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. 2015 Oct 15;13(10):e1002277.
doi: 10.1371/journal.pbio.1002277. eCollection 2015 Oct.

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

Justin R Hassler  1 , Donalyn L Scheuner  2 , Shiyu Wang  3 , Jaeseok Han  3 , Vamsi K Kodali  3 , Philip Li  3 , Julie Nguyen  3 , Jenny S George  4 , Cory Davis  4 , Shengyang P Wu  5 , Yongsheng Bai  6 , Maureen Sartor  7 , James Cavalcoli  7 , Harmeet Malhi  4 , Gregory Baudouin  3 , Yaoyang Zhang  8 , John R Yates III  8 , Pamela Itkin-Ansari  3 , Niels Volkmann  3 , Randal J Kaufman  9
Affiliations

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

Justin R Hassler et al. PLoS Biol. .

Abstract

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 (Xbp1) mRNA splicing in adult primary β cells. Using mRNA sequencing (mRNA-Seq), we show that unconventional Xbp1 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 Ire1α 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, Ire1α deletion did not alter insulin mRNA expression either in the presence or absence of glucose stimulation. Instead, β cell failure upon Ire1α 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, Ire1α 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 Ire1α deletion. The results demonstrate that glucose activates IRE1α-mediated Xbp1 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.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Tam-induced Ire1α deletion in adult β cells reduces proinsulin synthesis, insulin content, and insulin secretion, without altering insulin mRNA levels.
(A) Blood glucose levels for 16-wk-old male mice following 4 h of fasting with increasing weeks post-Tam. Respectively for 4, 8, and 20 wk post-Tam ([p = 0.042, 0.009, 0.031], [WT Fe/+ n = 8, KO Fe/-; Cre n = 10]). (B) Glucose tolerance tests (GTTs) performed on 16-wk-old male mice at 6 wk post-Tam and the areas under the curves 6 wk post-Tam. The values for statistical significance in Fig 1A and S1A Fig were calculated from areas under the GTT curves. The data and statistics for the GTTs and all other data except when indicated are within S1 Data. ([WT Fe/+, Het-I Fe/+; Cre, Het-B Fe/- and KO Fe/-; Cre n = 8], [p = 1.408 x 10−7, WT Fe/+ versus KO Fe/-; Cre]). (C) Serum insulin levels in mice 6 wk post-Tam: fed, 4 h fasted, 30 and 90 min after refeeding ([n = 4, all groups], student’s t test for significance for WT Fe/+ versus KO Fe/-; Cre [p = 0.044]). (D) Immunofluorescence microscopy of islets co-stained for insulin (red), proinsulin (green), and DAPI (blue); see S3B Fig for additional examples. Scale bar, 100 μm. (E) Percent islet areas were determined on 6-wk post-Tam pancreas by outlining 138, 153, 234, and 297 cross sections from 9, 9, 11, and 14 mice WT Fe/+, Het-I Fe/+; Cre, and KO Fe/-; Cre groups, respectively. (F and G) Insulin and proinsulin ELISAs of acid ethanol extracts from 6 wk post-Tam mouse pancreas (WT Fe/+ versus KO Fe/-; Cre; [insulin, p = 0.039, proinsulin, p = 0.031], [n = 5, all groups]). (H) Individual mouse proinsulin/insulin ratios were determined and averaged ([p = 0.009], [n = 5]). (I) Islets were shifted from 4 mM to 25 mM glucose for 30 min in [35S]-Cys/Met in order to determine the synthesis rate during high glucose by antiproinsulin immunoprecipitation IP ([n = 3] for WT Fe/+, Het-I Fe/+; Cre, and KO Fe/-; Cre), ([n = 6] for +/+ infected with Ad-Cre versus Ad-ΔR (p = 0.019)]. Since limiting amounts of a homemade proinsulin antibody was used for the first five lanes, the Ad experiments used a commercial antibody that produced consistent results. (J) Real-time PCR (quantitative real-time PCR [qRT-PCR]) of total RNA isolated from islets at 6 wk post-Tam ([n = 5], [p = 0.022**, 0.039*, and 0.047*]) for Ire1α deletion-specific, Xbp1 spliced-specific, and Mafa mRNAs, respectively.
Fig 2
Fig 2. KO islets exhibit ER stress.
(A) qRT-PCR of UPR genes in islets isolated 6 wk post-Tam and incubated in 11 mM glucose 16 h ([n = 5], [p ≤ 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 KO Fe/-; Cre 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 S3B Fig. (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 Xbp1 mRNA spliced by IRE1α from the islet complementary DNAs (cDNAs) used for mRNA-Seq analysis, 6 mM versus 18 mM glucose. Results representative of n = 5 per genotype. (E) Global heatmap for the ~22,000 mRNAs detected by mRNA-Seq for 18 mM KO Fe/-; Cre & WT Fe/+ samples; green and red indicate increased and decreased expression. The blue box indicates genes with inverse expression dependent on IRE1α and high glucose.
