Requirement for Aralar and Its Ca2+-binding Sites in Ca2+ Signal Transduction in Mitochondria from INS-1 Clonal β-Cells*

Aralar, the mitochondrial aspartate-glutamate carrier present in β-cells, is a component of the malate-aspartate NADH shuttle (MAS). MAS is activated by Ca2+ in mitochondria from tissues with aralar as the only AGC isoform with an S0.5 of ∼300 nm. We have studied the role of aralar and its Ca2+-binding EF-hand motifs in glucose-induced mitochondrial NAD(P)H generation by two-photon microscopy imaging in INS-1 β-cells lacking aralar or expressing aralar mutants blocked for Ca2+ binding. Aralar knock-down in INS-1 β-cell lines resulted in undetectable levels of aralar protein and complete loss of MAS activity in isolated mitochondria and in a 25% decrease in glucose-stimulated insulin secretion. MAS activity in mitochondria from INS-1 cells was activated 2-3-fold by extramitochondrial Ca2+, whereas aralar mutants were Ca2+ insensitive. In Ca2+-free medium, glucose-induced increases in mitochondrial NAD(P)H were small (1.3-fold) and unchanged regardless of the lack of aralar. In the presence of 1.5 mm Ca2+, glucose induced robust increases in mitochondrial NAD(P)H (∼2-fold) in cell lines with wild-type or mutant aralar. There was a ∼20% reduction in NAD(P)H response in cells lacking aralar, illustrating the importance of MAS in glucose action. When small Ca2+ signals that resulted in extremely small mitochondrial Ca2+ transients were induced in the presence of glucose, the rise in mitochondrial NAD(P)H was maintained in cells with wild-type aralar but was reduced by ∼50% in cells lacking or expressing mutant aralar. These results indicate that 1) glucose-induced activation of MAS requires Ca2+ potentiation; 2) Ca2+ activation of MAS represents a larger fraction of glucose-induced mitochondrial NAD(P)H production under conditions where suboptimal Ca2+ signals are associated with a glucose challenge (50 versus 20%, respectively); 3) inactivation of EF-hand motifs in aralar prevents activation of MAS by small Ca2+ signals. The results suggest a possible role for aralar and MAS in priming the β-cell by Ca2+-mobilizing neurotransmitter or hormones.

Glucose metabolism drives glucose-stimulated insulin secretion (GSIS) 4 in pancreatic ␤-cells. The increased glucose-induced energy production results from two processes: mass action of glucose (or glucose push) and signal-dependent potentiation of metabolism (1). It is believed that mass action of glucose is governed by glucokinase as glucose sensor and increased substrate pressure is the driving force for the generation of glycolysis-derived cytosolic NADH, pyruvate formation, redox transfer to mitochondria through shuttle activity, and the glucose-induced respiratory burst (2). After the closure of ATP-dependent K ϩ channels (K ATP channels), Ca 2ϩ enters in the ␤-cell (2)(3)(4) and potentiates metabolism activating mitochondrial dehydrogenases subsequent to Ca 2ϩ inflow into the mitochondrial matrix (1). Accordingly, the initial increase of cellular NAD(P)H/NAD ϩ (P), which is produced by mass action of glucose precedes the first cytosolic Ca 2ϩ signal recorded (5,6). On the other hand, glucose oxidation to CO 2 , which largely reflects Krebs cycle activity is markedly reduced in the absence of extracellular Ca 2ϩ , and thus requires Ca 2ϩ potentiation (1,7,8). Paradoxically, it has been reported that glucose-induced NAD(P)H production, which reflects mainly mitochondrial NAD(P)H, was not significantly reduced in the absence of extracellular Ca 2ϩ (9). Thus, the extent to which Ca 2ϩ signal potentiation is required for NAD(P)H production and the actual pathways involved is an open question.
Krebs cycle dehydrogenase activation may proceed through glucose mass action, as pyruvate derived from glucose is pushed into mitochondria (1) and is oxidized in the organelle forming mitochondrial NAD(P)H. However, Ca 2ϩ activates pyruvate, isocitrate, and ␣-ketoglutarate dehydrogenases and this is expected to potentiate mitochondrial NAD(P)H production. Redox shuttle systems, particularly the malate aspartate shuttle (MAS), are also pathways for glucose-induced NAD(P)H pro-duction in mitochondria (10), and MAS is now known to be activated by Ca 2ϩ (11)(12)(13)(14). The aspartate-glutamate carrier (AGC) is one of the two mitochondrial carriers involved in MAS and catalyzes the only irreversible step in the shuttle. Aralar (also named aralar1 (11,15)) is the AGC isoform expressed in excitatory tissues, including pancreatic islets and ␤-cell (11,12,(15)(16)(17). Aralar has Ca 2ϩ -binding motifs in a long N-terminal extension of the carrier, which faces the intermembrane space and allows activation of the shuttle by cytosolic, but not mitochondrial, Ca 2ϩ signals, in brain, skeletal muscle, and heart mitochondria (11)(12)(13)(14).
