Glycogen Synthase Kinase 3 Beta Controls Presenilin-1-Mediated Endoplasmic Reticulum Ca2+ Leak Directed to Mitochondria in Pancreatic Islets and β-Cells
Christiane Kleca Corina T. Madreiter-Sokolowskia Sarah Stryecka
Vinay Sachdeva,b Madalina Duta-Marea Benjamin Gottschalka Maria R. Depaolia Rene Rosta Jesse Haya,c Markus Waldeck-Weiermaira Dagmar Kratkya,d
Tobias Madla,d Roland Mallia,d Wolfgang F. Graiera,d
aMolecular Biology and Biochemistry, Gottfried Schatz Research Center for Cellular Signaling, Metabolism & Aging, Medical University of Graz, Graz, Austria, bDepartment of Medical Biochemistry, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands, cUniversity of Montana, Division of Biological Sciences, Center for Structural & Functional Neuroscience, Missoula, MT, USA, dBioTechMed, Graz, Austria
Key Words
Mitochondria • Ca2+ leak • Endoplasmic reticulum Ca2+ • Insulin release • Respiration • Presenilin-1
Abstract
Background/Aims: In pancreatic β-cells, the intracellular Ca2+ homeostasis is an essential regulator of the cells’ major functions. The endoplasmic reticulum (ER) as interactive intracellular Ca2+ store balances cellular Ca2+. In this study basal ER Ca2+ homeostasis was evaluated in order to reveal potential β-cell-specificity of ER Ca2+ handling and its consequences for mitochondrial Ca2+, ATP and respiration. Methods: The two pancreatic cell lines INS-1 and MIN-6, freshly isolated pancreatic islets, and the two non-pancreatic cell lines HeLA and EA.hy926 were used. Cytosolic, ER and mitochondrial Ca2+ and ATP measurements were performed using single cell fluorescence microscopy and respective (genetically-encoded) sensors/dyes. Mitochondrial respiration was monitored by respirometry. GSK3β activity was measured with ELISA. Results: An atypical ER Ca2+ leak was observed exclusively in pancreatic islets and β-cells. This continuous ER Ca2+ efflux is directed to mitochondria and increases basal respiration and organellar ATP levels, is established by GSK3β-mediated phosphorylation of presenilin-1, and is prevented by either knockdown of presenilin-1 or an inhibition/knockdown of GSK3β. Expression of a presenlin-1 mutant that mimics GSK3β-mediated phosphorylation established a β-cell-like ER Ca2+ leak in HeLa and EA.hy926 cells. The ER Ca2+ loss in β-cells was compensated at steady state by Ca2+ entry that is linked to the activity of TRPC3. Conclusion: Pancreatic β-cells establish a cell-specific ER Ca2+ leak that is under the control of GSK3β and directed to mitochondria, thus, reflecting a cell-specific intracellular Ca2+ handling for basal mitochondrial activity.
Introduction
The occurrence of type 2 diabetes mellitus (DM), characterized by insulin resistance and pancreatic β-cell dysfunction, is rapidly increasing worldwide and is associated with increased morbidity and mortality [1, 2]. The predominant function of pancreatic β-cells is the control of blood glucose levels by the regulated secretion of sufficient amounts of insulin. Insulin production and release are tightly controlled by mainly glucose, several peptide hormones, neurotransmitters and other nutrients under physiological conditions [3]. On the molecular level, elevations in blood glucose are sensed in pancreatic β-cells via efficient uptake and subsequent catabolism of glucose, leading to an increase in mitochondrial ATP production. Subsequently, elevated intracellular ATP levels inhibit KATP channels, leading to plasma membrane depolarization and Ca2+ influx via L-type Ca2+ channels. This stimulated Ca2+ entry directly results in exocytosis of insulin-containing secretory granules [4]. Recently, it was shown that mitochondrial Ca2+ uptake is also crucial for activation of glucose stimulated insulin secretion (GSIS) [5, 6]. Indeed, it was demonstrated that mitochondrial Ca2+ sequestration in clonal pancreatic β-cells supports GSIS [7, 8]. In line with this assumption, two mitochondrial proteins, namely the mitochondrial Ca2+ uniporter (MCU) [9, 10] and the mitochondrial Ca2+ uptake 1 (MICU1), known for their role in mitochondrial Ca2+ uptake [11], were shown to be essential for pancreatic β-cell function [12]. However, the current concepts explain only how elevated mitochondrial Ca2+ uptake could promote insulin secretion once Ca2+ entry is already activated by elevated ATP levels, and do not address its required role in the production of the elevated ATP at the triggering stage. It is, entirely unclear whether or not, and if so, how the resting mitochondrial Ca2+ homeostasis of pancreatic β-cells contributes to the initial glucose sensing process. Recently, it was speculated that a pre-activation of mitochondria in pancreatic β-cells during fasting is required for efficient glucose sensing once blood glucose levels increase [13]. In this context it was speculated that low levels of continuous glucose uptake and metabolism, other nutrients, hormones and/or neural stimulation weakly pre-activate pancreatic β-cell mitochondria during fasting. While this concept appears convincing as such and pre-stimulation of mitochondria would allow the organelle to immediately respond to increased rates of glycolysis, the underlying molecular mechanisms remain elusive.
