Bcl-2/Bcl-xL antagonism stimulates β-cell mitochondrial metabolism, KATP-dependent Ca2+ entry, and insulin secretion. A: Representative recording of ER Ca2+ changes in MIN6 β-cells exposed to C6 and carbachol (Cch) (n = 6 cells). Inset: MIN6 cell expressing the ER-targeted D1ER Ca2+ sensor. B: Lack of C6-induced Ca2+ influx in the absence of extracellular Ca2+. The basally active cell illustrates the rapid loss of Ca2+ entry upon Ca2+ removal. C: Nifedipine blocks ongoing C6-induced Ca2+ influx (n = 14 cells). D: Quantification of nifedipine, diazoxide (Dz), and CCCP-mediated suppression of cytosolic Ca2+ responses in mouse islet cells exposed to C6 or YC137 (n = 3). E: Insulin secretion from dispersed islet-cells treated with C6, diazoxide, and/or tolbutamide (Tolb) (n = 5). *P < 0.05 vs. 3 mmol/L glucose control. F: Percentage of PI-positive mouse islet cells following culture with C6 with or without the presence of nifedipine or Dz (n = 3). G: Reversible inhibition of C6-induced Ca2+ signaling in mouse islet cells by sodium azide (NaN3). H and I: Relative changes in ΔΨm of primary mouse β-cells exposed to stimulatory glucose and the Bcl-2 antagonist C6. In panel H, glucose was added prior to C6. The black line is representative of 38 cells exposed to 80 µmol/L, and the superimposed red
line is representative of 15 cells responding to a shorter stimulation with 20 μmol/L C6. Panel I illustrates the addition of glucose during the C6-induced response (representative of 17 cells). Loss of rhodamine 123 fluorescence
indicates mitochondrial hyperpolarization. J: MIN6 β-cell expressing the mitochondrial FRET-based Ca2+ sensor mt4D3cpv and examples of mitochondrial Ca2+ fluctuations induced by glucose or Bcl-2/Bcl-xL inhibition (n = 29 cells at 40 μmol/L C6; n = 34 cells at 80 μmol/L C6). K: Change in the cellular ATP-to-ADP ratio of MIN6 β-cells following 30 min culture in stimulatory 20 mmol/L glucose (20G)
or in 3 mmol/L glucose with 60 μmol/L C6, relative to 3 mmol/L glucose alone. The depletion seen with CCCP reflects the metabolic
pool of ATP (n = 3 cultures; *P < 0.05, **P < 0.001 vs. 3 mmol/L glucose; n.s., not significant). Data are mean ± SEM. Basal glucose is 3 mmol/L in all experiments.
(A high-quality color representation of this figure is available in the online issue.)