Aralar overexpression in the ␤-cell increases glucose-induced rises in NAD(P)H levels and mitochondrial membrane potential, and the increase in mitochondrial activation correlates with augmentation of GSIS (17). It appears that because aralar levels in INS-1 ␤-cell lines and islets are lower than those found in neurons or skeletal muscle cells, increasing aralar levels augmented MAS activity and GSIS. This may result from glucose-mass action, cytosolic redox transfer keeping pace with glycolysis.
Here we have studied the impact of aralar knock-down and the importance of the intermembrane space Ca 2ϩ binding motifs of aralar on metabolism-secretion coupling. MAS activity in mitochondria isolated from INS-1 cells was activated by extramitochondrial Ca 2ϩ in the same range as that of brain or skeletal muscle, but not if aralar Ca 2ϩ -binding motifs were mutated. Our results reveal that cytosolic Ca 2ϩ binding to aralar is required for glucose-stimulated NAD(P)H transfer to mitochondria through MAS. Moreover, Ca 2ϩ activation of shuttle activity in cells expressing wild-type but not mutated aralar accounted for most of the NAD(P)H produced in mitochondria under conditions in which glucose-induced entry of Ca 2ϩ in mitochondria was prevented, and a small cytosolic Ca 2ϩ signal was delivered together with glucose. The results suggest a possible role for aralar and MAS in priming the ␤-cell by Ca 2ϩ -mobilizing neurotransmitter or hormones.

EXPERIMENTAL PROCEDURES
Vector Construction-To knock-down aralar, we have followed previously described methods (18) to generate aralar shRNA.pSUPER or empty pSUPER constructs from pSUPER. retro. To generate aralar shRNA.pSUPER, oligonucleotides were designed containing the aralar target sequence (shRNA A6), the mouse U6 (mU6) promoter fused to a sense strand of a 20-nucleotide sequence (GTTAGCTTCTCCTACTTCAA) followed by a short spacer (TTCAAGAGA), the reverse complement of the sense strand, and five thymidines as an RNA polymerase III transcriptional stop signal. The selection of the coding sequences for shRNA was empirically determined but they started with G, contained 40 -55% GC, and were analyzed by BLAST research to ensure that they did not have significant sequence homology with other genes. Plasmid PGTEmU6 (Ambion) was used as a template for PCR isolation of the mU6 promoter. The PCR conditions were 96°C denaturation for 3 min followed by 30 cycles of 96°C for 1 min, 50°C for 1 min, and 72°C for 2 min; followed by one cycle of 72°C for 10 min. The resulting PCR product was cloned into pCRII-TOPO (Invitrogen) to generate shRNA.pCRII-TOPO. To generate aralar shRNA.pSUPER, pSUPER.retro was digested with EcoRI and the shRNA cassette digested with EcoRI/EcoRI from shRNA. pCRII-TOPO was ligated into the vector. The H1 promoter from pSUPER.retro was deleted with NspV/AccI. The configuration of the construct was verified by DNA sequencing. To reconstitute the expression of wild-type aralar on cells expressing aralar shRNA.pSUPER vector, we have introduced mutations in the shRNA target sequence. To this end, mutations in the third base of the codons for Phe 216 , Ser 217 , and Tyr 218 were introduced in the 1.04-kb BstEII fragment of FLAG-tagged pIRESaralar1 (11,12) by site-directed mutagenesis using the QuikChange site-directed mutagenesis kit (Stratagene). The resulting sequence escapes RNA interference effects while maintaining the amino acid sequence. To generate an aralar protein inactive in Ca 2ϩ -binding, amino acids Glu 27 (position x) and Asp 29 (position y) of EF-hand 1, and amino acids Asp 65 (position x) and Thr 67 (position y) of EF-hand 2 of the aralar sequence (11) were replaced by alanines by site-directed mutagenesis (FLAG-tagged pIRESaralar1 Mut vector). FLAG-tagged pIRESaralar1 and FLAG-tagged pIRESaralar1Mut vectors were confirmed by sequencing.
