Na+/Ca2+ Exchanger a Druggable Target to Promote ß-Cell Proliferation and Function.

An important feature of type 2 diabetes is a decrease in ß-cell mass. Therefore, it is essential to find new approaches to stimulate ß-cell proliferation. We have previously shown that heterozygous inactivation of the Na+/Ca2+ exchanger (isoform 1; NCX1), a protein responsible for Ca2+ extrusion from cells, increases ß-cell proliferation, mass, and function in mice. Here, we show that Ncx1 inactivation also increases ß-cell proliferation in 2-year-old mice and that NCX1 inhibition in adult mice by four small molecules of the benzoxyphenyl family stimulates ß-cell proliferation both in vitro and in vivo. NCX1 inhibition by small interfering RNA or small molecules activates the calcineurin/nuclear factor of activated T cells (NFAT) pathway and inhibits apoptosis induced by the immunosuppressors cyclosporine A (CsA) and tacrolimus in insulin-producing cell. Moreover, NCX1 inhibition increases the expression of ß-cell-specific genes, such as Ins1, Ins2, and Pdx1, and inactivates/downregulates the tumor suppressors retinoblastoma protein (pRb) and miR-193a and the cell cycle inhibitor p53. Our data show that Na+/Ca2+ exchange is a druggable target to stimulate ß-cell function and proliferation. Specific ß-cell inhibition of Na+/Ca2+ exchange by phenoxybenzamyl derivatives may represent an innovative approach to promote ß-cell regeneration in diabetes and improve the efficiency of pancreatic islet transplantation for the treatment of the disease.

The Na+/Ca2+ exchanger and the Plasma Membrane Ca2+-ATPase in ß-cell function and diabetes.

The rat pancreatic ß-cell expresses 6 splice variants of the Plasma Membrane Ca2+-ATPase (PMCA) and two splice variants of the Na+/Ca2+ exchanger 1 (NCX1). In the ß-cell Na+/Ca2+ exchange displays a high capacity, contributes to both Ca2+ outflow and influx and participates to the control of insulin release. Gain of function studies show that overexpression of PMCA2 or NCX1 leads to endoplasmic reticulum (ER) Ca2+ depletion with subsequent ER stress, decrease in ß-cell proliferation and ß-cell death by apoptosis. Loss of function studies show, on the contrary, that heterozygous inactivation of NCX1 (Ncx1+/-) leads to an increase in ß-cell function and a 5 fold increase in both ß-cell mass and proliferation. The mutation also increases ß-cell resistance to hypoxia, and Ncx1+/- islets show a 2-4 times higher rate of diabetes cure than Ncx1+/+ islets when transplanted in diabetic animals. Thus, down-regulation of the Na+/Ca2+ exchanger leads to various changes in ß-cell function that are opposite to the major abnormalities seen in diabetes. In addition, the ß-cell includes the mutually exclusive exon B in the alternative splicing region of NCX1, which confers a high sensitivity of its NCX splice variants (NCX1.3 & 1.7) to the inhibitory action of compounds like KBR-7943. Heterozygous inactivation of PMCA2 leads to apparented, though not completely similar results.These provide 2 unique models for the prevention and treatment of ß-cell dysfunction in diabetes and following islet transplantation.

Heterozygous inactivation of plasma membrane Ca(2+)-ATPase in mice increases glucose-induced insulin release and beta cell proliferation, mass and viability.

AIMS/HYPOTHESIS: Calcium plays an important role in the process of glucose-induced insulin release in pancreatic beta cells. These cells are equipped with a double system responsible for Ca(2+) extrusion--the Na/Ca exchanger (NCX) and the plasma membrane Ca(2+)-ATPase (PMCA). We have shown that heterozygous inactivation of NCX1 in mice increased glucose-induced insulin release and stimulated beta cell proliferation and mass. In the present study, we examined the effects of heterozygous inactivation of the PMCA on beta cell function. METHODS: Biological and morphological methods (Ca(2+) imaging, Ca(2+) uptake, glucose metabolism, insulin release and immunohistochemistry) were used to assess beta cell function and proliferation in Pmca2 (also known as Atp2b2) heterozygous mice and control littermates ex vivo. Blood glucose and insulin levels were also measured to assess glucose metabolism in vivo. RESULTS: Pmca (isoform 2) heterozygous inactivation increased intracellular Ca(2+) stores and glucose-induced insulin release. Moreover, increased beta cell proliferation, mass, viability and islet size were observed in Pmca2 heterozygous mice. However, no differences in beta cell glucose metabolism, proinsulin immunostaining and insulin content were observed. CONCLUSIONS/INTERPRETATION: The present data indicates that inhibition of Ca(2+) extrusion from the beta cell and its subsequent intracellular accumulation stimulates beta cell function, proliferation and mass. This is in agreement with our previous results observed in mice displaying heterozygous inactivation of NCX, and indicates that inhibition of Ca(2+) extrusion mechanisms by small molecules in beta cells may represent a new approach in the treatment of type 1 and type 2 diabetes.

