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. 2018 Apr 2;217(4):1287-1301.
doi: 10.1083/jcb.201707143. Epub 2018 Mar 5.

IRE1-XBP1 pathway regulates oxidative proinsulin folding in pancreatic β cells

Yuichi Tsuchiya  1 , Michiko Saito  1   2 , Hiroshi Kadokura  1   3 , Jun-Ichi Miyazaki  4 , Fumi Tashiro  4 , Yusuke Imagawa  1   5 , Takao Iwawaki  6 , Kenji Kohno  7
Affiliations

IRE1-XBP1 pathway regulates oxidative proinsulin folding in pancreatic β cells

Yuichi Tsuchiya et al. J Cell Biol. .

Erratum in

Abstract

In mammalian pancreatic β cells, the IRE1α-XBP1 pathway is constitutively and highly activated under physiological conditions. To elucidate the precise role of this pathway, we constructed β cell-specific Ire1α conditional knockout (CKO) mice and established insulinoma cell lines in which Ire1α was deleted using the Cre-loxP system. Ire1α CKO mice showed the typical diabetic phenotype including impaired glycemic control and defects in insulin biosynthesis postnatally at 4-20 weeks. Ire1α deletion in pancreatic β cells in mice and insulinoma cells resulted in decreased insulin secretion, decreased insulin and proinsulin contents in cells, and decreased oxidative folding of proinsulin along with decreased expression of five protein disulfide isomerases (PDIs): PDI, PDIR, P5, ERp44, and ERp46. Reconstitution of the IRE1α-XBP1 pathway restored the proinsulin and insulin contents, insulin secretion, and expression of the five PDIs, indicating that IRE1α functions as a key regulator of the induction of catalysts for the oxidative folding of proinsulin in pancreatic β cells.

