Christiane Kleca,c Corina T. Madreiter-Sokolowskia
Gabriela Ziomeka Sarah Stryecka Vinay Sachdeva,d
Madalina Duta-Marea Benjamin Gottschalka
Maria R. Depaolia Rene Rosta Jesse Haya,e Markus
Waldeck-Weiermaira Dagmar Kratkya,b Tobias Madla,b
Roland Mallia,b Wolfgang F. Graiera,b
aMolecular Biology and Biochemistry, Gottfried Schatz Research Center for Cellular Signaling, Metabolism & Aging, Medical University of Graz, Graz, bBioTechMed, Graz, cResearch Unit for Non-Coding RNAs and Genome Editing in Cancer, Division of Oncology, Department of Internal Medicine, Medical University of Graz, Graz, Austria, dDepartment of Medical Biochemistry, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands, eUniversity of Montana, Division of Biological Sciences, Center for Structural & Functional Neuroscience, Missoula, Montana, USA
Key Words
Mitochondria • Endoplasmic reticulum • Ca2+ spiking • Insulin secretion
Abstract
Background/Aims: In our recent work, the importance of GSK3β-mediated phosphorylation of presenilin-1 as crucial process to establish a Ca2+ leak in the endoplasmic reticulum and, subsequently, the pre-activation of resting mitochondrial activity in β-cells was demonstrated. The present work is a follow-up and reveals the importance of GSK3β-phosphorylated presenilin-1 for responsiveness of pancreatic islets and β-cells to elevated glucose in terms of cytosolic Ca2+ spiking and insulin secretion. Methods: Freshly isolated pancreatic islets and the two pancreatic β-cell lines INS-1 and MIN-6 were used. Cytosolic Ca2+ was fluorometrically monitored using Fura-2/AM and cellular insulin content and secretion were measured by ELISA. Results: Our data strengthened our previous findings of the existence of a presenilin-1-mediated ER-Ca2+ leak in β-cells, since a reduction of presenilin-1 expression strongly counteracted the ER Ca2+ leak. Furthermore, our data revealed that cytosolic Ca2+ spiking upon administration of high D-glucose was delayed in onset time and strongly reduced in amplitude and frequency upon siRNA-mediated knock-down of presenilin-1 or the inhibition of GSK3β in the pancreatic β-cells. Moreover, glucose-triggered initial insulin secretion disappeared by depletion from presenilin-1 and inhibition of GSK3β in the pancreatic β-cells and isolated pancreatic islets, respectively. Conclusion: These data complement our previous work and demonstrate that the sensitivity of pancreatic islets and β-cells to glucose illustrated as glucose-triggered cytosolic Ca2+ spiking and initial but not long-lasting insulin secretion crucially depends on a strong ER Ca2+ leak that is due to the phosphorylation of presenilin-1 by GSK3β, a phenomenon that might be involved in the development of type 2 diabetes.
Introduction
The elevation of blood glucose is sensed by pancreatic β-cells that take up and subsequently metabolize glucose, resulting in an increased mitochondrial production of ATP that, in turn, inhibits KATP channels yielding plasma membrane depolarization resulting in activation of L-type Ca2+ channels [1]. This stimulated Ca2+ entry triggers the exocytosis of insulin-containing secretory granules [2]. In the coupling between mitochondrial ATP production to elevated glucose and insulin secretion (i.e. glucose stimulated insulin secretion; GSIS) mitochondrial Ca2+ uptake has been found to play a crucial role [3–5]. However, all these reports convincingly demonstrate that mitochondrial Ca2+ uptake in response to the entering Ca2+ facilitates mitochondrial ATP production, most likely by Ca2+-mediated activation of mitochondrial dehydrogenases [6–9], thus, facilitating citric acid cycle and the subsequent ATP production. Notably, such effect boosts mitochondrial ATP formation in a second phase, when mitochondria sequester the Ca2+ that entered via L-type channels, thus, this effect follows a mitochondrial ATP production in response to elevated glucose.
