Endoplasmic Reticulum Chaperone Glucose-Regulated Protein 94 Is Essential for Proinsulin Handling

Abstract

Although endoplasmic reticulum (ER) chaperone binding to mutant proinsulin has been reported, the role of protein chaperones in the handling of wild-type proinsulin is underinvestigated. Here, we have explored the importance of glucose-regulated protein 94 (GRP94), a prominent ER chaperone known to fold insulin-like growth factors, in proinsulin handling within β-cells. We found that GRP94 coimmunoprecipitated with proinsulin and that inhibition of GRP94 function and/or expression reduced glucose-dependent insulin secretion, shortened proinsulin half-life, and lowered intracellular proinsulin and insulin levels. This phenotype was accompanied by post-ER proinsulin misprocessing and higher numbers of enlarged insulin granules that contained amorphic material with reduced immunogold staining for mature insulin. Insulin granule exocytosis was accelerated twofold, but the secreted insulin had diminished bioactivity. Moreover, GRP94 knockdown or knockout in β-cells selectively activated protein kinase R-like endoplasmic reticulum kinase (PERK), without increasing apoptosis levels. Finally, GRP94 mRNA was overexpressed in islets from patients with type 2 diabetes. We conclude that GRP94 is a chaperone crucial for proinsulin handling and insulin secretion.

Figure 1

GRP94 interacts with proinsulin. A: GRP94-proinsulin interaction was analyzed in silico by the docking computational modeling software (ZDOCK 3.0.2) with proinsulin (2KQP, purple) and three GRP94 crystal structures: one open (5ULS) and two closed (2O1U and 2O1V, all green/blue). The top-scored prediction model of each pair is presented. CD, C-terminal domain; MD, middle domain; ND, N-terminal domain. Next, binding of GRP94 to proinsulin was tested in B. C: GRINCH cells (INS-1 cells stably expressing hPro-CpepSfGFP) and INS-1E cells expressing GFP-tagged GRP94. Cells were lysed, and GRP94 (INS-1E) or proinsulin (GRINCH) was subjected to immunoprecipitation (via GFP-tag) and analyzed by SDS-PAGE and WB for the presence of proinsulin or GRP94, respectively. B and C: Representative blot of n = 3. IP, immunoprecipitation.

Figure 2

GRP94 KD or KO does not lead to β-cell death and induces ER stress via the PERK-ATF4-CHOP pathway. A: SDS-PAGE and WB analysis of GRP94 expression levels in INS-1E cells after lentiviral transduction with GRP94 targeting shRNA (cells lysed 2 weeks after viral transduction, KD) or CRISPR/Cas9 guide GRP94-directed RNA (clonal cell line shown 3 months after viral transduction, KO); n = 5. B: Apoptosis levels (representing internucleosomal degradation of genomic DNA) were analyzed in GRP94 KD and KO (clone 1 and 2) and control cells exposed for 24 h to 1 μmol/L of thapsigargin (Tg); n = 4. C: mRNA levels of ER stress pathways and Ins-1/2 genes were analyzed by quantitative RT-PCR in INS-1E control and GRP94 KD cells. The Ire1 inhibitor 4q8C (Ire1i) was used at 30 μmol/L for 4 h. Data represent the means ± SD analyzed by Bonferroni-corrected paired Student t test of treatments vs. control, *P < 0.02; ***P < 0.0005; n = 6. Ctrl, control.

Figure 3

Diminished intracellular proinsulin and insulin contents after GRP94 KD, KO, or pharmacological inhibition. SDS-PAGE and WB (A, C, and D) or ELISA (300,000 cells) (B) analysis of proinsulin and insulin expression levels in INS-1E cells and dispersed human islet cells (n = 2) (D) after 3 h in 2 or 20 mmol/L glucose–containing media after lentiviral transduction of GRP94-specific shRNA (cells collected 2 weeks after viral transduction, KD [shGRP94 1 and 2]), CRISPR/Cas9 guide GRP94-directed RNA (clonal cell lines shown 3 months after viral transduction, KO), and 4-h exposure to GRP94-specific ATPase inhibitor PU-WS13 (representative blot of n = 6 on the left and band quantification on the right). Data represent means ± SD analyzed by Bonferroni-corrected paired Student t test of treatments vs. control. Ctrl, control.

Figure 4

GRP94 KD cells show diminished, non-ER proinsulin staining accompanied by an increased number of larger secretory granules with amorphic content and increased exocytotic events. A: INS-1E cells (control [Ctrl] or GRP94 KD) were transfected with plasmids encoding for ER-mCherry, and 48 h later, cells were incubated for 3 h in 2 mmol/L glucose–containing medium. Next, cells were fixed with 2% paraformaldehyde and immunostained with mouse monoclonal antiproinsulin (GS-9A8) antibody followed by secondary antibody treatment (FITC). B: GRINCH cells were clonally derived from an INS-1 cell line stably expressing hProCpepSfGFP. The cells were incubated for 3 h in 2 mmol/L glucose–containing medium and fixed with 2% paraformaldehyde. A and B: Immunofluorescence was acquired by confocal laser microscopy, and illustrative images from three independent experiments are shown. C: Representative transmission EM of Ctrl and GRP94 KD INS-1E cells cultured for 3 h in 2 mmol/L glucose–containing medium. Images were obtained using a CM100 BioTWIN with tungsten emitter. Arrows point to secretory vesicles (black, vesicles with dark content; white, vesicles with gray/white content). D and E: EM-visualized vesicles were manually counted, marked, and measured in 19 images of Ctrl and 12 images of GRP94 KD cells. In total, 427 vesicles for Ctrl and 618 for GRP94 KD cells were analyzed. Each dot on the graph represents the number of vesicles in a single cell (D) (data represent the means ± SEM analyzed by Bonferroni-corrected nonpaired Student t test of treatments vs. Ctrl). Mean signal intensity of vesicles: dark <145, gray 146–170, and white >171. Correlation between vesicle size and intensity signal of its content in Ctrl and GRP94 KD cells (E). C–E: n = 3. F–J: Exocytosis of fluorescently labeled granules during application of 75 mmol/L K⁺. F: Representative TIRF microscopy images of Ctrl (WT) and GRP94 KO (1) INS-1 cells expressing the granule marker NPY-GFP. G: Average cumulative number of exocytotic events as a function of time and normalized to the cell area; K⁺ was elevated from 10 s. INS-1E (Ctrl), GRP94 KO, and GRP94i (20 μmol/L for 24 h). H: Total exocytosis (mean ± SEM) for three independent experiments as in I; number of cells is shown on bars. I and J: As in G and H, but for human β-cells from five donors with or without GRP94i pretreatment (20 μmol/L for 24 h). In H and J, the difference from Ctrl was tested with Student t tests.

