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Originally published In Press as doi:10.1074/jbc.M409303200 on October 19, 2004

J. Biol. Chem., Vol. 279, Issue 53, 55659-55666, December 31, 2004
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The Malate-Aspartate NADH Shuttle Member Aralar1 Determines Glucose Metabolic Fate, Mitochondrial Activity, and Insulin Secretion in Beta Cells*

Blanca Rubi{ddagger}, Araceli del Arco§, Clarissa Bartley{ddagger}, Jorgina Satrustegui¶, and Pierre Maechler{ddagger}||

From the {ddagger}Department of Cell Physiology and Metabolism, University Medical Centre, 1211 Geneva, Switzerland, the Departamento de Biología Molecular, Universidad Autónoma de Madrid, Centro de Biología Molecular Severo Ochoa, 28049 Madrid, Spain, and the §Facultad de Ciencias del Medio Ambiente, Universidad de Castilla-La Mancha, 45071 Toledo, Spain

Received for publication, August 13, 2004 , and in revised form, October 18, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The NADH shuttle system, which transports reducing equivalents from the cytosol to the mitochondria, is essential for the coupling of glucose metabolism to insulin secretion in pancreatic beta cells. Aralar1 and citrin are two isoforms of the mitochondrial aspartate/glutamate carrier, one key constituent of the malateaspartate NADH shuttle. Here, the effects of Aralar1 overexpression in INS-1E beta cells and isolated rat islets were investigated for the first time. We prepared a recombinant adenovirus encoding for human Aralar1 (AdCA-Aralar1), tagged with the small FLAG epitope. Transduction of INS-1E cells and isolated rat islets with AdCA-Aralar1 increased aralar1 protein levels and immunostaining revealed mitochondrial localization. Compared with control INS-1E cells, overexpression of Aralar1 potentiated metabolism secretion coupling stimulated by 15 mM glucose. In particular, there was an increase of NAD(P)H generation, of mitochondrial membrane hyperpolarization, ATP levels, glucose oxidation, and insulin secretion (+45%, p < 0.01). Remarkably, this was accompanied by reduced lactate production. Rat islets overexpressing Aralar1 secreted more insulin at 16.7 mM glucose (+65%, p < 0.05) compared with controls. These results show that aspartate-glutamate carrier capacity limits glucose-stimulated insulin secretion and that Aralar1 overexpression enhances mitochondrial metabolism.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Glucose metabolism, through glycolysis and mitochondria, drives stimulation of insulin secretion in pancreatic beta cells (1, 2). According to low lactate dehydrogenase activity in beta cells, glycolysis-derived electrons carried by NADH+H+ are mostly transferred to mitochondria through the NADH shuttle system. Therefore, NADH shuttles couple glycolysis to activation of mitochondrial energy metabolism, leading to insulin secretion. Moreover, low activity of NADH shuttles in beta cells has been found in type 2 diabetes models (3) and is also the cause of impaired glucose-stimulated insulin secretion (GSIS)1 in fetal islets (4).

In beta cells, the NADH shuttle system is composed essentially of the glycerophosphate and the malate-aspartate shuttles (5). The respective importance of these shuttles is illustrated in pancreatic islets of mice with abrogation of NADH shuttle activities. Mice lacking mitochondrial glycerol-phosphate dehydrogenase exhibit normal GSIS (6). However, additional inhibition of the malate-aspartate shuttle with aminooxyacetate strongly impairs the secretory response to glucose (6). This suggested that the malate-aspartate shuttle might play a key role in both mitochondrial metabolism and cytosolic redox state. Besides glycerophosphate and malate-aspartate shuttles, pyruvate-citrate shuttle also regenerates NAD+ necessary to maintain glycolysis. Pyruvate-citrate shuttle (7) contributes to the formation of malonyl-CoA and cytosolic NADPH, two molecules proposed as candidate coupling factors in GSIS (8, 9).

In the mitochondria, NADH electrons are transferred to the electron transport chain, which in turn supplies the energy necessary to pump protons across the inner mitochondrial membrane thereby creating a proton electrochemical gradient that drives ATP synthesis. In addition to ATP generation, mitochondrial membrane potential drives the transport of metabolites between mitochondrial and cytosolic compartments, including the transfer of mitochondrial factors participating in insulin secretion. Hyperpolarization of the mitochondrial membrane relates to the proton export from the mitochondrial matrix and directly correlates with insulin secretion stimulated by different secretagogues (10). However, nutrient-stimulated insulin secretion is limited by the inherent thermodynamic constraints of proton gradient formation (10). These findings suggest that, in beta cells, NADH shuttles might control mitochondrial proton gradient formation upon glucose stimulation.

