UPR pathways combine to prevent hepatic steatosis caused by ER stress-mediated suppression of transcriptional master regulators

Abstract

The unfolded protein response (UPR) is linked to metabolic dysfunction, yet it is not known how endoplasmic reticulum (ER) disruption might influence metabolic pathways. Using a multilayered genetic approach, we find that mice with genetic ablations of either ER stress-sensing pathways (ATF6alpha, eIF2alpha, IRE1alpha) or of ER quality control (p58(IPK)) share a common dysregulated response to ER stress that includes the development of hepatic microvesicular steatosis. Rescue of ER protein processing capacity by the combined action of UPR pathways during stress prevents the suppression of a subset of metabolic transcription factors that regulate lipid homeostasis. This suppression occurs in part by unresolved ER stress perpetuating expression of the transcriptional repressor CHOP. As a consequence, metabolic gene expression networks are directly responsive to ER homeostasis. These results reveal an unanticipated direct link between ER homeostasis and the transcriptional regulation of metabolism, and suggest mechanisms by which ER stress might underlie fatty liver disease.

Figure 1

ATF6α deletion reveals an intersection between ER stress and metabolic homeostasis. (A) Livers from wild-type, heterozygous, or Atf6α-null mice injected with vehicle or 1 mg/kg b.w. TM were probed by immunoblot for expression of tubulin (loading control) BiP, CHOP, GADD34, and the phosphorylated form of eIF2α. Efficacy of the TM was reflected in inhibition of TRAPα glycosylation, for which the glycosylated (closed arrow) and unglycosylated (open arrow) species are indicated. (B) Wild-type and Atf6α−/− mice were injected with 1 mg/kg TM and livers were visualized in situ at the indicated times post-injection. (C) Wild-type and Atf6α−/− mice were injected with TM. Cryosections (6 μM) of liver isolated 48 hours post injection were stained by Hematoxylin & Eosin (H&E) and visualized at 400× magnification. (D) Wild-type and Atf6α−/− mice were injected with vehicle or TM (1 mg/kg). Livers were surgically removed 48hrs post injection, fixed in 2.5% glutaraldehyde, then prepared for transmission electron microscopic analysis. Endoplasmic reticulum (ER), nuclei (N), mitochondria (M) and lipid droplets (LD) are indicated. White scale bar equals 500 nm. (E) Protein lysates from liver, isolated 8 or 48 hrs post-injection, were probed by immunoblot as labeled. RNA was prepared from the same liver tissue samples and assayed by RT-PCR that simultaneously detects both spliced (sp) and unspliced (us) Xbp1 mRNA. (F) Mice were injected with 1 mg/kg TM for 48 hours. Mice were fasted for 6 hours and then injected with insulin (2 U/kg) 20 minutes prior to sacrifice. Immunoblotting detected phosphorylated or total AKT.

Figure 2

Persistent ER stress in vivo suppresses expression of a subset of metabolic genes. (A) Cluster analysis of gene expression and transcriptional profiling analysis of liver RNAs were performed as described in the Experimental Procedures. Graphic representation of the average expression levels of 4262 differentially-expressed genes is shown. Each vertical bar represents a single gene. Green coloration indicates lower expression, and red indicates higher expression. (B, C) The expression levels of two subsets of mRNAs from microarray analysis are shown, normalized against vehicle-injected wild-type samples. Note that the differences in expression levels of these genes in the absence of ER stress were generally not significant. Error bars represent means ±S.D.M. from three animals in each group. For (B), all genes shown were significantly (p<0.05) less-induced by TM in Atf6α-null animals than wild-type. For (C), all genes shown were significantly (p<0.05) downregulated by stress in both genotypes. The ATF4-dependent tryptophanyl tRNA synthetase (Wars) is here Atf6α-dependent likely because of the failure of Atf6α-null mice to stimulate eIF2α phosphorylation in response to TM challenge. (D) Real-time RT-PCR analysis was used to assess the expression of selected mRNAs in wild-type or Atf6α-null livers 8 or 48 hours after TM injection. Error bars are means ±S.D.M. from 2 (vehicle) or 3 (TM) animals. Expression values were normalized against β-actin levels and are shown relative to the expression level in wild-type unchallenged animals. (E) Protein lysates from livers of wild-type or Atf6α−/− mice injected with vehicle or TM (1 mg/kg) were probed by immunoblot for either C/EBPα (which exists in a 42kd long form and 30kd short form) or C-JUN. (F, G) The expression of metabolic genes subdivided into various categories was determined 48 hours after TM injection by real-time RT-PCR. For (D, F, G, and H). Red lettering indicates genes significantly (p<0.05) different in expression comparing TM-challenged Atf6α-null animals to unchallenged.