Intracellular dyssynchrony of diastolic cytosolic [Ca²âº] decay in ventricular cardiomyocytes in cardiac remodeling and human heart failure.

RATIONALE: Synchronized release of Ca²âº into the cytosol during each cardiac cycle determines cardiomyocyte contraction. OBJECTIVE: We investigated synchrony of cytosolic [Ca²âº] decay during diastole and the impact of cardiac remodeling. METHODS AND RESULTS: Local cytosolic [Ca²âº] transients (1-µm intervals) were recorded in murine, porcine, and human ventricular single cardiomyocytes. We identified intracellular regions of slow (slowCaR) and fast (fastCaR) [Ca²âº] decay based on the local time constants of decay (TAUlocal). The SD of TAUlocal as a measure of dyssynchrony was not related to the amplitude or the timing of local Ca²âº release. Stimulation of sarcoplasmic reticulum Ca²âº ATPase with forskolin or istaroxime accelerated and its inhibition with cyclopiazonic acid slowed TAUlocal significantly more in slowCaR, thus altering the relationship between SD of TAUlocal and global [Ca²âº] decay (TAUglobal). Na⁺/Ca²âº exchanger inhibitor SEA0400 prolonged TAUlocal similarly in slowCaR and fastCaR. FastCaR were associated with increased mitochondrial density and were more sensitive to the mitochondrial Ca²âº uniporter blocker Ru360. Variation in TAUlocal was higher in pig and human cardiomyocytes and higher with increased stimulation frequency (2 Hz). TAUlocal correlated with local sarcomere relengthening. In mice with myocardial hypertrophy after transverse aortic constriction, in pigs with chronic myocardial ischemia, and in end-stage human heart failure, variation in TAUlocal was increased and related to cardiomyocyte hypertrophy and increased mitochondrial density. CONCLUSIONS: In cardiomyocytes, cytosolic [Ca²âº] decay is regulated locally and related to local sarcomere relengthening. Dyssynchronous intracellular [Ca²âº] decay in cardiac remodeling and end-stage heart failure suggests a novel mechanism of cellular contractile dysfunction.

Na(+)/Ca (2+) exchange and the plasma membrane Ca(2+)-ATPase in ß-cell function and diabetes.

The rat pancreatic ß-cell expresses two splice variants of the Na+/Ca(2+) exchanger 1 (NCX1) and six splice variants of the plasma membrane Ca(2+)-ATPase (PMCA). In the ß-cell, Na(+)/Ca(2+) exchange displays a high capacity, contributes to both Ca(2+) outflow and influx and participates to the control of insulin release. Gain of function studies show that overexpression of NCX1 or PMCA2 leads to endoplasmic reticulum (ER) Ca(2+) depletion with subsequent ER stress, decrease in ß-cell proliferation and ß-cell death by apoptosis. Interestingly, chronic exposure to cytokines or high free fatty acids concentration also induces ER Ca(2+) depletion and ß-cell death in diabetes. Loss of function studies shows, on the contrary, that heterozygous inactivation of NCX1 (Ncx1 ( +/- )) leads to an increase in ß-cell function (insulin production and release) and a fivefold increase in both ß-cell mass and proliferation. The mutation also increases ß-cell resistance to hypoxia, and Ncx1 ( +/- ) islets show a four to seven times higher rate of diabetes cure than Ncx1 ( +/+ ) islets when transplanted in diabetic animals. Thus, downregulation of the Na(+)/Ca(2+) exchanger leads to various changes in ß-cell function that are opposite to the major abnormalities seen in diabetes. In addition, the ß-cell, which is an excitable cell, includes the mutually exclusive exon B in the alternative splicing region of NCX1, which confers a high sensitivity of its NCX splice variants (NCX1.3 & 1.7) to the inhibitory action of compounds like KB-R7943. This provides a unique model for the prevention and treatment of ß-cell dysfunction in diabetes and following islet transplantation.