Here, the role of the key member of the malate-aspartate NADH shuttle (see Fig. 1) was investigated for the first time in beta cells. We studied the effects of elevated aralar1 expression on glucose fate, mitochondrial activation, and insulin secretion in INS-1E beta cells and rat islets. Aralar1 (or aspartate/glutamate carrier 1, AGC1), a Ca2+ sensitive member of the malate-aspartate shuttle (11), was overexpressed to levels comparable with the brain by means of a recombinant adenovirus. Aralar1 and citrin are members of the subfamily of Ca2+-binding mitochondrial carriers and correspond to two isoforms of the mitochondrial aspartate/glutamate carrier (AGC1). These proteins are activated by Ca2+, acting on the external side of the inner mitochondrial membrane (11-13). We show here that Aralar1 is the isoform expressed in beta cells. Adenovirus-mediated overexpression of Aralar1 induced a metabolic switch, increasing glucose-induced mitochondrial activation and insulin secretion, while reducing lactate release.



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  		FIG. 1.
The malate-aspartate NADH shuttle. NADH+H+ generated in the course of glycolysis can be re-oxidized to NAD+ through activity of the malate-aspartate shuttle, resulting in the transfer of reducing equivalents from the cytosolic compartment to the mitochondrial matrix. Cytosolic malate dehydrogenase (MDH) reduces oxaloacetate (OAA) to malate, thereby regenerating NAD+, and malate enters mitochondria in exchange of {alpha}-ketoglutarate ({alpha}-KG). In the mitochondrial matrix, MDH generates OAA and NADH+H+ from malate and NAD+. OAA is then transaminated to aspartate, using glutamate (Glu) as the amino group donor. Next, in exchange of Glu, aspartate is exported into the cytosol through the inner mitochondrial membrane (IMM) by aralar1 or citrin, key members of the shuttle activated by calcium (see white ovals representing EF-hand motifs). In the cytosol, the shuttle is completed by formation of OAA and Glu from aspartate and {alpha}-KG.

 

   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture and Materials—INS-1E cells (14) were used as a well differentiated beta cell clone derived from the parental INS-1 cells (15). INS-1E cells (passages 55-80) were cultured in a humidified atmosphere containing 5% CO2 in a medium composed of RPMI 1640 supplemented with 10 mM Hepes, 5% (v/v) heat-inactivated fetal calf serum, 2 mM glutamine, 100 units/ml penicillin, 100 µg/ml streptomycin, 1 mM sodium pyruvate, and 50 µM 2-mercaptoethanol (14). RPMI 1640 and Dulbecco's modified Eagle's medium used for HEK-293 cells were obtained from Invitrogen. Reagents commonly used for the experiments were from Sigma. Secondary antibody and radioactive compounds were obtained from Amersham Biosciences, and chemiluminescent reagent (Supersignal West Pico Chemiluminescent substrate) from Pierce. An antibody against rat insulin was purchased from Linco (St. Louis, MO).

Adenovirus Construction—Recombinant adenovirus was constructed as described previously (16). The expression unit to be introduced into the recombinant adenovirus was obtained from the pIRESAralar1, which contains the coding sequence of Aralar1 fused with a FLAG epitope at the C terminus and was cloned into the BamHI site of the pIRES expression vector (11). The Aralar1 cDNA was cut with SmaI flanking the cloned Aralar1 cDNA and subcloned into the cosmid pAdCA (where AdCA corresponds to adeno-chicken actin promoter) and inserted into the unique SwaI site of the E1 substitution-type full-length adenovirus genome cloned in a cassette cosmid. The cassette bearing the expression unit was then co-transfected into HEK-293 cells together with the adenovirus DNA-terminal protein complex digested at several sites with EcoT22I. The Aralar1 insert (nucleotides 1-2060) of the previously reported mRNA (11) contained Aralar1 cDNA tagged in 3' with the FLAG epitope. The presence of the insert was checked with the restriction enzyme ClaI and its correct orientation was checked by DNA sequencing. The cosmid containing the full coding sequence for human Aralar1 was called pAdCA-Aralar1. Cassette cosmid pAdCAAralar1 (8 µg) and 1 µg of the EcoT22I-digested DNA-terminal protein complex were co-transfected in a 6-cm dish using calcium phosphate (Cellfect Transfection Kit; Amersham Biosciences) in HEK-293 cells and 1 day later the cells were distributed into 96-well plates. The desired recombinant adenovirus was generated by overlapping recombination. At day 10 after transfection, the cell lysates from the selected viral clones were used to infect 24-well plates, the adenoviral DNA was extracted from these cells, and the DNA was analyzed by digestion with ClaI to check the presence of the Aralar1 insert. To amplify and purify the selected virus, the cell lysate containing the virus with full-length Aralar1 (AdCA-Aralar1) was used to infect two 138-mm dishes of HEK-293 cells. The AdCA-Aralar1 virus and the AdCA-LacZ virus (expressing the bacterial {beta}-galactosidase and used as a control) were amplified and purified by CsCl ultracentrifugation. Adenovirus amplification was performed in HEK-293 cells cultured in Dulbecco's modified Eagle's medium containing 5 or 10% (v/v) fetal calf serum.

