Bcl-2 and Bcl-x_L Suppress Glucose Signaling in Pancreatic β-Cells

Compound-6 (also known as Bcl-2 inhibitor) and YC137 (also known as Bcl-2 inhibitor II) from Calbiochem (La Jolla, CA) were prepared in dimethyl sulfoxide. Fura-2/AM, Rhodamine123, and MitoTracker were from Life Technologies/Invitrogen (Burlington, Ontario, Canada). Propidium iodide (PI), nifedipine, diazoxide, sodium azide, and tetramethylrhodamine ethyl ester perchlorate (TMRE) were from Sigma-Aldrich (St. Louis, MO).

Dispersed islet cells and intact islets were imaged following 24–48 h culture on glass coverslips. Changes in cytosolic Ca²⁺, mitochondrial membrane potential (ΔΨ_m), and NAD(P)H autofluorescence were imaged as described (12,13). Mitochondrial Ca²⁺, endoplasmic reticulum (ER) Ca²⁺, and the activation of caspase-3 were monitored in single MIN6 cells transfected with fluorescence resonance energy transfer (FRET)-based biosensors. MIN6 cell ΔΨ_m was estimated using TMRE and flow cytometry (14). Late-stage cell death was imaged using PI (14).

To examine changes in the ATP/ADP ratio, MIN6 cells in 96-well plates were equilibrated for 30 min in Krebs Ringer Buffer (KRB) containing (in mM unless otherwise noted): 119 NaCl, 4.7 KCl, 25 NaHCO₃, 2.5 CaCl₂, 1.2 MgSO₄, 1.2 KH₂PO₄, 5 mg/mL radioimmunoassay-grade bovine albumin, and 3 mmol/L glucose, followed by treatment for 30 min as indicated. ATP/ADP ratios were measured using the ApoSENSOR kit (BioVision, Mountain View, CA) using a TECAN M1000 luminometer (Tecan Group Ltd.).

Glucose oxidation rates were determined in cultures of dispersed islet cells by quantifying ¹⁴CO₂ generated from metabolized ¹⁴C-labeled glucose as described (15,16). Adherent islet cells in T-25 flasks were preincubated 1 h in 3 mmol/L glucose KRB. This was followed by 1 h incubation with KRB containing 1 μCi/mL [U-¹⁴C]glucose and test concentrations of nonlabeled glucose during which ¹⁴CO₂ was collected in a hyamine trap. Injection of 9 N H₂SO₄ stopped the reaction and released ¹⁴CO₂ captured in the media as [¹⁴C]bicarbonate. Flasks were gently agitated for 2 h at room temperature before measuring captured radioactivity using a Beckman LS6500 Liquid Scintillation Counter (Beckman Coulter). Glucose oxidation rates were normalized to total protein quantified from identical aliquots of similarly treated cells.

Bax^−/− (Jax stock number 002994) and Bak^−/− (Jax stock number 004183) mice and age- and background-matched wild-type controls were from The Jackson Laboratory (Bar Harbor, ME). Littermate Bcl-2^−/−, Bcl-2^+/−, and Bcl-2^+/+ mice were obtained by breeding mice heterozygous for the Bcl2^tm1Sjk mutation (Jax stock number 002265; The Jackson Laboratory). To generate Bcl-x^flox/flox:Pdx1-CreER mice and Bcl-x^flox/flox control littermates, we mated Pdx1-CreER mice (17) with Bcl-x^flox/flox mice (exons 1 and 2 flanked by Lox P sites) (18). To create mice with double Bax and Bak deletion in their β-cells, we bred Pdx1-CreER animals with Bak^−/−:Bax^flox/flox mice (Bax^tm2SjkBak1^tm1Thsn/J; Jax stock number 006329; The Jackson Laboratory) and obtained Bak^−/−:Bax^flox/flox:Pdx1-CreER and Bak^−/−:Bax^flox/flox littermates. Ablation of Bcl-x and Bax was achieved by injecting Pdx1-CreER–positive animals and littermate controls with 3 mg/40 g of tamoxifen (Sigma-Aldrich) on 5 consecutive days. Glucose tolerance and in vivo insulin secretion were assessed following intraperitoneal injection with glucose as indicated. Insulin tolerance was assessed after intraperitoneal injection of 0.75 units/kg insulin. Animals were fasted for 6 h prior to study. Studies were approved by the University of British Columbia Animal Care Committee.