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Figures

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Graphical abstract
Figure 1.
Figure 1.
Physiological activation of the IRE1α–XBP1 pathway in pancreatic islets. (A) Xbp1 mRNA splicing was analyzed by RT-PCR using total RNA isolated from the tissues of 8-wk-old WT male mice. The ratio of Xbp1 mRNA splicing was quantified. Error bars show the means and SD. n = 3. Xbp1 splicing (%) = Xbp1s/total Xbp1 × 100. Lane 1, Xbp1u_pcDNA3.1(+). Lane 2, Xbp1s_pcDNA3.1(+). (B) Expression levels of IRE1α and ER-resident proteins harboring the KDEL motif (KDEL (ER)) in mouse tissues from an 8-wk-old WT male mouse based on immunoblotting. The expression levels of the cytosolic chaperone HSP90 (HSP90 (Cyt)) and the mitochondrial chaperone HSP60 (HSP60 (Mit)) were also examined. Positions of molecular mass markers are indicated on the left.
Figure 2.
Figure 2.
Phenotypic analysis of pancreatic β cell–specific Ire1α CKO (IRE1αB(-/ΔR)) mice. (A) Serum glucose in male mice of the indicated ages (wk). Horizontal lines show the means for each group; points show glucose measurements for individual mice in each group (IRE1αB(+/ΔR) mice, blue points, n = 9; IRE1αB(-/ΔR) mice, red points, n = 9). (B) Glucose tolerance of 12-wk-old male mice. Points show the means and SD for each group (IRE1αB(+/ΔR) mice, n = 7; IRE1αB(-/ΔR) mice, n = 5). Values for IRE1αB(+/ΔR) and IRE1αB(-/ΔR) mice are indicated by a blue solid line and a red dashed line, respectively. (C) Serum insulin levels of individual mice at 20 wk of age were measured by ELISA and are shown as points. The mean in each group is shown as a horizontal line (IRE1αB(+/ΔR) mice, blue points, n = 23; IRE1αB(-/ΔR) mice, red points, n = 7). (D) Detection of proinsulin and insulin in the pancreatic islets of male mice at 20 wk of age by immunoblotting. n = 3. (E) Protein expression levels of proinsulin and insulin relative to that of GAPDH in D were quantified and normalized by the value obtained for IRE1αB(+/ΔR) mice. Blue bars indicate islets from IRE1αB(+/ΔR) mice. Red bars indicate islets from IRE1αB(-/ΔR) mice. Error bars show the means and SD for each group. n = 3. *, P < 0.05; **, P < 0.01 by Student’s t test.
Figure 3.
Figure 3.
Establishment of MIN6 cells lacking the IRE1α-RNase domain. (A–I) MIN6 (Ire1αfl/fl) cells were established and then infected with Ad-Cre to delete the RNase domain of Ire1α. MIN6 (Ire1αfl/fl) cells infected with Ad-GFP were used as a control for adenovirus infection. Non–adenovirus-infected MIN6 (Ire1αfl/fl) cells were also used as controls (Con). (A) Levels of IRE1α, proinsulin, insulin, and GAPDH were analyzed by immunoblotting in the indicated MIN6 cells. RT-PCR for Xbp1 mRNA relative to total RNA isolated from the indicated MIN6 cells. (B) Splicing ratios of Xbp1 mRNA in A were quantified. White bars indicate MIN6 (Ire1αfl/fl) cells as a control (Con). Blue bars indicate MIN6 (Ire1αfl/fl) cells infected with Ad-GFP. Red bars indicate MIN6 (Ire1αΔR/ΔR) cells infected with Ad-Cre. n = 3. (C) Protein expression levels of proinsulin relative to those of GAPDH in A were quantified and normalized by the value obtained for MIN6 (Ire1αfl/fl) cells without adenovirus infection as a control (Con). Each bar presents the indicated MIN6 cells as described in B. (D) Protein expression levels of insulin relative to those of GAPDH in A were quantified and normalized as described in C. Each bar presents the indicated MIN6 cells as described in B. (E) Insulin secretion in response to various glucose concentrations in indicated MIN6 (Ire1αfl/fl) cells was measured by ELISA. White bars indicate insulin secretion upon exposure to low glucose (LG). Orange bars indicate insulin secretion upon exposure to high glucose (HG). L indicates low glucose treatment (1.67 mM glucose/KRBH; 4 h). H indicates high glucose treatment (16.7 mM glucose/KRBH; 4 h). n = 3. (F and G) Sections of the indicated MIN6 cells were observed by transmission electron microscopy at 8,000×. (F) Sections of Ad-GFP, MIN6 (Ire1αfl/fl) cells. The right panel is an