26 research outputs found

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

    No full text
    <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

    Regulation of target gene sensitivity to miRNA suppression by 5’UTR.

    No full text
    <p>(<b>A</b>) An engineered psiCheck2 vector (psiCheck-2-pd) for investigating the effect of 5’UTR and 3’UTR on reporter gene expression. TSS, transcription start site. (<b>B</b>) Experimental scheme of reporter assays in primary B cells. FACS plots show electroporation efficiency using a GFP-expressing plasmid. (<b>C,D</b>) Dual luciferase reporter assay to determine the effect of 5’UTR and 3’UTR on the reporter gene protein (luciferase activity) (<b>C</b>) and mRNA (qRT-PCR) levels (<b>D</b>). Closed and open circles indicate reporters with wild-type (wt) and mutated (mut) <i>CD69</i> 3’UTR, respectively. A comparison of renilla luciferase activity normalized to firefly luciferase activity (hRluc/Fluc) between psiCheck-2-pd containing mut and wt <i>CD69</i> 3’UTR reveals the sensitivity of the renilla luciferase mRNA (hRluc) to miR-17~92-mediated suppression. Results of normalized hRlcu/Fluc (n = 10) are from three independent experiments. Each experiment contained 3–4 replicates.</p

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

    No full text
    <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

    Depression-like behavior in the tail suspension test (TST).

    No full text
    <p>High (HAB), normal (NAB) and low (LAB) anxious animals were fed with <sup>14</sup>N and/or <sup>15</sup>N enriched bacteria diets. <b>A</b>) No behavioral changes due to diet <i>per se</i> were found for animals fed with bacteria diet compared to the animals of the respective lines of the HAB/NAB/LAB standard breeding of the same generation (dotted lines and indicated in <b>B</b>) as percentage difference of the standard breeding). However, <sup>15</sup>N fed HAB animals showed a strongly reduced immobility, indicating depression-like behavior, compared to <sup>14</sup>N fed HABs (**p<0.01). B) This differed significantly from the standard HAB/NAB/LAB breeding (*p<0.05). Due to division by zero, no value is given for <sup>14</sup>N fed LAB animals in relation to the standard breeding: values are <sup>14</sup>N LAB 0.53±0.3 sec vs. standard LAB 4.06±1.5 sec.</p

    Quantification of miR-17~92 miRNAs and binding sites in primary B cells.

    No full text
    <p>(<b>A,B</b>) Quantitative Northern blot to determine miR-17~92 miRNA copy numbers. Indicated amounts of synthetic miR-17, miR-18a, miR-19b and miR-92 were added to naïve and activated TKO B cells before RNA extraction. The copy numbers of each miRNA subfamily were determined by Northern blot comparing WT B cells and TKO B cells with graded amounts of spike-in synthetic miRNAs, using a mixture of probes corresponding to all members of a miRNA subfamily (also see <b><a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006623#pgen.1006623.s029" target="_blank">S7 Table</a></b>). Naïve B cells were activated with LPS and IL-4 for indicated amounts of time (h, hour). (<b>C-E</b>) Summary of miR-17~92 family miRNA copy numbers (<b>C</b>), conserved miR-17~92 family miRNA binding sites (<b>D</b>) (also see <b><a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006623#pgen.1006623.s030" target="_blank">S8 Table</a></b>), and ratios of conserved miR-17~92 family miRNA binding sites to miRNAs (<b>E</b>) in naïve and activated B cells.</p

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

    No full text
    <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

    Depression-like behavior of HAB animals in the tail suspension test (TST).

    No full text
    <p>Animals fed with blue-green algae diet hat a strongly reduced immobility, indicating depression-like behavior, compared to standard fed animals. This effect was even more pronounced in animals fed with the <sup>15</sup>N isotope (*p<0.05; **p<0.01).</p

    Incorporation rate of <sup>15</sup>N in (A) plasma and (B) brain proteins.

    No full text
    <p>In 5, 14 and 28 day old animals (PND 5, 14, 28) bacteria diet feeding provides a significantly higher incorporation of <sup>15</sup>N in plasma proteins compared to blue-green algae diet; only at the age of 56 days, blue-green algae fed diet animals show the same <sup>15</sup>N incorporation. In the cerebellum, a faster incorporation in adolescence at PND 14 reached significance (**p<0.01; *p<0.05).</p

    Ribosome accumulation in 5’UTR correlates with translational repression of target genes.

    No full text
    <p>(<b>A-C</b>) Ribosome accumulation in 5’UTRs of ribo-upregulated TKO targets in WT B cells (<b>A</b>), ribo-downregulated TG targets in TG B cells (<b>B</b>), but not in 5’UTRs of other targets (<b>C</b>). Ribosome occupancy in 5’UTR was normalized to the overall ribosome footprint abundance of the same gene [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006623#pgen.1006623.ref096" target="_blank">96</a>]. The first nucleotide of start codon is set as position 0 (grey dashed line). (<b>D</b>) Inverse correlation between ribosome occupancy in 5’UTR and the overall ribosome density on target mRNA in WT B cells. (<b>E</b>) High GC content in 5’UTRs of ribo-upregulated TKO targets.</p
    corecore