Mouse islet isolation by collagenase digestion/filtration and culture have been described (19). Human islets were obtained from Dr. Garth Warnock (Vancouver General Hospital) after consent and cultured as described (12). MIN6 cells were cultured in Dulbecco’s modified Eagle’s medium (25 mmol/L glucose). Insulin release was measured from dispersed mouse islet cells in 48-well plates (20). Cells were equilibrated for 60 min in 3 mmol/L glucose KRB and then stimulated for 45 min with treatments as indicated (20). Insulin secretion from batches of size-matched islets was examined by perifusion and radioimmunoassay as previously described (12).

For immunoprecipitation, 500 µg of total protein was incubated overnight (4°C) with anti-Bad (catalog number 9292; Cell Signaling Technology; 1:200), followed by 3-h incubation (4°C) with protein A-agarose (Santa Cruz Biotechnology). Complexes were washed with PBS and protease inhibitors in Ultrafree-MC Filters (Millipore) and eluted using 4% SDS in PBS with protease inhibitors. In some studies, nuclear (PARIS; Ambion Inc.) and mitochondrial fractions (Mitochondrial/Cytosol Kit; BioVision, Inc.) were obtained. Proteins were resolved by SDS-PAGE, transferred to polyvinylidene difluoride membranes, and probed overnight (4°C) for Bcl-x_L, Bcl-2, Bax, cytochrome c (catalog numbers 2762, 2870, 2772, and 4272; Cell Signaling Technology); phospho-Bad Ser112, Ser136, and Ser155 (catalog numbers 9291, 4366, and 9297; Cell Signaling Technology), Bad, Bak (#06–536; Millipore), and Cre recombinase (Novagen).

Data are shown as mean ± SEM. Differences between two groups were compared by unpaired Student t test and multiple groups by one-way ANOVA followed by Bonferroni multiple comparison test. Differences were considered significant if P < 0.05.

We used small-molecule antagonists to probe moment-to-moment functions of Bcl-2 and Bcl-x_L in pancreatic β-cells. The structurally distinct inhibitors, compound 6 (C6) and YC137, were originally identified by their ability to bind both Bcl-2 and Bcl-x_L and displace proapoptotic members such as Bak and Bid (21,22). We first established that BH3-displacement could also be observed in intact β-cells. Indeed, C6 caused a rapid reduction in the amount of Bcl-x_L that was bound to the BH3-only protein Bad without affecting the total levels of Bad or Bcl-x_L protein within 1 h (Fig. 1A and B). This confirmed the expected mechanism of this antagonist on endogenous Bcl proteins in intact β-cells.

Displacement of BH3 domain proteins from Bcl-2 and Bcl-x_L by YC137 or C6 sensitizes tumor cells to apoptosis, an effect that is more potent with increasing Bcl-2 expression (21,22). Unlike many tumor cells, primary β-cells do not hyperexpress Bcl-2 or Bcl-x_L. We therefore tested if these inhibitors affect β-cell survival. Prolonged Bcl antagonism induced dose- and time-dependent cell death in human and mouse islet cells, as well as MIN6 β-cells (Fig. 1C–E). This involved mitochondrial apoptosis, as evidenced by redistribution of Bax from cytosol to mitochondria and release of mitochondrial cytochrome c (Fig. 1F). PI incorporation was preceded by the activation of caspase-3, imaged in real time (Fig. 1G). Of note, the ΔΨ_m of β-cells treated with C6 or YC137 underwent an initial hyperpolarization that suggested mitochondrial activation within the first half hour, well prior to any evidence of apoptosis. This was followed hours later by collapse of ΔΨ_m, demonstrating a late-stage loss of mitochondrial integrity (Fig. 1H). These results demonstrate that even in the absence of other stresses, combined and sustained antagonism of Bcl-2 and Bcl-x_L initiates mitochondrial apoptosis in β-cells. Importantly, apoptosis was not detected earlier than 2 h, indicating that cellular responses occurring less than an hour after Bcl-2/Bcl-x_L antagonism are separate from the central apoptotic events.