Viral Treatment of INS-1E Cells—INS-1E cells were seeded in 24- or 12-well plates and cultured for 3-4 days prior to viral treatment. For infection, cells were incubated with medium containing different amounts of the recombinant adenovirus/cell for 90 min, washed, and further cultured in RPMI 1640 medium for 18-20 h before experiments to allow transgene expression. AdCA-LacZ, which expresses bacterial {beta}-galactosidase, was used as a control adenovirus.

Immunoblot Analysis—For detection of citrin and aralar1 protein, rat tissues, INS-1E cells, isolated rat islets, and FACS-sorted beta cells (17) were collected and lysed in SDS sample buffer. Proteins were subjected to SDS-PAGE (10%) and transferred onto nitrocellulose membranes. The membranes were blocked for 1 h with Tris-buffered saline containing 0.1% Tween 20 and 5% (w/v) nonfat dried milk. The membranes were washed three times in Tris-buffered saline/Tween at 22 °C. Membranes were probed with the aralar1-specific antibody overnight at 4 °C (1:2000) or anti-citrin (1:2000) (which recognize amino acids 12-343 of human aralar1 protein and 9-278 of human citrin, respectively) (11), before incubation for 1 h at room temperature with anti-rabbit IgG conjugated to horseradish peroxidase (1:5000). The signal was detected by chemiluminescence.

Immunostaining—For immunofluorescence, INS-1E cells were grown on polyornithine-treated glass coverslips for 3 days prior to infection with AdCA-LacZ or AdCA-Aralar1 for 90 min. The next day, cells were fixed as described (18) before incubation with anti-aralar1 (1:200) and then goat anti-rabbit IgG fluorescein isothiocyanate (1:200) (Chemicon, Temecula, CA) antibodies. Cells were viewed using a Axiocam microscope (Carl Zeiss, Göttingen, Germany). For mitochondrion-specific staining, living cells were incubated with 100 nM Mitotracker red CMXRos (Molecular Probes) for 25 min at 37 °C and rinsed with prewarmed phosphate-buffered saline before fixation. For immunofluorescence on transduced islets, rat islets were isolated and cultured for 24 h. Islets were then exposed to the adenoviruses AdCA-LacZ or AdCA-Aralar1 during 90 min. The following day the islets were either used for insulin secretion experiments or trypsinized and seeded on glass coverslips. The same day, cells were fixed before incubation with anti-aralar1 antibody (1:200) or mouse monoclonal anti-human insulin (1:1000), followed by secondary antibody labeling. Cells were viewed using Axiocam microscope.

NAD(P)H Measurements—NAD(P)H generation was monitored as described (14) in attached cells stimulated with 15 mM glucose, after stabilization of the signal for 10 min in 2.5 mM glucose Krebs-Ringer bicarbonate Hepes buffer (KRBH, containing in mM: 135 NaCl, 3.6 KCl, 10 Hepes (pH 7.4), 5 NaHCO3, 0.5 NaH2PO4, 0.5 MgCl2, 1.5 CaCl2). Briefly, NAD(P)H autofluorescence was measured using excitation and emission filters set at 340 and 470 nm, respectively, in a plate reader fluorimeter (Fluostar Optima, BMG Labtechnologies, Offenburg, Germany) at 37 °C with automated injectors for glucose (addition of 13 mM on top of basal 2.5 mM). NAD(P)H autofluorescence was normalized over a 10-min stimulation period by setting the fluorescence at 100% for cells maintained in basal 2.5 mM glucose. Maximal fluorescence changes were recorded after the addition of 5 µM rotenone used to inhibit complex-1 of the electron transport chain.

Mitochondrial Calcium Concentrations ([Ca2+]m)—INS-1E cells were seeded in 24-well plates and 3-4 days later treated with AdCA-LacZ or AdCA-Aralar1 plus AdCA-mAeq adenoviruses. AdCA-mAeq enables expression of the Ca2+-sensitive photoprotein aequorin targeted to the mitochondria. Twenty hours after viral treatment, cells were loaded with coelenterazine (2.5 µM) in glucose-free RPMI 1640 for 2 h, washed with KRBH, 2.5 mM glucose and the luminescence was monitored in the plate reader luminometer (Fluostar Optima) (14).

Mitochondrial Membrane Potential and ATP Levels—Cultured INS-1E cells were infected with AdCA-Aralar1 or AdCA-LacZ for 90 min and used for experiments the next day. Mitochondrial membrane potential ({Delta}{psi}m) was measured in cells previously preincubated with 10 µg/ml rhodamine-123 for 20 min at 37 °C in KRBH. The {Delta}{psi}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. Cellular ATP concentrations were determined in cells transduced with the AdCA-LacZ and AdCA-Aralar1 adenovirus, following a 10-min glucose stimulation, using the ATP Bioluminiscent assay kit (Roche).