Plasma membrane Ca2+-ATPase overexpression depletes both mitochondrial and endoplasmic reticulum Ca2+ stores and triggers apoptosis in insulin-secreting BRIN-BD11 cells.

Ca(2+) may trigger apoptosis in ß-cells. Hence, the control of intracellular Ca(2+) may represent a potential approach to prevent ß-cell apoptosis in diabetes. Our objective was to investigate the effect and mechanism of action of plasma membrane Ca(2+)-ATPase (PMCA) overexpression on Ca(2+)-regulated apoptosis in clonal ß-cells. Clonal ß-cells (BRIN-BD11) were examined for the effect of PMCA overexpression on cytosolic and mitochondrial [Ca(2+)] using a combination of aequorins with different Ca(2+) affinities and on the ER and mitochondrial pathways of apoptosis. ß-cell stimulation generated microdomains of high [Ca(2+)] in the cytosol and subcellular heterogeneities in [Ca(2+)] among mitochondria. Overexpression of PMCA decreased [Ca(2+)] in the cytosol, the ER, and the mitochondria and activated the IRE1α-XBP1s but inhibited the PRKR-like ER kinase-eIF2α and the ATF6-BiP pathways of the ER-unfolded protein response. Increased Bax/Bcl-2 expression ratio was observed in PMCA overexpressing ß-cells. This was followed by Bax translocation to the mitochondria with subsequent cytochrome c release, opening of the permeability transition pore, and apoptosis. In conclusion, clonal ß-cell stimulation generates microdomains of high [Ca(2+)] in the cytosol and subcellular heterogeneities in [Ca(2+)] among mitochondria. PMCA overexpression depletes intracellular [Ca(2+)] stores and, despite a decrease in mitochondrial [Ca(2+)], induces apoptosis through the mitochondrial pathway. These data open the way to new strategies to control cellular Ca(2+) homeostasis that could decrease ß-cell apoptosis in diabetes.

Effects of plasma membrane Ca(2+)-ATPase overexpression upon D-glucose metabolism in insulin-producing BRIN-BD11 cells.

In order to investigate the possible link between PMCA (plasma-membrane Ca(2+)-ATPase) activity and D-glucose catabolism in insulin-producing cells, BRIN-BD11 cells were transfected with two isoforms of PMCA2. Transfection of insulin-producing BRIN-BD11 cells with PMCA2yb and PMCA2wb was documented by RT-PCR (reverse transcription-PCR), Western blot analysis, indirect immunofluorescence microscopy and (45)Ca(2+) uptake by microsomes. In the transfected cells, the overexpression of PMCA coincided with three major anomalies of D-glucose metabolism, namely a lower rate of D-[5-(3)H]glucose utilization prevailing at a low extracellular concentration of D-glucose (1.1 mM), a low ratio between D-[U-(14)C]oxidation and D-[5-(3)H]glucose utilization prevailing at a high extracellular glucose concentration (16.7 mM), and a high ratio between the net generation of (14)C-labelled acidic metabolites and amino acids and that of (3)H(2)O from D-[5-(3)H]glucose. These anomalies resulted in a decreased estimated rate of ATP generation (linked to the catabolism of the hexose) and a lowered ATP cell content, whether at low or high extracellular D-glucose concentrations. The net uptake of (45)Ca(2+) by intact cells was also decreased in the transfected cells, but to a greater extent than can apparently be attributed to the change in the ATP-generation rate. These findings document the relevance of PMCA activity to both D-glucose metabolism and Ca(2+) handling in insulin-producing cells, with emphasis on the key role of both cytosolic and mitochondrial Ca(2+) concentrations in the regulation of D-glucose catabolism. They also reveal that overexpression of PMCA leads, in insulin-producing cells, to an imbalance between ATP generation and consumption.

Carbamylcholine and ouabain effects on Ca2+ handling and insulin release in islets from rats depleted in long-chain polyunsaturated omega 3 fatty acids.

A number of metabolic, ionic and secretory variables were recently found to be affected in pancreatic islets obtained from second generation rats depleted in long-chain polyunsaturated omega 3 fatty acids (omega 3 rats). The present study further documents three sets of anomalies in such islets. First, after 90 min exposure to D-glucose (8.3 mM), the release of insulin from perifused islets, prelabelled with 45Ca, is lower in omega 3 rats than in control animals, despite comparable 45Ca fractional outflow rate. Second, over 15 min exposure to carbamylcholine (0.1 mM), in the presence of D: -glucose, the cytosolic concentration of Ca2+ is increased to a greater relative extent in dispersed islet cells from omega 3 rats, as compared to control animals. This coincides with a greater relative increase in insulin output from perifused islets during the second phase of the secretory response to the cholinergic agent. Last, the increase provoked by ouabain (1.0 mM) in cytosolic Ca2+ concentration, 45Ca fractional outflow rate and insulin release are all delayed in the omega 3 rats. Taking into account the decreased activity of Na+, K+-ATPase in the islets of omega 3 rats, these findings are interpreted as reflecting an impaired priming of insulin-producing cells when first exposed for 105 min to a physiological postprandial concentration of D-glucose.