Remarkably, Bcl-2/Bcl-x_L antagonists rapidly triggered marked Ca²⁺ fluctuations in mouse and human islet cells that resembled Ca²⁺ responses to glucose (Fig. 2A–C). Similar effects were observed in MIN6 β-cells (Fig. 2D). The percentage of cells activated within 30 min of Bcl inhibition was concentration-dependent (Fig. 2E). The Ca²⁺ signals ceased upon washout of the inhibitor, strongly suggesting a physiological basis rather than cell damage. C6 also increased average cytosolic Ca²⁺ in intact islets, although the rapid fluctuations were dampened (Fig. 2F). Together, these findings provide the first direct evidence that prosurvival Bcl proteins regulate moment-to-moment calcium homeostasis in β-cells.

Next, we sought to determine the cellular site where Bcl-2 and/or Bcl-x_L control Ca²⁺ homeostasis. In other cell types, Bcl-2 and Bcl-x_L reside on the membranes of Ca²⁺ handling organelles, including the mitochondria, ER, and the nuclear envelope (23,24). The subcellular location of Bcl-2 and Bcl-x_L in β-cells has not been reported. We found that Bcl-2:GFP displayed some colocalization with mitochondria, but also had a clear nonmitochondrial distribution, likely reflecting ER (Fig. 3A and D). In contrast, Bcl-x_L:yellow fluorescent protein (YFP) showed near-exclusive colocalization with mitochondria, which at high magnification could often be seen in an apparent association with the mitochondrial membrane (Fig. 3B and D) and minimal association with ER (Fig. 3C and D). A differential distribution of endogenous Bcl-2 and Bcl-x_L between mitochondrial and nonmitochondrial compartments was also found by subcellular fractionation (Fig. 3E). These findings suggest that β-cell Bcl-2 and Bcl-x_L may have overlapping, but distinct, roles.

Bcl-2 and Bcl-x_L influence ER Ca²⁺ release in other cell types (6,25,26). Imaging ER luminal Ca²⁺ directly (14,27), we found that ER Ca²⁺ levels were not affected by C6 treatment for up to 50 min (Fig. 4A; similar results seen with 20–80 μmol/L C6). As a positive control, inositol triphosphate generation with carbachol consistently mobilized ER Ca²⁺ (Fig. 4A). Although these results suggest that acutely antagonizing Bcl-2 and Bcl-x_L does not alter β-cell ER Ca²⁺, it remains possible that Bcl-2 or Bcl-x_L regulate β-cell ER Ca²⁺ homeostasis by mechanisms that are unaffected by these small-molecule antagonists.

Next, we sought to further elucidate the mechanism by which Bcl inhibitors increase cytosolic Ca²⁺. Similar to stimulatory glucose, Bcl inhibitors had no effect on cytosolic Ca²⁺ in the absence of extracellular Ca²⁺ (Fig. 4B). Moreover, the L-type Ca²⁺ channel antagonist nifedipine reversibly lowered cytosolic Ca²⁺ during Bcl inhibition (Fig. 4C). Pretreatment of islet cells with nifedipine, the K_ATP channel agonist diazoxide, or a low dose of the mitochondrial uncoupler carbonyl cyanide m-chlorophenyl hydrazone (CCCP) prevented Ca²⁺ entry (Fig. 4D). Incubating islet cells with Bcl antagonist in the presence of substimulatory glucose (3 mmol/L) induced a modest, but significant, elevation in insulin release similar in magnitude to that triggered by 100 μmol/L of the K_ATP channel blocker tolbutamide (Fig. 4E). These insulin-secretion responses were attenuated by diazoxide and were not additive with the response to tolbutamide (Fig. 4E), or with a maximally stimulatory concentration of glucose (fold increase: 3.99 ± 1.12 in 20 mmol/L glucose vs. 3.44 ± 1.06 in 20 mmol/L glucose plus 40 μmol/L C6; n = 7; P = 0.73). Despite their ability to block the acute Ca²⁺ signals, nifedipine and diazoxide did not alter the degree or the kinetics of islet cell death induced by C6 (Fig. 4F). Together, these data demonstrate that antagonizing Bcl-2/Bcl-x_L acutely induces Ca²⁺ entry and insulin secretion from β-cells by mechanisms similar to those involved in glucose signaling (28) and that these physiological Ca²⁺ signals are not mechanistically related to the late-stage apoptotic effects of sustained Bcl-2/Bcl-x_L coinhibition.

