Calcium-activated K+ channels of mouse beta-cells are controlled by both store and cytoplasmic Ca2+: experimental and theoretical studies

A novel calcium-dependent potassium current (K_slow) that slowly activates in response to a simulated islet burst was identified recently in mouse pancreatic β-cells (Göpel, S.O., T. Kanno, S. Barg, L. Eliasson, J. Galvanovskis, E. Renström, and P. Rorsman. 1999. J. Gen. Physiol. 114:759–769). K_slow activation may help terminate the cyclic bursts of Ca²⁺-dependent action potentials that drive Ca²⁺ influx and insulin secretion in β-cells. Here, we report that when [Ca²⁺]_i handling was disrupted by blocking Ca²⁺ uptake into the ER with two separate agents reported to block the sarco/endoplasmic calcium ATPase (SERCA), thapsigargin (1–5 μM) or insulin (200 nM), K_slow was transiently potentiated and then inhibited. K_slow amplitude could also be inhibited by increasing extracellular glucose concentration from 5 to 10 mM. The biphasic modulation of K_slow by SERCA blockers could not be explained by a minimal mathematical model in which [Ca²⁺]_i is divided between two compartments, the cytosol and the ER, and K_slow activation mirrors changes in cytosolic calcium induced by the burst protocol. However, the experimental findings were reproduced by a model in which K_slow activation is mediated by a localized pool of [Ca²⁺] in a subspace located between the ER and the plasma membrane. In this model, the subspace [Ca²⁺] follows changes in cytosolic [Ca²⁺] but with a gradient that reflects Ca²⁺ efflux from the ER. Slow modulation of this gradient as the ER empties and fills may enhance the role of K_slow and [Ca²⁺] handling in influencing β-cell electrical activity and insulin secretion.     

Small subpopulations of β-cells do not drive islet oscillatory [Ca2+] dynamics via gap junction communication

The islets of Langerhans exist as multicellular networks that regulate blood glucose levels. The majority of cells in the islet are excitable, insulin-producing β-cells that are electrically coupled via gap junction channels. β-cells are known to display heterogeneous functionality. However, due to gap junction coupling, β-cells show coordinated [Ca²⁺] oscillations when stimulated with glucose, and global quiescence when unstimulated. Small subpopulations of highly functional β-cells have been suggested to control [Ca²⁺] dynamics across the islet. When these populations were targeted by optogenetic silencing or photoablation, [Ca²⁺] dynamics across the islet were largely disrupted. In this study, we investigated the theoretical basis of these experiments and how small populations can disproportionality control islet [Ca²⁺] dynamics. Using a multicellular islet model, we generated normal, skewed or bimodal distributions of β-cell heterogeneity. We examined how islet [Ca²⁺] dynamics were disrupted when cells were targeted via hyperpolarization or populations were removed; to mimic optogenetic silencing or photoablation, respectively. Targeted cell populations were chosen based on characteristics linked to functional subpopulation, including metabolic rate of glucose oxidation or [Ca²⁺] oscillation frequency. Islets were susceptible to marked suppression of [Ca²⁺] when ~10% of cells with high metabolic activity were hyperpolarized; where hyperpolarizing cells with normal metabolic activity had little effect. However, when highly metabolic cells were removed from the model, [Ca²⁺] oscillations remained. Similarly, when ~10% of cells with either the highest frequency or earliest elevations in [Ca²⁺] were removed from the islet, the [Ca²⁺] oscillation frequency remained largely unchanged. Overall, these results indicate small populations of β-cells with either increased metabolic activity or increased frequency are unable to disproportionately control islet-wide [Ca²⁺] via gap junction coupling. Therefore, we need to reconsider the physiological basis for such small β-cell populations or the mechanism by which they may be acting to control normal islet function.     