In our recent work, we reported a β-cell-specific phenomenon of Ca2+-mediated pre-activation of mitochondria already under basal (i.e. resting glucose) conditions [10]. In particular, we described a large ER Ca2+ leak in pancreatic β-cell lines and freshly isolated mouse pancreatic islets [10]. Mechanistically, this leak is established by the phosphorylation of ER-localized presenilin-1 by glycogen synthase kinase 3 beta (GSK3β) and installs a unidirectional Ca2+ flux from the ER towards the mitochondrial matrix yielding enhanced resting mitochondrial Ca2+ levels and, subsequently, enhanced resting respiratory activity and mitochondrial ATP levels [10]. In extension to the Ca2+-facilitating function on mitochondrial ATP production in the second phase as described above, these data point to a pre-sensitization of β-cell mitochondria. Accordingly, one might hypothesize that such pre-sensitizing of β-cell mitochondria might facilitate the responsiveness of β-cell mitochondria to elevated glucose and, thus, for the initial insulin secretion.
The present work is designed to challenge this hypothesis and to elaborate the importance of GSK3β-mediated phosphorylation of presenilin-1 for responsiveness of β-cells to elevated glucose and GSIS. The two clonal pancreatic β-cell lines, INS-1 [11] and MIN-6 [12] and freshly isolated pancreatic islets were used and the importance of presenilin-1 and its phosphorylation by GSK3β for glucose triggered cytosolic Ca2+ spiking and consecutive insulin secretion was investigated.
Materials and Methods
Reagents
Cell culture materials were purchased from Greiner Bio-One (Kremsmünster, 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 obtained from Tocris (Bristol, UK). The selective JNK inhibitor (SP600125; PubChem CID: 8515) was obtained from Santa Cruz Biotechnology (Dallas, TX, USA). 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). Carbachol (PubChem CID: 5831), Thapsigargin (PubChem CID: 446378) and 2, 5-Di-Tert-Butylhydroquinone (BHQ; PubChem CID: 2374) were obtained from Sigma Aldrich (Vienna, Austria). All other buffer salts 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 [13]. Briefly, mice were euthanized by cervical dislocation. The common bile duct was cannulated using a 27G needle, and 2 ml of ice-cold Liberase II (Roche Diagnostics, Basel, Switzerland) was injected immediately. The perfused pancreas was dislodged from the intestine, spleen, and stomach. To facilitate complete digestion, the pancreas was further incubated in a water bath preheated at 37°C for predetermined time intervals (batch variation, average time 12 min and 30 s). After digestion, islets were purified by filtration and gradient separation. Islets were maintained in RPMI (GIBCO; Life Technologies, Carlsbad, CA) supplemented with 10% fetal bovine serum and 100 U/ml penicillin-streptomycin (GIBCO) for 24 h, and healthy islets were hand-picked for experiments. Islet GSIS was determined from ~200 hand-picked islets.
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), 50 µg of penicillin (PubChem CID: 5904) and 100 µg of streptomycin (PubChem CID: 19646). 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. Origin of cells was confirmed by STR-profiling by the cell culture facility of ZMF (Graz).
Buffers
2CaNa buffer: 2 mM CaCl2, 138 mM NaCl, 1 mM MgCl2, 5 mM KCl, 10 mM Hepes, 10 mM D-glucose, pH 7.4. 0 CaNa buffer: same as 2CaNa buffer but without CaCl2, plus 10 mM EGTA (Ca2+ chelator), pH 7.4. Experimental Buffer (EB): 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 D-glucose, 0.1% vitamins, 0.2% essential amino acids, and 1% penicillin-streptomycin (all v/v); pH adjusted to 7.4. Hank’s buffered salt solution (HBSS): 114 mM NaCl, 4.7 mM KCl, 2.5 mM CaCl2, 1.2 mM MgSO4, 2 mM Hepes, 1.2 mM KH2PO4, 25 mM NaHCO3, 0.2% BSA, 3 mM D-glucose.
