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Figure S3. Calcium imaging analysis of wild type and STIM1 KO INS-1 β cells. WT and STIM1KO cells were treated with or without 5 ng/ml IL-1β (A-B) or the combination of 0.5 mM palmitate + 25 mM glucose (GLT) for 24 hrs (C-D). Cells were loaded with Calcium 6, and Ca 2+ imaging experiments were performed to measure SOCE in the presence of 5.5 mM glucose, 200 µM diazoxide, and 10 µM verapamil (Dz+V). Representative Ca 2+ imaging traces are shown in Panels A and C. Quantitative results for the ΔF/F0 response to extracellular Ca 2+ supplementation are shown as the means ± S.E.M in Panels B, D, and E.; n=4-8 replicates for each condition as indicated by the open circles. *p<0.05 for comparison to WT control or for indicated comparisons. (F) WT and STIM1 KO cells were transduced with a D4ER adenovirus, and FLIM analysis was used to measure ER Ca 2+ levels. Shown is a representative lifetime map with look-up table indicating donor lifetime in nanoseconds (ns). Quantitated results are shown in Figure 5F. Scale bars = 50 µm.
Source publication
Store-operated calcium entry (SOCE) is a dynamic process that leads to refilling of ER Ca2+ stores through reversible gating of plasma membrane Ca2+ channels by the ER Ca2+ sensor, 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 expre...
Contexts in source publication
Context 1
... Kit and a FlexStation 3 plate reader (Molecular Devices, Sunnyvale, CA Figure 1A. Baseline (F0) fluorescence was measured for a minimum of 10 seconds under Ca 2+ free conditions and in the presence of 0.5 mM EGTA (Ca 2+ chelator), 10 µM verapamil (L-type voltage-dependent Ca 2+ channel [VDCC] blocker), and 200 µM diazoxide (K ATP channel opener applied to prevent VDCC activation). ...Context 2
... image β cell store-operated Ca 2+ entry (SOCE), INS-1 cells were loaded with Calcium 6; Ca 2+ imaging experiments were performed according to the schematic shown in Fig. 1A and described in the "Methods". As shown previously (26; 27), pharmacologic SOCE inhibitors, ML-9 and 2-APB, reduced the ∆F/F0 activated in response to ER Ca 2+ depletion (Fig. ...Context 3
... image β cell store-operated Ca 2+ entry (SOCE), INS-1 cells were loaded with Calcium 6; Ca 2+ imaging experiments were performed according to the schematic shown in Fig. 1A and described in the "Methods". As shown previously (26; 27), pharmacologic SOCE inhibitors, ML-9 and 2-APB, reduced the ∆F/F0 activated in response to ER Ca 2+ depletion (Fig. ...Context 4
... ML-9 and 2-APB reduced GSIS by 61% and 68%, respectively in INS-1 β cells (Fig. 1D). To test whether SOCE inhibitors similarly impacted glucose-stimulated Ca 2+ oscillations, islets from wild-type C57BL/6J mice were treated with either ML-9 or 2-APB and then loaded with Fura-2AM for analysis of glucose-stimulated Ca 2+ responses (Fig. 1E). While pharmacological inhibition of SOCE had no effect on the Phase 1 ∆F ...Context 5
... ML-9 and 2-APB reduced GSIS by 61% and 68%, respectively in INS-1 β cells (Fig. 1D). To test whether SOCE inhibitors similarly impacted glucose-stimulated Ca 2+ oscillations, islets from wild-type C57BL/6J mice were treated with either ML-9 or 2-APB and then loaded with Fura-2AM for analysis of glucose-stimulated Ca 2+ responses (Fig. 1E). While pharmacological inhibition of SOCE had no effect on the Phase 1 ∆F response to glucose (Fig. 1F), ML-9 and 2-APB reduced the average amplitude of the oscillatory response (∆F Phase 2) and increased the oscillatory period ( Fig. 1G-H). In islet perifusion experiments, the addition of 2-APB to the high glucose buffer ...Context 6
... test whether SOCE inhibitors similarly impacted glucose-stimulated Ca 2+ oscillations, islets from wild-type C57BL/6J mice were treated with either ML-9 or 2-APB and then loaded with Fura-2AM for analysis of glucose-stimulated Ca 2+ responses (Fig. 1E). While pharmacological inhibition of SOCE had no effect on the Phase 1 ∆F response to glucose (Fig. 1F), ML-9 and 2-APB reduced the average amplitude of the oscillatory response (∆F Phase 2) and increased the oscillatory period ( Fig. 1G-H). In islet perifusion experiments, the addition of 2-APB to the high glucose buffer significantly reduced both first and second phase insulin secretion ( Fig. 1I-J). Consistent with effects observed ...Context 7
