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. 2018 Nov;67(11):2293-2304.
doi: 10.2337/db17-1351. Epub 2018 Aug 21.

Impaired Store-Operated Calcium Entry and STIM1 Loss Lead to Reduced Insulin Secretion and Increased Endoplasmic Reticulum Stress in the Diabetic β-Cell

Tatsuyoshi Kono  1   2 , Xin Tong  3 , Solaema Taleb  4 , Robert N Bone  4 , Hitoshi Iida  4 , Chih-Chun Lee  4 , Paul Sohn  5 , Patrick Gilon  6 , Michael W Roe  7 , Carmella Evans-Molina  1   2   5   8   9
Affiliations

Impaired Store-Operated Calcium Entry and STIM1 Loss Lead to Reduced Insulin Secretion and Increased Endoplasmic Reticulum Stress in the Diabetic β-Cell

Tatsuyoshi Kono et al. Diabetes. 2018 Nov.

Abstract

Store-operated Ca2+ entry (SOCE) is a dynamic process that leads to refilling of endoplasmic reticulum (ER) Ca2+ stores through reversible gating of plasma membrane Ca2+ channels by the ER Ca2+ sensor Stromal Interaction Molecule 1 (STIM1). Pathogenic reductions in β-cell ER Ca2+ have been observed in diabetes. However, a role for impaired SOCE in this phenotype has not been tested. We measured the expression of SOCE molecular components in human and rodent models of diabetes and found a specific reduction in STIM1 mRNA and protein levels in human islets from donors with type 2 diabetes (T2D), islets from hyperglycemic streptozotocin-treated mice, and INS-1 cells (rat insulinoma cells) treated with proinflammatory cytokines and palmitate. Pharmacologic SOCE inhibitors led to impaired islet Ca2+ oscillations and insulin secretion, and these effects were phenocopied by β-cell STIM1 deletion. STIM1 deletion also led to reduced ER Ca2+ storage and increased ER stress, whereas STIM1 gain of function rescued β-cell survival under proinflammatory conditions and improved insulin secretion in human islets from donors with T2D. Taken together, these data suggest that the loss of STIM1 and impaired SOCE contribute to ER Ca2+ dyshomeostasis under diabetic conditions, whereas efforts to restore SOCE-mediated Ca2+ transients may have the potential to improve β-cell health and function.