Intrinsic Islet Heterogeneity and Gap Junction Coupling Determine Spatiotemporal Ca2+ Wave Dynamics

Insulin is released from the islets of Langerhans in discrete pulses that are linked to synchronized oscillations of intracellular free calcium ([Ca²⁺]_i). Associated with each synchronized oscillation is a propagating calcium wave mediated by Connexin36 (Cx36) gap junctions. A computational islet model predicted that waves emerge due to heterogeneity in β-cell function throughout the islet. To test this, we applied defined patterns of glucose stimulation across the islet using a microfluidic device and measured how these perturbations affect calcium wave propagation. We further investigated how gap junction coupling regulates spatiotemporal [Ca²⁺]_i dynamics in the face of heterogeneous glucose stimulation. Calcium waves were found to originate in regions of the islet having elevated excitability, and this heterogeneity is an intrinsic property of islet β-cells. The extent of [Ca²⁺]_i elevation across the islet in the presence of heterogeneity is gap-junction dependent, which reveals a glucose dependence of gap junction coupling. To better describe these observations, we had to modify the computational islet model to consider the electrochemical gradient between neighboring β-cells. These results reveal how the spatiotemporal [Ca²⁺]_i dynamics of the islet depend on β-cell heterogeneity and cell-cell coupling, and are important for understanding the regulation of coordinated insulin release across the islet.     

Linoleic Acid Stimulates [Ca2+]i Increase in Rat Pancreatic Beta-Cells through Both Membrane Receptor- and Intracellular Metabolite-Mediated Pathways

The role of the free fatty acid (FFA) receptor and the intracellular metabolites of linoleic acid (LA) in LA-stimulated increase in cytosolic free calcium concentration ([Ca²⁺]i) was investigated. [Ca²⁺]i was measured using Fura-2 as indicator in rat pancreatic β-cells in primary culture. LA (20 µM for 2 min) stimulated a transient peak increase followed by a minor plateau increase in [Ca²⁺]i. Elongation of LA stimulation up to 10 min induced a strong and long-lasting elevation in [Ca²⁺]i. Activation of FFA receptors by the non-metabolic agonist GW9508 (40 µM for 10 min) resulted in an increase in [Ca²⁺]i similar to that of 2-min LA treatment. Inhibition of acyl-CoA synthetases by Triacsin C suppressed the strong and long-lasting increase in [Ca²⁺]i. The increase in [Ca²⁺]i induced by 2 min LA or GW9508 were fully eliminated by exhaustion of endoplasmic reticulum (ER) Ca²⁺ stores or by inhibition of phospholipase C (PLC). Removal of extracellular Ca²⁺ did not influence the transient peak increase in [Ca²⁺]i stimulated by 2 min LA or GW9508. The strong and long-lasting increase in [Ca²⁺]i induced by 10 min LA was only partially suppressed by extracellular Ca²⁺ removal or thapsigargin pretreatment, whereas remaining elevation in [Ca²⁺]i was eliminated after exhaustion of mitochondrial Ca²⁺ using triphenyltin. In conclusion, LA stimulates Ca²⁺ release from ER through activation of the FFA receptor coupled to PLC and mobilizes mitochondrial Ca²⁺ by intracellular metabolites in β-cells.     

Glucose Decouples Intracellular Ca2+ Activity from Glucagon Secretion in Mouse Pancreatic Islet Alpha-Cells

The mechanisms of glucagon secretion and its suppression by glucose are presently unknown. This study investigates the relationship between intracellular calcium levels ([Ca²⁺]_i) and hormone secretion under low and high glucose conditions. We examined the effects of modulating ion channel activities on [Ca²⁺]_i and hormone secretion from ex vivo mouse pancreatic islets. Glucagon-secreting α-cells were unambiguously identified by cell specific expression of fluorescent proteins. We found that activation of L-type voltage-gated calcium channels is critical for α-cell calcium oscillations and glucagon secretion at low glucose levels. Calcium channel activation depends on K_ATP channel activity but not on tetrodotoxin-sensitive Na⁺ channels. The use of glucagon secretagogues reveals a positive correlation between α-cell [Ca²⁺]_i and secretion at low glucose levels. Glucose elevation suppresses glucagon secretion even after treatment with secretagogues. Importantly, this inhibition is not mediated by K_ATP channel activity or reduction in α-cell [Ca²⁺]_i. Our results demonstrate that glucose uncouples the positive relationship between [Ca²⁺]_i and secretory activity. We conclude that glucose suppression of glucagon secretion is not mediated by inactivation of calcium channels, but instead, it requires a calcium-independent inhibitory pathway.     