Glucose Utilization and Oxidation—Glucose utilization was measured in INS-1E cells as previously described (19). Briefly, cells were incubated for 30 min with KRBH containing 2.5 or 15 mM glucose traced with 1 µCi of D-[5-3H]glucose (specific activity 15 Ci/mmol), then stopped on ice. Supernatants were collected, centrifuged to remove any detached cells, and 3H2O separated from D-[5-3H]glucose using a Dowex column. Proteins were determined and glucose utilization was expressed as nanomoles per mg of protein per hour. For glucose oxidation, INS-1E cells in 12-well plates were placed in a water bath at 37 °C, washed once with 1 ml of KRBH/bovine serum albumin and then preincubated for 30 min with 500 µl of KRBH/bovine serum albumin. The rate of glucose oxidation over a 2-h period was measured in attached cells as previously described (20). We measured the amount of radiolabeled CO2 from cells, using [U-14C]glucose as substrate. Control incubations without INS-1E cells were run with each series. 14CO2 production was counted in an LS6500 liquid scintillation counter (Beckman Instruments Inc., Palo Alto, CA). Glucose oxidation was expressed as percent of the basal oxidation rate at 2.5 mM glucose.

Lactate and Glutamate Measurements—INS-1E cells cultured in 24-well plates were transduced with defined adenoviruses and media were changed the next day. Following a 24-h culture period, media were collected for lactate release measurements. Lactate concentrations were determined as NADH generated from NAD+ in the presence of an excess lactate dehydrogenase as described (21). NADH autofluorescence was monitored in a plate reader fluorimeter (Fluostar Optima) using excitation and emission filters set at 340 and 470 nm, respectively. Glutamate concentrations were determined following a 30-min incubation at 2.5 and 15 mM glucose as described previously (22).

Insulin Secretion Assay—INS-1E cells cultured in 24-well plates were infected over a 90-min period with either AdCA-Aralar1 or AdCALacZ adenovirus and assayed the next day. The cells were washed and preincubated in glucose-free KRBH supplemented with 0.1% bovine serum albumin (KRBH/bovine serum albumin). Next, cells were stimulated for 30 min with different glucose concentrations, 2.5, 7.5, or 15 mM or pyruvate (in the presence of 2.5 mM glucose). Isolated rat islets were infected the day following isolation with either AdCA-Aralar1 or AdCA-LacZ adenovirus over a 90-min period and further cultured for 24 h. Then, hand-picked islets were distributed into 3-ml tubes in KRBH for the 30-min secretion assay. Insulin secretion was determined by radioimmunoassay using rat insulin as standard (23).

Statistical Analysis—Unless otherwise indicated, data are the mean ± S.E. for at least three independent experiments performed in triplicate. Differences between groups were assessed by the Student's t test for unpaired data.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Expression of Aralar1 and Citrin in Beta Cells—The expression of aralar1 and citrin was studied by immunoblot on protein extracts of adult rat tissues, INS-1E cells, isolated rat islets, and sorted beta cells. In accordance with a previous report (24), there were high aralar1 levels in the brain, lower levels in kidney, lung, liver, and pancreatic islets, and hardly detectable levels in white adipose tissue (Fig. 2A). Citrin was present in kidney and liver, but also at very low levels in the lung. Aralar1 was the only isoform expressed in INS-1E cells, rat islets and, more specifically, FACS-sorted beta cells (Fig. 2B). This first set of data demonstrated that, between the two known forms of aspartate/glutamate carriers, aralar1 is the one expressed in beta cells. However, the levels of expression are lower compared with brain, another neuroendocrine tissue. This suggested the potential of increasing Aralar1 expression in beta cells within a physiological range.



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  		FIG. 2.
Immunoblot analysis of aralar1 and citrin in adult rat tissues, rat islets, beta cells, and INS-1E cells. A, distribution of aspartate/glutamate carrier isoforms in rat tissues; 15 µg of protein were used for all tissues except for white adipose (ad.) tissue (7.5 µg). Blots for citrin and aralar1 were performed in parallel, each blot also incubated with anti-actin. Aralar1 was detected with an antibody directed against aralar1 amino acids 12-343. Citrin was detected with an antibody directed against citrin amino acids 9-278. Bands correspond to 70 kDa (aralar1 and citrin) and 45 kDa (actin). B, immunoblot for aralar1 and citrin in islets, INS-1E cells, and FACS-sorted beta cells.

 
Aralar1 Overexpression and Subcellular Localization—The capacity of AdCA-Aralar1 to direct expression of Aralar1 in INS-1E cells was evaluated by immunoblot analysis on extracts from cells infected with AdCA-Aralar1. The conditions of infection are described under "Experimental Procedures." Immunoblotting revealed a band of the expected size (70 kDa) and the signal increased according to the viral titer, i.e. from 0 to 30 plaque forming units (pfu)/cell (Fig. 3D). An infection titer of 10 pfu/cell was used as optimum throughout the study. Next, we assessed the mitochondrial localization of the overexpressed Aralar1. Immunostaining with anti-aralar1 antibodies and visualization by fluorescence microscopy was carried out 20 h after transduction with the AdCA-Aralar1. The immunofluorescence (Fig. 3, A-C) revealed typical mitochondrial morphology. The presence of aralar1 in mitochondria was further confirmed by co-localization of the anti-aralar1 positive organelles with that of Mitotracker red CMXRos (Fig. 3, E-G), a mitochondrial-specific fluorescent vital dye (25), as previously described (11).