Our results to this point indicated that the events induced by Bcl protein inhibition are similar to those that underlie β-cell glucose signaling. Indeed, sodium azide blocked C6-stimulated Ca²⁺ signals, establishing a requirement for mitochondrial respiratory flux (Fig. 4G). Importantly, Ca²⁺ signaling upon Bcl antagonism was suppressed in the absence of glucose (Fig. 2E), suggesting that a minimum of metabolic substrate is required to support the Ca²⁺ responses. These findings indicate that blocking Bcl-2/Bcl-x_L increases basal mitochondrial glucose metabolism rather than acting independently of the sugar.

To further establish if Bcl antagonists promote mitochondrial activity, we directly imaged ΔΨ_m and mitochondrial Ca²⁺ (12,14,29). In agreement with our flow cytometry results, Bcl antagonism hyperpolarized the mitochondria of single primary mouse β-cells within minutes in a concentration-dependent manner (Fig. 4H). The ΔΨ_m changes were comparable to the β-cell response to glucose, and the presence of Bcl-2/Bcl-x_L antagonist did not prevent additional glucose-induced hyperpolarization (Fig. 4I). Bcl-2/Bcl-x_L inhibition also evoked reversible mitochondrial Ca²⁺ signals that resembled glucose stimulation (Fig. 4J), providing further evidence for mitochondrial activation. Of note, Bcl antagonism caused an increase in the cellular ratio of ATP to ADP similar to that evoked by 20 mmol/L glucose (Fig. 4K). Our results demonstrate that acute inhibition of Bcl-2/Bcl-x_L promotes mitochondrial activity in β-cells, resulting in the same events activated by stimulatory glucose. These findings implicate Bcl-2 and/or Bcl-x_L in the regulation of β-cell mitochondrial physiology and suggest that one of their day-to-day roles is to suppress basal glucose metabolism.

To validate our experiments with small molecule inhibitors, we examined the importance of Bcl-2 and Bcl-x_L in β-cell function in vitro using a genetic loss-of-function approach. First, we used islets from Bcl-2^−/− mice. These global knockout mice are approximately half the size of their wild-type or heterozygous littermates, insulin-hypersensitive, develop polycystic kidney disease, and die at various ages between 2 and 19 weeks (30) (D.S.L. and J.D.J., unpublished observations), precluding interpretable in vivo analysis of β-cell function. Nevertheless, we were able to isolate islets for in vitro analysis from a limited number of Bcl-2^−/− mice prior to any signs of illness and compare them to islets from phenotypically normal heterozygous and wild-type littermates (31). Real-time PCR confirmed the loss of Bcl-2 expression in Bcl-2^−/− and Bcl-2^+/− islets relative to Bcl-2^+/+ controls, with no compensatory increase in Bcl-x_L (Fig. 5A). Bcl-2^−/− and Bcl-2^+/− β-cells showed enhanced sensitivity to a stepwise glucose ramp stimulus relative to cells from wild-type littermates (Fig. 5B and C). Intact islets from Bcl-2^−/− mice also showed increased Ca²⁺ and metabolic NAD(P)H responses to 6 mmol/L glucose (Fig. 5E and F). In perifusion experiments, we observed significantly increased insulin secretion from Bcl-2^−/− islets in response to 10 and 15 mmol/L glucose, compared with Bcl-2^+/+ islets (Fig. 5G). Loss of Bcl-2 had no effect on the responses to depolarization with KCl, further indicating a change at the level of β-cell metabolism (Fig. 5D and H). Generally, the effects on intact islets were less pronounced than those in dispersed cells, suggesting that cell–cell coupling may dampen the amplified responses of individual Bcl-2^−/− β-cells. The intermediate augmentation of glucose-induced Ca²⁺ responses in Bcl-2 heterozygous β-cells (Fig. 5B and C) indicates that the effects were dependent on gene dosage and independent of pathological conditions that limit the number of healthy Bcl-2^−/− mice available for study. Throughout our studies, we did not notice any obvious differences in the viability of Bcl-2^−/− islets or β-cells in culture.

