Research ArticleFATTY LIVER DISEASE

IRE1α prevents hepatic steatosis by processing and promoting the degradation of select microRNAs

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Sci. Signal.  15 May 2018:
Vol. 11, Issue 530, eaao4617
DOI: 10.1126/scisignal.aao4617
  • Fig. 1 Hepatocyte-specific Ire1α−/− mice on an HFD exhibit NASH and insulin resistance.

    (A) Hepatic TGs in Ire1α−/− [knockout (KO)] and control (CTL) (Ire1αfl/fl) mice fed either NC or an HFD for 20 weeks. *P < 0.05 versus CTL. (B) Serum cholesterol (Chol), HDL, LDL, and TGs in the KO and CTL mice described in (A). (C) H&E staining, ADRP immunohistochemistry (IHC) staining, and Sirius red staining of collagen deposition with liver tissue sections from the KO and CTL mice described in (A). Scale bars, 5 μm. (D) Table showing the quantification (means ± SD) of hepatic inflammation and fibrosis in IRE1α-KO and CTL mice fed an HFD for 20 weeks. (E) Analysis of insulin tolerance tests (ITTs) in IRE1α-KO and CTL mice fed an HFD for 19 weeks, fasted for 4 hours, and then intraperitoneally injected with human insulin (0.75 mU/g body weight). (F) Analysis of glucose tolerance tests (GTTs) in IRE1α KO and CTL mice fed an HFD for 11 weeks, fasted for 14 hours, and then injected with 2 mg of glucose per gram body weight. Data are means ± SEM from n = 8 (KO) or 4 (CTL) mice per group; *P < 0.05, **P < 0.01 by unpaired two-tailed Student’s t test (A and B and D to F).

  • Fig. 2 IRE1α RNase activity is suppressed in the fatty livers of HFD-fed mice or human patients.

    (A) Western blot analysis of IRE1α protein abundance in the livers of hepatocyte-specific IRE1α-KO and CTL mice fed NC or an HFD for 20 weeks. (B) Western blot analysis through Phos-tag SDS-PAGE for phosphorylated IRE1α (P-IRE1α) and unphosphorylated IRE1α in the livers of IRE1α-KO and CTL mice fed NC or an HFD. Tm, tunicamycin, a positive control for ER stress–induced phosphorylation of IRE1α. (C) Quantitative electrophoretic analysis of spliced and unspliced Xbp1 mRNA in the livers of the IRE1α-KO and CTL mice fed NC or an HFD. Xbp1 complementary DNAs (cDNAs) were amplified by semiquantitative reverse transcription polymerase chain reaction (RT-PCR) from the total RNAs isolated from the mouse livers, followed by Pst I restriction enzyme digestion. Tunicamycin-treated hepatocytes (5 μg/ml) were a positive control. Xbp1(S), spliced Xbp1; Xbp1(U), unspliced Xbp1. (D) Western blot analysis of XBP1s protein abundance in IRE1α-KO and CTL mice fed NC or an HFD. GAPDH, glyceraldehyde-3-phosphate dehydrogenase (loading control). (E) Abundance of S-nitrosylated (SNO) IRE1α, total IRE1α, and β-actin in the livers of NC- and HFD-fed mice. SNO-IRE1α protein was enriched by a biotin switch method and detected by Western blot analysis. Mouse liver protein lysates incubated with GSNO (100 μM; +) or without (−) are positive and negative controls, respectively. (F and G) Representative images (63×) of staining for SNO (green) and IRE1α (red) in the livers from NC- and HFD-fed mice (F) and from patients with or without hepatic steatosis (G). Nuclei were counterstained with 4′,6-diamidino-2-phenylindole (blue). Arrows indicate colocalization of SNO and IRE1α, quantified as inferred SNO-IRE1α in the graphs (right). Ascorbate-omitted staining (−AS) are negative controls. Scale bars, 10 μm. Data are means ± SEM of n = 4 mice per group (A to E) or n = 8 mice or patients per group (F and G). *P < 0.05, **P < 0.01 by unpaired two-tailed Student’s t test.

  • Fig. 3 IRE1α deficiency results in an increased abundance of a subset of miRNA clusters in the steatotic livers of HFD-fed mice or human diabetic patients.

    (A and B) miRNA-qPCR analysis of miRNA cluster expression in the livers of IRE1α-KO and CTL mice fed NC (A) or an HFD (B) for 20 weeks. (C) miRNA-qPCR analysis of miRNA cluster expression in the livers of human patients with or without hepatic steatosis. Data in all panels are means ± SEM from n = 4 individuals per group. *P < 0.05, **P < 0.01 versus controls by unpaired two-tailed Student’s t test.

  • Fig. 4 IRE1α represses levels of miR-200 and miR-34 families in hepatocytes.

