19 research outputs found

    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

    Science and society: in the sixteenth and seventeenth centuries

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    eIF2α phosphorylation in hepatocytes is dispensable for survival of adult mice. (a) Diagram depicting the four genotypes of mice used in these experiments. S/A and A/A represent heterozygous and homozygous eIF2α Ser51Ala (*) mutation(s) in exon 2 of one eIF2α allele and both eIF2α alleles, respectively. fTg/0 represents the floxed wild type (WT) eIF2α transgene driven by the CMV enhancer and chicken β-actin promoter (Enh-Pro). The loxP sequences (black arrowheads) allow excision of the WT eIF2α floxed transgene (fTg) and expression of EGFP, an indicator of recombination. CRE Hep /0 represents the Cre recombinase transgene driven by the promoter (Alfp) of Alb1 (encoding albumin) and the enhancer of Afp (encoding alpha-fetoprotein). (b) Efficiency of deletion of the fTg in liver tissues. Results from quantitative RT-PCR analyses of transgenic and total eIF2α mRNAs are shown. Data are means ± SEM (n = 4 ~ 5 mice per group); ### p < 0.001; Cont. vs A/A Hep . (c) Western blot analysis of eIF2α protein expression driven by the fTg in liver tissues. To quantity expression of eIF2α, blots were incubated with anti-eIF2α antibody followed by IRDye-800 goat anti-rabbit IgG (LI-COR). Membranes were scanned on an Odyssey scanner (LI-COR) (lower two panels in left panels) and quantified with the Odyssey Software package. (d) Western blot analysis of liver lysates in Cont. and A/A Hep mice at the indicated times after Tm injection. Cont. mice and A/A Liv mice were injected with vehicle or tunicamycin (Tm, 1 mg/kg). (e) Body weight measurements of fTg -deleted A/A Liv mice. At the weeks, body weight was measured in both male and female mice. Data are means ± SEM (n = 6-14 mice per group). (PDF 1987 kb

    <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

    List of common significant differential expression genes among KO versus WT, koTG versus wtTG, and koTM versus wtTM (Figure 4b Venn diagram).

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    <p>List of common significant differential expression genes among KO versus WT, koTG versus wtTG, and koTM versus wtTM (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004348#pgen-1004348-g004" target="_blank">Figure 4b</a> Venn diagram).</p

    ATF4 transcriptional activity following oxygen glucose deprivation is Siah1/2 dependent.

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    <p>(A–C) ATF4 transcriptional activity under oxygen and glucose deprivation is Siah2 dependent. WT or <i>Siah1a<sup>−/−</sup>::Siah2<sup>−/−</sup></i> MEFs were subjected to glucose deprivation (GD) oxygen deprivation (1% O<sub>2</sub>; OD) or their combination (OGD) for 12 h followed by cell harvest and RNA analyses. qPCR analysis was performed for ATF3 (A), CHOP (B) and VEGFA (C) using qPCR. (D–E) MEF cells were either infected with scramble shRNA, Siah2 shRNA (D) or Siah1 shRNA (E) and subjected to glucose deprivation and 1% O<sub>2</sub> (OGD) for 12 h followed by cell harvest and RNA analysis using qPCR to determine mRNA levels Siah2, ATF3, and CHOP. (F) WT or <i>Siah1a<sup>−/−</sup>::Siah2<sup>−/−</sup></i> MEF were subjected to glucose deprivation (GD), oxygen deprivation (1% O<sub>2</sub>; OD) or their combination (OGD) for 12 h followed by cell harvest and RNA analysis using qPCR for ATF4 mRNA levels. (G) WT and <i>Siah1a<sup>−/−</sup>::Siah2<sup>−/−</sup></i> MEFs were exposed to glucose deprivation and 1% O<sub>2</sub> (OGD) for 12 h prior to the analysis for the expression of ATF4, PHD1, PHD3 and β-tubulin by Western blotting. Arrow points to the position of the endogenous PHD3 protein. (H) WT and <i>Siah1a<sup>−/−</sup>::Siah2<sup>−/−</sup></i> MEFs were subjected to glucose deprivation and 1% O<sub>2</sub> (OGD) for 12 h prior to the treatment with the protein synthesis inhibitor cycloheximide (40 µg/ml) for 15, 30, and 60 minutes. Cell lysates were subjected to Western blotting using ATF4 and PHD3 antibodies. β-tubulin served as the loading control. Arrow points to the position of the endogenous PHD3 protein. *** p<0.0005, ** p<0.005, * p<0.05 compared to Siah1a/2 WT (A–C, F) or scr. shRNA (D–E) in the same condition (student's t-test). The Western blot experiments were repeated three times and the qPCR results are shown as the mean values ± S.E. of three independent experiments.</p

    Mapping ATF4 and sXBP1 response elements on Siah1a/2 regulatory regions.