Global deletion of Bcl-x_L is embryonically lethal (32), so to assess the specific role of Bcl-x_L, we generated tissue-specific, tamoxifen-inducible Bcl-x_L knockout (BclxβKO) mice. Bcl-x_L was knocked out in β-cells of adult animals as early as 2 to 3 days after tamoxifen administration with no compensatory change in Bcl-2 expression (Fig. 6A). Bcl-x_L deletion was not observed in hypothalamus (Fig. 6B). Using BclxβKO islet cells, we confirmed a significant molecular contribution of Bcl-x_L to C6-induced Ca²⁺ signals (Fig. 6C). Like Bcl-2^−/− β-cells, BclxβKO β-cells showed significantly larger glucose-induced Ca²⁺ and NAD(P)H responses (Fig. 6D, E, and G), whereas Ca²⁺ responses to KCl were normal (Fig. 6F). These findings support a metabolic basis for the amplified Ca²⁺ responses, although we did not detect changes at the level of glucose oxidation in bulk cultures of dispersed BclxβKO islet cells (Fig. 6H). A modest tendency toward increases in insulin secretion was seen in BclxβKO islets perifused with 3, 6, and 10 mmol/L glucose and in mice injected with glucose in vivo, but these did not achieve statistical significance (Figs. 6I and 7A). Interestingly, glucose tolerance was nonetheless moderately improved in BclxβKO mice administered 2 g/kg glucose relative to littermate controls (Fig. 7B). The potentiation of β-cell Ca²⁺ signals at submaximal glucose prompted us to examine the in vivo response to a more moderate glucose challenge (0.5 g/kg), and indeed glucose tolerance was also improved under these conditions (Fig. 7C and D). This was not associated with improved insulin sensitivity (Fig. 7E). Together, our combined findings using chemical inhibitors and islets from knockout mice point to novel roles for both Bcl-2 and Bcl-x_L in the fine-tuning of glucose signaling in pancreatic β-cells.

Proapoptotic Bax and Bak have been reported to interact with two regulators of mitochondrial physiology: the voltage-dependent anion channel (VDAC) and the adenine nucleotide translocase (ANT) (33–35), but it is not clear if this occurs outside of apoptosis. Given the established roles of Bax and Bak downstream of Bcl-2/Bcl-x_L, we asked if they participate in Bcl-2/Bcl-x_L regulation of metabolism. Bax^−/− or Bak^−/− β-cells responded to Bcl-2/Bcl-x_L inhibition similar to wild-type β-cells (Fig. 8A). To conclusively exclude the involvement of Bax and Bak, we generated mice lacking both genes in their β-cells (Fig. 8B and C). Islet cells from these Bax–Bak β double-knockout (DKO) mice responded to Bcl-2/Bcl-x_L antagonism similar to islet cells from control mice (Fig. 8D and E). Together, these data establish that Bax and Bak do not mediate the effects of Bcl-2 or Bcl-x_L in β-cell function and further distinguish the physiological effects of Bcl inhibition from Bax/Bak-dependent apoptotic events.

