzed with electron microscopy. Bar, 10 μm (a–d) and 500 nm (e and f). (B) Tm was injected into ATF6α +/+ and −/− mice as described in Figure 1A. Whole liver obtained at the indicated time points after injection was homogenized and then subjected to sucrose gradient (5–39% from top to bottom) centrifugation. An aliquot of 12 collected fractions was analyzed by immunoblotting using anti-ADRP antibody.