In the present study we sought to explore i, the potential β-cell specific characteristics in the resting Ca2+ handling of the ER, ii, its impact on basal mitochondrial Ca2+, and, iii, their consequences on mitochondrial respiration. We used the two widely-used β-cell lines INS-1 [14] and MIN-6 [15], and freshly isolated mouse pancreatic islets and compared the Ca2+ tightness/leakage kinetics of their ER and basal mitochondrial Ca2+ homeostasis with that of the two non- β-cell lines, HeLa [16] and EA.hy926 [17]. Cytosolic, mitochondrial and ER Ca2+ measurements using either Fura-2 or organelle-targeted genetically encoded Ca2+ sensors on single cell fluorescence imaging microscopes were applied [12, 18]. The impact on mitochondrial respiration was examined using Seahorse® technology [19] and ELISA was used to verify phosphorylation and enzymatic activity. Applying such technological variety, we were able to identify β-cell specificities in resting ER Ca2+ handling that, subsequently, impacts mitochondrial basal Ca2+ levels and the organelle’s respiratory activity.
Materials and Methods
Reagents
Cell culture materials were obtained from Greiner Bio-One (Kremsmünster, Austria). Histamine (His; PubChem CID: 774), antimycin A (PubChem CID: 16218979), oligomycin A (PubChem CID: 5281899), carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP; PubChem CID: 3330), 2, 5-di-t-butyl-1, 4-benzohydroquinone (BHQ; PubChem CID: 16043), ethylene glycol tetraacetic acid (EGTA; PubChem CID: 6207), carbachol (Cch; PubChem CID: 5832), efonidipine hydrochloride monoethanolate (PubChem CID: 163838), N-[4-[3, 5-Bis(trifluoromethyl)-1H-pyrazol-1-yl]phenyl]-3-fluoro-4-pyridinecarboxamide (Pyr6; PubChem CID: 10596093) and N-(4-(3, 5-bis(trifluoromethyl)-1H-pyrazole-1-yl)phenyl)-4-methylbenzenesulfonamide (Pyr10; PubChem CID: 53475435) were purchased from Sigma Aldrich (Vienna, Austria). The selective GSK3β inhibitor 6-[[2-[[4-(2, 4-Dichlorophenyl)-5-(5-methyl-1H-imidazol-2-yl)-2- pyrimidinyl]amino]ethyl] amino]-3-pyridinecarbonitrile (CHIR99021; PubChem CID: 9956119) was purchased from Tocris (Bristol, UK). Acetyloxymethyl 2-[6-[bis [2-(acetyloxymethoxy)-2-oxoethyl]amino]-5-[2-[2-[bis [2-(acetyloxymethoxy)-2-oxoethyl]amino]-5-methylphenoxy]ethoxy]-1-benzofuran-2-yl]-1, 3-oxazole-5-carboxylate (Fura-2/AM; PubChem CID: 3364574) was from MoBiTec GmbH (Göttingen, Germany) or TEFLabs (Austin, TX, USA). Unless otherwise specified, genetically-encoded fluorescence sensors were purchased either from Addgene (Cambridge, MA 02139, USA) or Next Generation Fluorescence Imaging, NGFI, Graz, Austria (www.ngfi.eu). Other chemicals were from Carl Roth (Karlsruhe, Germany).
Isolation of murine pancreatic islets
For all experiments, age-matched male C57BL/6 mice purchased from Jackson Laboratory (Bar Harbor, ME) between 3-4 months of age were used. Mice were fed regular chow diet (11.9% caloric intake from fat; Altromin 1324, Lage, Germany) and maintained in a 12:12-h light-dark cycle in a temperature-controlled environment. All animal experiments were carried out in accordance with the guidelines set by the Division of Genetic Engineering and Animal Experiments and were approved by the Austrian Federal Ministry of Science, Research, and Economy (Vienna, Austria). Murine islets were isolated as described [20].
Cell culture and transfection
INS-1 832/13 (INS-1) cells were a generous gift from Prof. Dr. Claes B. Wollheim and Dr. Françoise Assimacopoulos-Jeannet (University Medical Center, Geneva, Switzerland). INS-1 cells were cultured in RPMI 1640 containing 11 mM glucose (PubChem CID: 5793) supplemented with 10 mM HEPES (PubChem CID: 23831), 10% fetal calf serum (FCS), 1 mM sodium pyruvate (PubChem CID: 23662274), 50 µM β-mercaptoethanol (PubChem CID: 1567), 1% (v/v) Pen Strep® (ThermoFischer, Vienna, Austria; 10.000 U/L). MIN-6 cells (ATCC® CRL-11506™) were cultured in DMEM supplemented with 25 mM glucose, 10 mM HEPES, 10% FCS, 1 mM sodium pyruvate, 50 µM β-mercaptoethanol, 50 µg penicillin and 100 µg streptomycin. HeLa and EA.hy926 cells were grown in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin and 100 µg/ml streptomycin as well as 2 mM glutamine (PubChem CID: 5961) (Gibco, Life Technologies, Vienna Austria), designated as full DMEM. Origin of cells was confirmed by STR-profiling by the cell culture facility of ZMF (Graz).