The current study was undertaken to examine the physiological roles of endogenous prosurvival Bcl proteins in pancreatic β-cells. Using small-molecule Bcl-2/Bcl-x_L antagonists and islets from KO mice, we provide the first loss-of-function evidence that Bcl-2 and Bcl-x_L acutely affect mitochondrial function, Ca²⁺ homeostasis, and insulin secretion. A previous report described apoptosis sensitivity in mice in which Bcl-x_L was deleted in embryonic β-cells, but did not provide information on glucose homeostasis or β-cell physiology (5). The effects of the chemical inhibitors, which target both Bcl-2 and Bcl-x_L, were partially distinct and generally more robust than the genetic ablation of either protein individually. It is likely that combined tissue-specific deletion of Bcl-2 and Bcl-x will be required to mimic the effects of the inhibitors on basal β-cell activity and viability. Our work demonstrates new physiological roles for two proteins previously presumed to function mainly in the control of apoptosis.

Sustained cytosolic Ca²⁺ rises and insulin release following glucose stimulation rely heavily on mitochondrial ATP synthesis, K_ATP channel-dependent β-cell depolarization, and voltage-gated Ca²⁺ influx (28). In this study, we report that acute coinhibition of antiapoptotic Bcl-2 and Bcl-x_L stimulates an identical cascade of events culminating in insulin secretion. Moreover, genetic deletion of Bcl-2 or Bcl-x_L increased the in vitro β-cell responses to glucose and improved in vivo glucose tolerance of the islet-specific Bcl-x_L KO mice. Our experiments suggest that this involves amplification of β-cell glucose metabolism and thus that Bcl-2 and Bcl-x_L restrict β-cell metabolic activity. Our findings conceptually agree with a previous study in which mice overexpressing Bcl-x_L 10-fold under the control of the rat insulin promoter exhibited impaired β-cell oxidative metabolism and glucose intolerance (9). Several groups have overexpressed Bcl-2 in pancreatic islets as part of efforts to block apoptosis, but we are only aware of a few studies that examined the impact on β-cell function, and these reported no impairment of insulin secretion (36,37). This could be interpreted as evidence for a saturation effect whereby excess levels of Bcl-2 protein do not negatively affect the stoichiometry of complexes associated with the metabolic machinery. Alternatively, the lower fraction of mitochondria-localized Bcl-2 relative to Bcl-x_L (Fig. 3) might require that correspondingly larger amounts of Bcl-2 are expressed to achieve detectable metabolic suppression.

We considered that antagonizing Bcl-2/Bcl-x_L might free Bax and/or Bak to indirectly activate mitochondria, possibly by affecting ANT or VDAC (33–35). We tested and eliminated this possibility using Bax KO, Bak KO, and Bax/Bak DKO islet cells. Work from the Danial group (3,38) is consistent with an alternative indirect model whereby Bcl-2 and/or Bcl-x_L might sequester Bad and limit its promotion of glucokinase activity. Bcl-2 and Bcl-x_L have not been detected in the Bad/glucokinase complex, but reducing their binding to Bad might release this brake on β-cell metabolism from afar. In preliminary studies in MIN6 cells, Bcl inhibition did not change Bad levels or phosphorylation within 60 min, the timescale corresponding to the acute Ca²⁺ signals and metabolic effects. Preliminary studies in Bcl-x_L KO islets revealed 58% increase in serine 155 phosphorylation, with no significant effects on the phosphorylation of Bad at serine 136 or serine 112. Thus, phosphorylation-dependent functions of Bad do not appear acutely involved in the effects of Bcl antagonism. However, a more chronic contribution from the Bad/glucokinase axis following chronic Bcl protein loss remains a possibility that might promote the amplification of insulin secretion in stimulatory glucose that is not apparent acutely following inhibition with small molecules.