... with either ML-9 or 2-APB and then loaded with Fura-2AM for analysis of glucose-stimulated Ca 2+ responses (Fig. 1E). While pharmacological inhibition of SOCE had no effect on the Phase 1 ∆F response to glucose (Fig. 1F), ML-9 and 2-APB reduced the average amplitude of the oscillatory response (∆F Phase 2) and increased the oscillatory period ( Fig. 1G-H). In islet perifusion experiments, the addition of 2-APB to the high glucose buffer significantly reduced both first and second phase insulin secretion ( Fig. 1I-J). Consistent with effects observed on glucose-stimulated Ca 2+ oscillations, a more striking effects was observed on second phase insulin ...Context 8
... no effect on the Phase 1 ∆F response to glucose (Fig. 1F), ML-9 and 2-APB reduced the average amplitude of the oscillatory response (∆F Phase 2) and increased the oscillatory period ( Fig. 1G-H). In islet perifusion experiments, the addition of 2-APB to the high glucose buffer significantly reduced both first and second phase insulin secretion ( Fig. 1I-J). Consistent with effects observed on glucose-stimulated Ca 2+ oscillations, a more striking effects was observed on second phase insulin ...Context 9
... amplitude of the ∆F Phase 1 and 2 responses and a decreased oscillatory period ( Fig. 2A-E). To test whether observed changes in calcium signaling may be related to changes in expression of the molecular components of the β cell SOCE complex, first, we compared expression levels of STIM and Orai isoforms in human and mouse islets and INS-1 cells (Fig. S1A-C). In human islets, mouse islets, and INS-1 cells, STIM1 was expressed at higher levels compared to STIM2. Orai2 was the most highly expressed Orai isoform in human islets. In mouse islets, Orai1 and 3 levels were nearly equivalent, and these were the most abundantly expressed Orai isoforms. Orai3 was the mostly highly expressed isoform ...Context 10
... the amplitude of the Phase 1 and 2 responses were reduced in pSTIM1KO islets ( Fig. 2J-M). Similar to results obtained in islets from STZ-treated mice, the oscillatory period was also reduced in pSTIM1KO islets (Fig. 2E, N). However, this was noted to be in contrast to the increased oscillatory period observed in islets treated with ML-9 or 2-APB (Fig. ...Context 11
... our data revealed that STIM1 reconstitution in human cadaveric islets from diabetic donors was sufficient to improve glucose-stimulated insulin secretion, while also protecting against pro-inflammatory cytokine- induced cell death in INS-1 cells. Figure S1. Expression analysis of STIM and Orai gene isoforms in human and mouse islets and INS-1 β cells. ...Similar publications
Mesencephalic astrocyte-derived neurotrophic factor (MANF) was originally identified as a secreted trophic factor for dopamine neurons in vitro. It protects and restores damaged cells in rodent models of Parkinson’s disease, brain and heart ischemia, spinocerebellar ataxia and retina in vivo. However, its exact mechanism of action is not known. MAN...
Citations
... A reduction in Ca 2+ ER leads to the oligomerization of STIM1 dimers at ER/plasmalemmal junctions, where STIM1 interacts with and activates Orai1 and TRPC1 channels, stimulating extracellular Ca 2+ influx, which is then pumped into the ER via SERCA [98,99]. SOCE was significantly impaired in INS-1 cells that expressed dominant negative mutants of either Orai1 or TRPC1 and the knockout of STIM1; SOCE was measured following Ca 2+ ER depletion by SERCA inhibition (with thapsigargin) or muscarinic M 3 receptor-mediated IP 3 R activation [100,101]. GSIS was reduced in mouse and rat islets treated with pharmacological SOCE inhibitors, which reduce glucose-induced Ca 2+ influx [100,101]. Long-chain free fatty acids also induce β-cell SOCE following FFAR1-mediated IP 3 R activation [17]. ...
... SOCE was significantly impaired in INS-1 cells that expressed dominant negative mutants of either Orai1 or TRPC1 and the knockout of STIM1; SOCE was measured following Ca 2+ ER depletion by SERCA inhibition (with thapsigargin) or muscarinic M 3 receptor-mediated IP 3 R activation [100,101]. GSIS was reduced in mouse and rat islets treated with pharmacological SOCE inhibitors, which reduce glucose-induced Ca 2+ influx [100,101]. Long-chain free fatty acids also induce β-cell SOCE following FFAR1-mediated IP 3 R activation [17]. MIN6 cells treated with the FFAR1 agonist fasiglifam displayed enhanced intracellular Ca 2+ and subsequent insulin secretion that was blunted when STIM1 or Orai1 was silenced or when IP 3 R was inhibited [17]. ...