Transfection with siRNAs and plasmids
Cells seeded on 30 mm glass coverslips (for Ca2+ measurements) or in 6-well plates were transiently transfected at 60 - 80% confluency 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. Knock-down efficiency for presenilin-1 is shown in the Supplementary Information to our previous work [10]. For inhibition of GSK3β or JNK cells were incubated in their respective media containing 2.5 µM CHIR99021 or 10 – 100 µM SP600125, respectively. All experiments were performed 24 - 48 h after transfection or treatment. siRNAs were obtained from Microsynth (Balgach, Switzerland) or Thermofisher Scientific (Vienna, Austria).
mRNA Isolation and qRT-PCR
24 h after treatment of INS-1 cells with different concentrations of SP600125 or DMSO control, total RNA isolation, reverse transcription, PCR and real-time-PCR were performed according to our recently published protocols [18]. Relative expression of specific genes was normalized to rat GAPDH, as a housekeeping gene. Primers for rat presenilin-1 and GAPDH were obtained from Qiagen (QuantiTect Rn_PSEN1 and QuantiTect Rn_GAPDH).
Cytosolic Ca2+ imaging in cultured β-cells
ER Ca2+ leak experiments: 24 h after treatment with 100 µM SP600125, cells were loaded with 2 µM Fura-2/AM (TEFLabs) in EB for 40 min as previously described [5] and were alternately illuminated at 340 and 380 nm, whereas fluorescence emission was recorded at 510 nm. Results of Fura-2/AM measurements are shown as the ratio of F340/F380. For measurements of cytosolic Ca2+ to indirectly visualize ER Ca2+ leak, after Fura-2/AM loading, cells were incubated for the indicated times (1 min or 20 min) in an experimental buffer without Ca2+ (0CaNa buffer) and subsequently stimulated with 100 µM of IP3-generating agonist carbachol in the presence of 15 µM of the SERCA inhibitor BHQ.
L-type Ca2+ channel and thapsigargin experiments
As indicated in the respective graphs different combinations of 2CaNa, 0CaNa buffers with or without 1 µM thapsigargin (non-competitive SERCA inhibitor) have been used. To evaluate the maximal Ca2+ uptake via L-type Ca2+ channels, cells were depolarized with a high K+ buffer (30 mM K+), where 25 mM NaCl (2 CaNa recipe) were substituted with KCl to reach a final K+ concentration of 30 mM.
Glucose-induced Ca2+ oscillations: After Fura-2/AM loading, cells were stabilized by a 20 min incubation in glucose free buffer (0G; 10 mM mannitol substitute the glucose) before imaging, to minimize the cytosolic oscillations and to improve the signal upon exposure to high glucose. On the microscope, cells were perfused with 0G buffer for 2 min before switching to 16 mM glucose (16G) during acquisition. Cells were alternately illuminated at 340 and 380 nm, whereas fluorescence emission was recorded at 510 nm. Results of Fura-2/AM measurements are shown as the ratio of F340/F380. 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.
For determination of insulin secretion rate INS-1 cells were routinely plated on 6-well plates and transfected at a confluency of 70 to 80% with either scrambled control siRNA or siRNA against presenilin-1 or treated with 100 µM JNK inhibitor (SP600125) or DMSO. Islets were treated with either 2.5 µM CHIR99021 or DMSO control for 24 h. 48 h after transfection or 24 h after treatment cells or islets were washed twice with HBSS buffer containing 3 mM D-glucose, followed by a 30 min incubation in this buffer to record basal insulin secretion. For stimulation of insulin secretion cells and islets were stimulated with HBSS buffer containing high D-glucose concentrations to reach a final concentration of 16 mM D-glucose. Samples were taken for the next 2 to 45 min. Samples were stored on ice and centrifuged at 3000 rpm for 5 min to remove residual cells. Supernatant was transferred into fresh tubes, stored at -80°C and assayed with Mercodia Rat Insulin ELISA (Mercodia, Uppsala, Sweden). Insulin secretion rate was calculated as described previously [5] following a standard procedure [14].
