12 research outputs found

    Mmu-miR-615-3p Regulates Lipoapoptosis by Inhibiting C/EBP Homologous Protein

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    <div><p>Lipoapoptosis occurring due to an excess of saturated free fatty acids such as palmitate is a key pathogenic event in the initiation of nonalcoholic fatty liver disease. Palmitate loading of cells activates the endoplasmic reticulum stress response, including induction of the proapoptotic transcription factor C/EBP homologous protein (CHOP). Furthermore, the loss of microRNAs is implicated in regulating apoptosis under conditions of endoplasmic reticulum (ER) stress. The aim of this study was to identify specific microRNAs regulating CHOP expression during palmitate-induced ER stress. Five microRNAs were repressed under palmitate-induced endoplasmic reticulum stress conditions in hepatocyte cell lines (miR-92b-3p, miR-328-3p, miR-484, miR-574-5p, and miR-615-3p). We identified miR-615-3p as a candidate microRNA which was repressed by palmitate treatment and regulated CHOP protein expression, by RNA sequencing and <i>in silico</i> analyses, respectively. There is a single miR-615-3p binding site in the 3′untranslated region (UTR) of the <i>Chop</i> transcript. We characterized this as a functional binding site using a reporter gene-based assay. Augmentation of miR-615-3p levels, using a precursor molecule, repressed CHOP expression; and under these conditions palmitate- or tunicamycin-induced cell death were significantly reduced. Our results suggest that palmitate-induced apoptosis requires maximal expression of CHOP which is achieved via the downregulation of its repressive microRNA, miR-615-3p. We speculate that enhancement of miR-615-3p levels may be of therapeutic benefit by inhibiting palmitate-induced hepatocyte lipoapoptosis.</p></div

    MiR-615-3p inhibits CHOP expression.

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    <p>(A) Representative western blot for CHOP in IRE-WT cells treated with 400 µM palmitate (PA) or 1 µg/mL tunicamycin (tuni) for 16 hours. The cells were transfected with either a negative control, or a precursor of miR-615-3p (pre-miR-615-3p). Molecular weights are indicated in kDa. The immune complexes were detected using an infrared fluorescent imaging system. Alpha-tubulin was used as loading control. (B) Quantification of CHOP protein levels normalized to tubulin, under the same conditions as A, expressed relative to the negative control mimic treated cells for each condition, respectively. ** P<0.01. (C) Representative western blot for CHOP in Hepa 1–6 cells treated with 400 µM palmitate (PA) or 1 µg/mL tunicamycin for 24 hours. The cells were transfected with either a negative control, or a precursor of miR-615-3p (pre-miR-615-3p). Molecular weights are indicated in kDa. The immune complexes were detected using enhanced chemiluminescence. Alpha-tubulin was used as loading control. The middle panel (**) depicts a longer film exposure of the top panel (*) (D) Quantification of CHOP protein levels normalized to tubulin, under the same conditions as C, expressed relative to the negative control mimic treated cells for each condition, respectively. * P<0.05, ** P<0.01 (E) Representative western blot for CHOP in IRE-WT and Hepa1-6 cells in cells transfected with either an antagomir to miR-615-p or a negative control antagomir. Alpha-tubulin was used as loading control. (F) Quantification of CHOP protein levels normalized to tubulin, under the same conditions as E, expressed relative to the negative control antagomir treated cells for each condition, respectively. P = ns.</p

    <i>Chop</i> mRNA is a direct target of miR-615-3p.

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    <p>A region of the <i>Chop</i> 3′UTR containing the putative miR-615-3p binding site was cloned into the pMIR-report vector downstream of the luciferase coding region (p-MIR-Chop). The 3′UTR segment with the putative miR-615-3p binding site mutated (p-MIR-Chop-mut) was also cloned into the pMIR-report vector. HEK293 cells were co-transfected with the respective reporter plasmid and precursor of miR-615-3p (pre-miR-615-3p) or a negative control precursor molecule. Relative luciferase activity (normalized to renilla) was measured 24 hours after the transfection. Data are expressed relative to the wild-type binding site transfected cells treated with a negative control precursor molecule and (n = 5 independent experiments), * P<0.05.</p

    Palmitate treatment decreases miR-615-3p levels.