... Dysfunction: SOCE is impaired in diabetic settings and promotes β-cell dysfunction. STIM1 mRNA and protein levels are significantly reduced in human islets from T2D donors, islets from hyperglycemic mice (low-dose streptozotocin-treated), and cytokine and palmitate-treated INS-1 cells [101]. This suggests the reduced STIM1-mediated activation of SOCE and Ca 2+ depletion under diabetogenic stress. ...
The β-cell workload increases in the setting of insulin resistance and reduced β-cell mass, which occurs in type 2 and type 1 diabetes, respectively. The prolonged elevation of insulin production and secretion during the pathogenesis of diabetes results in β-cell ER stress. The depletion of β-cell Ca2+ER during ER stress activates the unfolded protein response, leading to β-cell dysfunction. Ca2+ER is involved in many pathways that are critical to β-cell function, such as protein processing, tuning organelle and cytosolic Ca2+ handling, and modulating lipid homeostasis. Mutations that promote β-cell ER stress and deplete Ca2+ER stores are associated with or cause diabetes (e.g., mutations in ryanodine receptors and insulin). Thus, improving β-cell Ca2+ER handling and reducing ER stress under diabetogenic conditions could preserve β-cell function and delay or prevent the onset of diabetes. This review focuses on how mechanisms that control β-cell Ca2+ER are perturbed during the pathogenesis of diabetes and contribute to β-cell failure.
... Pharmacological inhibition of SOCE alters glucose-stimulated Ca 2+ oscillations and reduces insulin secretion in murine pancreatic islets and clonal β cell lines (5)(6)(7). Moreover, STIM1 gene deletion in mice leads to the loss of SOCE in islets and decreases glucose-stimulated insulin secretion (GSIS) that is mediated through the GPR40 agonist fasiglifam, a modulator of IP3 production and Ca 2+ release (7). ...
... Moreover, STIM1 gene deletion in mice leads to the loss of SOCE in islets and decreases glucose-stimulated insulin secretion (GSIS) that is mediated through the GPR40 agonist fasiglifam, a modulator of IP3 production and Ca 2+ release (7). Notably, lower STIM1 expression has been observed in human islets from donors with T2D and in mouse islets and INS-1 832/13 pancreatic β cells (INS-1 cells) treated with proinflammatory cytokines and free fatty acids, showing that reduced insulin secretion, ER Ca 2+ depletion, and increased β cell ER stress are associated with lower STIM1 levels (6). Conversely, STIM1 overexpression increases insulin secretion in human islets isolated from organ donors with T2D (6). ...
... Notably, lower STIM1 expression has been observed in human islets from donors with T2D and in mouse islets and INS-1 832/13 pancreatic β cells (INS-1 cells) treated with proinflammatory cytokines and free fatty acids, showing that reduced insulin secretion, ER Ca 2+ depletion, and increased β cell ER stress are associated with lower STIM1 levels (6). Conversely, STIM1 overexpression increases insulin secretion in human islets isolated from organ donors with T2D (6). However, at present, the pathways leading to altered SOCE in the diabetic β cell remain largely unexplored. ...
Histone deacetylase inhibitors (HDIs) modulate β cell function in preclinical models of diabetes; however, the mechanisms underlying these beneficial effects have not been determined. In this study, we investigated the impact of the HDI sodium butyrate (NaB) on β cell function and calcium (Ca2+) signaling using ex vivo and in vitro models of diabetes. Our results show that NaB significantly improved glucose-stimulated insulin secretion in islets from human organ donors with type 2 diabetes and in cytokine-treated INS-1 β cells. Consistently, NaB partially rescued glucose-stimulated Ca2+ oscillations in mouse islets treated with proinflammatory cytokines. Because the oscillatory phenotype of Ca2+ in the β cell is governed by changes in endoplasmic reticulum (ER) Ca2+ levels, next we explored the relationship between NaB and store-operated calcium entry (SOCE), a rescue mechanism that acts to refill ER Ca2+ levels through STIM1-mediated gating of plasmalemmal Orai channels. We found that NaB treatment preserved basal ER Ca2+ levels and restored SOCE in IL-1β-treated INS-1 cells. Furthermore, we linked these changes with the restoration of STIM1 levels in cytokine-treated INS-1 cells and mouse islets, and we found that NaB treatment was sufficient to prevent β cell death in response to IL-1β treatment. Mechanistically, NaB counteracted cytokine-mediated reductions in phosphorylation levels of key signaling molecules, including AKT, ERK1/2, glycogen synthase kinase-3α (GSK-3α), and GSK-3β. Taken together, these data support a model whereby HDI treatment promotes β cell function and Ca2+ homeostasis under proinflammatory conditions through STIM1-mediated control of SOCE and AKT-mediated inhibition of GSK-3.
... Kono et al. described a specific reduction in STIM1 expression level in type 2-diabetic islets where insulin-induced glucose metabolism was impaired [138]. Sabourin et al. observed that prolonged exposure to supraphysiological glucose concentration impaired SOCE without the changes of STIM1 and Orai1 expression levels in pancreatic β-cells [139]. ...