Statistical analyses
Data shown represent the mean ± SEM. ‘n’ values refer to the number of individual experiments performed. For life 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 assuming unequal variances was used, where applicable using GraphPad Prism 5.0f (GraphPad Software, La Jolla, CA, USA).
Data availability
Original data are available from the corresponding author upon request.
Results
Reduced presenilin-1 expression by treatment with the JNK inhibitor SP600125 counteracts ER Ca2+ leak in pancreatic β-cells
In order to corroborate our previous findings of the presenilin-1-mediated ER Ca2+ leak in β-cells, we used another approach to reduce presenilin-1 expression by treating INS-1 cells with the selective c-Jun N-terminal kinase (JNK) inhibitor SP600125. Notably, besides its multiple substrates [15], amongst other in pancreatic β-cells [16], inhibition of JNK was shown to reduce presenilin-1 expression in the neuroblastoma cell line SK-N-SH [17]. Accordingly, we treated INS-1 cells with different concentrations of the inhibitor for 24 h and evaluated presenilin-1 expression on mRNA level. Presenilin-1 expression was reduced at high concentrations of the JNK inhibitor (i.e. 100 µM) (Fig. 1A). Next, we performed ER Ca2+ leak experiments according to our ER Ca2+ leakage protocol [10]. Extracellular Ca2+ was removed from the cells and the ER Ca2+ content was indirectly measured as cytosolic Ca2+ increase upon ER Ca2+ mobilization with an inositol1, 4,5-trisphosphate- (IP3) generating agonist (100 µM carbachol) in the presence of the reversible SERCA inhibitor tert-butylhydroxyquinone (BHQ) to avoid refilling of the ER. Under control conditions a massive ER Ca2+ loss was detected after 20 min pre-incubation in Ca2+-free buffer (Fig. 1B, D). After 24 h treatment with the JNK inhibitor this ER Ca2+ loss was strongly reduced (Fig. 1C, D). These data indicate that JNK-inhibitor-mediated reduction of presenilin-1 expression yields reduction of the ER Ca2+ leak in pancreatic β-cells, and, thus, strongly supporting our previous findings [10].
Influence of presenilin-1 depletion on plasma membrane Ca2+ uptake via L-type Ca2+ channels
In order to validate whether or not the depletion of presenilin-1 affects the Ca2+ entry pathways existing in pancreatic β-cells, the Ca2+ entry due to Ca2+ store (ER) depletion (store-operated Ca2+ entry, SOCE) and depolarization (L-type Ca2+ channels) was assessed in control and presenilin-1 depleted cells. Therefore, cells were treated in the absence of extracellular Ca2+ with the irreversible SERCA inhibitor thapsigargin to empty the ER followed by the re-addition of extracellular Ca2+. The Ca2+ upon the re-addition of extracellular Ca2+ reflects the SOCE activity. Our experiments did not reveal any effect of the knock-down of presenilin-1 in SOCE activity in pancreatic β-cells (Fig. 2A).
In order to investigate a possible influence of presenilin-1 knock-down on the activity of voltage-gated L-type Ca2+ channels, we measured cytosolic Ca2+ in the presence of extracellular Ca2+ and depolarized the pancreatic β-cells with extracellular 30 mM K+. Knock-down of presenilin-1 failed to affect the increases in cytosolic Ca2+ upon cell depolarization (Fig. 2B), thus, indicating that the lack of presenilin-1 did not affect the activity of L-type Ca2+ channels.
Presenilin-1 is prerequisite for robust Ca2+ responsiveness of β-cells to elevated glucose
Because D-glucose-stimulated Ca2+ spiking is known to link mitochondrial activity with insulin secretion in β-cells, we examined the putative involvement of presenilin-1 on glucose-triggered cytosolic Ca2+ spiking in the two pancreatic β-cell lines INS-1 and MIN-6 cells under control conditions and after siRNA-mediated depletion from presenilin-1. β-cells were exposed to elevated glucose to trigger cytosolic Ca2+ transients, which were observed in both β-cell lines (Fig. 3A), demonstrating their normal responsiveness. Depletion of presenilin-1 by siRNA-mediated knock-down reduced the Ca2+ responses of the β-cells to elevated glucose (Fig. 3B). Statistical analyses of the kinetics of glucose-induced Ca2+ transients revealed that in both β-cell lines the depletion of presenilin-1 delayed the response times until cytosolic Ca2+ elevations occurred (Fig. 3D), reduced mean amplitudes (Fig. 3E) and numbers of individual Ca2+ transients (Fig. 3F).