Our data also allow for a model whereby Bcl proteins directly affect mitochondrial proteins in the β-cell, provided such interactions are changed by Bcl antagonists. Studies of the antiapoptotic activities of Bcl-2 and Bcl-x_L have reported that they can interact with mitochondrial ANT and VDAC via their BH4 domains (35). We have found that a cell-permeant Bcl-x_L BH4 domain peptide triggers cytosolic and mitochondrial Ca²⁺ fluctuations in β-cells (D.S.L. and J.D.J. unpublished observations). This could result from direct mitochondrial actions of the BH4 domain and/or ER Ca²⁺ release (39). Of note, it was recently reported that Bcl-x_L can lower acetyl-CoA levels independently of Bax and Bak (40). Another study suggested that Bcl-x_L suppresses O₂ consumption, although promoting ATP synthesis in neurons by interacting with the mitochondrial F₁F₀ ATPase (8), indicating that Bcl-x_L can have opposing metabolic effects in a cell. Conceivably, changes in the relative contributions of these effects may shape the net metabolic impact of Bcl-x_L in a given cell type and might complicate the analysis of complex mechanisms such as insulin secretion that involve multiple metabolic pathways.

Our finding that antiapoptotic Bcl-2 family proteins can modulate β-cell function has intriguing implications for our understanding of the pathophysiology of diabetes. The signal transduction machinery of β-cells is optimized for maximal delivery of glycolytic intermediates for oxidative phosphorylation (41). However, β-cells are remarkably sensitive to the deleterious effects of reactive oxygen species (42). Metabolic suppression may provide a means by which Bcl proteins protect pancreatic β-cells against metabolic stress. One might also speculate that the reduction in Bcl-2 and Bcl-x_L seen under prodiabetic conditions (4,43–45) can affect β-cell function. In this regard, it is noteworthy that insulin hypersecretion is an early marker of human diabetes (46), and chronic hyperinsulinemia a persistent feature of diabetic animal models, including the Zucker Diabetic Fatty rat (47), which has 70% less Bcl-2 protein (48). In extension of this, our results suggest caution may be prudent in efforts to treat diabetes by augmenting β-cell metabolic flux using agents such as glucokinase activators (49).

In summary, we demonstrate novel roles of endogenous antiapoptotic Bcl proteins in the physiology of pancreatic β-cells. Specifically, our data suggest that endogenous Bcl-2 and Bcl-x_L suppress the β-cell response to glucose. Our findings add to emerging evidence that places Bcl family proteins at the intersection of β-cell function and survival. The involvement of apoptosis-regulating proteins in the normal function of primary cells promises to provide fertile grounds for future insights into the pathophysiology of diabetes and other diseases.

This work was supported by operating grants from the Canadian Institutes of Health Research (CIHR) (MOP-86559 to J.D.J. and MOP-119537 to D.S.L.). J.D.J. was supported by salary awards from the CIHR, the Juvenile Diabetes Research Foundation (JDRF), the Canadian Diabetes Association (CDA), and the Michael Smith Foundation for Health Research (MSFHR). D.S.L. received fellowship support from the JDRF, the Carlsberg Foundation, CDA, and the MSFHR.

No potential conflicts of interest relevant to this article were reported.

D.S.L. designed experiments, performed research, and wrote the manuscript. S.A.W., S.B.W., and V.V.S. performed research, contributed to discussion, and reviewed and edited the manuscript. F.T. performed research. X.H. performed research. M.F.A. contributed to discussion and reviewed and edited manuscript. J.D.J. designed research and wrote the manuscript. J.D.J. is the guarantor of this work and, as such, had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.

The authors thank Dr. Douglas Melton, Harvard University, for the Pdx1-CreER mice and Dr. You-Wen He, Duke University, for the Bcl-x^flox mice. The authors also thank Dr. Atsushi Miyawaki, RIKEN Japan, for the MiCy-DEVD-mKO probe; Dr. Roger Tsien, University of California San Diego, and Dr. Amy Palmer, University of Colorado, for the mt4D3cpv and D1ER Ca²⁺ sensors; and Karen Groden, Suzen Lines, and the University of British Columbia rederivation facility for help with husbandry of the Bcl-2 mice.