Cell Culture and Transfection of INS-1 Cells-INS-1 cells were cultured in a humidified atmosphere containing 5% CO 2 in a medium composed of RPMI 1640 supplemented with 10 mM Hepes, 10% (v/v) heat-inactivated fetal calf serum, 2 mM glutamine, 100 units/ml penicillin, 100 g/ml streptomycin, 1 mM sodium pyruvate, and 50 M 2-mercaptoethanol (19). The aralar shRNA.pSUPER and the empty pSUPER vector as control were transfected into the cell line INS-1 (1 g of DNA/1 ϫ 10 6 cells). Stable cell lines expressing aralar shRNA.pSUPER (aralar KD) were selected for puromycyn resistance by plating for 3 weeks in media containing 1 g/ml puromycin (Sigma). The FLAG-tagged pIRESaralar1 and pIRESaralar1Mut vectors were transfected into the aralar KD cell line (1 g DNA/1 ϫ 10 6 cells). Stable cell lines expressing either FLAG-tagged pIRESaralar1 (Wt 24) or pIRESaralar1Mut (Mut 37) were selected for hygromycin resistance by plating for 3 weeks in media containing 200 g/ml hygromycin (Calbiochem) and 1 g/ml puromycin. Clonal cell lines were grown and maintained in the presence of 1 g/ml puromycin or 1 g/ml puromycin plus 100 g/ml hygromycin. For all transfections Lipofectamine Plus (Invitrogen) was used. Clonal lines were isolated with cloning cylinders (Sigma) and verified by Western blotting.
Western Blotting-To determine the protein levels of aralar, FLAG-tagged aralar, or its Ca 2ϩ -binding mutant, cells were homogenized in lysis buffer (250 mM sucrose, 20 mM Hepes, 10 mM KCl, 1.5 mM MgCl 2 , 1 mM EDTA, 1 mM dithiothreitol, pH 7.4, 1 mM iodoacetate, and 1 mM phenylmethylsulfonyl fluoride). Proteins were determined by the Bradford method. Homogenates (20 g) were subjected to electrophoresis on a 8% polyacrylamide gel in the presence of SDS, transferred to nitrocellulose membranes (Schleicher and Schuell), and analyzed by Western blotting. Antibodies specific against aralar1 peptide-(12-343) (N-terminal extension) (15) and FLAG peptide (Sigma) were used as first antibodies (1:5,000) with goat anti-rabbit and horse anti-mouse peroxidase-conjugated IgG secondary antibodies and processed with the luminescence technique ECL (Amersham Biosciences). As control, an anti-body against the ␤-subunit of F 1 -ATPase (generous gift of Professor J. M. Cuezva) was used (1:10,000). Quantitation of the bands with respect to ␤-subunit of F 1 -ATPase was carried out by densitometry (GS-800 Calibrated densitometer Bio-Rad, with Bio-Rad Quantity One software).
Isolation of Mitochondria from INS-1 Clonal ␤-Cells-Cells were washed twice in homogenization buffer (250 mM sucrose, 20 mM Hepes, 2 mM EGTA, 10 mM KCl, 1.5 mM MgCl 2 , and 0.1% bovine serum albumin, pH 7.4), harvested from the dish using a cell scraper and sedimented (350 ϫ g for 10 min). Cells were homogenized in homogenization buffer supplemented with protease inhibitors (1 mM phenylmethylsulfonyl fluoride, 1 mM iodoacetamide) and nuclei, cell debris and intact cells were removed by centrifugation for 3 min at 1,090 ϫ g. Mitochondrial fractions were then spun down (11 min, 12,100 ϫ g), resuspended in MSK (75 mM mannitol, 25 mM sucrose, 5 mM potassium phosphate, 20 mM Tris-HCl, 0.5 mM EDTA, 100 mM KCl, and 0.1% bovine serum albumin, pH 7.4) and kept on ice until used. Proteins were measured by the Bradford method. The respiratory competence of the mitochondrial preparation was assessed by verifying succinate-dependent oxygen consumption and ADP-stimulated respiration with Clark-type electrode (about 20 and 60 nanoatoms O 2 /min/mg of protein for mitochondria in State 4 and State 3, respectively).
Reconstitution of the Malate-Aspartate NADH Shuttle Activity in Mitochondria-MAS activity was reconstituted in mitochondria isolated from INS-1 clonal ␤-cells as described (13,14,20), except that aspartate was omitted from the assay. Mitochondrial fractions (0.4 mg of protein) were suspended in 2 ml of MSK and the shuttle was reconstituted in the presence of 4 units/ml glutamate-oxalacetate transaminase, 6 units/ml malate dehydrogenase, 66 M NADH, 5 mM malate, 0.5 mM ADP, 200 nM ruthenium red, and appropriate CaCl 2 additions. MAS activity was started by the addition of 5 mM glutamate and measured as a decrease in NADH fluorescence (excitation at 340 nm, emission at 465 nm) at 37°C under constant stirring, which was calibrated with appropriate NADH standards.
Mitochondrial Membrane Potential-Cells were cultured in polylysine-coated 24-well plates 48 h before the experiment. Cells were preincubated with 10 g/ml rhodamine 123 for 20 min at 37°C in Krebs-Ringer bicarbonate Hepes buffer (KRBH buffer; 140 mM NaCl, 3.6 mM KCl, 0.5 mM NaH 2 PO 4 , 0.5 mM MgSO 4 , 1.5 mM CaCl 2 , 2 mM NaHCO 3 , and 10 mM Hepes, pH 7.4) and washed in the same buffer. The ⌬⌿ m was monitored at 37°C in a plate reader fluorimeter (Fluostar Optima) with excitation and emission filters set at 485 and 520 nm, respectively.