GSK3β activity is essential for the initiation of Ca2+ spiking to elevated glucose in β-cells
Since in our previous work presenilin-1 is essentially needed to be phosphorylated by GSK3β in order to establish ER Ca2+ leak that yields pre-activation of mitochondria in the β-cells, we next examined the effect of an inhibition of GSK3β with its inhibitor CHIR99021 [18] on glucose-triggered cytosolic Ca2+ spiking in the two pancreatic β-cell lines. Compared with controls (Fig. 3A), in cells treated with CHIR99021 glucose-induced Ca2+ spiking was strongly attenuated (Fig. 3C). Hence, the statistical analyses of the kinetics of glucose-induced Ca2+ transients showed that inhibition of GSK3β delayed cytosolic Ca2+ spiking (Fig. 3C), lowered mean amplitudes (Fig. 3D) and the count of Ca2+ transients (Fig. 3E).
Presenilin-1 is essential for the initial insulin secretion upon elevated glucose in β-cells
We obtained a time course of GSIS in the pancreatic β-cell lines INS-1 that were transfected with scrambled siRNA or specific siRNA against presenilin-1 to prevent the ER Ca2+ leak as previously shown [10]. In control cells, the administration of elevated glucose triggered an initial burst of GSIS within 6-8 min of glucose exposure followed by a sustained phase of insulin secretion (Fig. 4A). However, in cells that were depleted of presenilin-1 the initial burst of GSIS was absent while the sustained phase of insulin secretion was unaffected by the presenilin-1 knockdown (Fig. 4A). Notably, the absolute amount of insulin secreted remained delayed over the entire 50 min observation time while insulin secretion after 10 min glucose administration was comparable in control cells and that depleted from presenilin-1 (Fig. 4B). Notably, depletion of presenilin-1 did not affect the insulin content in the INS-1 cells (controls: 60.1 ± 10.9 ng insulin/mg protein; presenilin-1 depleted cells: 66.5 ± 9.3 ng insulin/mg protein; n=5) (Fig. 4C).
Moreover, experiments were conducted using INS-1 cells and in freshly isolated pancreatic islets that were treated with the GSK3β inhibitor CHIR99021 to avoid the enhanced ER Ca2+ leak recently described in freshly isolated pancreatic islets [10]. Similar to our findings in the INS-1 cells where presenilin-1 expression was reduced by siRNA-mediated knock-down, the inhibition of ER Ca2+ leak by administration of the GSK3β inhibitor prevented the initial GSIS (Fig. 5A, D) but not the long-lasting insulin secretion (Fig. 5B, E) in response to elevated glucose. Despite an inhibition of GSK3β by CHIR99021 the insulin content in INS-1 cells and the freshly isolated pancreatic islets remained only marginally affected (islets: controls: 263.6 ± 11.1 ng insulin/mg protein; CHIR99021: 290.7 ± 2.8 ng insulin/mg protein INS-1 cells: controls: 30.5 ± 2.9 ng insulin/mg protein; CHIR99021: 30.3 ± 1.8 ng insulin/mg protein) (Fig. 5C, F).
