5 research outputs found
The IRE1Ī±/XBP1s Pathway Is Essential for the Glucose Response and Protection of Ī² Cells
<div><p>Although glucose uniquely stimulates proinsulin biosynthesis in Ī² 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 (IRE1Ī±) to initiate X-box-binding protein 1 (<i>Xbp1</i>) mRNA splicing in adult primary Ī² 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>Ire1Ī±</i> deletion, mice develop hyperglycemia and hypoinsulinemia, due to defective Ī² cell function that was exacerbated upon feeding and glucose stimulation. Although previous reports suggest IRE1Ī± degrades insulin mRNAs, <i>Ire1Ī±</i> deletion did not alter insulin mRNA expression either in the presence or absence of glucose stimulation. Instead, Ī² cell failure upon <i>Ire1Ī±</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>Ire1Ī±</i> deletion in Ī² 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 Ī² cell-specific <i>Ire1Ī±</i> deletion. The results demonstrate that glucose activates IRE1Ī±-mediated <i>Xbp1</i> splicing to expand the secretory capacity of the Ī² cell for increased proinsulin synthesis and to limit oxidative stress that leads to Ī² cell failure.</p></div
<i>KO</i> islets exhibit ER stress.
<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> ā¤ 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 Ī¼m, 1,000x = 10 Ī¼m, 5,180x = 2 Ī¼m and 10,500x = 1 Ī¼M. 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 Ī² cells from mice 2 wk post-Tam. Scale bars, both panels, 1 Ī¼m. Distended mitochondria are outlined with yellow dashes. (D) Conventional PCR flanking the 26 nt intron in <i>Xbp1</i> mRNA spliced by IRE1Ī± 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 IRE1Ī± and high glucose.</p
mRNA sequencing identifies IRE1Ī±- and glucose-dependent mRNAs in islets.
<p>(A) mRNA-Seq data on Ī² 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 ā¤ 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>Ire1Ī±</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>Ire1Ī±</i>- and glucose-dependent trends of interest are labeled āInductionā and āRepression.ā (C) Combined <b>DAVID</b> (the Database for Annotation, Visualization and Integrated Discovery) and āConceptGenā GO analysis of <i>Ire1Ī±-</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-IREĪ±-K907A (Ad-ĪR)</i> versus <i>Ad-Ī²-Galactosidase</i> (<i>Ī²-Gal</i>). Proteins with ā„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 IRE1Ī± 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
<i>KO</i> islets accumulate oxidative stress, inflammation, and fibrosis.
<p>(A) mRNA-Seq expression values for 25/368 of the mRNAs identified by Venn analysis (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002277#pbio.1002277.g003" target="_blank">Fig 3C</a>; right panel, underlined) that are reduced by <i>Ire1Ī±</i> because of glucose that accumulates in the <i>KO</i><sup><i>Fe/-; Cre</i></sup> ([<i>n</i> = 5], [<i>p</i>-values ā¤ 0.05]). (B) Oxidized lipid (hydroxyl-octadecadienoic acids, HODEs) from islets of the indicated genotypes infected with <i>Ad-Cre Ad-GFP</i> or no virus control ([<i>n</i> = 2; controls versus <i>n</i> = 3; <i>Ad-Cre</i>], [<i>p</i> = 0.00434]). (C) Antinitrotyrosine immunohistochemistry (IHC) of islets from 8-mo-old <i>WT</i><sup><i>Fe/Fe</i></sup> and <i>KO</i><sup><i>Fe/Fe; Cre</i></sup> mice 15 wk post-Tam with or without BHA diet for 3 wk. (Scale bar, 50 Ī¼m) (<i>WT</i><sup><i>Fe/Fe</i></sup> [<i>n</i> = 4 with BHA], [<i>n</i> = 5 regular chow]), (<i>KO</i><sup><i>Fe/Fe; Cre</i></sup> [<i>n</i> = 5 with BHA], [<i>n</i> = 