Transfection with siRNAs and plasmids
For Ca2+ imaging, cells were plated on 30 mm glass coverslips in 6-well plates and transiently transfected at 60 - 80% confluency with 1.5 µg plasmid DNA alone or with 100 nM siRNA using 2.5 µl TransFast transfection reagent (Promega, Madison, WI, USA) in 1 ml of serum- and antibiotic-free medium. Cells were maintained in a humidified incubator (37°C, 5% CO2, 95% air) for 16-20 h. Thereafter, the transfection mix was replaced by full culture medium. For treatment with the GSK3β inhibitor CHIR99021 cells were incubated in their respective media containing 2.5 µM CHIR99021. All experiments were performed 24 - 48 h after transfection or treatment. siRNAs were obtained from Microsynth (Balgach, Switzerland) or Thermofisher Scientific (Vienna, Austria). Sequences are listed in Supplementary Table 1. Presenilin-1 wild type as well as the mutant versions presenilin-1S353/357A and presenilin-1S353/357D overexpression plasmids were designed by us and synthesized by General Biosystems (Morrisville, USA). All presenilin-1 versions were cloned into a pcDNA3.1 backbone and are flanked by XbaI and EcoRI restriction sites. Cells were transfected with an excessive amount of siRNA vs. the respective fluorescent sensor (ratio of 2.5x109 mol siRNA: 1 mol plasmid) to ensure the transfection of the siRNA in cells positively transfected with the fluorescent sensor as the cells used for single cell recordings.
mRNA Isolation, real time and detection PCRs
Total RNA isolation, reverse transcription, PCR and real-time-PCR were performed according our recently published protocols [18]. Relative expression of specific genes was normalized to human, rat or mouse GAPDH, as a housekeeping gene. Primers for real-time PCR and detection PCR were obtained from Invitrogen (Vienna, Austria). The respective primer sequences are listed in Supplementary Table 1.
Total and pS9 GSK3β enzyme-linked immunosorbent assay (ELISA)
To determine the levels of total and pS9 (inactive) GSK3β the ELISA kit ab205711 (Abcam, Cambridge, UK) was used. In short, MIN-6, INS-1, HeLa and EA.hy926 cells were cultured until they reached approx. 80% confluency and lysed with the lysis buffer supplied with the kit. Protein concentration was measured using the PierceTM BCA Protein Assay Kit (Thermofisher Scientific) and 5 µg protein were used for the ELISA. The remaining procedure was performed according to the manufacturer’s protocol.
Mitochondrial respiration measurements
INS-1, MIN-6, HeLa and EA.hy926 cells, treated or not with siRNA against presenilin-1 or GSK3β inhibitor CHIR99021, were plated in XF96 polystyrene cell culture microplates (Seahorse Bioscience) at a density of 60.000 cells/well for HeLa and EA.hy926, 120.000 cells/well for MIN-6 and 140.000 cells/well for INS-1 cells. After overnight incubation, mitochondrial respiration was performed using an XF96 extracellular flux analyzer (Seahorse Bioscience®) as previously described [21]. Oxygen consumption was normalized to protein content (pmol O2/min × μg protein).
Calcium imaging of isolated murine pancreatic islets
Islets were treated with 2.5 µM CHIR99021 or DMSO as control as described above. The next day islets were washed once with experimental buffer (EB; containing 135 mM NaCl, 5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 10 mM Hepes, 2.6 mM NaHCO3, 440 µM KH2PO4, 340 µM Na2HPO4, 10 mM glucose, 0.1% vitamins, 0.2% essential amino acids, and 1% penicillin-streptomycin (all v/v); pH adjusted to 7.4) and loaded with 2 µM Fura-2/AM in EB for 40 min as previously described [12]. After washing once with EB buffer, islets were transferred on a 35 mm ibidi single imaging dish with glass bottom (ibidi, Munich, Germany) in 1.5 ml of 0 Ca buffer (i.e. EB buffer without CaCl2 added plus 1 mM EGTA) and immediately imaged or kept on 0 Ca buffer for 20 min and imaged afterwards. To initiate ER Ca2+ release islets were stimulated by adding 500 µl 0 Ca buffer containing carbachol and BHQ to reach final concentrations of 100 µM and 15 µM, respectively.
Cytosolic Ca2+ imaging in cultured β-cells
Cells were loaded with 2 µM Fura-2/AM (TEFLabs) in EB for 40 min and were alternately illuminated at 340 and 380 nm, whereas fluorescence emission was recorded at 510 nm {Alam:2012bo}[12]{Alam:2012bo}. Results of Fura-2/AM measurements are shown as the ratio of F340/F380. For titration of cytosolic Ca2+ to visualize ER Ca2+ leak, after Fura-2/AM loading, cells were incubated for the indicated times in an experimental buffer without Ca2+ consisting of 138 mM NaCl, 1 mM MgCl2, 5 mM KCl, 10 mM Hepes, 10 mM glucose and 1 mM EGTA and subsequently stimulated with 100 µM of IP3-generating agonists (INS-1 and MIN-6: carbachol, HeLa and EA.hy926: histamine) in the presence of 15 µM of the SERCA inhibitor BHQ. For glucose-induced cytosolic Ca2+ measurements, cells were loaded with 2 µM Fura-2/AM as described previously [12]. Briefly, prior imaging cells were incubated for 20 min incubation in glucose free buffer (0G; 10 mM mannitol to isosmotically substitute the glucose). On the microscope, cells were perfused with 0G buffer for 2 min before switching to 16 mM glucose (16G) during acquisition. To evaluate the maximal Ca2+ uptake via L-type Ca2+ channels, cells were depolarized with a high K+ buffer, where 25 mM NaCl were substituted with KCl.