... In addition, exercise can improve insulin resistance [159]; how-ever, STIM1 ablation in cardiomyocytes reduced exercise-induced AKT phosphorylation at Ser473 [160]. Together with the fact that reduced STIM1 expression is also associated with impaired insulin-induced glucose uptake in other types of cells [127,138], we guess that decreased STIM1 might contribute to impaired glucose uptake by inhibiting the insulin pathway in diabetic cardiomyopathy, while increased STIM1 may upregulate insulin-induced glucose uptake via enhancing AKT phosphorylation in cardiac hypertrophy (Figure 4y). cardiomyopathy [125,126], the reduction in STIM1 could also contribute to the decrease in glucose utilization by downregulating GLUT4 expression in diabetic cardiomyopathy. ...
... In addition, exercise can improve insulin resistance [159]; however, STIM1 ablation in cardiomyocytes reduced exercise-induced AKT phosphorylation at Ser473 [160]. Together with the fact that reduced STIM1 expression is also associated with impaired insulin-induced glucose uptake in other types of cells [127,138], we guess that decreased STIM1 might contribute to impaired glucose uptake by inhibiting the insulin pathway in diabetic cardiomyopathy, while increased STIM1 may upregulate insulin-induced glucose uptake via enhancing AKT phosphorylation in cardiac hypertrophy ( Figure 4②). ...
The heart requires a variety of energy substrates to maintain proper contractile function. Glucose and long-chain fatty acids (FA) are the major cardiac metabolic substrates under physiological conditions. Upon stress, a shift of cardiac substrate preference toward either glucose or FA is associated with cardiac diseases. For example, in pressure-overloaded hypertrophic hearts, there is a long-lasting substrate shift toward glucose, while in hearts with diabetic cardiomyopathy, the fuel is switched toward FA. Stromal interaction molecule 1 (STIM1), a well-established calcium (Ca2+) sensor of endoplasmic reticulum (ER) Ca2+ store, is increasingly recognized as a critical player in mediating both cardiac hypertrophy and diabetic cardiomyopathy. However, the cause–effect relationship between STIM1 and glucose/FA metabolism and the possible mechanisms by which STIM1 is involved in these cardiac metabolic diseases are poorly understood. In this review, we first discussed STIM1-dependent signaling in cardiomyocytes and metabolic changes in cardiac hypertrophy and diabetic cardiomyopathy. Second, we provided examples of the involvement of STIM1 in energy metabolism to discuss the emerging role of STIM1 in the regulation of energy substrate preference in metabolic cardiac diseases and speculated the corresponding underlying molecular mechanisms of the crosstalk between STIM1 and cardiac energy substrate preference. Finally, we briefly discussed and presented future perspectives on the possibility of targeting STIM1 to rescue cardiac metabolic diseases. Taken together, STIM1 emerges as a key player in regulating cardiac energy substrate preference, and revealing the underlying molecular mechanisms by which STIM1 mediates cardiac energy metabolism could be helpful to find novel targets to prevent or treat cardiac metabolic diseases.
... Mouse islet Ca 2+ imaging Direct measurement of ER Ca 2+ was performed as previously described [26,38]. Briefly, isolated mouse islets were incubated for 24 h with an adenovirus encoding the ER-targeted D4ER Cameleon probe expressed under transcriptional control of the rat insulin promoter [25,38]. ...
... Mouse islet Ca 2+ imaging Direct measurement of ER Ca 2+ was performed as previously described [26,38]. Briefly, isolated mouse islets were incubated for 24 h with an adenovirus encoding the ER-targeted D4ER Cameleon probe expressed under transcriptional control of the rat insulin promoter [25,38]. Transduced islets were incubated for an additional 24 h and transferred for imaging to a glass-bottom plate containing Hanks' balanced salt solution (HBSS) supplemented with 0.2% (vol./vol.) ...
... For cytosolic Ca 2+ imaging, islets were incubated in HBSS buffer supplemented with 2 mmol/l Ca 2+ and loaded with the ratiometric Ca 2+ indicator fura-2-acetoxymethylester (Fura-2 AM). Baseline measurements were performed at 5.5 mmol/l glucose and spontaneous intracellular Ca 2+ transients were measured in response to 16.7 mmol/l glucose using a Zeiss Lightsheet Z.1 microscope, as previously reported [25,38]. ...
Aims/hypothesis:
Increased circulating levels of incompletely processed insulin (i.e. proinsulin) are observed clinically in type 1 and type 2 diabetes. Previous studies have suggested that Ca2+ signalling within beta cells regulates insulin processing and secretion; however, the mechanisms that link impaired Ca2+ signalling with defective insulin maturation remain incompletely understood.