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    <p>(A) Neonatal IRE1α wild-type hepatocyte cells (IRE-WT) were treated with 400 µM palmitate (PA) or 1 µg/mL tunicamycin (tuni) for 16 hours. Control cells were treated with vehicle alone (VC). * P<0.05 compared to VC. (B) Neonatal IRE1α knockout (IRE-KO) cells were treated with 400 µM palmitate (PA) or 1 µg/mL tunicamycin for 16 hours. Control cells were treated with vehicle alone (VC). * P<0.05 compared to VC (C) Primary mouse hepatocytes (PMH) were treated with 400 µM palmitate (PA) or 1 µg/mL tunicamycin for 16 hours. Control cells were treated with vehicle alone (VC). * P<0.05 compared to VC. (D) Hepa1-6 cells were treated with 400 µM palmitate (PA) or 1 µg/mL tunicamycin for 24 hours. Control cells were treated with vehicle alone (VC). * P<0.05 compared to VC. (E) Huh7 cells were treated with 400 µM palmitate (PA) or 1 µg/mL tunicamycin for 30 hours. Control cells were treated with vehicle alone (VC). * P<0.05 compared to VC.</p

    MiR-615-3p reduces palmitate-induced cell death.

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    <p>(A) Neonatal liver wild-type cells were transfected with either the negative control precursor or precursor of miR-615-3p, both fluorescently labeled with Cy3. 8 hours after transfection, cells were treated with 400 µM palmitate (PA) or 1 µg/mL tunicamycin for 18 hours. DAPI stained nuclei were counted in each condition. * P<0.05. (B) Neonatal liver wild-type cells were transfected with either the negative control precursor or precursor of miR-615-3p. 8 hours after transfection, cells were treated with 400 µM palmitate (PA) or 1 µg/mL tunicamycin for 18 hours. Caspase 3/7 activity was measured as described. * P<0.05.</p

    MicroRNAs downregulated by palmitate and tunicamycin treatment in IRE1α wild-type and knockout cells.

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    <p>MicroRNAs downregulated by palmitate and tunicamycin treatment in IRE1α wild-type and knockout cells.</p

    MiR-615-3p and CHOP levels in nonalcoholic steatohepatitis.

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    <p>(A) MiR-615-3p levels were measured in mouse liver from chow fed mice (n = 7) and mice with nonalcoholic steatohepatitis induced by feeding a diet high in fructose, fat and cholesterol (FFC) for 6 months (n = 13). Bars depict mean ±S.E.M, * <i>P</i><0.05. (B) Immunoblots for p-eIF2α, total-eIF2 α, ATF4, CHOP and GAPDH for loading control from whole liver protein extracts from chow fed mice (n = 3) and FFC-fed mice (n = ). The arrow points to the predicted CHOP band in the immunoblot.</p

    The IRE1α/XBP1s Pathway Is Essential for the Glucose Response and Protection of β Cells

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    <div><p>Although glucose uniquely stimulates proinsulin biosynthesis in β cells, surprisingly little is known of the underlying mechanism(s). Here, we demonstrate that glucose activates the unfolded protein response transducer inositol-requiring enzyme 1 alpha (IRE1α) to initiate X-box-binding protein 1 (<i>Xbp1</i>) mRNA splicing in adult primary β cells. Using mRNA sequencing (mRNA-Seq), we show that unconventional <i>Xbp1</i> mRNA splicing is required to increase and decrease the expression of several hundred mRNAs encoding functions that expand the protein secretory capacity for increased insulin production and protect from oxidative damage, respectively. At 2 wk after tamoxifen-mediated <i>Ire1α</i> deletion, mice develop hyperglycemia and hypoinsulinemia, due to defective β cell function that was exacerbated upon feeding and glucose stimulation. Although previous reports suggest IRE1α degrades insulin mRNAs, <i>Ire1α</i> deletion did not alter insulin mRNA expression either in the presence or absence of glucose stimulation. Instead, β cell failure upon <i>Ire1α</i> deletion was primarily due to reduced proinsulin mRNA translation primarily because of defective glucose-stimulated induction of a dozen genes required for the signal recognition particle (SRP), SRP receptors, the translocon, the signal peptidase complex, and over 100 other genes with many other intracellular functions. In contrast, <i>Ire1α</i> deletion in β cells increased the expression of over 300 mRNAs encoding functions that cause inflammation and oxidative stress, yet only a few of these accumulated during high glucose. Antioxidant treatment significantly reduced glucose intolerance and markers of inflammation and oxidative stress in mice with β cell-specific <i>Ire1α</i> deletion. The results demonstrate that glucose activates IRE1α-mediated <i>Xbp1</i> splicing to expand the secretory capacity of the β cell for increased proinsulin synthesis and to limit oxidative stress that leads to β cell failure.</p></div

    <i>KO</i> islets exhibit ER stress.