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  		FIG. 3.
Expression analysis and cellular localization of aralar1 in transduced INS-1E cells. Immunolocalization of aralar1 in INS-1E cells transduced with AdCA-Aralar1 at titers 0 pfu/cell (A), 15 pfu/cell (B), and 30 pfu/cell (C). Effects of the adenovirus dose response on immunoreactive aralar1 signal in transduced INS-1E cells 18-20 h after a 90-min infection period. Immunoblot analysis following SDS-PAGE 10% protein separation of cell lysates (D). INS-1E cells transduced with the AdCA-Aralar1 adenovirus were incubated with the mitochondrial dye Mitotracker, fixed, and incubated with anti-aralar1 and FITC-conjugated secondary antibodies. Identical fields revealing anti-aralar1 (E), Mitotracker (F), and overlay of both (G) are presented.

 
Effects of Aralar1 on NAD(P)H Levels and [Ca2+]m—In beta cells, glucose-induced increases in NAD(P)H are mostly of mitochondrial origin (26). Exposure of control INS-1E cells to stimulatory 15 mM glucose raised NAD(P)H levels (+53%, p < 0.05). Blockade of the respiratory chain complex-1 by 5 µM rotenone, used to maximally increase mitochondrial NADH, further enhanced autofluorescence and revealed the mitochondrial origin of the signal. In cells overexpressing Aralar1 glucose-induced elevations of NAD(P)H were potentiated by +37% (p < 0.05) versus AdCA-LacZ (Fig. 4A). Mitochondrial matrix Ca2+ concentrations are mostly determined by Ca2+ uniporter and Na+/Ca2+ exchanger activities. Mitochondrial Ca2+ regulates NADH+H+ generating enzymes, such as isocitrate dehydrogenase and {alpha}-ketoglutarate dehydrogenase (27, 28). As expected, glucose (15 mM) stimulation of control cells resulted in elevations of [Ca2+]m (Fig. 4B). There were no significant differences in [Ca2+]m when comparing control and aralar1 overexpressing cells. Therefore, augmentation of NAD(P)H associated with aralar1 overexpression cannot be attributed to mitochondrial Ca2+ changes.



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  		FIG. 4.
Effects of Aralar1 overexpression on NAD(P)H levels and mitochondrial Ca2+ in INS-1E cells. A, NAD(P)H autofluorescence in INS-1E cells stimulated with 15 mM glucose (Glc 15) for 10 min before addition of 5 µM rotenone to maximally increase mitochondrial NADH. Data are the mean ± S.E. of five independent experiments performed in quadruplicate. B, the increases in mitochondrial Ca2+ concentrations in response to 15 mM glucose (Glc) were measured in cells co-transduced with AdCA-mAeq and AdCA-Aralar1. Traces are the mean ± S.E. of six independent experiments.

 
Effects of Aralar1 on Mitochondrial Membrane Potential—In control LacZ cells expressing {beta}-galactosidase, the mitochondrial membrane was hyperpolarized by raising glucose from 2.5 to 15 mM, or by the addition of the mitochondrial substrate pyruvate (1 mM). The protonophore carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) collapsed {Delta}{psi}m (Fig. 5). INS-1E cells overexpressing Aralar1 exhibited marked potentiation of mitochondrial membrane hyperpolarization upon glucose (15 mM) stimulation (Fig. 5A). Glycolysis and the associated NADH shuttles can be by-passed by the use of the mitochondrial substrate pyruvate. Stimulation with 1 mM pyruvate revealed no difference between the AdCA-LacZ- and AdCA-Aralar1-transduced cells (Fig. 5B).



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  		FIG. 5.
Effects of Aralar1 overexpression on mitochondrial membrane potential ({Delta}{psi}m). The {Delta}{psi}m was measured on attached INS-1E cells using rhodamine-123 fluorescence. Electron transport chain activation, observed as hyperpolarization of {Delta}{psi}m, was induced by 15 mM glucose (A) or by 1 mM pyruvate (B) followed by control {Delta}{psi}m depolarization using 1 µM of the uncoupler FCCP. Values are mean ± S.D. (n = 6) of one of six independent experiments.