Discussion
The predominant function of pancreatic β-cells is the control of blood glucose levels by 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, 19]. Importantly, after glucose metabolization and ATP formation, the inhibition of KATP channels yields opening of L-type Ca2+ channels and, subsequently, Ca2+ influx into the β-cell as the final trigger for insulin secretion [20–22]. Notably, during the cytosolic Ca2+ elevations, the sequestration of these ions into the mitochondrial matrix via MCU [23]/MICU1 [5]-dependent pathway is fundamental for GSIS [3, 24–26]. While the crucial role of mitochondrial Ca2+ has been clearly presented [4, 5,23], these reports focus on the importance of mitochondrial Ca2+ uptake of entering Ca2+, thus, describing the later phase that succeeds mitochondrial ATP production, membrane depolarization by ATP-mediated inhibition of KATP channels, and the subsequent opening of L-type voltage-gated Ca2+ channels. Notably, in our recent work [10], elevated resting mitochondrial Ca2+ levels were described in pancreatic β-cells that builds on an increased ER Ca2+ leak due to GSK3β-mediated phosphorylation of presenilin-1 [10]. Our present findings that inhibition of JNK kinase that decreases the expression of presenilin-1 [35] reduced the ER Ca2+ leak further support our previous hypothesis on the crucial role of presenilin-1 on the ER Ca2+ leak in pancreatic β-cells. These findings shed a light on the resting situation of mitochondria in pancreatic β-cells and reveal a cell specific setting of enhanced basal mitochondrial activity under resting conditions. The present work represents a follow-up work that explores the physiological consequences of such elevated basal mitochondrial activity in terms of the β-cell most prominent function: the glucose-stimulated insulin secretion (GSIS).
Matrix Ca2+ is a major determinant for mitochondrial activity [4]. According to the current concept of GSIS, glycolysis-derived pyruvate fuels matrix citric acid cycle required for mitochondrial ATP production. The ATP inhibits plasma membrane KATP channels resulting in plasma membrane depolarization and opening of L-type Ca2+ channels. Subsequently, entering Ca2+ initiates fusion of insulin-containing granules with the cell membrane, thus, releasing insulin [2]. Notably, mitochondrial Ca2+ increase is known to serve as a key trigger for insulin release in β-cells [27], because of its stimulatory effect on the matrix dehydrogenases of the citric acid cycle [6, 22, 28]. Missing from the current concept, however, is the exact time point of the contribution of mitochondrial Ca2+ to GSIS. In theory, a mitochondrial Ca2+ increase would occur upon Ca2+ entry due to the opening of L-type Ca2+ channels, however, this would be already downstream of mitochondrial activation by glycolysis-derived pyruvate and ATP production and hence be of questionable importance in triggering GSIS. Since an increase in mitochondrial Ca2+ stimulates Ca2+-dependent dehydrogenases of the citric acid cycle [28], we envisaged that a ER Ca2+ leak evoked pre-stimulation of β-cell mitochondrial dehydrogenases, which are ultimately responsible for the necessary ATP production, would be of metabolic advantage by ensuring a fast and precise insulin response solely controlled by glucose.
Our data on the enhanced basal respiratory activity in β-cells [10] support such new perspective on GSIS that involves a continuous priming of β-cells based on a weak mitochondrial stimulation to ensure accurate responsiveness to elevated blood glucose sensing [29]. This conclusion was previously supported by our recent findings that inhibition of ER Ca2+ leak, either by knock-down of presenilin-1 expression or an inhibition of GSK3β by CHIR99021 [18] abolished enhanced mitochondrial resting metabolic activity and elevation in basal mitochondrial ATP production [10]. The importance of GSK3β/presenilin-1-provided ER Ca2+ for the responsiveness of pancreatic islets and β-cells to elevated glucose is further illustrated by our present experiments measuring glucose-induced cytosolic Ca2+ spiking and GSIS, the ultimate readout for pancreatic islet/β-cell function. Notably, the lack of ER Ca2+ leak yielded a delayed onset time of cytosolic Ca2+ oscillations, reduced their mean amplitude and total number of spikes upon elevated glucose in the β-cells, thus, pointing to a reduced responsiveness of the cells to elevated glucose. Our findings, that the depletion of presenilin-1 did neither affect the depolarization-induced