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Figures

Figure 1
Figure 1
Pharmacologic inhibition of SOCE led to impaired insulin secretion and glucose-induced Ca2+ oscillations. A­: INS-1 832/13 β-cells were loaded with Calcium 6, and Ca2+ imaging experiments were performed to measure SOCE, according to the strategy shown. B and C: Ca2+ imaging was performed with or without known SOCE inhibitors, 50 μmol/L ML-9 and 200 μmol/L 2-APB, in the presence of 5.5 mmol/L glucose, 200 μmol/L diazoxide, and 10 μmol/L verapamil (Dz + V). ER Ca2+ depletion was induced with 3 μmol/L TG, and SOCE was quantified as the cytosolic Ca2+ increase after supplementation with 2 mmol/L Ca2+. Results were displayed as the ΔF/F0 after Ca2+ supplementation. D: GSIS was measured in INS-1 cells pretreated with or without ML-9 and 2-APB and normalized to total protein content. E–H: Glucose-stimulated Ca2+ imaging was performed using Fura-2 AM in islets isolated from C57BL/6J mice and pretreated with ML-9 or 2-APB. E: Representative cytosolic Ca2+ recording after stimulation with 15 mmol/L glucose. Quantification of the average phase 1 amplitude (F), phase 2 (oscillation) amplitude (G), and oscillation period (H). I: Mean insulin concentration profiles during islet perifusion at basal 2.8 mmol/L glucose concentrations (0–20 min) and in response to 16.7 mmol/L glucose stimulation (20–60 min). DMSO or 200 μmol/L 2-APB was added to the 16.7 mmol/L glucose buffer. Fifty islets were loaded per chamber, and data are reported as ng/mL insulin normalized to islet DNA content (n = 3). J: Relative area under curve (AUC) values for first phase (20–30 min) and second phase (30–60 min) insulin secretion with DMSO and 2-APB treatment. The numbers of replicates for each experiment are indicated by the open circles. Results are displayed as the means ± SEM; *P < 0.05 compared with control (Cont) conditions.
Figure 2
Figure 2
Loss of STIM1 expression led to impaired islet glucose–stimulated Ca2+ oscillations. Islets were isolated from saline and STZ-treated C57BL/6J mice and loaded with Fura-2 AM for Ca2+ imaging experiments in the presence of 5.5 mmol/L glucose. A and B: Representative cytosolic Ca2+ recording of islets after stimulation with 15 mmol/L glucose. Quantification of the average phase 1 amplitude (C), phase 2 (oscillation) amplitude (D), and phase 2 period (E). F–H: Islets were isolated from saline and STZ-treated C57BL/6J mice. F: Total islet RNA was subjected to qRT-PCR for the quantification of STIM and Orai isoform mRNA expression levels (normalized to actin mRNA expression). G and H: Immunoblot was performed using antibodies against STIM1 and actin. Shown is a representative immunoblot and the mean ± SEM of STIM1 protein levels from multiple replicates. *P < 0.05 compared with islets from saline-treated mice. I–N: Islets isolated from STIM1flox/flox mice were transduced with a Cre-expressing adenovirus (pSTIM1KO) or empty viral control (WT). I: Reduced STIM1 protein expression in pSTIM1KO islets was confirmed by immunoblot. J–N: pSTIM1KO islets were loaded with Fura-2 AM, and Ca2+ imaging was performed. J and K: Representative cytosolic Ca2+ recording after stimulation with 15 mmol/L glucose. Quantification of the average phase 1 amplitude (L), phase 2 (oscillation) amplitude (M), and oscillation period (N); n = 4–5 per group. *P < 0.05 compared with WT islets. Numbers of replicates for each experiment are indicated by the open circles. Cont, control.
Figure 3
Figure 3
STIM1 expression was reduced in human islets from donors with T2D. Human islets were obtained from seven to nine donors without diabetes (ND donors) and 7–11 donors with T2D. A: Total RNA was isolated and subjected to qRT-PCR for quantification of STIM and Orai isoform expression levels. B and C: Total islet protein was isolated and immunoblot was performed using antibodies against STIM1 and actin. *P < 0.05 compared with ND donors. D: Correlation analysis between islet STIM1 mRNA levels and donor BMI.
Figure 4
Figure 4