6 regular chow]). (<i>p</i> = 0.00698; <i>WT</i><sup><i>Fe/Fe</i></sup> versus <i>WT</i><sup><i>Fe/Fe</i></sup> with BHA), (<i>p</i> = 0.04018; <i>WT</i><sup><i>Fe/Fe</i></sup> versus <i>KO</i><sup><i>Fe/Fe; Cre</i></sup>) and (<i>p</i> = 0.04420; <i>KO</i><sup><i>Fe/Fe; Cre</i></sup> versus <i>KO</i><sup><i>Fe/Fe; Cre</i></sup> with BHA). (D) Massonās trichrome stain (blue) for collagens. Results demonstrate increased staining surrounding <i>KO</i><sup><i>Fe/Fe; Cre</i></sup> islets with haemotoxylin (red) and eosin (black) co-stains. Quantification of percent strong collagen stain is shown below the images. Scale bar, 50 Ī¼m. (<i>WT</i><sup><i>Fe/Fe</i></sup> [<i>n</i> = 4 with BHA], [<i>n</i> = 5 regular chow]), (<i>KO</i><sup><i>Fe/Fe; Cre</i></sup> [<i>n</i> = 5 with BHA], [<i>n</i> = 6 without BHA]). Percent strong collagen stain significance for <i>WT</i><sup><i>Fe/Fe</i></sup> without BHA versus <i>KO</i><sup><i>Fe/Fe; Cre</i></sup> without BHA <i>p</i> = 0.01049). (E) 8-mo-old male mice carrying the doubly floxed allele (<i>Ire1Ī±</i><sup><i>Fe/Fe</i></sup><i>)</i> with and without RIP-Cre 12 wk post-Tam had their pre-BHA GTTs taken, and then half were fed the antioxidant BHA supplemented chow diet for 3 wk or not before examining the mice by GTT again. (<i>WT</i><sup><i>Fe/Fe</i></sup> [<i>n</i> = 11 with BHA], [<i>n</i> = 12 regular chow], [<i>p</i> = 0.035]), (<i>KO</i><sup><i>Fe/Fe; Cre</i></sup> [<i>n</i> = 18 with BHA], [<i>n</i> = 16 without BHA], [<i>p</i> = 0.041]). <i>P</i>-values were calculated by one-tailed studentās <i>t</i> test comparison of the areas under the GTT curves for the biological replicates of control group <i>WT</i><sup><i>Fe/Fe</i></sup> versus the Tam-induced <i>KO</i><sup><i>Fe/Fe; Cre</i></sup> group.</p
IRE1Ī± mediated <i>Xbp1</i> splicing is necessary for proper signal peptide cleavage of preproinsulin and ribosome distribution.
<p>(A) mRNA-Seq expression values of 24/141 mRNAs identified (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002277#pbio.1002277.g003" target="_blank">Fig 3Bā3D</a>) to be both <i>Ire1Ī±-</i> and high glucose-dependent for their induction ([<i>n</i> = 5], [<i>p</i>-values ā¤ 0.01]). (B) Autoradiograph from whole cell islet lysates prepared by steady-state (18 h) [<sup>35</sup>S]-Cys/Met radiolabeling from 12 wk post-Tam mice (<i>n</i> = 3) following peptide gel electrophoresis. (C) Western blotting for proinsulin/preproinsulin, IRE1Ī±, SEC11C, SSR1, and tubulin after peptide gel electrophoresis of lysates prepared 72 h after COS-1 cells were coinfected with adenoviruses expressing <i>WT</i> preproinsulin, the Akita mutant (A), and/or the (G) GFP, (X) XBP1s, or (Ī) the dominant negative IRE1Ī±-RNase mutant K907A representative results shown (<i>n</i> = 4). (D) Ribosomes and their relative position to one another were measured from electron micrographs at a magnification of 25,000x. Total numbers of ribosomes analyzed are shown below the images ([<i>n</i> = 3], [<i>p</i> = 2.2 x 10<sup>ā15</sup>]). (E) Subcellular fractionation and western blot analysis for ribosomal small subunit 9 and tubulin of the <i>Ire1Ī±</i><sup><i>Fe/Fe</i></sup> Ī² cell insulinoma line at after 2-h glucose shift from 12 mM to either 4 mM or 36 mM with or without infection by the indicated adenoviruses were blotted representative of (<i>n</i> = 3). The 12 mM condition western blots and the quantified results normalized to tubulin membranous/cytosolic are shown in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002277#pbio.1002277.s010" target="_blank">S6D Fig</a>.</p