    IRE1α-KO and wild-type CTL mouse hepatocytes were incubated with OA (500 μM) or vehicle [Veh; 0.5% bovine serum albumin (BSA)] for 24 hours. (A) Western blot analysis of IRE1α abundance in IRE1α-KO and CTL hepatocytes. β-Actin, loading control. (B) Quantitative electrophoretic analysis of spliced and unspliced Xbp1 mRNA in IRE1α-KO and CTL mouse hepatocytes incubated with OA or vehicle. Xbp1 cDNAs were amplified by semiquantitative RT-PCR, followed by Pst I restriction enzyme digestion. (C) Western blot analysis of XBP1s protein abundance in IRE1α-KO and CTL hepatocytes incubated with OA or vehicle. GAPDH, loading control. (D and E) miRNA-qPCR analysis of miRNA cluster expression in IRE1α-KO and CTL hepatocytes loaded with OA or vehicle. Data in all panels are means ± SEM of n = 3 biological replicates *P < 0.05, **P < 0.01 versus controls by unpaired two-tailed Student’s t test.

  • Fig. 5 IRE1α-deficient hepatocytes accumulate more neutral lipids and represses miR-200 and miR-34 in a manner independent of XBP1.

    (A and B) Overexpression of IRE1α, activated XBP1, or GFP control in IRE1α-KO and CTL hepatocytes using adenoviral-based expression system [multiplicity of infection (MOI), 100] for 48 hours. miRNA-qPCR analysis of levels of miR-200 and miR-34 family members in the hepatocytes. (C) Representative images and quantification of Oil Red O staining of lipid droplets in IRE1α-KO and CTL hepatocytes after incubation with OA or vehicle (0.5% BSA) for 24 hours. Scale bars, 5 μm. The quantification was determined by eluting the Oil Red O dye in isopropanol, and the optical density (OD) was read at 500 nm. Data are means ± SEM of n = 3 biological replicates. *P < 0.05 versus CTL + adenovirus (Ad)–GFP (A and B) or CTL + vehicle (C), #P < 0.05 versus CTL + OA (C).

  • Fig. 6 IRE1α processes pre-miR-200 and pre-miR-34 and leads to their degradation.

    (A) The sequence motif and stem-loop structures for the IRE1α cleavage sites within human XBP1 mRNA, pre-miR-34 and pre-miR-200. Lower pictures show the predicted secondary structures for miR-34 and miR-200 with their potential IRE1α cleavage sites (G/C sites marked by red arrows). (B and C) qPCR analyses of expression levels of pri-miR-34, pri-miR-200, pre-miR-34, and pre-miR-200 in the livers of IRE1α-KO and CTL mice under NC or HFD for 20 weeks. Data are means ± SEM (n = 4 mice per group). *P < 0.05 versus CTL + NC; #P < 0.05 versus CTL + HFD by unpaired two-tailed Student’s t test. (D) miRNA-qPCR analysis of the abundance of mature miR-34 and miR-200 in IRE1α-KO and CTL hepatocytes transfected with plasmid vector expressing pre-miR-34, pre-miR-200, or scramble oligonucleotides (Scr). Data are means ± SEM (n = 3 biological replicates); *P < 0.05 versus CTL + Scr; #P < 0.05 versus CTL + pre-miR-34 or pre-miR-200. (E) In vitro cleavage of pre-miR-200 and pre-miR-34 by recombinant IRE1α protein (1 μg each), incubated at 37°C with ATP (2 mM), or not as controls, and resolved on a 1.2% agarose gel. N-Ctl, negative control reaction containing no pre-miRNA; VH, vehicle buffer containing no IRE1α protein. The image is representative of three experiments.

  • Fig. 7 IRE1α deficiency represses expression of PPARα and SIRT1 through increased abundance of miR-34 and miR-200.

    (A to D) Abundance of PPARα and SIRT1 mRNA (A and B; by qPCR) and protein (C and D; by Western blot) in livers from hepatocyte-specific IRE1α-KO and CTL mice fed NC or an HFD diet for 20 weeks. Data are means ± SEM (n = 4 mice per group); *P < 0.05 versus CTL + NC; #P < 0.05 versus CTL + HFD by unpaired two-tailed Student’s t test (A to G). (E) IP-Western blot analysis of acetylated PGC1α abundance in the livers from IRE1α-KO and control mice fed NC or an HFD. Mouse liver protein lysates were immunoprecipitated with the anti-PGC1α antibody, followed by immunoblotting (IB) with the anti–acetyl-lysine (Ace-K) antibody. Data are means ± SEM (n = 4 mice per group); *P < 0.05 versus CTL + NC. (F and G) Luciferase reporter assay of suppressive activities of miR-200 or miR-34 family members on the reporter driven by the human PPARα mRNA 3′UTR (F) or SIRT1 mRNA 3′UTR (G). Attenuation of luciferase activity was interpreted as direct binding of miRNAs to the 3′UTR. Data are means ± SEM (n = 3 biological replicates); *P < 0.05, **P < 0.01 versus scramble cotransfection with PPARα or SIRT1 mRNA 3′UTR reporter vector.