enlarged part of the left panel. Blue arrowheads indicate the ER. (G) Sections of Ad-Cre, MIN6 (Ire1αΔR/ΔR) cells. The right panel is an enlarged part of the left panel. Red arrowheads indicate the ER. Bars, 2 µm. (H) Number of insulin granules per µm2 in MIN6 (Ire1αfl/f or Ire1αΔR/ΔR) cells was quantified by using electron microscope images and calculated using ImageJ software. Error bars show means and SD. n = 10. (I) Sizes of insulin granules (µm2) of MIN6 (Ire1αfl/f or Ire1αΔR/ΔR) cells were quantified as described in H. Results are summarized in box plots, and dots indicate individual size estimates of insulin granules. n = 1,000. **, P < 0.01.
Figure 4.
Figure 4.
Decreased oxidative folding of proinsulin in IRE1α-RNase domain KO MIN6 cells. (A) Levels of Ins genes and transcription factors that regulate Ins genes relative to Gapdh expression levels in the indicated MIN6 cells were measured by qRT-PCR. White bars indicate control MIN6 (Ire1αfl/fl) cells (Con). Blue bars indicate MIN6 (Ire1αfl/fl) cells infected with Ad-GFP. Red bars indicate MIN6 (Ire1αΔR/ΔR) cells infected with Ad-Cre. (B) To detect newly synthesized preproinsulin and proinsulin in the indicated MIN6 cells, MIN6 cells were pulsed with [35S]Met/Cys for 30 min. Radiolabeled preproinsulin and proinsulin were then immunoprecipitated, separated by NuPAGE, and detected by autoradiography. The ratio of the translation of preproinsulin and proinsulin in MIN6 (Ire1αΔR/ΔR) cells to that of MIN6 (Ire1αfl/fl) cells was quantified. Blue bar, MIN6 (Ire1αfl/fl) cells; red bar, MIN6 (Ire1αΔR/ΔR) cells. (C) To compare the rate of conversion from preproinsulin to proinsulin in MIN6 (Ire1αfl/fl or Ire1αΔR/ΔR) cells, the cells were pulsed for 1 min with [35S]Met/Cys and then chased for the indicated times. The ratio of proinsulin to the sum of preproinsulin and proinsulin was quantified. The values for MIN6 (Ire1αfl/fl) and MIN6 (Ire1αΔR/ΔR) cells are indicated by a blue solid line and by a red broken line, respectively. The values obtained did not differ significantly among time points by Student’s t test. (D) To compare the rate of oxidative folding of proinsulin in MIN6 (Ire1αfl/fl or Ire1αΔR/ΔR) cells, the cells were pulsed for 5 min with [35S]Met/Cys and then chased for the indicated times. Radiolabeled proinsulin was immunoprecipitated with an antiproinsulin antibody, boiled with (Reducing) or without (Nonreducing) DTT, separated by NuPAGE, and detected by autoradiography. The closed diamond indicates preproinsulin. (E) The ratio of oxidized proinsulin (left, red arrowhead) to total proinsulin (right, white arrowhead) in D was quantified. Error bars represent means and SD for each group. n = 3. *, P < 0.05; **, P < 0.01.
Figure 5.
Figure 5.
Expression levels of ER chaperones and PDI family proteins in MIN6 (Ire1αΔR/ΔR) cells and pancreatic islets of IRE1αB(-/ΔR) mice. (A) Expression levels of PDI family proteins and ER chaperones in the MIN6 (Ire1αfl/fl or Ire1αΔR/ΔR) cells were analyzed by immunoblotting. (B) Expression levels of PDI family proteins and ER chaperones relative to the GAPDH expression in the MIN6 (Ire1αfl/fl or Ire1αΔR/ΔR) cells in A were measured by immunoblotting and normalized by the value obtained for MIN6 (Ire1αfl/fl) cells without adenovirus infection (Con). White bars indicate control MIN6 (Ire1αfl/fl) cells (Con). Blue bars indicate MIN6 (Ire1αfl/fl) cells infected with Ad-GFP. Red bars indicate MIN6 (Ire1αΔR/ΔR) cells infected with Ad-Cre. (C) Expression levels of PDI family proteins and ER chaperones in pancreatic islets from the indicated mice at 24 wk of age were analyzed by immunoblotting. (D) Expression levels of PDI family proteins and ER chaperones relative to those of GAPDH in C were quantified and normalized by the value of control mice. Blue bars indicate islets from IRE1αB(+/ΔR) mice. Red bars indicate islets from IRE1αB(-/ΔR) mice. Error bars show means and SD for each group. n = 3. *, P < 0.05; **, P < 0.01.
Figure 6.
Figure 6.