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    <p>(A) Overexpression of ATF4 decreases ectopic PHD3 and OGDH protein levels. 293T cells were transfected with myc-tagged PHD3 or flag-tagged OGDH, and 12 h later were infected with ATF4 virus for 24 h. Whole cell lysates were analyzed by Western blotting with the indicated antibodies. Three independent experiments were carried out and Western blot bands corresponding to PHD3 and OGDH were quantified and normalized against β-actin (graph on the right). (B) Overexpression of ATF4 decreases AKAP121 and increases HIF-1α protein level. <i>Atf4<sup>−/−</sup></i> MEFs were infected with ATF4 virus for 24 h and then exposed to hypoxia (1% O<sub>2</sub>) for 8 h. Whole cell lysates were analyzed by Western blotting with the indicated antibodies. (C) Schematic diagram of the human Siah2 promoter, showing the sequence of ATF4 and sXBP1 response elements. The transcription start site is marked as +1. (D) Mapping ATF4 response element on Siah2 promoter. A 2 Kb fragment containing the upstream and the 5′-UTR domains were cloned into luciferase constructs, in which the putative ATF4 binding site (ATF4M; +308 bp) within the 5′-UTR region was mutated. Siah2 promoter activity was monitored in MEFs infected with either GFP or ATF4 expressing adenovirus and 24 h later cells were harvested and proteins were used to perform luciferase assays and to perform Western blotting with the indicated antibodies. (E) ChIP confirms ATF4 occupancy of corresponding Siah2 promoter site. MEFs were exposed to TG for 5 h and ChIP analysis was performed using antibodies to ATF4 or control IgG as indicated. A set of primers was used to quantify a 214 bp fragment containing the ATF4 site in position +308 using qPCR. (F) Mapping the XBP1 response element in the Siah2 promoter. A 2 Kb fragment containing the upstream and the 5′-UTR domains was cloned into luciferase constructs, in which the putative sXBP1 binding site (XBP1M; +287) within the 5′-UTR region was mutated. Siah2 promoter activity was monitored in MEFs infected with adenoviruses expressing either GFP or sXBP1. After 24 h, cells were harvested and proteins were used to perform luciferase assays. (G) ChIP confirms XBP1 occupancy of corresponding Siah2 promoter sites. MEFs were exposed to TG for 5 h and ChIP analysis was performed using antibodies to XBP1 or control IgG as indicated. A set of primers was used to quantify a 214-bp fragment containing the Xbp1 site in position +287 using qPCR. (H) Mapping the ATF4 response element in the Siah1a promoter. ChIP analysis confirms ATF4 occupancy of corresponding Siah1a intron site. MEFs were exposed to TG for 5 h and ChIP analysis was performed using antibodies to ATF4 or control IgG as indicated. A set of primers was used to quantify a 154-bp fragment containing the ATF4 site in position +4,710 using qPCR. *** p<0.0005, ** p<0.005, * p<0.05 compared to ad GFP (D, F) or to control in the same condition (student's t test). The Western blot experiments were repeated three times and the qPCR results are shown as the mean values ± S.E. of three independent experiments.</p

    UPR induction of Siah1/2 RNA and protein is ATF4-dependent.

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    <p>(A) TM induces Siah2 protein levels. Litter-matched MEF WT and <i>Siah1a<sup>−/−</sup>::Siah2<sup>−/−</sup></i> cells were treated with TM (1 µg/ml) for 10 h. Whole cell lysates were prepared and analyzed by immunoblotting with the indicated antibodies. (B) TM induces Siah1 protein. Litter-matched MEF WT and <i>Siah1a<sup>−/−</sup>::Siah2<sup>−/−</sup></i> cells were treated with TM (1 µg/ml) for 10 h. Whole cell lysates were prepared and analyzed by immunoblotting with the indicated antibodies. Right panel: ER stress does not induce Siah1b mRNA levels. MEFs from <i>Siah1a<sup>−/−</sup>::Siah2<sup>−/−</sup></i> cells were treated with TM (2 µg/ml) for 6 h and the relative expression of Siah1b mRNA was measured by quantitative real time PCR (qPCR). (C) ER stress induces Siah2 mRNA levels. MEFs from WT animals were treated with the indicated concentrations of TM for 6 h and the relative expression of Siah2 mRNA was measured by qPCR. (D) HeLa and Lu1205 cells were treated with the indicated concentrations of TM for 6 h and the relative expression of Siah2 mRNA was measured by qPCR. (E) ER-stress induction of Siah2 mRNA is attenuated in <i>Atf4<sup>−/−</sup></i> MEFs. Littermate-matched MEFs of the indicated genotypes were subjected to treatment with TM (2 µg/ml), hypoxia (H), or both. RNA prepared 6 h later was used for qPCR to quantify Siah2 transcripts, relative to levels of H3.3A mRNA. (F) CHOP mRNA levels are ATF4 but not hypoxia dependent. MEFs from <i>Atf4</i> WT and <i>Atf4<sup>−/−</sup></i> genotypes were subjected to TM or hypoxia or combined treatment and RNA prepared for qPCR analysis of CHOP transcripts. (G) VEGFA mRNA levels are ATF4 dependent under normoxia. Experiment was performed as indicated in panel E, except that qPCR analysis used the VEGFA primers. (H) <i>Atf4</i> WT and <i>Atf4<sup>−/−</sup></i> MEFs were infected with ATF4-expressing adenovirus. Cell lysates were prepared and the level of ATF4 protein was detected in immunoblots with the respective antibody. β-actin served as the loading control. (I) Ectopic ATF4 expression rescues Siah2 mRNA levels in TM-treated <i>Atf4<sup>−/−</sup></i> MEFs. MEFs of the indicated genotypes were infected with control (β-Gal) or with ATF4 adenoviruses for 24 h, followed by 6 h exposure to TM (2 µg/ml), as indicated. Relative expression of Siah2 mRNA was quantified by qPCR. *** p<0.0005, ** p<0.005, * p<0.05 compared to non treated (C–D) or to ATF4 WT in the same condition (student's t test). The Western blot experiments were repeated three times and the qPCR results are shown as the means ± S.E. of three independent experiments.</p
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