Dimensionality and Size Scaling of Coordinated Ca2+ Dynamics in MIN6 β-cell Clusters

Pancreatic islets of Langerhans regulate blood glucose homeostasis by the secretion of the hormone insulin. Like many neuroendocrine cells, the coupling between insulin-secreting β-cells in the islet is critical for the dynamics of hormone secretion. We have examined how this coupling architecture regulates the electrical dynamics that underlie insulin secretion by utilizing a microwell-based aggregation method to generate clusters of a β-cell line with defined sizes and dimensions. We measured the dynamics of free-calcium activity ([Ca²⁺]_i) and insulin secretion and compared these measurements with a percolating network model. We observed that the coupling dimension was critical for regulating [Ca²⁺]_i dynamics and insulin secretion. Three-dimensional coupling led to size-invariant suppression of [Ca²⁺]_i at low glucose and robust synchronized [Ca²⁺]_i oscillations at elevated glucose, whereas two-dimensional coupling showed poor suppression and less robust synchronization, with significant size-dependence. The dimension- and size-scaling of [Ca²⁺]_i at high and low glucose could be accurately described with the percolating network model, using similar network connectivity. As such this could explain the fundamentally different behavior and size-scaling observed under each coupling dimension. This study highlights the dependence of proper β-cell function on the coupling architecture that will be important for developing therapeutic treatments for diabetes such as islet transplantation techniques. Furthermore, this will be vital to gain a better understanding of the general features by which cellular interactions regulate coupled multicellular systems.     

Mitochondrial Free Ca2+ Levels and Their Effects on Energy Metabolism in Drosophila Motor Nerve Terminals

Mitochondrial Ca²⁺ uptake exerts dual effects on mitochondria. Ca²⁺ accumulation in the mitochondrial matrix dissipates membrane potential (_ΔΨ_m), but Ca²⁺ binding of the intramitochondrial enzymes accelerates oxidative phosphorylation, leading to mitochondrial hyperpolarization. The levels of matrix free Ca²⁺ ([Ca²⁺]_m) that trigger these metabolic responses in mitochondria in nerve terminals have not been determined. Here, we estimated [Ca²⁺]_m in motor neuron terminals of Drosophila larvae using two methods: the relative responses of two chemical Ca²⁺ indicators with a 20-fold difference in Ca²⁺ affinity (rhod-FF and rhod-5N), and the response of a low-affinity, genetically encoded ratiometric Ca²⁺ indicator (D4cpv) calibrated against known Ca²⁺ levels. Matrix pH (pH_m) and _ΔΨ_m were monitored using ratiometric pericam and tetramethylrhodamine ethyl ester probe, respectively, to determine when mitochondrial energy metabolism was elevated. At rest, [Ca²⁺]_m was 0.22 ± 0.04 μM, but it rose to ∼26 μM (24.3 ± 3.4 μM with rhod-FF/rhod-5N and 27.0 ± 2.6 μM with D4cpv) when the axon fired close to its endogenous frequency for only 2 s. This elevation in [Ca²⁺]_m coincided with a rapid elevation in pH_m and was followed by an after-stimulus _ΔΨ_m hyperpolarization. However, pH_m decreased and no _ΔΨ_m hyperpolarization was observed in response to lower levels of [Ca²⁺]_m, up to 13.1 μM. These data indicate that surprisingly high levels of [Ca²⁺]_m are required to stimulate presynaptic mitochondrial energy metabolism.     