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    <p>(A) qRT-PCR of UPR genes in islets isolated 6 wk post-Tam and incubated in 11 mM glucose 16 h ([<i>n</i> = 5], [<i>p</i> ≤ 0.05]). (B) Immunofluorescence microscopy of pancreas sections stained for KDEL (BIP and GRP94) (green), the plasma membrane protein GLUT2 (red), and nuclei DAPI (blue). Overlap of red/green channels represents defective compartmentalization that was found to be increased in the <i>KO</i><sup><i>Fe/-; Cre</i></sup> as shown in yellow. Scale bars, 400x = 50 μm, 1,000x = 10 μm, 5,180x = 2 μm and 10,500x = 1 μM. Additional examples are shown in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002277#pbio.1002277.s007" target="_blank">S3B Fig</a>. (C) EM of adult mouse (16 wk old) islets and their β cells from mice 2 wk post-Tam. Scale bars, both panels, 1 μm. Distended mitochondria are outlined with yellow dashes. (D) Conventional PCR flanking the 26 nt intron in <i>Xbp1</i> mRNA spliced by IRE1α from the islet complementary DNAs (cDNAs) used for mRNA-Seq analysis, 6 mM versus 18 mM glucose. Results representative of <i>n</i> = 5 per genotype. (E) Global heatmap for the ~22,000 mRNAs detected by mRNA-Seq for 18 mM <i>KO</i><sup><i>Fe/-; Cre</i></sup> & <i>WT</i><sup><i>Fe/+</i></sup> samples; green and red indicate increased and decreased expression. The blue box indicates genes with inverse expression dependent on IRE1α and high glucose.</p

    mRNA sequencing identifies IRE1α- and glucose-dependent mRNAs in islets.

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    <p>(A) mRNA-Seq data on β cell-specific mRNAs. The results show no significant change to INS1 or INS2 in the <i>KO</i><sup><i>Fe/-; Cre</i></sup> samples, while MAFA, GCG, and PC5 are increased by deletion ([<i>n</i> = 5], [18 mM <i>KO</i><sup><i>Fe/-; Cre</i></sup>, <i>p</i>-values ≤ 0.05]). mRNA-Seq expression fold changes were normalized relative to the 6 mM <i>WT</i><sup><i>Fe/+</i></sup> islet context. (B) Four-way Venn diagrams of <i>WT</i><sup><i>Fe/+</i></sup> versus <i>KO</i><sup><i>Fe/-; Cre</i></sup> islets during 6 mM versus 1 8mM glucose exposur<i>e</i> for 72 h. <i>Ire1α</i>-dependent mRNAs are in bold italics, while those also dependent on high glucose are in bold, italicized, and underlined font. At the center, bar graphs representing the <i>Ire1α</i>- and glucose-dependent trends of interest are labeled “Induction” and “Repression.” (C) Combined <b>DAVID</b> (the Database for Annotation, Visualization and Integrated Discovery) and “ConceptGen” GO analysis of <i>Ire1α-</i> and glucose-dependent mRNAs. Categories shown are specifically found in the genotype, while the shared categories have been omitted for simplicity, although no single mRNA was common between the groups. (D) Mass spectrometry of murine islets infected with <i>Ad-IREα-K907A (Ad-ΔR)</i> versus <i>Ad-β-Galactosidase</i> (<i>β-Gal</i>). Proteins with ≥5 unique peptides detected per protein increased or decreased upon infection in triplicate were analyzed for GO using ConceptGen and DAVID web resources (<i>n</i> = 3). The proteins shown (Fig 3D) exhibit the same expression dependence for IRE1α as measured by mRNA-Seq (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002277#pbio.1002277.s002" target="_blank">S2 Data</a>).</p
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