FRET measurements using genetically encoded sensors
[Ca2+]mito, [Ca2+]ER and [ATP]mito were measured in cells expressing 4mtD3cpv, D1ER and mtAT1.03, as previously described [12, 22].
Statistical analyses
Data shown represent the mean ± SEM. ‘n’ values refer to the number of individual experiments performed. For live cell imaging numbers indicate the numbers of cells/independent repeat. If applicable, analysis of variance (ANOVA) was used for data evaluation and statistical significance of differences between means was estimated by Bonferroni post hoc test or two-tailed Student’s t-test using GraphPad Prism 5.0f (GraphPad Software, La Jolla, CA, USA).
Results
Pancreatic islets and β-cells display an atypical ER Ca2+ leakage
To seek for potential β-cell specific characteristics in the resting Ca2+ handling of the ER, we compared the potential ER Ca2+ leakage in freshly isolated murine pancreatic islets and the two β-cell lines, INS-1 [14] and MIN-6 [15], with that of the two distinct widely studied non-β-cell lines, HeLa [16] and EA.hy926 [17]. To this end, extracellular Ca2+ was removed from cells and the ER Ca2+ content was indirectly estimated from cytosolic Ca2+ elevations upon ER Ca2+-mobilization with an inositol-1, 4,5-trisphosphate- (IP3-) generating agonist (either by 100 µM carbachol [Cch] in pancreatic islets and β-cells or 100 µM histamine [His] in the non- β-cells) in presence of the reversible SERCA inhibitor tert-butylhydroxyquinone (BHQ, to avoid ER Ca2+ refilling) (Fig. 1A). By applying this protocol we detected a massive ER Ca2+ loss after 20 min in Ca2+-free buffer in isolated pancreatic islets (Fig. 1B) and both β-cell lines (Fig. 2A), whereas there was no detectable net ER Ca2+ loss during 20 min in a Ca2+-free environment in HeLa and EA.hy926 cells (Fig. 2B). Determination of the ER Ca2+ content utilizing the ER-targeted genetically encoded sensor, D1ER [23], confirmed the existence of a β-cell-specific enhanced ER Ca2+ loss upon removal of extracellular Ca2+ (Fig. 3A).
In contrast to the robust ER Ca2+ maintenance seen in HeLa and EA.hy926, maintenance of ER Ca2+ in β-cells was entirely dependent upon extracellular Ca2+ (Fig. 3B). Furthermore, upon removal of BHQ and Cch, and re-addition of extracellular Ca2+, ER Ca2+ levels were rapidly restored in pancreatic β-cells. These results reveal a pancreatic islets/β-cell-specific strong ER Ca2+ leakage that is normally compensated by continuous ER Ca2+ replenishment, which is in turn fueled by extracellular Ca2+ entry (Fig. 3B).
β-cells specific ER Ca2+ leakage is compensated by continuous TRPC3-associated Ca2+ entry
To determine the mechanisms involved in the compensation of enhanced ER Ca2+ depletion in clonal pancreatic β-cells by extracellular Ca2+, the potential contribution of store-operated Ca2+ entry (SOCE) via Orai1, the transient receptor potential channel 3 (TRPC3) and the L-type Ca2+ channels (L-TCC) was examined. Initially, we approached this issue by using pharmacological inhibitors. Specifically, we determined the ER Ca2+ content of pancreatic β-cells in Ca2+-containing buffer in the presence of 20 µM efonidipine [24], 1 or 40 µM Pyr6, and 3 µM Pyr10 [25] to inhibit Ca2+ entry via L-TCC, Orai1 and TRPC3, respectively. In contrast to efonidipine and 1 µM Pyr6 treatment, that did not affect the ER Ca2+ content of the β-cells (Suppl. Fig. 1), the inhibition of TRPC3 by 40 µM Pyr6 or 3 µM Pyr10 yielded a consistent ER Ca2+ depletion (Fig. 4A). To support these findings, TRPC3 expression was specifically reduced with siRNA and the ER Ca2+ content was evaluated. Treatment with siRNA yielded significantly reduced TRPC3 expression levels by 12 and 18 h post transfection with siRNA (Suppl. Fig. 2). The diminution of TRPC3 expression was associated with a reduction of ER Ca2+ content 12 and 18 h after transfection (Fig. 4B). Longer transfection periods negatively impacted cell survival, probably due to the continuous ER Ca2+ depletion and initiation of ER stress-induced cell death. These results suggest that in the presence of extracellular Ca2+, ER Ca2+ leakage is compensated by TRPC3 channel activity. Moreover, these findings imply that SERCA activity counteracts the ER Ca2+ leak in pancreatic β-cells by continuously sequestering entering Ca2+ into the ER lumen. To exclude that a reduced SERCA activity is itself responsible for the observed increased ER Ca2+ leak, ER Ca2+ refilling was determined directly after maximal ER Ca2+ depletion by a combination of histamine (in HeLa and EA.hy926 cells) or Cch (in INS-1 and MIN-6 cells) with the reversible SERCA inhibitor BHQ. In line with this, the ER Ca2+ refilling kinetics upon Ca2+ re-addition was comparable in all cell types tested (Fig. 4C, D). These findings exclude a reduced SERCA activity as the cause of the enhanced ER Ca2+ leakage in pancreatic β-cells.