Mycobacterial HBHA induces endoplasmic reticulum stress-mediated apoptosis through the generation of reactive oxygen species and cytosolic Ca2+ in murine macrophage RAW 264.7 cells

Mycobacterial heparin-binding haemagglutinin antigen (HBHA) is a virulence factor that induces apoptosis of macrophages. Endoplasmic reticulum (ER) stress-mediated apoptosis is an important regulatory response that can be utilised to study the pathogenesis of tuberculosis. In the present study, HBHA stimulation induced ER stress sensor molecules in a caspase-dependent manner. Pre-treatment of RAW 264.7 cells with an IκB kinase 2 inhibitor reduced not only C/EBP homology protein expression but also IL-6 and monocyte chemotactic protein-1 (MCP-1) production. BAPTA-AM reduced both ER stress responses and caspase activation and strongly suppressed HBHA-induced IL-6 and MCP-1 production in RAW 264.7 cells. Enhanced reactive oxygen species (ROS) production and elevated cytosolic [Ca²⁺]_i levels were essential for HBHA-induced ER stress responses. Collectively, our data suggest that HBHA induces cytosolic [Ca²⁺]_i, which influences the generation of ROS associated with the production of proinflammatory cytokines. These concerted and complex cellular responses induce ER stress-associated apoptosis during HBHA stimulation in macrophages. These results indicate that the ER stress pathway has an important role in the HBHA-induced apoptosis during mycobacterial infection.     

Ca microdomains and the control of insulin secretion

Nutrient-induced increases in intracellular free Ca²⁺ concentrations are the key trigger for insulin release from pancreatic islet β-cells. These Ca²⁺ changes are tightly regulated temporally, occurring as Ca²⁺ influx-dependent baseline oscillations. We explore here the concept that locally high [Ca²⁺] concentrations (i.e. Ca²⁺ microdomains) may control exocytosis via the recruitment of key effector proteins to sites of exocytosis. Importantly, recent advances in the development of organelle- and membrane-targeted green fluorescent protein (GFP-) or aequorin-based Ca²⁺ indicators, as well as in rapid imaging techniques, are providing new insights into the potential role of these Ca²⁺ microdomains in β-cells. We summarise here some of the evidence indicating that Ca²⁺ microdomains beneath the plasma membrane and at the surface of large dense core vesicles may be important in the normal regulation of insulin secretion, and may conceivably contribute to “ATP-sensitive K⁺-channel independent” effects of glucose. We also discuss evidence that, in contrast to certain non-excitable cells, direct transfer of Ca²⁺ from the ER to mitochondria via localised physical contacts between these organelles is relatively less important for efficient mitochondrial Ca²⁺ uptake in β-cells. Finally, we discuss evidence from single cell imaging that increases in cytosolic Ca²⁺ are not required for the upstroke of oscillations in mitochondrial redox state, but may underlie the reoxidation process.     

Plasma Membrane Ca2+-ATPase Isoforms Composition Regulates Cellular pH Homeostasis in Differentiating PC12 Cells in a Manner Dependent on Cytosolic Ca2+ Elevations

Plasma membrane Ca²⁺-ATPase (PMCA) by extruding Ca²⁺ outside the cell, actively participates in the regulation of intracellular Ca²⁺ concentration. Acting as Ca²⁺/H⁺ counter-transporter, PMCA transports large quantities of protons which may affect organellar pH homeostasis. PMCA exists in four isoforms (PMCA1-4) but only PMCA2 and PMCA3, due to their unique localization and features, perform more specialized function. Using differentiated PC12 cells we assessed the role of PMCA2 and PMCA3 in the regulation of intracellular pH in steady-state conditions and during Ca²⁺ overload evoked by 59 mM KCl. We observed that manipulation in PMCA expression elevated pH_mito and pH_cyto but only in PMCA2-downregulated cells higher mitochondrial pH gradient (ΔpH) was found in steady-state conditions. Our data also demonstrated that PMCA2 or PMCA3 knock-down delayed Ca²⁺ clearance and partially attenuated cellular acidification during KCl-stimulated Ca²⁺ influx. Because SERCA and NCX modulated cellular pH response in neglectable manner, and all conditions used to inhibit PMCA prevented KCl-induced pH drop, we considered PMCA2 and PMCA3 as mainly responsible for transport of protons to intracellular milieu. In steady-state conditions, higher TMRE uptake in PMCA2-knockdown line was driven by plasma membrane potential (Ψp). Nonetheless, mitochondrial membrane potential (Ψm) in this line was dissipated during Ca²⁺ overload. Cyclosporin and bongkrekic acid prevented Ψ_m loss suggesting the involvement of Ca²⁺-driven opening of mitochondrial permeability transition pore as putative underlying mechanism. The findings presented here demonstrate a crucial role of PMCA2 and PMCA3 in regulation of cellular pH and indicate PMCA membrane composition important for preservation of electrochemical gradient.     