 
Glucose-induced ATP and Glutamate Generation in Aralar1 Overexpressing Cells—Activation of the electron transport chain, measured in Fig. 5 as the mitochondrial membrane potential, leads to the formation of ATP. Elevation of glucose from 2.5 to 15 mM glucose increased cellular ATP (+18%, p < 0.05) in control INS-1E cells (Fig. 6A). In Aralar1 overexpressing cells the -fold increase in cellular ATP concentrations upon glucose stimulation was (+36%, p < 0.01) higher compared with control cells (p < 0.05 versus LacZ). Therefore, Aralar1 overexpression amplified glucose-evoked augmentations of cellular ATP. In control cells, glutamate concentrations were increased 2.03-fold by glucose stimulation (15 mM) compared with low glucose (2.5 mM) exposure during 30 min (Fig. 6B). Aralar1 overexpression potentiated glucose-induced glutamate elevation by 23% (p < 0.05).



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  		FIG. 6.
ATP levels, glucose oxidation, and glutamate concentrations in INS-1E cells overexpressing Aralar1. A, cellular ATP levels were measured in cells incubated for 10 min with glucose as indicated. Values are means of four experiments. B, cellular glutamate concentrations determined following a 30-min incubation period. Values are mean ± S.E. of five independent experiments. C, [U-14C]glucose oxidation was measured in INS-1E cells overexpressing LacZ or Aralar1. Data are the mean ± S.E. of four independent experiments performed in triplicate, expressed as percentage of basal glucose oxidation. *, p < 0.05, and **, p < 0.01 versus 2.5 mM Glc; §, p < 0.05 versus control LacZ group at corresponding Glc.

 
Glucose Oxidation and Glucose Utilization in INS-1E Cells—[U-14C]Glucose oxidation was measured in cells over a 2-h stimulation period. In control cells, 15 mM glucose prompted 14CO2 production 5.1-fold (p < 0.01) versus 2.5 mM glucose. Overexpression of Aralar1 increased glucose oxidation (+33%, p < 0.05) at 15 mM glucose (Fig. 6C) compared with control cells. In AdCA-Aralar1-treated INS-1E cells, glucose utilization was not modified as measured by 3H2O formation from D-[5-3H]glucose (Fig. 7A). This suggests that the glycolytic rate was unchanged by Aralar1 overexpression.



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  		FIG. 7.
Glucose utilization and lactate release in cells overexpressing Aralar1. A, glucose utilization was measured at the indicated glucose concentrations using D-[5-3H]glucose. Data are the mean ± S.E. of three independent experiments. B, immunoblot showing Aralar1 overexpression at 10 pfu/cell multiplicity of infection. C, lactate release over a 24-h period at 11.1 mM glucose in control and Aralar1 overexpressing cells. §, p < 0.05 versus control LacZ group.

 
Lactate Production from INS-1E Cells—Having established the action of AdCA-Aralar1 in increasing glucose-induced mitochondrial activation as well as glucose oxidation, we evaluated the effect of Aralar1 on lactate release and insulin secretion. INS-1E cells were maintained in culture for 3-4 days, infected with AdCA-LacZ and AdCA-Aralar1, and further cultured for 20 h prior to the experiments or control of Aralar1 expression (Fig. 7B). Lactate release in the medium (containing 11.1 mM glucose) over a 24-h period was reduced by 68% (p < 0.05) in cells overexpressing Aralar1 compared with control cells (Fig. 7C).

Effects of Aralar1 on Insulin Secretion in INS-1E Cells—Insulin secretion was determined over a stimulation period of 30 min (Fig. 8). In control INS-1E cells, 7.5 and 15 mM glucose caused 4.0- and 6.6-fold increases in insulin secretion, respectively, relative to a basal release at 2.5 mM glucose (p < 0.05). Transduction of INS-1E cells with the control virus AdCA-LacZ did not modify reported secretory responses of non-transduced INS-1E cells at the same glucose concentrations (14). Cells treated with AdCA-Aralar1 exhibited 4.3- and 9.6-fold secretory responses to 7.5 and 15 mM glucose, respectively, showing that overexpression of Aralar1 modified GSIS. Indeed, at 15 mM glucose, the secretory response was increased by 45% (p < 0.01) versus control cells (Fig. 8). Next, pyruvate was used as a mitochondrial substrate by-passing glycolysis and the NADH shuttle system. Pyruvate (1 mM) stimulated insulin secretion in control cells, but the effect was not modified by Aralar1 overexpression. Cellular insulin content was not affected by Aralar1 overexpression in INS-1E cells (2.53 ± 0.3 versus 2.40 ± 0.98 µg of insulin/106 cells for LacZ and Aralar1 overexpressing cells). These values are in the range of insulin content reported for non-infected INS-1E cells (14).



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  		FIG. 8.
Effects of Aralar1 overexpression on insulin secretion from INS-1E cells. INS-1E cells cultured in 24-well plates were infected over a 90-min period with either AdCA-Aralar1 or AdCA-LacZ adenovirus and assayed the next day. Following a preincubation period, cells were challenged for 30 min with 2.5, 7.5, and 15 mM glucose (Glc), or 1 mM pyruvate (pyr) (at 2.5 mM Glc). Values are the mean ± S.E. of 11 independent experiments. *, p < 0.05 and **, p < 0.01 versus 2.5 mM Glc; §, p < 0.01 versus control LacZ group at the corresponding Glc.