STIM1 expression was decreased under proinflammatory cytokine stress and GLT. Islets from human donors without diabetes (A) and C57BL/6J mice (B) were treated with 5 ng/mL IL-1β, 100 ng/mL IFN-γ, and 10 ng/mL TNF-α for 24 h. Immunoblot was performed using antibodies against STIM1 and actin. C: INS-1 β-cells were treated with 5 ng/mL IL-1β for 24 h; total RNA was isolated and subjected to qRT-PCR for quantification of STIM and Orai isoform mRNA expression levels (normalized to actin mRNA expression). D–H: INS-1 β-cells were treated with 0, 0.05, 0.5, or 5 ng/mL IL-1β, for 24 h. Total RNA and protein were isolated and subjected to qRT-PCR for quantification of STIM mRNA (D) and protein levels (E). F and G: Cytosolic Ca2+ imaging was performed to quantitate SOCE in IL-1β–treated INS-1 cells in the presence of 5.5 mmol/L glucose, 200 μmol/L diazoxide, and 10 μmol/L verapamil (Dz + V). H: ER Ca2+ levels were indirectly estimated by quantitating the ΔF/F0 response to TG. I–L: INS-1 β-cells were treated with a combination of 0.5 mmol/L palmitate + 25 mmol/L glucose (GLT), 0.5 mmol/L palmitate + 11 mmol/L glucose (Palmitate), or vehicle control + 11 mmol/L glucose (Cont) for 24 h (I). Total RNA was isolated from control and GLT-treated INS-1 cells and subjected to qRT-PCR for quantification of STIM and Orai mRNA expression levels. J: Immunoblot for STIM1 and actin was performed in INS-1 cells treated with GLT for 24 h. K and L: Cytosolic Ca2+ imaging was performed to quantitate SOCE in GLT and palmitate-treated INS-1 cells. The number of replicates for each experiment are indicated by the open circles. *P < 0.05 compared with control conditions or for indicated comparisons.
Figure 5
Figure 5
STIM1 deletion led to impaired SOCE, reduced ER Ca2+ levels, and decreased GSIS. CRISPR/Cas-9 genomic editing was used to create an INS-1 832/13 STIM1KO cell line. A: Total RNA was isolated and subjected to qRT-PCR for quantification of STIM and Orai isoforms and SERCA2b mRNA levels. Numbers of replicates are indicated by the open circles and black squares. Cytosolic Ca2+ imaging was performed to quantitate SOCE in STIM1KO and WT cells in response to 3 μmol/L TG (B) and 200 μmol/L carbachol (CCh) (C), which were used to empty ER Ca2+ stores in the presence of 5.5 mmol/L glucose, 200 μmol/L diazoxide, and 10 μmol/L verapamil (Dz + V). D: Quantitative assessment of SOCE in WT and KO cells; the numbers of replicates are indicated by the open circles. E and F: ER Ca2+ levels were compared in WT and STIM1KO cells transduced with a D4ER adenovirus, using a confocal microscope imaging system. E: FRET/CFP ratios were acquired in HBSS buffer containing 2 mmol/L Ca2+ in WT cells (n = 65) and KO cells (n = 53). F: Average donor lifetime from FLIM analysis of at least 20 cells from each group. For A, D, E, and F, *P < 0.05 compared with control WT cells. G: GSIS in WT and KO cells was measured and normalized to total protein levels. The number of replicates are indicated by the open circles. *P < 0.05 compared with 2.5 mmol/L glucose; #P < 0.05 compared with WT cells treated with 16.7 mmol/L glucose.
Figure 6
Figure 6
STIM1 deficient cells exhibited increased susceptibility to ER stress. A: STIM1KO and WT INS-1 cells were treated with or without 25 mmol/L glucose + 0.5 mmol/L BSA-conjugated palmitate (GLT) or 10 μmol/L TM for 3 h. Total RNA was isolated and subjected to qRT-PCR. Shown are the average spliced/unspliced XBP-1 ratios. *P < 0.05, compared with control (Cont) conditions; #P < 0.05, for indicated comparisons. B and C: STIM1KO and WT INS-1 cells were treated with or without 5 ng/mL IL-1β for 24 h. Immunoblot analysis was performed using antibodies against STIM1, cleaved caspase-3, and actin. Quantitative protein levels are shown graphically. *P < 0.05 compared with control conditions; #P < 0.05 compared with IL-1β–treated WT group. D and E: INS-1 cells were transfected with siRNAs against STIM1 (siSTIM1) or an siControl. Total RNA and protein were isolated and subjected to qRT-PCR and immunoblot to confirm reduced STIM1 mRNA and protein expression. F: siSTIM1- and siControl-transfected cells were treated with 10 μmol/L TM for 3 h, and the spliced/unspliced XBP-1 mRNA ratios were quantitated using qRT-PCR. *P < 0.05 compared with control conditions or for indicated comparisons. G: STIM1KO and WT INS-1 cells were treated with 10 μmol/L TM for 24 h, fixed, and analyzed by electron microscopy. Representative images of the β-cell ER ultrastructure, indicated by dotted lines in WT and KO cells (scale bars = 2 μm). For each experiment, the numbers of replicates are indicated by the open circles.