Figure 5

GRP94 KD/KO causes proinsulin misprocessing and increased cellular turnover. A: Control (Ctrl) and GRP94 KD INS-1E cells were cultured for 3 h in 2 mmol/L glucose containing Krebs-Ringer bicarbonate HEPES (KRBH) buffer and lysed. In order to preserve intramolecular disulfide bonds and visualize proinsulin folding intermediates, cell lysates were analyzed by nonreducing SDS-PAGE (Nu-Page 4–12% Bis-Tris Protein Gel), and proinsulin and insulin were visualized through WB with antiproinsulin and anti-insulin antibody (L6B10; Cell Signaling); n = 3. B: INS-1E Ctrl or GRP94 KO cells were pulse labeled with ³⁵S-Met/Cys for 1 h (C0) and chased for 1 h (C1 and M1) in 11 mmol/L glucose. Cell lysates and culture supernatants were immunoprecipitated with anti-insulin antibody, and newly synthesized proinsulin was analyzed by nonreducing Tris-tricine-urea SDS-PAGE and phosphorimaging. Numbers 1–4 identify proinsulin 1 and 2 conversion intermediates. C, cells; M, media. n = 3. C: INS-1E Ctrl and GRP94 KD cells were cultured for 3 h in 2 mmol/L glucose–containing KRBH buffer, and subsequently 100 μmol/L of the protein synthesis inhibitor cycloheximide was added and cells were lysed at the indicated time points; n = 3. D: Ctrl and GRP94 KO cells were treated similarly, but 200 nmol/L of the inhibitor of exocytosis brefeldin A was added to the culture media from the start of the experiment. Cell lysates were analyzed via reducing SDS-PAGE, and proinsulin and insulin were visualized through WB with antiproinsulin antibody; n = 3. Quantification of proinsulin bands was performed with ImageJ and normalized to tubulin bands (C and D, right panels). The quantification data are presented as means ± SEM analyzed by Bonferroni-corrected paired Student t test of treatments vs. Ctrl. AUC, area under the curve.

Figure 6

Glucose-stimulated secretion of bioactive insulin is impaired in GRP94-deficient cells, and GRP94 mRNA is overexpressed in islet cells from patients with T2D. INS-1E control (Ctrl) and GRP94 KD or INS-1E cells exposed to 20 μmol/L of GRP94i for 24 h (A [n = 5] and B [n = 4]) as well as dispersed human islet cells (C) (1 week after transduction with lentivirus carrying nontargeting or GRP94-targeting shRNA coding plasmids; n = 4) were tested for their ability to secrete insulin in response to the given glucose concentrations in Krebs-Ringer bicarbonate HEPES buffer. Supernatants were analyzed by ELISAs specifically detecting mature insulin (A–C, left graphs) or proinsulin (A–C, right graphs) only. The bars represent the means ± SD. D: Accumulated secretion of proinsulin and insulin over the period of 6 h in INS-1E Ctrl and 20 μmol/L GRP94i 24 h pretreated, GRP94 KO Ctrl, and KO cells were analyzed by SDS-PAGE and WB (n = 3). One milliliter of cell supernatants was concentrated using 10-kDa molecular weight cutoff filters to remove salts and reduce volume to 15 μL. E and F: TIRF recordings of PIP3 from a single MIN6 cell stimulated with 30 mmol/L K⁺ and 0.5 μmol/L insulin. Cells were preincubated without or with 20 μmol/L of the GRP94i for 0.5, 4, or 8 h. F: Means ± SEM of the amplitudes of the PIP3 responses to endogenous K⁺-triggered insulin secretion (left) and exogenous insulin (right) from experiments as in E with the indicated treatments and numbers of cells. **P < 0.01 for difference from Ctrl (one-way ANOVA with post hoc Tukey honestly significant difference test). Means ± SEM for the ratio of the PIP3 amplitude in response to K⁺ over that of insulin in individual cells (right). F: Means ± SEM for the time to half-maximal PIP3 increase after K⁺ stimulation. **P < 0.01 for difference from Ctrl (one-way ANOVA with post hoc Tukey honestly significant difference test). G: Expression levels of GRP94 in dispersed donor human islets α- and β-cells from healthy individuals (n = 6) and patients with T2D (n = 4) were determined by single-cell RNA sequencing and quantified by expression values per cell. Statistical analysis was performed using ANOVA with Bonferroni correction for multiple comparisons of T2D vs. healthy donors. **P < 0.01; ****P < 0.0001.