Animal experiments were carried out according to international legislations (European Communities Council Directive of 24 November 1986 (86/609/EEC)) and Norwegian (1996-01-15 no 23) and British (UK Animals (Scientific Procedures) Act 1986) regulations. Formal approval to conduct the experiments was obtained from the animal subjects review boards of our institutions. Every effort was made to minimize suffering and the number of animals used. The animals were maintained under standard colony conditions in a temperature- (23±1°C) and humidity- (40%) controlled animal room under a 12 h light/dark cycle (7:00–19:00), with ad libitum access to food and water.
Polyclonal antibodies against L-glutamate and D-aspartate were raised in rabbits as originally described 
. The no. 607 glutamate and the no. 482 D-aspartate antisera have been extensively characterized 
. The rabbit anti-73kDa antibodies to EAAT2 were raised against the EAAT2 protein isolated from rat brain 
. A monoclonal EAAT2-antibody (9C4) binding to residues 518–525 (rat numbering) was produced in mouse 
, and a mouse monoclonal EAAT2 antibody was purchased from Novo Castra, UK. Anti-peptide antibodies against glutamate transporters were prepared as described 
. Different EAAT2 antibodies were produced in rabbits against residues 12–26 (anti-B12) 
, residues 518–536 (anti-B518) 
, residues 493–517 (anti-B493) 
, or residues 563–573 (anti-B563). These, as well as the EAAT1 
, EAAT3 
and EAAT4 
antibodies have been extensively charcterized. In addition, goat anti-EAAT2 antibodies (Santa Cruz Biotechnology, Santa Cruz, USA) were used in double labelling immunogold experiments.
Rabbit VGLUT1, VGLUT2 and VGLUT3 antibodies were gifts from R. H. Edwards (University of California, San Francisco, USA) 
. Guinea pig VGLUT3 antibody was from Chemicon (Temecula, CA, USA).
A monoclonal mouse anti-glucagon antibody and polyclonal guinea pig anti-insulin antibodies were from Sigma (St. Louis, MO, USA), a monoclonal synaptophysin anitbody (mouse) from Boehringer Mannheim Biochemica (Mannheim, Germany), and a monoclonal mouse anti-insulin was from Zymed Laboratories, Inc. (San Francisco, USA). The anti-Myc mouse monoclonal antibody was purified from the culture medium of myeloma cells (clone 9E10) using an IgG affinity column.
Tissue and cells
Adult Wistar rats were fed ad libitum and perfused 
through the left cardiac ventricle with one of the following mixtures of fixatives in 0.1 M sodium phosphate buffer (pH 7.4) (NaPi): 2.5% glutaraldehyde and 1% formaldehyde (amino acid immunoperoxidase and immunogold cytochemistry), 4% formaldehyde and 0.1% glutaraldehyde (protein immunogold cytochemistry and immunofluorescence) or 4% formaldehyde (immunofluorescence). Wild type and KO mice 
were fed ad libitum. After cervical dislocation (at P14), pancreatic and brain tissue from EAAT2 KO mice and wt littermates were immediately immersion fixed in a mixture of 4% formaldehyde and 0.1% glutaraldehyde. INS-1E and COS7 cells were cultured as previously described 
Cellular uptake sites for D-aspartate
Pancreatic slices (0.5 mm thick) from adult Wistar rats were prepared by hand cutting, before they were transferred to vials with Krebs solution (pH 7.4 with 140 mM NaCl, 15 mM NaPi, 5 mM KCl, 5 mM glucose, 1.2 mM CaCl2, 1.2 mM MgSO4) that were continuously gassed with O2 at 30°C. The slices were preincubated for 30 minutes before incubation in the presence and absence of 100 µM D-aspartate for 20 minutes.
D-asparate uptake in secretory granule
As D-aspartate is a substrate for EAAT2 
, but not for the VGLUTs 
, we used D-aspartate as a tracer to explore whether secretory granules in β-cells show EAAT2 uptake activity. INS-1E cells were permeabilized with 1.5 IU/ml streptolysin-O
and incubated with 0, 0.5, 0.3 and 1.0 mM D-aspartate in an intracellular medium containing 20 mM HEPES, pH 7.0, 140 mM KCl, 5 mM NaCl, 7 mM MgSO4
, 5 mM Na2
ATP, 10.2 mM EGTA and 0.1 mM CaCl2
for 7 min at 37°C.