The enhanced ER Ca2+ leakage in β-cells is mediated by presenilin-1
As it is known that presenilin-1 forms ER Ca2+ leak channels in several cell types [26, 27], we tested whether or not this protein is responsible for the enhanced ER Ca2+ leakage in β-cells. To this end, we examined the expression of presenilin-1 in the two pancreatic β-cell lines, INS-1 and MIN-6, as well as in HeLa and EA.hy926 using gene specific primers (Suppl. Table 1). Applying standard PCR the expression of presenilin-1 was verified in all cells used (Fig. 5A). Real-time PCR analysis revealed an equal gene expression of presenilin-1 in all four cell lines tested (Fig. 5B). To test whether or not presenilin-1 is functionally involved in the enhanced ER Ca2+ leakage in β-cells, we designed specific siRNAs to knockdown presenilin-1 in murine (MIN-6) and rat (INS-1) β-cells (Suppl. Table 1). Cell transfection with these siRNAs yielded an approximately 50 to 70 percent knockdown efficiency (Fig. 5C). Notably, siRNA-mediated knockdown of presenilin-1 abolished the enhanced ER Ca2+ leak in both β-cell lines (INS: Fig. 5D, F; Min-6: Fig. 5E,G) while no effect was observed in HeLa and EA.hy926 cells (Suppl. Fig. 3). To further verify the involvement of presenilin-1 in the enhanced ER Ca2+ leak in β-cells, we tested the effect of presenilin-1 overexpression. While an overexpression of presenilin-1 did not affect ER Ca2+ content in the presence of extracellular Ca2+ in none of the β-cells (Suppl Fig. 4A), overexpression of presenlin-1 resulted in a slight increase in the ER Ca2+ leakage in both β-cell lines (INS: Fig. 5D, F; Min-6: Fig. 5E, G) while the ER Ca2+ content in HeLa and EA.hy926 cells overexpressing presenilin-1 remained unaffected (Suppl. Fig. 4B).
Presenilin-1 dependent ER Ca2+ leak is independent of IP3R expression
The involvement of inositol 1, 4,5-trisphosphate receptors (IP3Rs) in presenilin-1-dependent ER Ca2+ leak is currently a matter of controversy [28-31]. Therefore, we tested for a possible involvement of IP3Rs in the presenilin-1-dependent ER Ca2+ leak in INS-1. An initial expression analysis of all IP3Rs indicated comparable expression of all three IP3Rs in HeLa cells, while in the β-cell line, INS-1, the IP3R type 3 was the by far most abundant IP3R isoform (Suppl. Fig. 5). Based on these findings, we next attenuated the expression of IP3R type 1 and 2, or that of IP3R type 3 by specific siRNAs (Suppl. Fig. 6; for sequences see Suppl. Table 2). The efficiency of IP3R knockdown was functionally confirmed by measuring Cch- or histamine-induced intracellular Ca2+ release using the Fura-2 technique (Suppl. Fig. 7). Neither a depletion of IP3R type 1 and type 2 nor type 3 affected the ER Ca2+ leak in pancreatic β-cells, as shown by ER Ca2+ leak experiments (Fig. 5H). Likewise, knockdown of individual IP3Rs also did not introduce an ER Ca2+ leak in HeLa cells (Suppl. Fig. 8). These findings suggest that none of the IP3R subtypes is involved in the presenilin-1-dependent ER Ca2+ leak, which we found to be characteristic for β-cells and isolated islets.
GSK3β-mediated presenilin-1 phosphorylation drives the enhanced ER Ca2+ leakage in β-cells
Because expression levels of presenilin-1 in HeLa and EA.hy926 cells were comparable with that found in β-cells (Fig. 5B) and the overexpression of presenilin-1 did not introduce an enhanced ER Ca2+ leak in HeLa or EA.hy926 cells (Suppl. Fig. 4), we hypothesized that a post-translational modification of presenilin-1 was responsible for establishing the ER Ca2+ leakage in isolated pancreatic islets and β-cells. Notably, glycogen synthase kinase 3 beta (GSK3b)-mediated phosphorylation at serine 353 and serine 357 [32] of presenilin-1 is known to modulate its Ca2+ leak function [33]. Therefore, we explored the putative involvement of GSK3b in the enhanced ER Ca2+ leak found in the pancreatic islets and the two β-cell lines. Comparison of the expression level and activity of GSK3b in β-cells and the two non-β-cell lines revealed only slightly higher GSK3b expression in the two β-cell lines (Suppl. Fig. 9). However, an enzyme-linked immunosorbent assay [34] revealed strongly elevated GSK3b activity in both β-cell lines compared to the two other cell types (Fig. 6A). Accordingly, we hypothesized that an increased activity of GSK3b results in a hyperphosphorylation of presenilin-1 that, in turn, establishes increased ER Ca2+ leak.