Figure 7
Figure 7
STIM1 overexpression restored ER Ca2+ levels and improved insulin secretion in human islets from donors with T2D. A: STIM1KO and WT INS-1 cells were transduced with an adenovirus encoding human STIM1 in increasing concentrations (shown as plaque-forming units [pfu]/mL). Immunoblot was performed using antibodies against STIM1 and actin. B and C: SOCE was measured in STIM1KO cells and WT INS-1 cells that had been transduced with STIM1-expressing adenovirus (Ad-STIM1) or an empty adenoviral control (EV) in the presence of 5.5 mmol/L glucose, 200 μmol/L diazoxide, and 10 μmol/L verapamil (Dz + V). Quantitative results are shown as the ΔF/F0; *P < 0.05 compared with WT+EV; #P < 0.05 compared with EV-treated STIM1KO cells. D–H: ER Ca2+ analysis using FRET and FLIM was performed in D4ER-expressing STIM1KO cells and WT INS-1 cells that had been transduced with Ad-STIM1 or EV. D: Quantitation of FRET/CFP ratio of the D4ER probe at baseline in 2 mmol/L Ca2+ containing HBSS buffer. E: FLIM analysis for the D4ER donor probe is shown graphically as the relative average donor lifetime. For D and E, *P < 0.05 compared with WT cells. #P < 0.05 compared with STIM1 KO cells + EV. F: FRET analysis was performed in WT and STIM1 KO cells transduced with Ad-STIM1 or EV in the presence of 5.5 mmol/L glucose. Cch, carbachol. G and H: Carbachol-induced reductions in ER Ca2+ levels and ER Ca2+ refilling after the addition of 2 mmol/L Ca2+; results are shown quantitatively as the change in the FRET ratio (ΔR); *P < 0.05 compared with WT cells; #P < 0.05 compared with STIM1 KO cells + EV. I: STIM1KO cells and WT INS-1 cells were transduced with STIM1-expressing adenovirus or EV control and treated with or without 5 ng/mL IL-1β for 24 h. Immunoblot analysis was performed using antibodies against STIM1, cleaved caspase-3, and actin. J: Relative expression levels of cleaved caspase 3 are shown graphically; *P < 0.05, compared with control conditions; #P < 0.05 compared for indicated comparisons. K and L: Human islets from two donors with T2D were transduced with STIM1-expressing adenovirus (3 × 106 pfu/mL) or EV. K: Immunoblot was performed to verify STIM1 overexpression. L: GSIS from two separate experiments of two donors with four replicates each was measured and normalized to total protein content; *P < 0.05 compared with high-glucose conditions in EV-treated islets. For each experiment, the numbers of replicates are indicated by the open circles.
Figure 8
Figure 8
Overall model. Under normal conditions, reductions in ER Ca2+ levels are sensed by STIM1, leading to STIM1 oligomerization and translocation to the ER/plasmalemmal junctional regions. Here, STIM1 complexes with selective Orai and TRPC1 channels allow Ca2+ influx from the extracellular space, with subsequent transfer of Ca2+ into the ER lumen, leading to ER Ca2+ restoration. Our study revealed a preferential loss of STIM1 expression under diabetic stress conditions, including exposure to proinflammatory cytokines and elevated free fatty acids. Genetic as well as acquired loss of STIM1 was sufficient to impair β-cell SOCE, reduce ER Ca2+ levels, increase β-cell susceptibility to ER stress and death, and cause abnormal glucose-stimulated calcium oscillations and insulin secretory defects. Moreover, our data revealed that STIM1 reconstitution in human cadaveric islets from donors with diabetes was sufficient to improve GSIS, while also protecting against proinflammatory cytokine–induced cell death in INS-1 cells.
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