Insulin Secretion Assay-Cells were cultured in 24-well plates coated with polyornithine 48 h before the experiment. The cells were washed and preincubated in glucose-free KRBH supple-mented with 0.1% bovine serum albumin (KRBH/bovine serum albumin). Next, cells were stimulated for 30 min with 2.5 or 15 mM glucose or 30 mM KCl (in the presence of 2.5 mM glucose). Insulin secretion was determined by insulin enzyme-linked immunosorbent assay kit (SPI-BIO). Total insulin content was extracted with 10% acetic acid in ethanol (v/v). Secreted insulin was expressed as a percentage of total cellular insulin content.
Imaging Measurements of Cytosolic Ca 2ϩ in INS-1 Cells-Cells growing on coverslips coated with polylysine were loaded with 2 M Fura-2 AM (Molecular Probes) for 40 min at 37°C in glucose-free RPMI 1640 medium, and washed for 5-10 min in KRBH. Then coverslips were mounted in a perifusion chamber on the microscope stage as described earlier (23) and Fura-2 fluorescence was imaged ratiometrically using alternate excitation at 340 and 380 nm and a 510-nm emission filter with a Neofluar 40X/0.75 objective (13,23) at 37°C. Additions as indicated were made as a bolus. Single cell analysis of the changes in [Ca 2ϩ ] i were expressed as the ratio of fluorescence intensity at 340 (F 340 ) and 380 nm (F 380 ) (F 340 /F 380 ). Image acquisition and analysis were performed with the Aquacosmos 2.5 software (Hamamatsu).
Mitochondrial Ca 2ϩ Measurements in INS-1 Cells-INS-1 cells were seeded onto poly-L-ornithine-coated Thermanox plastic coverslips (13 mm diameter, Nalge Nunc). Attached cells were infected with an adenovirus carrying mitochondrially targeted Aequorin (24). Cells were analyzed 48 h after viral infection. The aequorin measurements were performed as described (25). Cells were loaded with coelenterazine (2.5 M) in glucose-free RPMI 1640 for 2 h. They were then perifused with KRBH buffer (1 ml/min) at 37°C and the Ca 2ϩ signal-dependent luminescence was monitored.
Two-photon Excitation Microscopy in INS-1 Cells-INS-1 cells were seeded on 24 ϫ 50-mm glass coverslips at the bottom of plastic dishes that were mounted in a perifusion chamber or on glass coverslips sealed at the bottom of plastic wells (4-well LabTek chamber slide systems, NUNC). In both cases the coverslips were coated with polylysine. Cells were washed once and incubated in KRBH for 1 h before experimentation. Two-photon excitation microscopy was performed by using Nikon TE300 inverted microscope with a S Fluor ϫ40/1.30 oil objective and an additional digital magnification of ϫ2, coupled to a RTS 2000 MP (Bio-Rad) confocal/multiphoton microscopy system. An infrared multiphoton laser (Coherent Mira 690 -1000 nm) provided excitation of intrinsic NADH fluorescence with a long wave excitation at 735 nm with 150-fs pulses. Images were collected with a 480/50 nm emission filter. Cells were maintained at 37°C with a temperature-controlled microscopic stage. Additions as indicated were made as a bolus. Images (512 ϫ 512 pixels per frame, 0.178 m/pixel) were taken every 10 s for 400 -1300 s. Image analysis was carried out with MetaMorph software (Universal Imaging). Mitochondrial intensities were determined in individual cells following procedures used in islet ␤-cells (26,27) or in neurons (13). Individual cells were outlined, and intensity thresholds were set that highlighted the bright areas that corresponded to in-focus mitochondria. With these thresholds, which may underestimate the mitochondrial fluorescence but avoid contamination from the cytoplasmic compartment, the intensity of these bright areas was calculated. Changes in mitochondrial NAD(P)H fluorescence were then quantified and normalized as F/F 0 fluorescence values.

RESULTS
Knock-down of Aralar-The aralar shRNA.pSUPER and the empty pSUPER vector as control were transfected into INS-1 clonal ␤-cells and stable cell lines were established. One of the different clones for aralar shRNA.pSUPER in which the levels of aralar protein was undetectable at all passage numbers tested was selected as the aralar knock-down INS-1 cell line used in this study (aralar KD) (Fig. 1A).
To obtain wild-type aralar in cells knocked-down for aralar (Wt 24), a vector carrying FLAG-tagged aralar (pIRESaralar1Wt-FLAG) was transfected into aralar KD cells and stable cell lines were established. The levels of aralar protein in the Wt 24 cell line were similar to those of normal INS-1 cells as tested by Western blotting using the anti-aralar antibody (Fig. 1B).