Enhanced ER Ca2+ store filling by overexpression of SERCA2b promotes IP3-evoked puffs

Liberation of Ca²⁺ from the endoplasmic reticulum (ER) through inositol trisphosphate receptors (IP₃R) is modulated by the ER Ca²⁺ content, and overexpression of SERCA2b to accelerate Ca²⁺ sequestration into the ER has been shown to potentiate the frequency and amplitude of IP₃-evoked Ca²⁺ waves in Xenopus oocytes. Here, we examined the effects of SERCA overexpression on the elementary IP₃-evoked puffs to elucidate whether ER [Ca²⁺] may modulate IP₃R function via luminal regulatory sites in addition to simply determining the size of the available store and electrochemical driving force for Ca²⁺ release. SERCA2b and Ca²⁺ permeable nicotinic plasmalemmal channels were expressed in oocytes, and hyperpolarizing pulses were delivered to induce Ca²⁺ influx and thereby load ER stores. Puffs evoked by photoreleased IP₃ were significantly potentiated in terms of numbers of responding sites, frequency and amplitude following transient Ca²⁺ influx in SERCA-overexpressing cells, whereas little change was evident with SERCA overexpression alone or following Ca²⁺ influx in control cells not overexpressing SERCA. Intriguingly, we observed the appearance of a new population of puffs that arose after long latencies and had prolonged durations supporting the notion of luminal regulation of IP₃R gating kinetics.     

JNK1/2 regulates ER–mitochondrial Ca2+ cross-talk during IL-1β–mediated cell death in RINm5F and human primary β-cells

Elevated interleukin-1β (IL-1β) induces apoptosis in pancreatic β-cells through endoplasmic reticulum (ER) stress induction and subsequent c-jun-N-terminal kinase 1/2 (JNK1/2) activation. In earlier work we showed that JNK1/2 activation is initiated before ER stress and apoptotic induction in response to IL-1β. However, the detailed regulatory mechanisms are not completely understood. Because the ER is the organelle responsible for Ca²⁺ handling and storage, here we examine the effects of IL-1β on cellular Ca²⁺ movement and mitochondrial dysfunction and evaluate the role of JNK1/2. Our results show that in RINm5F cells and human primary β-cells, IL-1β alters mitochondrial membrane potential, mitochondrial permeability transition pore opening, ATP content, and reactive oxygen species production and these alterations are preceded by ER Ca²⁺ release via IP₃R channels and mitochondrial Ca²⁺ uptake. All these events are prevented by JNK1/2 small interfering RNA (siRNA), indicating the mediating role of JNK1/2 in IL-1β–induced cellular alteration. This is accompanied by IL-1β–induced apoptosis, which is prevented by JNK1/2 siRNA and the IP₃R inhibitor xestospongin C. This suggests a regulatory role of JNK1/2 in modulating the ER-mitochondrial-Ca²⁺ axis by IL-1β in apoptotic cell death.     

Mitochondrial Ca2+ uptake with and without the formation of high-Ca2+ microdomains

