Figure 1.
NCLX is expressed in mitochondria of pancreatic β cells and mediates mitochondrial Ca2+ transport.
A. Immunoblot analysis of NCLX expression in total lysate and isolated mitochondria in MIN6 cells (20 µg). B. Immunoblot analysis of NCLX expression in siNCLX vs. siControl (20 µg) transfected MIN6 cell lysates. VDAC and β Actin were used as mitochondrial and cytosolic markers, respectively. C. Knock down of NCLX expression increases Ca2+ influx and inhibits mitochondrial Ca2+ efflux. At the indicated time, cells were superfused with high K+ Ringer solution while monitoring mitochondrial Ca2+ in MIN6 cells transfected with mito-pericam and either siNCLX or siControl. D. Dominant negative NCLX construct increases Ca2+ influx and inhibits mitochondrial Ca2+ efflux. Representative fluorescent traces of pancreatic MIN6 cells co-transfected with mito-pericam and either dnNCLX or control vector (pcDNA), while applying the same experimental paradigm described in Fig. 1C. Insert. Representative images of MIN6 cells co-transfected with mito-pericam. The scale bar is 10 µm. E. Averaged rates of mitochondrial Ca2+ influx of Fig. 1C, D, n = 9 (*P<0.05). F. Averaged rates of mitochondrial Ca2+ efflux of Fig. 1C, D, n = 9 (*P<0.05). G. Silencing of NCLX expression inhibits mitochondrial Ca2+ efflux following a metabotropic cytosolic Ca2+ response. Cells were co-transfected with mito-pericam and either siNCLX or siControl and superfused with Ca2+ free Ringer solution containing 50 µM ATP, while monitoring the Ca2+ response. H. Averaged rates of mitochondrial Ca2+ influx of Fig. 1G, n = 7 (*P<0.05). I. Averaged rates of mitochondrial Ca2+ efflux of Fig. 1G, n = 7 (*P<0.05).
Figure 2.
NCLX mediates glucose dependent mitochondrial Ca2+ transport and modulates the basal mitochondrial membrane potential and calcium resting levels.
A. Silencing of NCLX expression blocks the glucose dependent mitochondrial Ca2+ efflux. The mitochondrial Ca2+ transport was monitored in MIN6 cells co-transfected with mito-pericam and either siNCLX or siControl. Cells were first superfused with low glucose (3 mM) Ringer followed by high glucose (20 mM) Ringer solution. B. Averaged rates of mitochondrial Ca2+ influx of Fig. 2A, n = 11 (*P<0.05). C. Averaged rates of mitochondrial Ca2+ efflux of Fig. 2A, n = 11 (*P<0.05). D. Silencing NCLX modulates the basal but not glucose dependent change in mitochondrial membrane potential. Changes in mitochondrial membrane potential were monitored in MIN6 cells transfected with siNCLX or siControl, superfused continuously with 0.05 µM TMRM. FCCP 5 µM was added in the indicated times to calibrate the signal. E. Effect of knock down of NCLX expression on mitochondrial resting Ca2+ in MIN6 cells transfected with siNCLX vs. siControl. Averaged mitochondrial Ca2+ basal signals, n = 10 (*P<0.05).
Figure 3.
Role of NCLX in cytosolic Ca2+ responses.
A. Silencing of NCLX expression inhibits cytosolic Ca2+ responses. MIN6 cells either transfected with siNCLX or siControl were loaded with Fura 2 AM and depolarized with high K+ Ringer solution, while monitoring cytosolic Ca2+ responses. B. Dominant negative mutant NCLX, dnNCLX inhibits cytosolic Ca2+ responses. MIN6 cells transfected with either dnNCLX or control vector (pcDNA) were loaded with Fura 2 AM and cytosolic Ca2+ was monitored as described in Fig. 3A. C. Averaged rates of cytosolic Ca2+ responses of Fig. 3A, n = 12 (*P<0.05). D. Averaged rates of cytosolic Ca2+ responses of Fig. 3B, n = 12 (*P<0.05). E. Averaged cytosolic Ca2+ response amplitude of Fig. 3A, n = 12 (*P<0.05). F. Averaged cytosolic Ca2+ response amplitude of Fig. 3B, n = 12 (*P<0.05).
Figure 4.
Effect of NCLX expression or activity on glucose dependent cytosolic calcium responses.
A. Real time PCR analysis of mRNA NCLX expression normalized to GAPDH in pancreatic primary β cells transfected with siNCLX vs. siControl, n = 3 (*P<0.05). B. Silencing NCLX expression inhibits glucose-induced Ca2+ entry in primary β cells. Representative fluorescent traces of cytosolic Ca2+ in pancreatic primary β cells transfected with either siNCLX or siControl loaded with Fura 2 AM and stimulated with high glucose following the same experimental paradigm described in Fig. 2A. Insert. Shows representative images of MIN6 cells co-transfected with the Dharmacon siGLO Red transfection reagent. The scale bar is 10 µm. C. NCLX dominant negative construct inhibits glucose dependent cytosolic Ca2+ changes in primary β cells. Representative fluorescent traces of primary β cells transfected with dnNCLX or control vector (pcDNA) loaded with Fura 2 AM and treated with high glucose when indicated. D. Averaged rates of cytosolic Ca2+ responses of Fig. 4B, n = 10 (*P<0.05). E. Averaged rates of cytosolic Ca2+ responses of Fig. 4C, n = 10 (*P<0.05). F. Averaged cytosolic Ca2+ amplitudes of Fig. 4B, n = 10 (*P<0.05). G. Averaged cytosolic Ca2+ amplitudes of Fig. 4C, n = 10 (*P<0.05).
Figure 5.
Effect of NCLX on mitochondrial Ca2+ transport, metabolic rate in resting and high glucose dependent manner.
A. Knocked down of NCLX modulates mitochondrial calcium transport. Pancreatic primary β cells were infected with lenti-pericam viral particles and transfected with either siNCLX or siControl and superfused with the indicated high glucose Ringer solution. Insert. Representative image of pancreatic primary β cell infected with lenti-pericam. The scale bar is 10 µm. B. Averaged mitochondrial Ca2+ influx rates of pancreatic primary β cells of Fig. 5A, n = 3 (*P<0.05). C. Averaged mitochondrial Ca2+ efflux rates of Fig. 5A, n = 3 (*P<0.05). D. Effect of NCLX on respiratory chain activity determined by monitoring NAD(P)H intrinsic fluorescence in pancreatic primary β cells, transfected with either siNCLX or siControl before and after application of high glucose Ringer solution. FCCP or high glucose Ringer's solution was added where indicated.
Figure 6.
Effect of NCLX silencing expression on ATP production and insulin secretion.
A. Effect of NCLX silencing expression on ATP production. The ATP content was determined in pancreatic primary β cells lysates transfected with either siNCLX or siControl and stimulated with high glucose in the indicated times (see Experimental Procedures), n = 3 (*P<0.05). B. Effect of NCLX knocked down expression on glucose dependent insulin secretion. Cultured pancreatic primary β cells were transfected with either siNCLX or siControl and amounts of secreted insulin were determined in the indicated times, n = 3 (*P<0.05).