To verify that aralar knock-down was effective MAS activity was reconstituted in mitochondria isolated from aralar KD and Wt 24 cell lines. Fig. 1C shows that MAS activity was essentially absent in mitochondrial fractions from aralar KD cells.
Effects of Aralar KD on Mitochondrial Membrane Potential (⌬⌿ m )-Having confirmed that knock-down of aralar was effective in abolishing MAS activity, we tested the effects of aralar deficiency on glucose-induced mitochondrial hyperpolarization. In both aralar KD and Wt 24 cells, the mitochondrial membrane was hyperpolarized by 15 mM glucose ( Fig. 2A). The protonophore carbonyl cyanide p-trifluoromethoxyphenylhydrazone collapsed ⌬⌿ m . There were no significant differences between the Wt 24 and aralar KD cells.
Effects of Aralar KD on Glucose-induced Insulin Secretion-Insulin secretion in response to a stimulatory glucose concentration (15 mM) was assayed in Wt 24 and aralar KD cells over a stimulation period of 30 min and expressed as % of insulin content (Fig. 2B). At basal glucose concentration (2.5 mM glucose) 2.28 Ϯ 0.8% insulin was released from Wt 24 cells. Stimulatory glucose (15 mM) caused an increase in insulin secretion to 9.86 Ϯ 0.92% of content (p Ͻ 0.001). Compared with Wt 24 cells the insulin secretory response in aralar KD cells was attenuated by 25.7% (p Ͻ 0.05) (from 2.20 Ϯ 0.43 at 2.5 mM glucose to 7.33 Ϯ 0.64% of content, at 15 mM glucose, p Ͻ 0.001) (Fig. 2B). However, insulin release stimulated by 30 mM KCl, which rap-  A and B). C, MAS activity in mitochondrial preparations from Wt 24 and aralar KD cell lines was measured as a decrease in NADH fluorescence in the absence of glutamate addition or after glutamate (Glu) addition where indicated by the arrows. Traces correspond to representative experiments. The upward reflection of the trace upon glutamate addition reflects an initial reversal of the malate dehydrogenase reaction in the direction of NADH production, which lasts until aspartate coming out from mitochondria enables the transaminase reaction to proceed in the direction of oxaloacetate production coupled to NADH oxidation. In the case of mitochondria from aralar KD cells, the lack of AGC prevents aspartate to come out from mitochondria and thus the initial reversion of malate dehydrogenase in the direction of NADH production continues along the length of the recording. idly depolarizes the ␤-cell membrane and increases Ca 2ϩ influx without depending on mitochondrial metabolism (28), was the same in the two cell lines (Fig. 2B). In addition, insulin secretion in response to glucose and KCl was not significantly different in Wt 24 and the native INS-1 cells transfected with empty pSUPER vector (results not shown). Cellular insulin content was unaffected by aralar knock-down compared with Wt 24 cells (62.8 Ϯ 4.4 versus 69.1 Ϯ 10.2 ng of insulin/ml for Wt 24 and aralar KD cells). These results show that the lack of aralar causes a complete loss of MAS activity, and attenuates GSIS in INS-1 ␤-cells.
Mutation of EF-hand 1 and EF-hand 2 of Aralar-Aralar/ AGC1 and citrin/AGC2 contain four pairs of EF-hands, and a single nonfunctional hypothetical EF9 in its N-terminal half (14). Deletion studies with citrin/AGC2 indicate that Ca 2ϩ binding is mostly conferred by the EF1-EF2 pair, which is the only canonical EF-hand pair in aralar and citrin (14,15).
To study Ca 2ϩ regulation of aralar, specific mutations were introduced to interfere with its ability to bind Ca 2ϩ in the intermembrane space. This is illustrated in Fig. 3A. The amino acids that contribute to the octahedral Ca 2ϩ co-ordination cage are labeled x, y, z, -x, -y, and -z (11,15). To block Ca 2ϩ binding to aralar, the conserved glutamate (Glu) and aspartate (Asp) at positions 27 and 29 of EF-1 (positions x and y, respectively) and the conserved aspartate (Asp) and threonine (Thr) at positions 65 and 67 of EF-hand 2 (positions x and y, respectively) of aralar were replaced by alanines (Ala) by site-directed mutagenesis to yield a FLAG-tagged aralar mutant protein (Fig. 3A).
FLAG-tagged-pIRESaralar1Mut vector was transfected into aralar KD and stable cells were established (Mut 37). The levels of aralar protein in the Mut 37 cell line were similar to those of Wt 24 cells as tested by Western blotting using the anti-FLAG antibody (Fig. 3B). Verification of aralar levels in the Mut 37 cell line expressing the mutant form of the aralar protein was not possible, as the mutant protein was no longer recognized by the anti-aralar antibody (Fig. 3B).