To evaluate this hypothesis, isolated murine pancreatic islets and β-cells were pre-incubated for 24 or 48 h with the GSK3b inhibitor CHIR99021 [35] and the consequences thereof on the ER Ca2+ leak were investigated. Strikingly, inhibition of GSK3b prevented enhanced ER Ca2+ leak in the isolated pancreatic islets (Fig. 6B) and in both β-cell lines (Fig. 6C) but did not affect the ER Ca2+ content of the other cell types (Fig. 6D). Testing pancreatic islets viability revealed no toxic effect of CHIR99021 at the concentration and duration used (Suppl. Fig. 10). To further investigate the crucial role of GSK3b-mediated phosphorylation of presenilin-1 in the enhanced ER Ca2+ leak in β-cells we expressed a constitutively inactive presenilin-1 mutant with two alanines replacing the serine residues at position 353 and 357 (presenilin-1S353/357A). Notably, presenilin-1S353/357A cannot be phosphorylated by GSK3b. Expression of presenilin-1S353/357A in the β-cells abolished the ER Ca2+ leak (Fig. 6E), thus, supporting our concept that GSK3b-mediated serine phosphorylation of presenilin-1 accounts for the enhanced ER Ca2+ leak in this particular cell type.
To further substantiate the regulatory role of GSK3b a constitutively active presenilin-1 mutant was used, in which the two serine residues at positions 353 and 357 were replaced by aspartic acids that mimic phosphorylated serines (presenilin-1S353/357D). Expression of presenilin-1S353/357D slightly enhanced the constitutive ER Ca2+ leak in INS-1 but not MIN-6 cells (Fig. 6F). However, in contrast to the findings in wild-type INS-1 and MIN-6 cells (Fig. 4C), the GSK3b inhibitor CHIR99021 did not abolish the enhanced ER Ca2+ leak in β-cells expressing the phosphomimetic presenilin-1S353/357D mutant (Fig. 6G). Notably, expression of constitutively active presenilin-1S353/357D in HeLa and EA.hy926 cells introduced an enhanced ER Ca2+ leak, which was, in case of EA.hy926, comparable to that of wild-type β-cells (Fig. 6H). Together, these data demonstrate that GSK3b-mediated phosphorylation of the two serine residues at positions 353 and 357 of presenilin-1 activates an ER Ca2+ leak and that hyperphosphorylated presenilin-1 establishes the augmented ER Ca2+ leak in pancreatic β-cells.
Mitochondria sequester Ca2+ leaked from the ER in β-cells
Our data above indicate that, despite a greatly enhanced ER Ca2+ leak in the pancreatic islets and β-cells, basal cytosolic Ca2+ does not differ from that found in the two other lines. However, basal ER Ca2+ levels were decreased in both β-cell lines compared to HeLa and EA.hy926 cells (Fig. 7A), despite SERCA activity is similar in all cell types (Fig. 4C,D). Considering the close proximity between the ER and mitochondria in nearly all cells including β-cells [36], it is tempting to speculate that mitochondria serve as “Ca2+ receiver” for the enhanced ER Ca2+ leak. In line with this hypothesis, resting mitochondrial Ca2+ levels were increased in the pancreatic β-cells compared to non-β-cells (Fig. 7B).
To investigate whether mitochondria indeed sequester most of the Ca2+ which leaks from the ER in pancreatic β-cells, mitochondrial Ca2+ uptake was prevented by siRNA-mediated knockdown of the mitochondrial Ca2+ uniporter (MCU) (Suppl. Fig. 11). In MCU depleted β-cells, the resting cytosolic Ca2+ levels were elevated compared to control cells (Fig. 7C). Moreover, resting mitochondrial Ca2+ levels and mitochondrial Ca2+ elevations upon stimulation with Cch and BHQ were reduced in MCU depleted β-cells (Suppl. Fig. 12). In contrast, reduction of MCU expression had no effect on basal cytosolic Ca2+ levels in non-β-cell lines (Fig. 7D). Notably, the inhibition of the β-cell-specific ER Ca2+ leakage by knockdown of presenilin-1, prevented the increase in basal cytosolic Ca2+ upon MCU knockdown in both β-cells (Fig. 7E). These data support our hypothesis that mitochondria effectively sequester the Ca2+ that leaks from the ER in pancreatic β-cells.
The tight dependency of resting mitochondrial Ca2+ from a continuous transfer of extracellular Ca2+, presumably via the enhanced ER Ca2+ leak was further illustrated by our findings that the removal of extracellular Ca2+ resulted in a small, biphasic yet significant decrease in mitochondrial Ca2+ levels in the INS-1 β-cell line (Fig. 7F, H) but not in non-β-cells (Fig. 7G, H).
The basal ER-to-mitochondria Ca2+ flux yields metabolic pre-activation of mitochondria in β-cells
To further reveal the functional consequences of an enhanced ER-to-mitochondria Ca2+ flux in β-cells, we measured mitochondrial respiration using the Seahorse® technology. Since, several dehydrogenases of the citric acid cycle are regulated by matrix Ca2+ elevation [37-39], we predicted that the constant Ca2+ uptake by mitochondria under resting conditions has a stimulatory effect on respiration. In line with this prediction, β-cells showed a considerably higher basal and maximal mitochondrial respiration compared to the non-β-cell lines (Fig. 8A). Manipulating the ER Ca2+ leakage by siRNA-mediated knockdown of presenilin-1 resulted in decreased basal and maximal respiration in the pancreatic β-cells (Fig. 8B), while the depletion of presenilin-1 had no effect on basal and maximal respiration in the other cell types (Suppl. Fig. 13).