Methods:
We generated mice with beta cell-specific sarcoendoplasmic reticulum Ca2+ ATPase-2 (SERCA2) deletion (βS2KO mice) and used an INS-1 cell line model of SERCA2 deficiency. Whole-body metabolic phenotyping, Ca2+ imaging, RNA-seq and protein processing assays were used to determine how loss of SERCA2 impacts beta cell function. To test key findings in human model systems, cadaveric islets were treated with diabetogenic stressors and prohormone convertase expression patterns were characterised.
Results:
βS2KO mice exhibited age-dependent glucose intolerance and increased plasma and pancreatic levels of proinsulin, while endoplasmic reticulum (ER) Ca2+ levels and glucose-stimulated Ca2+ synchronicity were reduced in βS2KO islets. Islets isolated from βS2KO mice and SERCA2-deficient INS-1 cells showed decreased expression of the active forms of the proinsulin processing enzymes PC1/3 and PC2. Additionally, immunofluorescence staining revealed mis-location and abnormal accumulation of proinsulin and proPC2 in the intermediate region between the ER and the Golgi (i.e. the ERGIC) and in the cis-Golgi in beta cells of βS2KO mice. Treatment of islets from human donors without diabetes with high glucose and palmitate concentrations led to reduced expression of the active forms of the proinsulin processing enzymes, thus phenocopying the findings observed in βS2KO islets and SERCA2-deficient INS-1 cells. Similar findings were observed in wild-type mouse islets treated with brefeldin A, a compound that perturbs ER-to-Golgi trafficking.
Conclusions/interpretation:
Taken together, these data highlight an important link between ER Ca2+ homeostasis and proinsulin processing in beta cells. Our findings suggest a model whereby chronic ER Ca2+ depletion due to SERCA2 deficiency impairs the spatial regulation of prohormone trafficking, processing and maturation within the secretory pathway.
Data availability:
RNA-seq data have been deposited in the Gene Expression Omnibus (GEO; accession no.: GSE207498).
... Recent studies have implicated SOCE in glucose-and GPR40-mediated potentiation of insulin secretion [14]. We have previously demonstrated reduced STIM1 mRNA and protein expression in islets from donors with T2D and in human islets and INS-1 β cells treated with proinflammatory cytokines and palmitate, and we have shown that STIM1 overexpression improves insulin secretion [15]. However, whether STIM1 loss impacts in vivo responses to metabolic stressors, including diet-induced obesity, remains unexplored. ...
... Mouse pancreatic islets were isolated at indicated time points by collagenase digestion, as previously described [15]. Islets were cultured in phenol red-free, low-glucose DMEM supplemented with 10% charcoal-stripped fetal bovine serum, 100 U/mL penicillin, 100 µg/mL streptomycin, and 2 mM L-glutamine. ...
... cDNA was subjected to RT-qPCR using SensiFAST SYBR Lo-ROX (Bioline) and a QuantStudio 3 thermocycler (Applied Biosystems). Relative RNA levels were established against β-actin mRNA using comparative ΔΔCt method, as previously described [15]. Primer sequences are provided in the Key Resource Table. ...
Altered endoplasmic reticulum (ER) Ca2+ signaling has been linked with β-cell dysfunction and diabetes development. Store-operated Ca2+ entry replenishes ER Ca2+ through reversible gating of plasma membrane Ca2+ channels by the ER Ca2+ sensor, stromal interaction molecule 1 (STIM1). For characterization of the in vivo impact of STIM1 loss, mice with β-cell–specific STIM1 deletion (STIM1Δβ mice) were generated and challenged with high-fat diet. Interestingly, β-cell dysfunction was observed in female, but not male, mice. Female STIM1Δβ mice displayed reductions in β-cell mass, a concomitant increase in α-cell mass, and reduced expression of markers of β-cell maturity, including MafA and UCN3. Consistent with these findings, STIM1 expression was inversely correlated with HbA1c levels in islets from female, but not male, human organ donors. Mechanistic assays demonstrated that the sexually dimorphic phenotype observed in STIM1Δβ mice was due, in part, to loss of signaling through the noncanonical 17-β estradiol receptor (GPER1), as GPER1 knockdown and inhibition led to a similar loss of expression of β-cell maturity genes in INS-1 cells. Together, these data suggest that STIM1 orchestrates pancreatic β-cell function and identity through GPER1-mediated estradiol signaling.
Article Highlights
Store-operated Ca2+ entry replenishes endoplasmic reticulum (ER) Ca2+ through reversible gating of plasma membrane Ca2+ channels by the ER Ca2+ sensor, stromal interaction molecule 1 (STIM1). β-Cell–specific deletion of STIM1 results in a sexually dimorphic phenotype, with β-cell dysfunction and loss of identity in female but not male mice. Expression of the noncanonical 17-β estradiol receptor (GPER1) is decreased in islets of female STIM1Δβ mice, and modulation of GPER1 levels leads to alterations in expression of β-cell maturity genes in INS-1 cells.