Ca2+ entry nor the store-operated Ca2+ entry, let us speculate that the reduced Ca2+ spiking in presenilin-1 depleted cells is not due to a direct effect on the existing Ca2+ entry pathways. Hence these findings are in line with our previous experiments [10] and support our hypothesis that the presenilin-1 established ER Ca2+ leak is fundamental for glucose sensing/metabolism to ensure proper β-cell responsiveness to elevated glucose. Moreover, our present data reveal that in isolated pancreatic islets and in the two β-cell lines used for this study the GSK3β/presenilin-1-induced ER Ca2+ leak is a prerequisite for the initial insulin release that occurs 10 min after exposure to elevated glucose. Notably, the inhibition of enhanced ER Ca2+ leak had no effect on the second phase of insulin secretion after 12 min of exposure to elevated glucose. These data point to a delayed responsiveness to elevated glucose of pancreatic β-cells and pancreatic islets that lack the enhanced ER Ca2+ leak while the long-lasting insulin secretion remains unaffected. Several studies showed that a loss of the first phase of the biphasic insulin response [30] is associated with a delayed β-cell response/insulin secretion [31] as early irregularity in type 2 DM and to worsening post-prandial hyperglycemia [32]. Therefore, the lack of the initial phase of insulin secretion serves as predictive marker for the risk of developing DM [33]. Our data showing that a decreased ER Ca2+ leak ultimately results in reduced mitochondrial metabolism [10] as well as reduced glucose induced cytosolic Ca2+ oscillations (this study), demonstrate the molecular basis for such loss of the first phase insulin response. In line with our findings, recent studies point to the importance of ER-mitochondrial Ca2+ transfer for an adequate insulin release [34–38]. Accordingly, we hypothesize that disturbances in the presenilin-1 dependent ER Ca2+ leak in pancreatic islets/β-cells hamper the cell’s fast and precise first insulin response which, in turn, may contribute to postprandial hyperglycemia and the development of type 2 DM (Fig. 6).
Conclusion
Our present data together with our previous report [10] unveil an important phenomenon that builds on a GSK3/presenilin-1-established ER Ca2+ leak that yields basal activation of mitochondria as prerequisite for responsiveness of pancreatic β-cells to elevated glucose (i.e. GSIS). Besides its physiological importance for GSIS, disturbances of this very β-cell-specific mechanism might be of pathological importance because its possible contribution to the development of type 2 DM.
Acknowledgements
The authors wish to thank Dr. C.J.S. Edgell (University of North Carolina, Chapel Hill, NC, USA) for providing us the EA.hy926 cells. This work was funded by the Austrian Science Funds (FWF, DKplus W 1226-B18). Microscopic equipment is part of the Nikon-Center of Excellence, Graz that is supported by the Austrian infrastructure program 2013/2014, Nikon Austria Inc. and BioTechMed. C.K. and B.G. are fellows of the Doctoral College “Metabolic and Cardiovascular Disease” at the Medical University of Graz and were funded by the FWF (W 1226-B18, DKplus Metabolic and Cardiovascular Disease) and Nikon Austria. This work was supported by the President’s International Fellowship Initiative of CAS (No. 2015VBB045, to T.M.), the National Natural Science Foundation of China (No. 31450110423, to T.M.), the Austrian Science Fund (FWF; P28529-B27 and I3716-B27 to R.M.; P28854 to T.M.), the Austrian Research Promotion Agency (FFG: 864690 to T.M.), the Integrative Metabolism Research Center Graz, the Austrian infrastructure program 2016/2017, BioTechMed/Graz, and the OMICS center Graz.
Authors’ contributions
C.K., G.Z. M.W.-W. and M.R.D. performed cytosolic, mitochondrial and endoplasmic reticulum calcium measurements. C.K. and CTMS performed oscillation experiments as well as immunosorbent assay and expression evaluation. B.G. performed analysis of cytosolic Ca2+ oscillation data. V.S., M.D.-M. and D.K. isolated the murine pancreatic islets, and, R.R. was responsible for cell preparation, culture and transfection. W.F.G. supervised research and project planning, and together with R.M., T.M., D.K. and J.H. performed data interpretation and prepared the manuscript. All authors discussed the results and implications and commented on the manuscript at all stages.
Disclosure Statement
The authors declare no competing financial interests.
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