Both the tissue slices and the INS-1E cells were fixed by either a mixture of 1% formaldehyde and 2.5% glutaraldehyde (for immunperoxidase and immunogold cytochemistry) or 4% formaldehyde and 0.5% glutaraldehyde (for immunfluorescence).
Immunofluorescence and immunoperoxidase
After fixation, pancreas and brain specimens were cryostat sectioned (5–15 µm) after cryoprotection in 30% sucrose at 4°C overnight. The sections were processed with the antibodies according to an immunofluorescence or a streptavidin-biotin-peroxidase method as previously described 
. The following antibody dilutions were used: rabbit anti-D-aspartate 482 (1
1000), anti-glutamate 607 (1
3000), and the anti-EAAT2 immunoglobulins anti-B12 (0.3–3 µg/ml), anti-B518 (1 µg/ml), anti-B493 (3 µg/ml), anti-B563 (0.3–1 µg/ml), mouse monoclonal EAAT2 antibody (1
30), rabbit anti-EAAT1 (1–6 µg/ml), anti-EAAT3 (1–6 µg/ml), anti-EAAT4 (1–6 µg/ml), anti-VGLUT1 (1
1000), anti-VGLUT2 (1
2000), guinea-pig anti-VGLUT2 (1
3000), anti-VGLUT3 (1
300), guinea-pig anti-VGLUT3 (1
1500), anti-insulin (1
100), anti-glucagon (1
2000), mouse anti-c-Myc (1
10). Primary antibodies were visualized with FITC-, Cy3- or Alexa (488 or 555)- fluorescent secondary antibodies.
In control immunofluorescence experiments in which the primary antibodies were omitted or substituted with preimmunesera, there was no significant staining of the tissue sections, but some experiments produced a diffuse fluorescence background that was subtracted from the labelling produced by the primary antibodies. In immunoperoxidase experiments, the glutamate and D-aspartate antisera were used with the addition of 0.2 mM complexes of glutaraldehyde/formaldehyde treated L-aspartate plus glutamine and L-aspartate, respectively. Addition of 0.3 mM complexes of L-glutamate and D-aspartate to the primary antibodies abolished the glutamate and D-aspartate labeling.
Sections were observed with a Zeiss Pascal laser scanning microscope or a Zeiss fluorescence microscope at wavelengths of 488 nm and 568 nm.
Immunogold staining and quantitation
Tissue for electron microscopy was embedded in Lowicryl HM20 according to a freeze substitution protocol, and immunogold labelling was performed as described 
. Ultrathin sections were processed with the primary antibodies at the following dilutions: anti-D-aspartate (1
300), anti-glutamate (1
3000), anti-EAAT2 B12 (6–40 µg/ml) anti-EAAT2 73 kDa (5 µg/ml) and anti-VGLUT3 from guinea pig (1
100). In single labelling experiments the primary antibodies were visualized with anti-rabbit or anti-guniea pig Ig specific secondary antibodies coupled to 15 nm gold particles (Europrobe, Lyon, France). In the double labelling experiments of KO and wt tissue, rabbit anti-glutamate (1
3000) and goat anti-EAAT2 antibodies (2 µg/ml) were visualized with goat anti-rabbit (10 nm gold particles (BBI, UK)) and donkey anti-goat (15 nm gold particles (Aurion, The Netherlands)) secondary antibodies. In each amino acid experiment ultrathin test sections 
were processed along with the islet sections, ascertaining the specificity of the labelling produced. When tested on ultrathin test sections containing known concentrations of glutamate and D-aspartate, both the glutamate and the D-aspartate antisera have been shown to produce a close to linear relation between the immunogold labelling densities and the concentrations of amino acids in the test sections 
The sections were viewed in a Philips CM10 electron microscope and α- and β-cells were identified on morphological grounds 
. Immunogold particles signalling glutamate, EAAT2 and VGLUT3, and grid points for area determination 
(see below) were recorded over secretory granules, SLMVs, plasma membranes, cytosol and mitochondria (background labelling). Particles and grid points were included in the SLMV when the particle centres or grid points were within a 30 nm distance from the outer border of the SLMV. This is a distance within which most of the immunogold particles are expected to occur 
. Similarily, EAAT2 and VGLUT3 gold particles with centres within 30 nm on either side of plasma membranes and membranes limiting secretory granules were recorded as belonging to these membranes (for details, see 
). The EAAT2 and VGLUT3 values over tissue membranes were corrected for background labelling (measured over mitochondrial outer membranes: average 3.7 and 1.3 gold particles/µm2
), while EAAT2 and VGLUT3 values over non-membrane compartments (cytosol, secretory granule cores) were corrected for background labelling measured over the mitochondrial matrix (average 4.9 and 2.7 gold particles/µm2
). Similar quantifications were done for gold particles representing glutamate. The glutamate values were corrected for background labelling over empty resin (average 1.7 gold particles/µm2
). The results were statistically evaluated by a non-parametric test (Mann-Whitney-U, two tails) (Statistica) and a paired-sample t-test (Excel).