Reconstitution of the IRE1α–XBP1 pathway in MIN6 (Ire1αΔR/ΔR) cells. (A–G) To examine whether the phenotype of MIN6 (Ire1αΔR/ΔR) cells is restored by reconstituting the IRE1α–XBP1 pathway, WT Ire1α and Xbp1s were stably expressed by retroviruses harboring WT Ire1α and Xbp1s in MIN6 (Ire1αΔR/ΔR) cells, respectively. In B, C, D, and G, black bars indicate MIN6 (Ire1αfl/fl) cells infected with retrovirus harboring an empty vector (Vec). Red bars indicate MIN6 (Ire1αΔR/ΔR) cells infected with retrovirus harboring an empty vector. Orange bars indicate MIN6 (Ire1αΔR/ΔR) cells infected with retrovirus harboring WT Ire1α. Yellow bars indicate MIN6 (Ire1αΔR/ΔR) cells infected with retrovirus harboring Xbp1s. (A) Expression levels of IRE1α, proinsulin, insulin, and GAPDH were analyzed by immunoblotting in the indicated MIN6 cells. RT-PCR for Xbp1 mRNA of total RNA isolated from the indicated MIN6 cells. (B) The ratio of Xbp1 mRNA splicing in A was quantified. Xbp1 splicing (%) = Xbp1s/total Xbp1 × 100. (C) Expression levels of proinsulin relative to the GAPDH expression in A were quantified and normalized by the value obtained for MIN6 (Ire1αfl/fl) cells infected with retrovirus harboring an empty vector. (D) Expression levels of insulin relative to those of GAPDH in A were quantified and normalized as described in C. (E) Insulin secretion in response to glucose concentration in the indicated MIN6 cells for 4 h was measured by ELISA. White and black bars indicate insulin secretion upon exposure to low (L; 1.67 mM glucose/KRBH; 4 h) and high (H; 16.7 mM glucose/KRBH; 4 h) glucose, respectively. HG, high glucose; LG, low glucose. (F) Expression levels of PDI family proteins and ER chaperones in the indicated MIN6 cells were analyzed by immunoblotting. (G) Protein expression levels of PDI family proteins and ER chaperones relative to those of GAPDH in F were quantified and normalized as described in C. Error bars represent means and SD. n = 3. *, P < 0.05; **, P < 0.01.
Figure 7.
Figure 7.
XBP1s directly binds to the promoter regions of the five PDI family genes. (A) ChIP assay using normal IgG and anti–XBP1s IgG. PCR of the WT MIN6 cell extracts were performed using a set of primers outside of the promoter regions containing XBP1s binding sites. (B) ChIP assay followed by quantitative PCR to quantify the binding of XBP1s to proximal promoter regions of the indicated genes. Error bars indicate means and SD. n = 3.
Figure 8.
Figure 8.
Detection of mixed-disulfide complexes between proinsulin and PDI family members in MIN6 cells and reconstitution of the five PDIs in MIN6 (Ire1αΔR/ΔR) cells. (A) Cys-mediated interaction between PDI family proteins and newly synthesized proinsulin during proinsulin folding. WT MIN6 cells were transiently transfected with indicated FLAG-tagged PDI family proteins, pulsed for 5 min with [35S]Met/Cys, directly treated with TCA, and subjected to alkylation with NEM. Mixed-disulfide complexes between PDIs and proinsulin were collected by consecutive immunoprecipitation (first immunoprecipitation by anti-FLAG antibody; second immunoprecipitation by antiproinsulin antibody). After elution, samples were treated with (Reducing) or without (Nonreducing) DTT, separated by NuPAGE, and detected by autoradiography. (B) MIN6 (Ire1αΔR/ΔR) cells were transiently transfected with WT five PDIs or five CM PDIs. The expression of insulin was induced by culturing cells for 4 h in the presence of low glucose (LG; L; 1.67 mM glucose/KRBH; 4 h) or high glucose (HG; H; 16.7 mM glucose/KRBH; 4 h), and the amounts of secreted insulin were measured by ELISA. White bars indicate insulin secretion upon exposure to low glucose. Black bars indicate insulin secretion upon exposure to high glucose. (C) MIN6 (Ire1αfl/fl) cells were transiently transfected with WT PDI/PDIA1 or WT five PDIs. The expression of insulin was induced, and amounts of secreted insulin were measured as described in B. n = 3. **, P < 0.01. EV, empty vector.
Figure 9.
Figure 9.
Schematic diagram illustrating the function of IRE1α as a key regulator of oxidative proinsulin folding and PDI family levels in pancreatic β cells.
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