In line with our experiments above that indicate that GSK3β-induced phosphorylation of presenilin-1 accounts for the enhanced ER Ca2+ leak, the GSK3β inhibitor CHIR99021 strongly decreased basal in both pancreatic β-cell lines (Fig. 8C, D). Similarly, CHIR99021 reduced maximal respiration in both pancreatic β-cell lines although only statistical significant in the INS-1 but not the MIN-6 cells (Fig. 8C, D). No effect on respiration was observed in the other cell types (Suppl. Fig. 14). These findings highlight that the functional coupling between enhanced ER Ca2+ leak and mitochondrial Ca2+ sequestration stimulates respiratory activity in the pancreatic β-cells.
To test whether or not the enhanced mitochondrial respiration that is due to the increased ER Ca2+ leak in β-cells is coupled to an enhanced basal ATP production in the mitochondria, basal mitochondrial ATP levels were measured using the genetically encoded and mitochondria-targeted ATP sensor, mtAT1.03 [40]. Compared to HeLa and EA.hy926 cells, both β-cell types showed elevated mitochondrial ATP levels under resting conditions (Fig. 8E). The siRNA-mediated reduction of the expression of presenilin-1 as well as the inhibition of GSK3β with CHIR99021 reduced basal mitochondrial ATP levels in the pancreatic β-cells (Fig. 8F,G), whereas ATP levels within mitochondria of the non-β-cells cell types remained unaffected (Suppl. Fig. 15). Taken together, these data demonstrate that the enhanced ER Ca2+ leak and consequently increased mitochondrial Ca2+ sequestration yields an intrinsic pre-activation of mitochondrial respiration already under basal conditions.
Discussion
In the present study, we describe a cell type-specific, atypical ER Ca2+ leak in pancreatic β-cells under basal conditions. This continuous Ca2+ efflux from the ER in insulin secreting cells is established by GSK3β-mediated phosphorylation of presenilin-1, is independent from IP3-receptors, and is compensated by TRPC3-associated Ca2+ entry. Moreover, the Ca2+ leaking the ER is directly channeled towards the mitochondria in pancreatic β-cells and, subsequently, enhances basal Ca2+, respiration and ATP levels in mitochondria. Accordingly, these data illustrate a tight functional coupling of ER Ca2+ leak with mitochondrial basal activity that appears specific for pancreatic β-cells.
Comparing isolated murine pancreatic islets and the two β-cell lines INS-1 and MIN-6 with two distinct, non-β cells (HeLa and EA.hy926) in this study, revealed strong ER Ca2+ leak only in the pancreatic islets and β-cells. The present study revealed that the ER Ca2+ loss in freshly isolated pancreatic islets and the two β-cell lines tested is compensated by ER refilling fueled by extracellular Ca2+. The pharmacological profiling, together with our findings that a diminution of TRPC3 expression yields ER Ca2+ loss even in the presence of extracellular Ca2+ point to an involvement of TRPC3 in the continuous ER refilling process in the β-cells.
Our data show that the kinetics of Ca2+ refilling of emptied ER in the two β-cell lines and freshly isolated pancreatic islets are comparable to that found in the two cell lines that did not show enhanced ER Ca2+ leak (i.e. HeLa and EA.hy926). Therefore, a reduced SERCA activity as cause for the enhanced ER Ca2+ leakage in pancreatic islets/β-cells can be excluded. There are several proteins known to establish an ER Ca2+ leak including Bcl-2 [41], pannexin 1 [42], TRPC1 [43], Sec61 [44] and presenilins [26, 27, 45]. Presenilins are enriched in mitochondrial-associated membranes (MAMs) [46] and have also been shown to foster ER-mitochondria coupling in a mitofusin-2-dependent manner [47]. There is an existing controversy concerning whether the presenilin-1-dependent ER Ca2+ leak occurs directly [26, 27] through presenilin-1 or whether the protein triggers flash-openings of IP3Rs, especially of type 1 IP3R [28-30]. Our data presented herein indicate that in β-cells the effect of presenilin-1 on ER Ca2+ leak is independent from the presence of any IP3R. These findings favor either a direct function of presenilin-1 in β-cell ER Ca2+ leak or interactions with other ER Ca2+ channels. The lack of signs for involvement of IP3Rs in the enhanced ER Ca2+ leak in β-cells might be due to the fact that all respective studies indicate type 1 IP3R to be involved in presenilin-1-induced ER Ca2+ leak [30, 48, 49] while in pancreatic β-cells this particular type of IP3R has lower expression compared to the predominantly expressed IP3R type 3.
While our data strongly point to presenilin-1 being responsible for the ER Ca2+ leak found in pancreatic islets/β-cells in line with our hypothesis, our data revealed and enhanced activity of GSK3b, which is known to stimulate presenilin-1 Ca2+ leak [32], in the β-cells compared to non-β-cells. Although further studies are necessary, the various GSK3b activities might be due to the differences in the energetic setting between the β-cells (INS-1, MIN-6), as being highly dependent on oxidative metabolism as basis of the sensory system for insulin secretion [3], and the highly glycolytic immortalized cell lines (HeLa, EA.hy926), which effectively operate under the Warburg effect [50]. Our data showing that GSK3b inhibition abolished the ER Ca2+ leak in both β-cell lines and in isolated murine pancreatic islets, point to the involvement of GSK3b-in the presenilin-1-mediated ER Ca2+ leak in β-cells. This assumption is further demonstrated by the utilization of constitutively active/inactive presenilin-1 mutants of serines at position 353 and 357 [32]. Similar findings were shown in cardiac myocytes where GSK3b activity was found to establish an enhanced SR/ER-mitochondria Ca2+ crosstalk during reperfusion injury [49].