Ca 2ϩ Activation of the Malate-Aspartate NADH Shuttle in Isolated Mitochondria from INS-1 Cells-In tissues where aralar is the only AGC isoform, such as brain and skeletal muscle, MAS activity is activated by extramitochondrial Ca 2ϩ with S 0.5 values for Ca 2ϩ activation of around 300 nM. Ca 2ϩ stimulation of MAS leads to an increase in V max rather than to changes in the affinity for its substrate glutamate (14).
To study Ca 2ϩ activation of MAS in ␤-cell mitochondria, shuttle activity was assayed in the presence of external Ca 2ϩ and ruthenium red, which blocks Ca 2ϩ uptake by the Ca 2ϩ uniporter in isolated mitochondria (13). Any activation caused by extramitochondrial Ca 2ϩ in these conditions can be attributed to the regulation of aralar in the external face of the inner mitochondrial membrane (13,14). Fig. 4A shows that MAS activity in mitochondria from Wt 24 cells increased when Ca 2ϩ was available in the incubation medium. The maximal activation was about 2.5-fold in response to extramitochondrial Ca 2ϩ (from 4.49 Ϯ 1.11 to 12.37 Ϯ 1.14 nmol of NADH min Ϫ1 mg of protein Ϫ1 , in the absence of Ca 2ϩ (below 10 nM) or presence of ϳ20 M free Ca 2ϩ , respectively, (p Ͻ 0.01) (Fig. 4B), similar to results obtained with brain mitochondria (13,14). The activation by external Ca 2ϩ was the same in the presence or absence of 200 nM ruthenium red (Fig. 4C), in agreement with the fact that the Ca 2ϩ activation sites are in the intermembrane space.
To study the role of EF-hand motifs of aralar in the activation of MAS, shuttle activity was assayed in mitochondria from the Mut 37 cell line (Fig. 4D). Interestingly, Ca 2ϩ stimulation of MAS activity was abolished (6.64 Ϯ 0.77 to 5.54 Ϯ 1.0 nmol of NADH min Ϫ1 mg of protein Ϫ1 , in the absence or in the presence of ϳ20 M free Ca 2ϩ , respectively) (Fig. 4B). However, the basal activity of the shuttle in a Ca 2ϩ -free medium was the same as that of the Wt 24 cell line (Fig. 4B). These results clearly show that the N-terminal EF-hand pair in aralar is responsible for Ca 2ϩ activation of shuttle activity, but it is not required for the basal activity of MAS in Ca 2ϩ -free conditions.  lines or ␤-cells (29). Thus, under these conditions glucose-induced redox transfer can be studied in the absence of Ca 2ϩ signals.

Glucose-induced NAD(P)H Redox Transfer to Mitochondria in INS-1 Cells in a Ca
The percentage of cells showing increases in mitochondrial NAD(P)H in response to a stimulatory (15 mM) glucose concentration in these conditions was only about 25%, whereas about 70% of those cells responded to glucose in the presence of 1.5 mM extracellular Ca 2ϩ (see below). The fraction of cells responding in Ca 2ϩ -free conditions was similar in Wt 24, Mut 37, and aralar KD cells (Table 1). Moreover, the glucose-induced mitochondrial NAD(P)H peak response was also much smaller in Ca 2ϩ -free than in a Ca 2ϩ -containing medium (about 1.35 Ϯ 0.02-fold in Ca 2ϩ -free versus 1.94 Ϯ 0.11-fold in Ca 2ϩcontaining medium; see below), and the same in all three cell lines ( Table 1). The mitochondrial NAD(P)H increase is clearly dependent on cytosolic and/or mitochondrial Ca 2ϩ . Because this increase is not reduced in aralar KD cell lines, it appears that MAS is not activated under these conditions and the increase in mitochondrial NAD(P)H in Ca 2ϩ -free medium largely corresponds to glucose-derived pyruvate oxidation in mitochondria.