The mitochondrial Ca²⁺ uniporter has low affinity for Ca²⁺, therefore it has been assumed that submicromolar Ca²⁺ signals cannot induce mitochondrial Ca²⁺ uptake. The close apposition of the plasma membrane or the endoplamic reticulum (ER) to the mitochondria and the limited Ca²⁺ diffusion in the cytoplasm result in the formation of perimitochondrial high-Ca²⁺ microdomains (HCMDs) capable of activating mitochondrial Ca²⁺ uptake. The possibility of mitochondrial Ca²⁺ uptake at low submicromolar [Ca²⁺]_c has not yet been generally accepted.Earlier we found in permeabilized glomerulosa, luteal and pancreatic β cells that [Ca²⁺]_m increased when [Ca²⁺]_c was raised from 60 nM to less than 200 nM. Here we report data obtained from H295R (adrenocortical) cells transfected with ER-targeted GFP. Cytoplasmic Ca²⁺ response to angiotensin II was different in mitochondrion-rich and mitochondrion-free domains. The mitochondrial Ca²⁺ response to angiotensin II correlated with GFP fluorescence indicating the vicinity of ER. When the cells were exposed to K⁺ (inducing Ca²⁺ influx), no correlation was found between the mitochondrial Ca²⁺ signal and the vicinity of the plasma membrane or the ER. The results presented here provide evidence that mitochondrial Ca²⁺ uptake may occur both with and without the formation of HCMDs within the same cell.     

Mcl-1 downregulation by pro-inflammatory cytokines and palmitate is an early event contributing to β-cell apoptosis

Pancreatic β-cell apoptosis is a key feature of diabetes mellitus and the mitochondrial pathway of apoptosis is a major mediator of β-cell death. We presently evaluated the role of the myeloid cell leukemia sequence 1 (Mcl-1), an antiapoptotic protein of the Bcl-2 family, in β-cells following exposure to well-defined β-cell death effectors, for example, pro-inflammatory cytokines, palmitate and chemical endoplasmic reticulum (ER) stressors. All cytotoxic stresses rapidly and preferentially decreased Mcl-1 protein expression as compared with the late effect observed on the other antiapoptotic proteins, Bcl-2 and Bcl-xL. This was due to ER stress-mediated inhibition of translation through eIF2α phosphorylation for palmitate and ER stressors and through the combined action of translation inhibition and JNK activation for cytokines. Knocking down Mcl-1 using small interference RNAs increased apoptosis and caspase-3 cleavage induced by cytokines, palmitate or thapsigargin, whereas Mcl-1 overexpression partly prevented Bax translocation to the mitochondria, cytochrome c release, caspase-3 cleavage and apoptosis induced by the β-cell death effectors. Altogether, our data suggest that Mcl-1 downregulation is a crucial event leading to β-cell apoptosis and provide new insights into the mechanisms linking ER stress and the mitochondrial intrinsic pathway of apoptosis. Mcl-1 is therefore an attractive target for the design of new strategies in the treatment of diabetes.     

Exendin-4 Protects Mitochondria from Reactive Oxygen Species Induced Apoptosis in Pancreatic Beta Cells

Objective

Mitochondrial oxidative stress is the basis for pancreatic β-cell apoptosis and a common pathway for numerous types of damage, including glucotoxicity and lipotoxicity. We cultivated mice pancreatic β-cell tumor Min6 cell lines in vitro and observed pancreatic β-cell apoptosis and changes in mitochondrial function before and after the addition of Exendin-4. Based on these observations, we discuss the protective role of Exendin-4 against mitochondrial oxidative damage and its relationship with Ca²⁺-independent phospholipase A2.

Methods

We established a pancreatic β-cell oxidative stress damage model using Min6 cell lines cultured in vitro with tert-buty1 hydroperoxide and hydrogen peroxide. We then added Exendin-4 to observe changes in the rate of cell apoptosis (Annexin-V-FITC-PI staining flow cytometry and DNA ladder). We detected the activity of the caspase 3 and 8 apoptotic factors, measured the mitochondrial membrane potential losses and reactive oxygen species production levels, and detected the expression of cytochrome c and Smac/DLAMO in the cytosol and mitochondria, mitochondrial Ca₂-independent phospholipase A2 and Ca²⁺-independent phospholipase A2 mRNA.

Results

The time-concentration curve showed that different percentages of apoptosis occurred at different time-concentrations in tert-buty1 hydroperoxide- and hydrogen peroxide-induced Min6 cells. Incubation with 100 µmol/l of Exendin-4 for 48 hours reduced the Min6 cell apoptosis rate (p<0.05). The mitochondrial membrane potential loss and total reactive oxygen species levels decreased (p<0.05), and the release of cytochrome c and Smac/DLAMO from the mitochondria was reduced. The study also showed that Ca²⁺-independent phospholipase A2 activity was positively related to Exendin-4 activity.