  • Fig. 8 Inhibition of miR-34 or miR-200 reduces hepatic steatosis caused by IRE1 deficiency.

    (A) qPCR analysis of Pparα or Sirt1 mRNA abundance in OA-treated IRE1α-KO and CTL hepatocytes transfected with scramble oligonucleotides (Scr), miR-34 antagomir (miR-34i), or miR-200 family antagomir (miR-200i). Data are means ± SEM (n = 3 biological replicates. *P < 0.05 versus CTL + Scr; #P < 0.05 versus KO + Scr by unpaired two-tailed Student’s t test. (B) Western blot analysis of PPARα and SIRT1 in OA-treated IRE1α-KO and CTL hepatocytes transfected with Scr, miR-34i, or miR-200i. GAPDH, loading control. Data are means ± SEM (n = 3 biological replicates); *P < 0.05. (C) Oil Red O staining of neutral lipids in OA-incubated IRE1α-KO and CTL hepatocytes transfected with Scr, miR-34i, or miR-200i for 24 hours. Scale bars, 5 μm. Data are means ± SEM (n = 3 biological replicates). *P < 0.05 versus CTL + Scr; #P < 0.05 versus KO + Scr. (D) Oil Red O staining of neutral lipids in IRE1α-KO hepatocytes and CTL overexpressing PPARα, SIRT1, or GFP, after incubation with OA for 24 hours. Scale bars, 5 μm. Data are means ± SEM (n = 3 biological replicates). *P < 0.05, **P < 0.01 versus CTL + GFP; #P < 0.05 versus KO + GFP. (E) Illustration of the IRE1α-miRNA pathway in hepatic steatosis.

Supplementary Materials

  • www.sciencesignaling.org/cgi/content/full/11/530/eaao4617/DC1

    Fig. S1. Metabolic phenotype of IRE1α-KO and control mice fed NC or an HFD.

    Fig. S2. Immunofluorescent staining of IRE1α and S-nitrosylation signals in mouse liver tissues.

    Fig. S3. miRNA profiles in IRE1α-KO and control livers from NC- or HFD-fed mice and in OA-loaded mouse hepatocytes.

    Fig. S4. Palmitate represses IRE1α activity in processing select miRNAs.

    Fig. S5. Titration and duration analyses for the effect of XBP1 overexpression on modulating miR-200 and miR-34.

    Fig. S6. Expression of the genes involved in lipid and glucose metabolism in IRE1α-KO and control mice fed NC or an HFD.

    Fig. S7. miRNA-binding sequences of miR-200 and miR-34 family members in the 3′UTRs of human PPARα and SIRT1 genes.

    Fig. S8. Inhibition of miR-34 or miR-200 rescues Pparα and Sirt1 expression and reduces hepatic steatosis caused by IRE1 deficiency and palmitate treatment.

    Fig. S9. Overexpression of PPARα or SIRT1 reduces hepatic steatosis caused by IRE1 deficiency with palmitate treatment.

    Table S1. miRNA functional clusters and previously identified targets.

    References (5170)

  • Supplementary Materials for:

    IRE1α prevents hepatic steatosis by processing and promoting the degradation of select microRNAs

    Jie-Mei Wang,* Yining Qiu, Zhao Yang, Hyunbae Kim, Qingwen Qian, Qinghua Sun, Chunbin Zhang, Lei Yin, Deyu Fang, Sung Hong Back, Randal J. Kaufman, Ling Yang,* Kezhong Zhang*

    *Corresponding author. Email: kzhang{at}med.wayne.edu (K.Z.); jiemei.wang{at}wayne.edu (J.-M.W.); ling-yang{at}uiowa.edu (L.Y.)

    This PDF file includes:

    • Fig. S1. Metabolic phenotype of IRE1α-KO and control mice fed NC or an HFD.
    • Fig. S2. Immunofluorescent staining of IRE1α and S-nitrosylation signals in mouse liver tissues.
    • Fig. S3. miRNA profiles in IRE1α-KO and control livers from NC- or HFD-fed mice and in OA-loaded mouse hepatocytes.
    • Fig. S4. Palmitate represses IRE1α activity in processing select miRNAs.
    • Fig. S5. Titration and duration analyses for the effect of XBP1 overexpression on modulating miR-200 and miR-34.
    • Fig. S6. Expression of the genes involved in lipid and glucose metabolism in IRE1α-KO and control mice fed NC or an HFD.
    • Fig. S7. miRNA-binding sequences of miR-200 and miR-34 family members in the 3′UTRs of human PPARα and SIRT1 genes.
    • Fig. S8. Inhibition of miR-34 or miR-200 rescues Pparα and Sirt1 expression and reduces hepatic steatosis caused by IRE1 deficiency and palmitate treatment.
    • Fig. S9. Overexpression of PPARα or SIRT1 reduces hepatic steatosis caused by IRE1 deficiency with palmitate treatment.
    • Table S1. miRNA functional clusters and previously identified targets.
    • References (5170)

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