Role of Cytosolic and Mitochondrial Ca 2ϩ in Glucose-induced NAD(P)H Redox Transfer to Mitochondria in INS-1 Cells-
To further study the regulation of glucose-stimulated MAS activity by Ca 2ϩ in INS-1 cells, we have employed two types of Ca 2ϩ signals. A very small Ca 2ϩ signal delivered at the time of glucose addition was obtained by adding glucose (15 mM) together with 250 M ATP in the presence of 100 M EGTA in Ca 2ϩ -free KRBH. By acting on purinergic receptors, ATP evokes an inositol 1,4,5-trisphosphate-dependent release of Ca 2ϩ from the endoplasmic reticulum, which results in a small Ca 2ϩ signal in the absence of extracellular Ca 2ϩ (29,30). The stimulation of glucose-induced mitochondrial NAD(P)H production obtained under these conditions was compared with that obtained under physiological conditions, in which glucose was added in a medium containing 1.5 mM Ca 2ϩ .  Fig. 5, A-C, that correspond to individual cells). It also caused large increases in mitochondrial Ca 2ϩ in the whole cell population to peak levels 4 -7-fold over the basal (Fig. 5, D-F), as shown earlier with mitochondrial aequorin vectors in INS-1 cell lines (25). These large increases in [Ca 2ϩ ] m were also essentially the same in Wt 24, Mut 37, and aralar KD cell lines. In contrast, when INS-1 cells were exposed to 15 mM glucose together with 250 M ATP plus 100 M EGTA in Ca 2ϩ -free KRBH, chelation of external Ca 2ϩ abolished the Ca 2ϩ influx caused by glucose, and ATP addition resulted in a single, Ca 2ϩ transient in individual INS-1 cells (Fig. 5, A-C). In terms of mitochondrial Ca 2ϩ , the response was strikingly smaller than that obtained in the presence of Ca 2ϩ (Fig. 5, D-F), indicating that these conditions greatly limit Ca 2ϩ entry into mitochondria. There were no significant differences in peak [Ca 2ϩ ] i or peak [Ca 2ϩ ] m obtained under these conditions when comparing aralar KD (Fig. 5, A and D), Wt 24 (Fig. 5,  B and E), and Mut 37 (Fig. 5, C and F) INS-1 cells.

Glucose-induced peak increase in mitochondrial (Mit) NAD(P)H in the absence of extracellular Ca 2؉
Glucose-induced increases in mitochondrial NAD(P)H were measured as changes in F/F 0 in aralar KD, Wt 24, and Mut 37 cells incubated in a Ca 2ϩ -free medium. The fraction of cells responding to glucose, and the peak elevation of F/F 0 over the basal F/F 0 were computed in the three cell lines. Data are mean Ϯ S.E. of 7-9 independent experiments (n ϭ 200 -250 cells).  . 5H shows the large increases in mitochondrial NAD(P)H elicited by glucose in single Wt 24 INS-1 cells in the presence of Ca 2ϩ and in the Ca 2ϩ -free condition. Quite surprisingly, the two responses (both peak response and area under the peak) were almost identical (Fig. 5H, Table 2) even though cytosolic and especially mitochondrial Ca 2ϩ signals are far smaller in the Ca 2ϩ -free condition (Fig. 5, B and E). The comparison between Fig. 5, G and H, shows that knock-down of aralar results in a modest decrease in the mitochondrial NAD(P)H response elicited by glucose in the presence of Ca 2ϩ but a much greater decrease in mitochondrial NAD(P)H production obtained when glucose and ATP plus EGTA were added together in a Ca 2ϩ -free medium. Table 2 shows that the mitochondrial NAD(P)H response in the former condition was about 16 (peak) or 30% (area under the peak) lower than that observed in Wt 24 cells. The peak response in the Ca 2ϩ -free condition was about 49 (peak) or 60% (area under the peak) lower than that in Wt 24 cells.

Cell
Taken together these results clearly show that the aralar-MAS pathway accounts for about 50% of the glucose-stimulated NAD(P)H supply to mitochondria when Ca 2ϩ entry in mitochondria is strongly restricted. However, it only accounts for about ϳ20% of that when robust glucose-induced mitochondrial Ca 2ϩ signals are generated. Fig. 5I shows that mutations that block Ca 2ϩ binding in aralar, whereas maintaining its transport activity, produce the same increase in mitochondrial NAD(P)H in response to glu- and Mut 37 cells were exposed to glucose (Glc) in the presence of 1.5 mM Ca 2ϩ , and after a washoff, exposed to glucose and ATP in the presence of Ca 2ϩ -free KRBH and 100 M EGTA at 37°C. Individual additions are indicated by the arrowheads. Each trace represents the [Ca 2ϩ ] i of a single cell from a representative experiment. D-F, mitochondrial Ca 2ϩ dynamics of the responses to glucose (Glc) in mitochondrially targeted aequorin expressing cells from aralar KD, Wt 24, and Mut 37 lines. Cells were exposed to glucose in the presence of 1.5 mM Ca 2ϩ , and after a washoff, exposed to glucose ϩ ATP in the presence of Ca 2ϩ -free KRBH and 100 M EGTA. Each trace represents the [Ca 2ϩ ] m of the whole cell population from a representative experiment. G-I, aralar KD, Wt 24, and Mut 37 cells were preincubated in glucose-free KRBH, 1.5 mM Ca 2ϩ for 1 h at 37°C and then challenged twice with glucose as described in A-C. Two-photon microscopy imaging of mitochondrial NAD(P)H was performed. Traces in G-I correspond to changes in normalized (F/F 0 ) mitochondrial NAD(P)H autofluorescence in individual responsive cells from a representative experiment.
cose in a Ca 2ϩ -containing medium, as in the Wt 24 cell line ( Table 2). However, when glucose was added in the Ca 2ϩ -free condition the increase in mitochondrial NAD(P)H was clearly reduced, representing a ϳ42% decrease (peak or area under the peak) relative to Wt 24 cells (see Table 2). The response was only slightly larger than that obtained in aralar KD cells.