Conclusion

Exendin-4 reduces Min6 cell oxidative damage and the cell apoptosis rate, which may be related to Ca²-independent phospholipase A2.     

Spatiotemporal Correlations between Cytosolic and Mitochondrial Ca2+ Signals Using a Novel Red-Shifted Mitochondrial Targeted Cameleon

The transfer of Ca²⁺ from the cytosol into the lumen of mitochondria is a crucial process that impacts cell signaling in multiple ways. Cytosolic Ca²⁺ ([Ca²⁺]_cyto) can be excellently quantified with the ratiometric Ca²⁺ probe fura-2, while genetically encoded Förster resonance energy transfer (FRET)-based fluorescent Ca²⁺ sensors, the cameleons, are efficiently used to specifically measure Ca²⁺ within organelles. However, because of a significant overlap of the fura-2 emission with the spectra of the cyan and yellow fluorescent protein of most of the existing cameleons, the measurement of fura-2 and cameleons within one given cell is a complex task. In this study, we introduce a novel approach to simultaneously assess [Ca²⁺]_cyto and mitochondrial Ca²⁺ ([Ca²⁺]_mito) signals at the single cell level. In order to eliminate the spectral overlap we developed a novel red-shifted cameleon, D1GO-Cam, in which the green and orange fluorescent proteins were used as the FRET pair. This ratiometric Ca²⁺ probe could be successfully targeted to mitochondria and was suitable to be used simultaneously with fura-2 to correlate [Ca²⁺]_cyto and [Ca²⁺]_mito within same individual cells. Our data indicate that depending on the kinetics of [Ca²⁺]_cyto rises there is a significant lag between onset of [Ca²⁺]_cyto and [Ca²⁺]_mito signals, pointing to a certain threshold of [Ca²⁺]_cyto necessary to activate mitochondrial Ca²⁺ uptake. The temporal correlation between [Ca²⁺]_mito and [Ca²⁺]_cyto as well as the efficiency of the transfer of Ca²⁺ from the cytosol into mitochondria varies between different cell types. Moreover, slow mitochondrial Ca²⁺ extrusion and a desensitization of mitochondrial Ca²⁺ uptake cause a clear difference in patterns of mitochondrial and cytosolic Ca²⁺ oscillations of pancreatic beta-cells in response to D-glucose.     

Dual Effect of Phosphate Transport on Mitochondrial Ca2+ Dynamics

The large inner membrane electrochemical driving force and restricted volume of the matrix confer unique constraints on mitochondrial ion transport. Cation uptake along with anion and water movement induces swelling if not compensated by other processes. For mitochondrial Ca²⁺ uptake, these include activation of countertransporters (Na⁺/Ca²⁺ exchanger and Na⁺/H⁺ exchanger) coupled to the proton gradient, ultimately maintained by the proton pumps of the respiratory chain, and Ca²⁺ binding to matrix buffers. Inorganic phosphate (P_i) is known to affect both the Ca²⁺ uptake rate and the buffering reaction, but the role of anion transport in determining mitochondrial Ca²⁺ dynamics is poorly understood. Here we simultaneously monitor extra- and intra-mitochondrial Ca²⁺ and mitochondrial membrane potential (ΔΨ_m) to examine the effects of anion transport on mitochondrial Ca²⁺ flux and buffering in P_i-depleted guinea pig cardiac mitochondria. Mitochondrial Ca²⁺ uptake proceeded slowly in the absence of P_i but matrix free Ca²⁺ ([Ca²⁺]_mito) still rose to ∼50 μm. P_i (0.001–1 mm) accelerated Ca²⁺ uptake but decreased [Ca²⁺]_mito by almost 50% while restoring ΔΨ_m. P_i-dependent effects on Ca²⁺ were blocked by inhibiting the phosphate carrier. Mitochondrial Ca²⁺ uptake rate was also increased by vanadate (V_i), acetate, ATP, or a non-hydrolyzable ATP analog (AMP-PNP), with differential effects on matrix Ca²⁺ buffering and ΔΨ_m recovery. Interestingly, ATP or AMP-PNP prevented the effects of P_i on Ca²⁺ uptake. The results show that anion transport imposes an upper limit on mitochondrial Ca²⁺ uptake and modifies the [Ca²⁺]_mito response in a complex manner.     