... The cartoon has been created with BioRender.com 36], suggesting an active role of this ER protein in the initiating/sustaining ER stress and UPR signaling. The activation of PERK-dependent arm of the UPR, induces the phosphorylation of the downstream effector eIF2α, which reduced translation of misfolded proteins thus alleviating ER workload and promoting cell survival. ...
Background
We previously demonstrated that an Italian family affected by a severe dilated cardiomyopathy (DCM) with history of sudden deaths at young age, carried a mutation in the Lmna gene encoding for a truncated variant of the Lamin A/C protein (LMNA), R321X. When expressed in heterologous systems, such variant accumulates into the endoplasmic reticulum (ER), inducing the activation of the PERK-CHOP pathway of the unfolded protein response (UPR), ER dysfunction and increased rate of apoptosis. The aim of this work was to analyze whether targeting the UPR can be used to revert the ER dysfunction associated with LMNA R321X expression in HL-1 cardiac cells.
Methods
HL-1 cardiomyocytes stably expressing LMNA R321X were used to assess the ability of 3 different drugs targeting the UPR, salubrinal, guanabenz and empagliflozin to rescue ER stress and dysfunction. In these cells, the state of activation of both the UPR and the pro-apoptotic pathway were analyzed monitoring the expression levels of phospho-PERK, phospho-eIF2α, ATF4, CHOP and PARP-CL. In addition, we measured ER-dependent intracellular Ca²⁺ dynamics as indicator of proper ER functionality.
Results
We found that salubrinal and guanabenz increased the expression levels of phospho-eIF2α and downregulated the apoptosis markers CHOP and PARP-CL in LMNA R321X-cardiomyocytes, maintaining the so-called adaptive UPR. These drugs also restored ER ability to handle Ca²⁺ in these cardiomyocytes. Interestingly, we found that empagliflozin downregulated the apoptosis markers CHOP and PARP-CL shutting down the UPR itself through the inhibition of PERK phosphorylation in LMNA R321X-cardiomyocytes. Furthermore, upon empagliflozin treatment, ER homeostasis, in terms of ER ability to store and release intracellular Ca²⁺ was also restored in these cardiomyocytes.
Conclusions
We provided evidence that the different drugs, although interfering with different steps of the UPR, were able to counteract pro-apoptotic processes and to preserve the ER homeostasis in R321X LMNA-cardiomyocytes. Of note, two of the tested drugs, guanabenz and empagliflozin, are already used in the clinical practice, thus providing preclinical evidence for ready-to-use therapies in patients affected by the LMNA R321X associated cardiomyocytes.
... lacking voltage-gated Ca 2+ channels), but is also functionally important in excitable cells [23]. Islets from type 2 diabetics were shown to have reductions in STIM1, and deletion of STIM1 from INS-1 pancreatic β-cells was shown to impair SOCE [24]. Pharmacological inhibition of SOCE inhibits glucose-stimulated insulin secretion, but inhibitors of SOCE have many off-target effects [24][25][26]. ...
... Islets from type 2 diabetics were shown to have reductions in STIM1, and deletion of STIM1 from INS-1 pancreatic β-cells was shown to impair SOCE [24]. Pharmacological inhibition of SOCE inhibits glucose-stimulated insulin secretion, but inhibitors of SOCE have many off-target effects [24][25][26]. RyRs and IP 3 Rs may play a role in activation of SOCE. In pulmonary artery smooth muscle cells, stimulation of RyR2 can activate SOCE via a mechanism dependent on ER/SR Ca 2+ depletion and a specific conformation of RyR2 [27]. ...
... However, the magnitude of the SOCE response in IRBIT KO cells was not different from control INS-1 cells, and was strongly inhibited 2-APB ( Fig 4D). STIM1 is an essential component of SOCE, and decreases in STIM1 lead to impairments in SOCE [23,24]. Impairments in SOCE and reduced expression of STIM1 have been associated with β-cell dysfunction [24]. ...