Role of Na/Ca exchange and the plasma membrane Ca2+-ATPase in beta cell function and death.

Recent progresses concerning the Na/Ca exchanger (NCX) and the plasma membrane Ca2+-ATPase (PMCA) in the pancreatic beta cell are reviewed. The rat beta cell expresses two splice variants of NCX1 and six splice variants of the 4 PMCA isoforms. At the protein level, the most abundant forms are PMCA2 and PMCA3, providing the first evidence for the presence of these two isoforms in a non-neuronal tissue. Overexpression of NCX1 in an insulinoma cell line altered the initial rise in cytosolic-free Ca2+ concentration ([Ca2+]i) induced by membrane depolarization and the return of the [Ca2+]i to the baseline value on membrane repolarization, indicating that NCX contributes to both Ca2+ inflow and outflow in the beta cell. In contrast, overexpression of the PMCA markedly reduced the global rise in Ca2+ induced by membrane depolarization, indicating that the PMCA has a capacity higher than expected to extrude Ca2+. Glucose, the main physiological stimulus of insulin release from the beta cell, has opposite effect on NCX and PMCA transcription, expression and activity, inducing an increase in the case of NCX and a decrease in the case of the PMCA. This indicates that when exposed to glucose, the beta cell switches from a low-efficiency Ca2+ extruding mechanism, the PMCA, to a high-capacity system, the NCX, in order to better face the increase in Ca2+ inflow induced by the sugar. To our knowledge, this is the first demonstration of a reciprocal change in PMCA and NCX1 expression and activity in response to a given stimulus in any tissue.

Cytokines downregulate the sarcoendoplasmic reticulum pump Ca2+ ATPase 2b and deplete endoplasmic reticulum Ca2+, leading to induction of endoplasmic reticulum stress in pancreatic beta-cells.

Cytokines and free radicals are mediators of beta-cell death in type 1 diabetes. Under in vitro conditions, interleukin-1beta (IL-1beta) + gamma-interferon (IFN-gamma) induce nitric oxide (NO) production and apoptosis in rodent and human pancreatic beta-cells. We have previously shown, by microarray analysis of primary beta-cells, that IL-1beta + IFN-gamma decrease expression of the mRNA encoding for the sarcoendoplasmic reticulum pump Ca(2+) ATPase 2b (SERCA2b) while inducing expression of the endoplasmic reticulum stress-related and proapoptotic gene CHOP (C/EBP [CCAAT/enhancer binding protein] homologous protein). In the present study we show that cytokine-induced apoptosis and necrosis in primary rat beta-cells and INS-1E cells largely depends on NO production. IL-1beta + IFN-gamma, via NO synthesis, markedly decreased SERCA2b protein expression and depleted ER Ca(2+) stores. Of note, beta-cells showed marked sensitivity to apoptosis induced by SERCA blockers, as compared with fibroblasts. Cytokine-induced ER Ca(2+) depletion was paralleled by an NO-dependent induction of CHOP protein and activation of diverse components of the ER stress response, including activation of inositol-requiring ER-to-nucleus signal kinase 1alpha (IRE1alpha) and PRK (RNA-dependent protein kinase)-like ER kinase (PERK)/activating transcription factor 4 (ATF4), but not ATF6. In contrast, the ER stress-inducing agent thapsigargin triggered these four pathways in parallel. In conclusion, our results suggest that the IL-1beta + IFN-gamma-induced decrease in SERCA2b expression, with subsequent depletion of ER Ca(2+) and activation of the ER stress pathway, is a potential contributory mechanism to beta-cell death.

Opposite effects of glucose on plasma membrane Ca2+-ATPase and Na/Ca exchanger transcription, expression, and activity in rat pancreatic beta-cells.