 
Effects of Aralar1 Overexpression on GSIS in Rat Islets—Rat islets were infected with AdCA-LacZ or AdCA-Aralar1 for 90 min and the following day the islets were either trypsinized and then fixed for immunolocalization studies or used directly for insulin secretion experiments. Immunolocalization was performed using anti-aralar1 and anti-insulin antibodies to estimate the efficiency of transduction. The immunolocalization of aralar1 and insulin showed that 50-80% of insulin-positive cells overexpressed Aralar1 (Fig. 9A). In parallel, insulin secretion studies were performed from islets transduced under the same conditions (Fig. 9B). Control islets responded to 16.7 mM glucose stimulation 4.8-fold (p < 0.01). In rat islets overexpressing Aralar1, the secretory response was increased by 61% (p < 0.05) versus corresponding control. Control secretory responses to non-nutrient secretagogue were performed using the Ca2+ raising agent glibenclamide. Insulin secretion in islets stimulated with the sulfonylurea glibenclamide (3.3-fold increase, n = 4, p < 0.05) was not significantly affected by Aralar1 overexpression.



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  		FIG. 9.
Aralar1 overexpressing and insulin secretion in rat islets. A, immunolocalization of Aralar1 and insulin in isolated rat islet cells transduced with control virus (AdCA-LacZ) or Aralar1 virus (AdCA-Aralar1). B, effects of glucose (Glc) on insulin secretion. Isolated and transduced rat islets were incubated in the presence of 2.8 or 16.7 mM glucose concentrations for 30 min. Data are mean of 8 independent experiments ± S.E., *, p < 0.001 versus corresponding basal 2.5 mM Glc; §, p < 0.05 versus LacZ 16.7 mM Glc.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
It is reported here that Aralar1 is the only aspartate-glutamate carrier isoform expressed in beta cells. The levels of aralar1 in INS-1E cells and sorted beta cells were similar, but rather low compared with its expression in the brain. The lack of citrin in rat islets, although present in pancreas (24), indicates that citrin expression is probably restricted to the exocrine pancreas. By investigating the effects of Aralar1 overexpression in INS-1E cells, our data show a direct relationship between changes in NAD(P)H levels, mitochondrial activation, and insulin secretion.

Activity of NADH shuttles may limit glycolysis (29, 30). Here, Aralar1 overexpression enhanced glucose evoked NAD(P)H generation, electron transport chain activity, and ATP generation, correlating with elevation of glucose oxidation, thereby demonstrating increased mitochondrial metabolism. These metabolic potentiations were accompanied by increased insulin secretion. Surprisingly, glucose utilization, reflecting glycolytic rate, was not changed by aralar1 overexpression. This unexpected absence of correlation between glucose oxidation and utilization is reminiscent of previous observations made under opposite conditions and lacking interpretation. Indeed, islets with impaired NADH shuttles exhibited decreased GSIS and glucose oxidation, but preserved glucose utilization (6). The paradox reported by Eto et al. (6) and commented by others (31) could now eventually be explained. Indeed, our data show that for a similar glycolytic rate glucose oxidation and lactate production can be modulated according to NADH shuttle capacity. Previous observations provided indirect evidence for preferential use of NADH shuttles over lactate dehydrogenase to oxidize glycolysis-derived NADH in beta cells (32). Accordingly, adenovirus-mediated lactate dehydrogenase overexpression does not affect lactate output nor GSIS in isolated islets, suggesting that efficient metabolic channeling of glucose-derived pyruvate to the mitochondria is chiefly controlled by shuttle activity (33). Moreover, in insulin secreting cells, enhanced lactate production was related to decreased insulin secretion without affecting glucose utilization (34). NMR studies, enabling tracking of metabolites generated by glucose, showed inverse correlation between lactate production and secretory responses to glucose according to beta cell lines (22, 35). Here, upon Aralar1 overexpression, lactate release was decreased at 11.1 mM glucose. This strongly suggests that at similar rates of pyruvate generation from glucose, the relative fraction of pyruvate being either oxidized in mitochondria or reduced to lactate, may vary according to NADH shuttle activity. Hence, the elevated glucose oxidation observed in Aralar1 overexpressing cells could be explained by increased provision of glucose-derived pyruvate into the mitochondria. In accordance with such interpretation, the proton gradient generated by NADH shuttles upon glucose stimulation has been shown to limit pyruvate entry into the mitochondria (10). Conversely, inhibitors of the malate-aspartate shuttle activity decrease both carbon flow through the tricarboxylic acid cycle and oxidative metabolism (36).