The ER Ca2+ channel ryanodine receptor 2 (RyR2) is required for maintenance of insulin content and glucose-stimulated insulin secretion, in part, via regulation of the protein IRBIT in the insulinoma cell line INS-1. Here, we examined store-operated and depolarization-dependent Ca2+entry using INS-1 cells in which either RyR2 or IRBIT were deleted. Store-operated Ca2+ entry (SOCE) stimulated with thapsigargin was reduced in RyR2KO cells compared to controls, but was unchanged in IRBITKO cells. STIM1 protein levels were not different between the three cell lines. Basal and stimulated (500 μM carbachol) phospholipase C (PLC) activity was also reduced specifically in RyR2KO cells. Insulin secretion stimulated by tolbutamide was reduced in RyR2KO and IRBITKO cells compared to controls, but was potentiated by an EPAC-selective cAMP analog in all three cell lines. Cellular PIP2 levels were increased and cortical f-actin levels were reduced in RyR2KO cells compared to controls. Whole-cell Cav channel current density was increased in RyR2KO cells compared to controls, and barium current was reduced by acute activation of the lipid phosphatase pseudojanin preferentially in RyR2KO cells over control INS-1 cells. Action potentials stimulated by 18 mM glucose were more frequent in RyR2KO cells compared to controls, and insensitive to the SK channel inhibitor apamin. Taken together, these results suggest that RyR2 plays a critical role in regulating PLC activity and PIP2 levels via regulation of SOCE. RyR2 also regulates β-cell electrical activity by controlling Cav current density and SK channel activation.
... However, the role of STIM1 in diabetic vascular calcification in human has not been reported. STIM1 expression was reduced in islets from human donor with type II diabetes and STZ-induced diabetic mice [39]. By generating SMC-specific STIM1 deletion mouse model, we have provided the first evidence linking a causative regulation of STIM1 in VSMC calcification in vitro and diabetic vascular calcification in vivo. ...
... As STIM1 mutations have been linked to human diseases but the function of STIM1 in vascular disease are not fully understood, our funding of a causative effect of STIM1 deficiency on VSMC calcification has shed lights on the novel function of STIM1 in the pathogenesis of vascular disease. Of note, reduction of STIM1 expression has been demonstrated in islets from human donor with type II diabetes [39]. In addition, decreased STIM1 expression has been determined in aged arteries, such as rat mesenteric and mouse cerebral arteries [52,53], which may contribute to increased vascular calcification and stiffness with aging. ...
Vascular calcification is accelerated in patients with diabetes mellitus and increases risk of cardiovascular events and mortality. Vascular smooth muscle cells (VSMC) play a key role in regulating vascular tone and contribute significantly to the development of diabetic vasculopathy. In this study, the function of stromal interaction molecule 1 (STIM1), an important regulator for intracellular calcium homeostasis, in diabetic vascular calcification was investigated, and the underlying molecular mechanisms were uncovered.
A SMC-specific STIM1 deletion mouse model (STIM1Δ/Δ) was generated by breeding the STIM1 floxed mice (STIM1f/f) with SM22α-Cre transgenic mice. Using aortic arteries from the STIM1Δ/Δ mice and their STIM1f/f littermates, we found that SMC-specific STIM1 deletion induced calcification of aortic arteries cultured in osteogenic media ex vivo. Furthermore, STIM1 deficiency promoted osteogenic differentiation and calcification of VSMC from the STIM1Δ/Δ mice. In the low-dose streptozotocin (STZ)-induced mouse model of diabetes, SMC-specific STIM1 deletion markedly enhanced STZ-induced vascular calcification and stiffness in the STIM1Δ/Δ mice. The diabetic mice with SMC-specific STIM1 ablation also exhibited increased aortic expression of the key osteogenic transcription factor, Runx2, and protein O-GlcNAcylation, an important post-translational modulation that we have reported to promote vascular calcification and stiffness in diabetes. Consistently, elevation of O-GlcNAcylation was demonstrated in aortic arteries and VSMC from the STIM1Δ/Δ mice. Inhibition of O-GlcNAcylation with a pharmacological inhibitor abolished STIM1 deficiency-induced VSMC calcification, supporting a critical role of O-GlcNAcylation in mediating STIM1 deficiency-induced VSMC calcification. Mechanistically, we identified that STIM1 deficiency resulted in impaired calcium homeostasis, which activated calcium signaling and increased endoplasmic reticulum (ER) stress in VSMC, while inhibition of ER stress attenuated STIM1-induced elevation of protein O-GlcNAcylation.
In conclusion, the study has demonstrated a causative role of SMC-expressed STIM1 in regulating vascular calcification and stiffness in diabetes. We have further identified a novel mechanisms underlying STIM1 deficiency-induced impairment of calcium homeostasis and ER stress in upregulation of protein O-GlcNAcylation in VSMC, which promotes VSMC osteogenic differentiation and calcification in diabetes.
... Because SOCE has been implicated in diabetes development (Estrada et al., 2012;Kono et al., 2018;Mamenko et al., 2016), and because diabetes is a common side effect of chronic immunosuppression (Azzi et al., 2013;Bamgbola, 2016;Chakkera et al., 2017), we next tested whether hypomorph mice develop diabetes. Glucose tolerance tests show no changes in glucose clearance rates or resting glucose levels between ConKI and KI mice (Fig. 4D), arguing that chronic SOCE inhibition is not sufficient by itself to predispose to diabetes. ...