Fig 3
Fig 3. mRNA sequencing identifies IRE1α- and glucose-dependent mRNAs in islets.
(A) mRNA-Seq data on β cell-specific mRNAs. The results show no significant change to INS1 or INS2 in the KO Fe/-; Cre samples, while MAFA, GCG, and PC5 are increased by deletion ([n = 5], [18 mM KO Fe/-; Cre, p-values ≤ 0.05]). mRNA-Seq expression fold changes were normalized relative to the 6 mM WT Fe/+ islet context. (B) Four-way Venn diagrams of WT Fe/+ versus KO Fe/-; Cre islets during 6 mM versus 1 8mM glucose exposure for 72 h. Ire1α-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 Ire1α- and glucose-dependent trends of interest are labeled “Induction” and “Repression.” (C) Combined DAVID (the Database for Annotation, Visualization and Integrated Discovery) and “ConceptGen” GO analysis of Ire1α- 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 Ad-IREα-K907A (Ad-ΔR) versus Ad-β-Galactosidase (β-Gal). 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 (n = 3). The proteins shown (Fig 3D) exhibit the same expression dependence for IRE1α as measured by mRNA-Seq (S2 Data).
Fig 4
Fig 4. KO islets accumulate oxidative stress, inflammation, and fibrosis.
(A) mRNA-Seq expression values for 25/368 of the mRNAs identified by Venn analysis (Fig 3C; right panel, underlined) that are reduced by Ire1α because of glucose that accumulates in the KO Fe/-; Cre ([n = 5], [p-values ≤ 0.05]). (B) Oxidized lipid (hydroxyl-octadecadienoic acids, HODEs) from islets of the indicated genotypes infected with Ad-Cre Ad-GFP or no virus control ([n = 2; controls versus n = 3; Ad-Cre], [p = 0.00434]). (C) Antinitrotyrosine immunohistochemistry (IHC) of islets from 8-mo-old WT Fe/Fe and KO Fe/Fe; Cre mice 15 wk post-Tam with or without BHA diet for 3 wk. (Scale bar, 50 μm) (WT Fe/Fe [n = 4 with BHA], [n = 5 regular chow]), (KO Fe/Fe; Cre [n = 5 with BHA], [n = 6 regular chow]). (p = 0.00698; WT Fe/Fe versus WT Fe/Fe with BHA), (p = 0.04018; WT Fe/Fe versus KO Fe/Fe; Cre) and (p = 0.04420; KO Fe/Fe; Cre versus KO Fe/Fe; Cre with BHA). (D) Masson’s trichrome stain (blue) for collagens. Results demonstrate increased staining surrounding KO Fe/Fe; Cre islets with haemotoxylin (red) and eosin (black) co-stains. Quantification of percent strong collagen stain is shown below the images. Scale bar, 50 μm. (WT Fe/Fe [n = 4 with BHA], [n = 5 regular chow]), (KO Fe/Fe; Cre [n = 5 with BHA], [n = 6 without BHA]). Percent strong collagen stain significance for WT Fe/Fe without BHA versus KO Fe/Fe; Cre without BHA p = 0.01049). (E) 8-mo-old male mice carrying the doubly floxed allele (Ire1α Fe/Fe ) 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. (WT Fe/Fe [n = 11 with BHA], [n = 12 regular chow], [p = 0.035]), (KO Fe/Fe; Cre [n = 18 with BHA], [n = 16 without BHA], [p = 0.041]). P-values were calculated by one-tailed student’s t test comparison of the areas under the GTT curves for the biological replicates of control group WT Fe/Fe versus the Tam-induced KO Fe/Fe; Cre group.
Fig 5
Fig 5. IRE1α mediated Xbp1 splicing is necessary for proper signal peptide cleavage of preproinsulin and ribosome distribution.
(A) mRNA-Seq expression values of 24/141 mRNAs identified (Fig 3B–3D) to be both Ire1α- and high glucose-dependent for their induction ([n = 5], [p-values ≤ 0.01]). (B) Autoradiograph from whole cell islet lysates prepared by steady-state (18 h) [35S]-Cys/Met radiolabeling from 12 wk post-Tam mice (n = 3) following peptide gel electrophoresis. (C) Western blotting for proinsulin/preproinsulin, IRE1α, SEC11C, SSR1, and tubulin after peptide gel electrophoresis of lysates prepared 72 h after COS-1 cells were coinfected with adenoviruses expressing WT preproinsulin, the Akita mutant (A), and/or the (G) GFP, (X) XBP1s, or (Δ) the dominant negative IRE1α-RNase mutant K907A representative results shown (n = 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 ([n = 3], [p = 2.2 x 10−15]). (E) Subcellular fractionation and western blot analysis for ribosomal small subunit 9 and tubulin of the Ire1α Fe/Fe β 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 (n = 3). The 12 mM condition western blots and the quantified results normalized to tubulin membranous/cytosolic are shown in S6D Fig.
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