Glucose-Dependent Insulin Secretion in Pancreatic β-Cell Islets from Male Rats Requires Ca2+ Release via ROS-Stimulated Ryanodine Receptors

Glucose-stimulated insulin secretion (GSIS) from pancreatic β-cells requires an increase in intracellular free Ca²⁺ concentration ([Ca²⁺]). Glucose uptake into β-cells promotes Ca²⁺ influx and reactive oxygen species (ROS) generation. In other cell types, Ca²⁺ and ROS jointly induce Ca²⁺ release mediated by ryanodine receptor (RyR) channels. Therefore, we explored here if RyR-mediated Ca²⁺ release contributes to GSIS in β-cell islets isolated from male rats. Stimulatory glucose increased islet insulin secretion, and promoted ROS generation in islets and dissociated β-cells. Conventional PCR assays and immunostaining confirmed that β-cells express RyR2, the cardiac RyR isoform. Extended incubation of β-cell islets with inhibitory ryanodine suppressed GSIS; so did the antioxidant N-acetyl cysteine (NAC), which also decreased insulin secretion induced by glucose plus caffeine. Inhibitory ryanodine or NAC did not affect insulin secretion induced by glucose plus carbachol, which engages inositol 1,4,5-trisphosphate receptors. Incubation of islets with H₂O₂ in basal glucose increased insulin secretion 2-fold. Inhibitory ryanodine significantly decreased H₂O₂-stimulated insulin secretion and prevented the 4.5-fold increase of cytoplasmic [Ca²⁺] produced by incubation of dissociated β-cells with H₂O₂. Addition of stimulatory glucose or H₂O₂ (in basal glucose) to β-cells disaggregated from islets increased RyR2 S-glutathionylation to similar levels, measured by a proximity ligation assay; in contrast, NAC significantly reduced the RyR2 S-glutathionylation increase produced by stimulatory glucose. We propose that RyR2-mediated Ca²⁺ release, induced by the concomitant increases in [Ca²⁺] and ROS produced by stimulatory glucose, is an essential step in GSIS.     

Inhibition of Pancreatic β-Cell Ca2+/Calmodulin-dependent Protein Kinase II Reduces Glucose-stimulated Calcium Influx and Insulin Secretion,Impairing Glucose Tolerance

Glucose-stimulated insulin secretion (GSIS) from pancreatic β-cells is caused by Ca²⁺ entry via voltage-dependent Ca²⁺ channels. CaMKII is a key mediator and feedback regulator of Ca²⁺ signaling in many tissues, but its role in β-cells is poorly understood, especially in vivo. Here, we report that mice with conditional inhibition of CaMKII in β-cells show significantly impaired glucose tolerance due to decreased GSIS. Moreover, β-cell CaMKII inhibition dramatically exacerbates glucose intolerance following exposure to a high fat diet. The impairment of islet GSIS by β-cell CaMKII inhibition is not accompanied by changes in either glucose metabolism or the activities of K_ATP and voltage-gated potassium channels. However, glucose-stimulated Ca²⁺ entry via voltage-dependent Ca²⁺ channels is reduced in islet β-cells with CaMKII inhibition, as well as in primary wild-type β-cells treated with a peptide inhibitor of CaMKII. The levels of basal β-cell cytoplasmic Ca²⁺ and of endoplasmic reticulum Ca²⁺ stores are also decreased by CaMKII inhibition. In addition, CaMKII inhibition suppresses glucose-stimulated action potential firing frequency. These results reveal that CaMKII is a Ca²⁺ sensor with a key role as a feed-forward stimulator of β-cell Ca²⁺ signals that enhance GSIS under physiological and pathological conditions.