Our data further demonstrate that the leaked Ca2+ is rapidly sequestered by neighboring mitochondria and does not affect global cytosolic Ca2+ levels. This assumption builds on the increased resting Ca2+ levels in the mitochondrial matrix in β-cells and that the inhibition of mitochondrial Ca2+ uptake by knockdown of MCU yielded increase in resting cytosolic Ca2+ levels only in the β-cells. Hence, inhibition of β-cell ER Ca2+ leak by either presenilin-1 knockdown or inhibition of GSK3b by CHIR99021 prevented changes in cytosolic Ca2+ levels upon MCU depletion in both β-cell lines. Matrix Ca2+ is a major determinant for mitochondrial activity [6]. Notably, mitochondrial Ca2+ increase is known to stimulate matrix dehydrogenases of the citric acid cycle [37-39], thus, serving as a key trigger for insulin release in β-cells [51]. Our data that the ER Ca2+ leak yields increase in basal respiratory activity and mitochondrial ATP levels in β-cells are in line with this concept. Moreover, our data point to a continuous priming of β-cells based on a weak mitochondrial stimulation by the continuous Ca2+ flux from the ER as prerequisite for accurate responsiveness to elevated blood glucose sensing [13]. This conclusion is supported by our findings that inhibition of ER Ca2+ leak, either by diminution of presenilin-1 expression or inhibition of GSK3β, abolished the enhanced respiratory activity and elevated mitochondrial ATP levels exclusively in the β-cells.
Familial Alzheimer’s disease (AD) -linked presenlilin-1 mutations have been described to disturb the Ca2+ leak function of the protein [27, 31, 52]. Notably, in several Alzheimer models, mutations in presenilins have been shown to yield Ca2+ accumulation in the ER [53], thus, pointing to a lack of ER Ca2+ leak function by the mutated presenilin-1. Accordingly, one may speculate that some cases of type 2 DM and familiar AD might share the same molecular background: i.e. the lack of presenilin-1 established ER Ca2+ leak due to mutations either on the motifs responsible for the Ca2+ leak or on the phosphorylation sites for its regulator GSK3β. In fact, the majority of familial AD patients also suffer from type 2 DM leading to the current understanding of type 2 DM as risk factor of AD [54]. Multiple studies raised epidemiological and experimental evidence for such possible shared pathophysiology between type 2 DM and AD [55] and link both diseases with mitochondrial dysfunction [56, 57]. So far many mechanisms have been discussed as common molecular causes of AD and type 2 DM [58]. Our present data indicate that certain mutations of presenilin-1, which alter the ER Ca2+ leak in neurons and pancreatic β-cells, impair respective cell functions and, hence, might cause the frequent coincidence of these severe diseases. Several reports describe disrupted interorganellar communication between the ER and mitochondria (MAMs) in AD [59-61]. Accordingly, though further studies are necessary, our present data and other reports point to deranged subcellular Ca2+ homeostasis due to mutations of presenilin-1 as a common molecular mechanism for reduced β-cells responsiveness leading to type 2 DM [62], as well as dysfunctional amyloid degradation causing AD [63].
Conclusion
Our data presented herein describe a GSK3β/presenilin-1-dependent continuous ER Ca2+ leak in β-cells that yields priming of mitochondria by elevating organellar Ca2+, increased basal respiration and ATP production. The physiological importance of such inter-organelle Ca2+ transfer and mitochondrial pre-activation awaits further investigations but might be related to β-cell responsiveness and/or insulin secretion.
Acknowledgements
We thank Mrs. Anna Schreilechner, BSc for expert assistance in cell culture.
Funding: This work was supported by the Austrian Science Funds (FWF; DKplus W 1226-B18 to W.F.G., P28529-B27 and I3716-B27 to R.M.; P28854 to T.M.), the Austrian Research Promotion Agency (FFG; 864690 to T.M.), the Austrian infrastructure program (HSRM 2016/201), the President’s International Fellowship Initiative of CAS (No. 2015VBB045 to T.M.), and the National Natural Science Foundation of China (No. 31450110423 to T.M.). Microscopic equipment is part of the Nikon-Center of Excellence, Graz, supported by the HSRM 2013/2014, Nikon Austria, and BioTechMed. C.K. and B.G. are fellows of the Doctoral College “Metabolic and Cardiovascular Disease” at the Medical University of Graz funded by the FWF (W 1226-B18). W.F.G. is the guarantor of this work and, as such, had full access to all data in the study and takes responsibility for integrity of data and accuracy of data analyses. Author Contributions: C.K., B.G., M.W.-W. and M.R.D. performed calcium and ATP measurements, PCRs and ELISAs. C.T.M.S. performed respiration measurements. S.S. and T.M. performed and interpreted NMR data, V.S., M.D.-M. and D.K. isolated murine pancreatic islets, and, R.R. was responsible for cell culture. W.F.G. planned and supervised this work, and together with R.M. and J.H. prepared the manuscript. All authors discussed the results and implications and commented on the manuscript at all stages. Data availability: Supporting data are provided in the Supplementary Materials. Original data are available from the corresponding author upon request.
Disclosure Statement
The authors declare no competing financial interests.
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