When stimulated by glucose the pancreatic beta-cell displays large oscillations of the intracellular free Ca2+concentration, resulting from intermittent Ca2+ entry from the outside and outflow from the inside, the latter process being mediated by the plasma membrane Ca2+-ATPase (PMCA) and the Na+/Ca2+ exchanger (NCX). To understand the respective role of these two mechanisms, we studied the effect of glucose on PMCA and NCX transcription, expression, and activity in rat pancreatic islet cells. Glucose (11.1 and 22.2 mm) induced a parallel decrease in PMCA transcription, expression, and activity. In contrast the sugar induced a parallel increase in NCX transcription, expression, and activity. The effects of the sugar were mimicked by the metabolizable insulin secretagogue alpha-ketoisocaproate and persisted in the presence of the Ca2+-channel blocker nifedipine. The above results are compatible with the view that, when stimulated, the beta-cell switches from a low efficiency Ca2+-extruding mechanism, the PMCA, to a high capacity system, the Na/Ca exchanger, to better face the increase in Ca2+ inflow. These effects of glucose do not result from a direct effect of the sugar itself and are not mediated by the increase in intracellular free Ca2+ concentration induced by the sugar.

Na/Ca exchanger overexpression induces endoplasmic reticulum stress, caspase-12 release, and apoptosis.

This paper describes a possible strategy to control Ca(2+) homeostatsis as a possible approach to prevent or enhance beta-cell apoptosis

Plasma membrane Ca(2+)-ATPase overexpression reduces Ca(2+) oscillations and increases insulin release induced by glucose in insulin-secreting BRIN-BD11 cells.

In the mouse beta-cell, glucose generates large amplitude oscillations of the cytosolic-free Ca(2+) concentration ([Ca(2+)](i)) that are synchronous to insulin release oscillations. To examine the role played by [ Ca(2+)](i) oscillations in the process of insulin release, we examined the effect of plasma membrane Ca(2+)-ATPase (PMCA) overexpression on glucose-induced Ca(2+) oscillations and insulin release in BRIN-BD11 cells. BRIN-BD11 cells were stably transfected with PMCA2wb. Overexpression could be assessed at the mRNA and protein level, with appropriate targeting to the plasma membrane assessed by immunofluorescence and the increase in PMCA activity. In response to K(+), overexpressing cells showed a markedly reduced rise in [Ca(2+)](i). In response to glucose, control cells showed large amplitude [Ca(2+)](i) oscillations, whereas overexpressing cells showed markedly reduced increases in [Ca(2+)](i) without such large oscillations. Suppression of [Ca(2+)](i) oscillations was accompanied by an increase in glucose metabolism and insulin release that remained oscillatory despite having a lower periodicity. Hence, [Ca(2+)] (i) oscillations appear unnecessary for glucose-induced insulin release and may even be less favorable than a stable increase in [ Ca(2+)](i) for optimal hormone secretion. [Ca(2+)](i) oscillations do not directly drive insulin release oscillations but may nevertheless intervene in the fine regulation of such oscillations.

Na/Ca exchanger overexpression induces endoplasmic reticulum-related apoptosis and caspase-12 activation in insulin-releasing BRIN-BD11 cells.

Ca(2+) may trigger programmed cell death (apoptosis) and regulate death-specific enzymes. Therefore, the development of strategies to control Ca(2+) homeostasis may represent a potential approach to prevent or enhance cell apoptosis. To test this hypothesis, the plasma membrane Na/Ca exchanger (NCX1.7 isoform) was stably overexpressed in insulin-secreting tumoral cells. NCX1.7 overexpression increased apoptosis induced by endoplasmic reticulum (ER) Ca(2+)-ATPase inhibitors, but not by agents increasing intracellular calcium concentration ([Ca(2+)](i)), through the opening of plasma membrane Ca(2+)-channels. NCX1.7 overexpression reduced the rise in [Ca(2+)](i) induced by all agents, depleted ER Ca(2+) stores, sensitized the cells to Ca(2+)-independent proapoptotic signaling pathways, and reduced cell proliferation by approximately 40%. ER Ca(2+) stores depletion was accompanied by the activation of the ER-specific caspase (caspase-12), and the activation was enhanced by ER Ca(2+)-ATPase inhibitors. Hence, Na/Ca exchanger overexpression, by depleting ER Ca(2+) stores, triggers the activation of caspase-12 and increases apoptotic cell death. By increasing apoptosis and decreasing cell proliferation, overexpression of Na/Ca exchanger may represent a new potential approach in cancer gene therapy. On the other hand, our results open the way to the development of new strategies to control cellular Ca(2+) homeostasis that could, on the contrary, prevent the process of apoptosis that mediates, in part, beta-cell autoimmune destruction in type 1 diabetes.