In the present study, when pyruvate was used as a fuel in place of glucose, Aralar1 overexpression did not modify mitochondrial hyperpolarization or insulin secretion. This shows that aralar1 primarily contributes upstream of mitochondria to efficiently couple glycolysis to mitochondrial metabolism.

Aralar1 overexpression in INS-1E cells increased glucose-induced mitochondrial hyperpolarization, NAD(P)H-levels, and insulin secretion. Cellular ATP levels at high glucose were higher in Aralar1 overexpressing cells. It has been shown that Aralar1 overexpression in Chinese hamster ovary cells potentiated the increase in mitochondrial ATP levels obtained after stimulation with Ca2+ mobilizing agents (37). In the present study, Ca2+ handling in the mitochondria was not affected by Aralar1 overexpression. The elevated mitochondrial membrane potential in Aralar1 overexpressing cells did not modify Ca2+ dynamics in mitochondria. Therefore, in these experimental conditions, [Ca2+]m did not account for the increase in tricarboxylic acid cycle activity, reflected by oxidative metabolism.

What are the factors responsible for the observed potentiation of glucose-evoked insulin release in Aralar1 overexpressing cells? Cytosolic [Ca2+] levels were not measured in the present study, although [Ca2+] elevations in the cytosol are well known to be translated into the mitochondrial matrix (38). According to the lack of effects of Aralar1 overexpression on [Ca2+]m, it is unlikely that cytosolic [Ca2+] changes were affected. Among the factors that have been proposed to date to participate in metabolism-secretion coupling, three of them measured here were elevated further upon glucose stimulation in Aralar1 overexpressing cells. All of them, apart or synergistically, could hypothetically explain potentiation of insulin release: (i) NADH was proposed as a coupling factor per se in glucose-stimulated insulin secretion using toadfish islets (39, 40). A direct effect of NADH was reported on the release of insulin from isolated secretory granules (41), NADH being possibly bound or taken up by granules (42). Cytosolic NADPH, formed by the pyruvate-citrate and the pyruvate-malate shuttles, is also a candidate coupling factor (9). (ii) ATP is primarily implicated in KATP-channel regulation (43). However, ATP is also necessary for secretory granule movement (44, 45) and therefore implicated in sustained insulin release. (iii) Glutamate was shown to directly trigger insulin exocytosis in conditions of permissive cytosolic [Ca2+] (23, 46), possibly by modifying secretory vesicles pH (47). Further investigations should specifically address the potential contribution of these factors in aralar1-dependent modulation of GSIS.

Taken together, our data show that adenoviral-mediated overexpression of Aralar1 in insulin-secreting cells, to levels comparable with the brain, increased glucose-induced mitochondrial activation. Consequently, aspartate-glutamate carrier capacity appears to set a limit for NADH shuttle function and mitochondrial metabolism. This is supported by the reported increase in mitochondrial activation correlating with an increase in glucose metabolism and insulin secretion.

Here, the importance of the malate-aspartate shuttle member aralar1 was investigated for the first time in beta cells. The results demonstrate the crucial role of the malate-aspartate NADH shuttle in glucose-stimulated insulin secretion. An up-regulation of the malate-aspartate shuttle has been described in some pathological situations (48, 49), including type 2 diabetes (50). This suggests a possible compensatory role of the malate-aspartate shuttle in conditions where other NADH shuttles exhibit reduced activity. Our data also confirm previous results pointing to the predominant role of the malateaspartate shuttle versus the glycerophosphate shuttle in the beta cell (51), as opposed to other tissues (52). Therefore, aralar1 influences glucose-stimulated insulin secretion and modulation of aralar1 activity might have therapeutic applications for the treatment of diabetes.


   FOOTNOTES
 
* This work was supported by Swiss National Science Foundation Grant 31-67023.01, the Roche Research Foundation, the European Foundation for the Study of Diabetes/Johnson & Johnson, and Dr. Max Cloetta Foundation (Zurich) (to P. M.), and the Spanish Ministerio de Ciencia y Tecnología (to J. S.) and the Fundación Ramón Areces to the Centro de Biología Molecular Severo Ochoa. This study was part of the Geneva Programme for Metabolic Disorders. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

|| To whom correspondence should be addressed: Dept. of Cell Physiology and Metabolism, University Medical Centre, 1 rue Michel-Servet, CH-1211 Geneva 4, Switzerland. Tel.: 41-22-379-55-54; Fax: 41-22-379-55-43; E-mail: Pierre.Maechler{at}medecine.unige.ch.

1 The abbreviations used are: GSIS, glucose-stimulated insulin secretion; pfu, plaque forming unit; [Ca2+]m, mitochondrial Ca2+ concentration; KRBH, Krebs-Ringer bicarbonate Hepes; FCCP, carbonyl cyanide p-trifluoromethoxyphenylhydrazone; FACS, fluorescence-activated cell sorter. Back


   ACKNOWLEDGMENTS
 
We thank Dr. P. Halban (Geneva) and his team for generous supply of sorted beta cells.



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