Figure 3

Each arm of the canonical UPR contributes to protection from metabolic dysregulation. (A) Wild-type mice and mice with a hepatocyte-specific deletion of Ire1α were injected with vehicle or TM (2 mg/kg). Protein lysates from liver isolated 30 hours post-injection were probed by immunoblot as indicated. The long (open arrowhead) and short (closed arrowhead) forms of C/EBPα are indicated. Note that the apparent difference in inhibition of TRAPα glycosylation is likely a consequence of this protein itself being a UPR target at least partially regulated by IRE1α (Nagasawa et al., 2007), rather than any difference in the pharmacological efficacy of TM between genotypes. (B) RNA was prepared from wild-type and Ire1α−/− liver tissue samples from (A) and analyzed by real-time RT-PCR. n = 3 animals per group. (C) eIF2α S51A heterozygous or homozygous knockin mutant mice, rescued by a constitutively expressed wild-type eIF2α floxed transgene, were mated with mice expressing CRE recombinase under control of the albumin promoter to delete the transgene in hepatocytes. “eIF2α genomic” denotes whether the mice were heterozygous or homozygous for the S51A genomic allele, and “Δtg” indicates whether or not the transgene was deleted by CRE expression. Protein lysates from liver isolated 30 hours post-injection (2 mg/kg) were probed by immunoblot as labeled. Here inhibition of glycosylation was followed using the Transferrin Receptor (TfR) protein. Asterisk indicates non-specific background bands. (D) Expression of metabolic mRNAs from the animals shown in (C) was quantitated by real-time RT-PCR as in (B). n = 2-4 animals per group. While the difference in upregulation of Adrp mRNA was not statistically significant comparing SA and AA mice after TM challenge, more robust upregulation of ADRP protein was evident (data not shown). Red lettering indicates different expression levels comparing Ire1α−/− or AA TM-challenged animals to unchallenged, p<0.1.

Figure 4

Disruption of ER protein processing suppresses metabolic gene expression. (A) FaO rat hepatoma cells were treated in duplicate with 500 nM TG or 5 μg/ml TM for 8 or 24 hrs, followed by cell lysis and immunoblot for GRP94, BiP, C/EBPα, and C-JUN. In hepatocytes, slower-migrating phospho-C-JUN, induced by ER stress, is also observed. (B, C) Wild-type and Atf6α−/− mice were injected with vehicle or TM (1 mg/kg bodyweight). Protein lysates from liver isolated at the indicated time points after injection were probed by immunoblot for intracellular SAP (B); Plasma level of SAP was assayed by ELISA (C). (D) Total cholesterol and triglyceride levels were measured by enzymatic assay from plasma samples of mice injected with vehicle or TM for 24 hours, with a 6 hour fast preceding sacrifice. (E) Cholesterol and triglyceride represented either as total content, or in the form of LDL and VLDL particles purified by differential precipitation, was measured as in (D). In this case samples were collected 48 hours after TM injection rather than 24 hours. Note that the absolute values are somewhat different comparing panels (D) and (E), likely due to experimental variation. However, in panel (D) there is no significant difference in either cholesterol or triglyceride levels between genotypes after TM injection, while cholesterol levels recover to a greater extent in wild-type animals than Atf6α-null animals in panel (E). The fact that TM has a much greater effect on cholesterol-rich lipoprotein particles than triglyceride-rich particles suggests that TM inhibits liver lipoprotein production but not intestinal production. * = p<0.05

Figure 5

Disrupted ER protein folding leads to steatosis despite an intact UPR. (A, B, C) Wild-type and p58^IPK−/− mice were injected with vehicle or TM (1 mg/kg) and sacrificed 48 hrs post-injection. (A) Livers from TM challenged mice were visualized in situ. (B) Protein lysates from livers were probed by immunoblot as indicated. (C) RNA was analyzed by real-time RT-PCR. Red lettering represents p<0.05 comparing p58−/− treated to untreated.

Figure 6

CHOP suppresses expression of metabolic transcription factors. (A) FaO rat hepatoma cells were treated in duplicate with 5 μg/ml ActD and either no stress, 500 nM TG, or 5 μg/ml TM for 4 hours. Protein lysates were then harvested and probed by immunoblot as indicated. Note that ActD treatment completely blocks the upregulation of BiP and CHOP as expected. (B, C) Wild-type and Chop−/− mice were injected with vehicle or TM (1 mg/kg) and sacrificed 24 hours after injection. (B) RNA was analyzed by real-time RT-PCR as Figure 2D. n = 4-10 animals per group. Red lettering represents p<0.05 comparing Chop−/− treated to wild-type treated. (C) Protein lysates from liver were probed by immunoblot as indicated. (D) Nuclear lysates from TM-injected wild-type or Atf6α-null animals (48 hours) were probed for immunoblot as indicated.

Figure 7

Diverse ER stresses lead to lipid accumulation. (A) Normal mice were injected intravenously with the proteasome inhibitor Velcade (bortezomib) at a dose of 1mg/kg and sacrificed 8 hours after injection. Lysates were probed for upregulation of CHOP, ADRP, and C-JUN. (B) A schematic diagram depicting the joint role of the ATF6, PERK, and IRE1 pathways in maintaining lipid homeostasis. Failure of the UPR to adequately protect the ER results in ongoing production of CHOP, which suppresses metabolic genes via C/EBPα and ultimately leads to disruption of fatty acid oxidation, lipoprotein secretion, gluconeogenesis, and other metabolic processes.