Loss of function mutations in store‐operated Ca²⁺ entry (SOCE) are associated with severe paediatric disorders in humans, including combined immunodeficiency, anaemia, thrombocytopenia, anhidrosis and muscle hypotonia. Given its central role in immune cell activation, SOCE has been a therapeutic target for autoimmune and inflammatory diseases. Treatment for such chronic diseases would require prolonged SOCE inhibition. It is, however, unclear whether chronic SOCE inhibition is viable therapeutically. Here we address this issue using a novel genetic mouse model (SOCE hypomorph) with deficient SOCE, nuclear factor of activated T cells activation, and T cell cytokine production. SOCE hypomorph mice develop and reproduce normally and do not display muscle weakness or overt anhidrosis. They do, however, develop cardiovascular complications, including hypertension and tachycardia, which we show are due to increased sympathetic autonomic nervous system activity and not cardiac or vascular smooth muscle autonomous defects. These results assert that chronic SOCE inhibition is viable therapeutically if the cardiovascular complications can be managed effectively clinically. They further establish the SOCE hypomorph line as a genetic model to define the therapeutic window of SOCE inhibition and dissect toxicities associated with chronic SOCE inhibition in a tissue‐specific fashion. image
Key points
A floxed stromal interaction molecule 1 (STIM1) hypomorph mouse model was generated with significant reduction in Ca²⁺ influx through store‐operated Ca²⁺ entry (SOCE), resulting in defective nuclear translocation of nuclear factor of activated T cells, cytokine production and inflammatory response. The hypomorph mice are viable and fertile, with no overt defects.
Decreased SOCE in the hypomorph mice is due to poor translocation of the mutant STIM1 to endoplasmic reticulum–plasma membrane contact sites resulting in fewer STIM1 puncta.
Hypomorph mice have similar susceptibility to controls to develop diabetes but exhibit tachycardia and hypertension. The hypertension is not due to increased vascular smooth muscle contractility or vascular remodelling.
The tachycardia is not due to heart‐specific defects but rather seems to be due to increased circulating catecholamines in the hypomorph.
Therefore, long term SOCE inhibition is viable if the cardiovascular defects can be managed clinically.
... A study in T cells suggested that junctate acts as a calcium-sensing ER protein regulating the STIM1-Orai1 protein complex (Srikanth et al., 2012), which is critical to activate store-operated calcium entry (SOCE), a cellular response whereby extracellular calcium enters the cytosol following ER calcium depletion. Importantly, SOCE has been implicated in insulin secretion from β-cells (Sabourin et al., 2015), with loss of STIM1 leading to reduced insulin secretion and increased ER stress (Kono et al., 2018). These observations prompted us to investigate whether junctate γ-carboxylation would affect cellular calcium flux and SOCE. ...
... Thus, we cannot exclude, at this point, that carboxylated junctate also regulates calcium homeostasis in β-cell through SERCA2 or IP3R. STIM1 and SOCE have previously been shown to positively regulate insulin secretion and to reduce ER stress in β-cells (Kono et al., 2018;Sabourin et al., 2015). Other studies have shown that alteration in either ER or cytosolic free calcium levels can induce ER stress and β-cell death (Sabatini et al., 2019). ...
... Other studies have shown that alteration in either ER or cytosolic free calcium levels can induce ER stress and β-cell death (Sabatini et al., 2019). In addition, the expression level and the activity of STIM1 and SERCA2 were found to be reduced in human or mouse T2D islets, and to correlate with altered cytosolic calcium in response to glucose (Kono et al., 2018;Liang et al., 2014). In line with these findings, it was recently reported that tunicamycin-induced ER stress decreases ER calcium levels and increases SOCE in β-cells resulting in increased basal insulin secretion (Zhang et al., 2020). ...
Vitamin K (VK) is a micronutrient necessary for the γ-carboxylation of glutamic acids. This post- translational modification occurs in the endoplasmic reticulum (ER) and affects secreted proteins. Clinical studies have recently implicated VK in the pathophysiology of diabetes, but the underlying molecular mechanism remains unknown. Here, we show that β-cells lacking γ-carboxylation fail to adapt their insulin secretion in response to glucose in the context of age-related insulin resistance or diet-induced β-cell stress. Conversely, VK supplementation protects β-cells from ER stress-induced apoptosis. We identified junctate as a γ-carboxylated ER-resident protein expressed in β-cells, whose carboxylation is dysregulated in diabetic mouse models. Mechanistically, γ-carboxylation of junctate maintains basal cytosolic calcium levels and restrains store-operated calcium entry, by diminishing STIM1 and Orai1 puncta formation at the plasma membrane. These results reveal a critical role for γ- carboxylation in the regulation of calcium flux in β-cells and in their capacity to adapt to metabolic stress.