These results show that Ca 2ϩ binding to aralar N-terminal extension is critical under the conditions in which the aralar-MAS pathway plays a major role in supplying NAD(P)H to mitochondria, i.e. when cytosolic Ca 2ϩ signals are small and Ca 2ϩ entry in mitochondria is restricted at the time of glucose addition. In fact, blocking Ca 2ϩ binding to these domains results in an almost total block of the aralar-MAS pathway.

DISCUSSION
Aralar and mitochondrial glycerol-phosphate dehydrogenase, a mitochondrial transporter and a mitochondrial dehydrogenase, respectively, catalyze key steps in the malate-aspartate and glycerol-phosphate NADH shuttles. These shuttles are responsible for the transfer of reducing equivalents from cytosolic NADH to mitochondrial NADH (MAS) or mitochondrial FADH2 (glycerol phosphate shuttle). The role of MAS in insulin secretion was made evident in mice lacking mitochondrial glycerol-phosphate dehydrogenase, in which GSIS in the presence, but not absence, of aminooxyacetate was strongly impaired (32). Moreover, aralar overexpression in ␤-cells increases GSIS (17). Here we have studied the effect of aralar deficiency in INS-1 ␤-cells. Our results indicate that the lack of aralar in INS-1 cells causes a complete loss of MAS activity, and a 25% decrease in GSIS, supporting a role of MAS in insulin secretion.
Aralar and glycerol-phosphate dehydrogenase are embedded in the inner mitocondrial membrane and both have sites for interaction with substrates (aspartate/glutamate and glycerol phosphate/dihydroxyacetone phosphate, respectively) in the intermembrane space. In addition, both have Ca 2ϩ binding motifs also facing the intermembrane space. We have further addressed the role of Ca 2ϩ regulation of MAS by studying its influence in glucose-induced increase in mitochondrial NAD(P)H by two-photon microscopy imaging.
The Aralar-MAS Pathway in Intact ␤-Cells Has an Absolute Requirement for Ca 2ϩ -By studying the glucose-induced increase in mitochondrial NAD(P)H in Ca 2ϩ -free medium, in the absence of glucose-dependent cytosolic or mitochondrial Ca 2ϩ signals, it was observed that the increase in mitochondrial NAD(P)H is about 60% smaller than obtained in the presence of Ca 2ϩ . This difference matches the decrease in CO 2 production from D-[6-14 C]glucose in Ca 2ϩ -free medium reported previously (8). However, it contrasts with the results of Gilon and Henquin (9) who reported that the glucose-stimulated increase in NAD(P)H in mouse islets persists in the absence of extracellular Ca 2ϩ . The mitochondrial but not whole cell, glucose-induced NAD(P)H increase is clearly dependent on cytosolic and/or mitochondrial Ca 2ϩ signals. Because the increase in mitochondrial NAD(P)H in Ca 2ϩ -free medium is not reduced in aralar KD cell lines, it appears that MAS does not participate in this process. Therefore, the increase in mitochondrial NAD(P)H in Ca 2ϩ -free medium largely corresponds to glucose-derived pyruvate oxidation in mitochondria (glucose push).
At glucose concentrations above 10 mM, in which the limiting step of glycolysis is no longer set by glucokinase, pyruvate oxidation becomes rate-limiting in the formation of ATP or glucose-derived intermediates (1,33,34). Positive regulation of mitochondrial metabolism may require Ca 2ϩ potentiation. In the absence of Ca 2ϩ , pyruvate oxidation may limit mitochondrial NADH production, regardless of MAS activity explaining the lack of effect following aralar knock-down under these conditions. In other words, it appears that the glucose push is insufficient to activate MAS. Ca 2ϩ seems to be required to make it operative by increasing the V max of the aralar-MAS pathway (14).
Cytosolic Ca 2ϩ Signals Are Required to Couple MAS Activity to Glucose Utilization-The initial increase in cytosolic Ca 2ϩ produced by glucose is preceded by mitochondrial activation through mass action and is followed by Ca 2ϩ entry in mitochondria through the Ca 2ϩ uniporter. Thus, these potentiating signals, cytosolic and mitochondrial Ca 2ϩ , are preceded by mitochondrial activation through mass action. We have shown that the aralar-MAS pathway is important for NAD(P)H redox transfer to ␤-cell mitochondria during glucose utilization, in particular under conditions where glucose-stimulated Ca 2ϩ entry in mitochondria is prevented but ATP-induced small cytosolic Ca 2ϩ transients are evoked at the time of glucose utilization. The single cytosolic Ca 2ϩ transient observed under these conditions elicited a very small mitochondrial Ca 2ϩ peak, much smaller than that obtained as a consequence of glucose-