Pancreatic islets were acutely isolated as described 
. Homogenates of isolated islets and COS-7 cells were separated by SDS-PAGE, blotted onto nitrocellulose, and proteins were immunodetected by primary antibodies, HRP-conjugated secondary antibodies and chemiluminescence reagents. COS-7 cell homogenates were also processed by secondary antibodies coupled to alkaline phosphatase (AP) and detected with AP-substrates.
Cloning of rat EAAT2 and generation of EAAT2 silencers
The open reading frame of rat EAAT2 was cloned by PCR from an INS-1E cDNA library in pBK vector (Stratagene). The PCR reaction was performed using the forward primer 5′_CGCGGATCCATGGCATCAACCGAGGGT_3′
and the reverse primer 5′_GCTCTAGATTATTTTTCACGTTTCCA _3′
. Both primers were designed according to the sequence of rat EAAT2 (NM_017215) 
. For sequencing and expression experiments the PCR products were inserted in the BamHI and Xba I cloning sites of myc-pcDNA3. Sequence analysis of the inserts was performed by MWG Biotech Company (Germany). Mammalian expression vectors directing the synthesis of small interfering RNAs (siRNAs) targeted against EAAT2 were prepared according to the method of Brummelkamp 
. Two cDNA fragments encoding a 19-nucleotide sequence derived from the target transcript and separated from its reverse 19-nucleotide complement by a short spacer were synthesized by MWG Biotech Company (Ebersberg, Germany), annealed and cloned in front of the H1-RNA promoter in the pSUPER vector16
nucleotides: 5′_GATCCCCCCGAGGGTGCCAACAATATTTCAAGAGAATATTGTTGGCACCC CGGTTTTTGGAAA_3′
and 5′_AGCTTTTCCAAAAACCGAGGGTGCCAACAATATTCTCTTGAAATATTGTT GCACCCTCGGGGG_3′
(SilA) and 5′_GATCCCCGATGCTCATCCTCCCTCTCTTCAAGAGAGAGAGGGAGGATGAGCATCTTTTTGGAAA _3′
(SilB). To test for the silencing activity of SilA and SilB, each plasmid was transiently co-transfected with the plasmid encoding rat myc-tagged EAAT2 in COS 7 cells. The expression of EAAT2 was assessed two days later by Western blotting.
INS-1E cells were transiently cotransfected with a plasmid encoding human growth hormone (hGH) and with plasmids encoding the siRNAs or EAAT2-c-Myc or VGLUT2-green fluorescent protein (GFP) constructs. Three days later, the cells were preincubated for 30 min in 20 mM HEPES, pH 7.4, 128 mM NaCl, 5 mM KCl, 1 mM MgCl2
, 2.7 mM CaCl2
and 2 mM glucose. The cells were then incubated for 45 min at 37°C either in the same buffer or stimulated with the 20 mM HEPES buffer containing 40 mM KCl, 20 mM glucose, 1 µM forskolin, and 1 mM 3-isobutyl-L-methylxantine (IBMX). In some experiments the cells were stimulated with either 20 mM glucose or 40 mM K+
alone. Exocytosis from transfected cells was determined by measuring by ELISA the amount of hGH (Roche, Rotkreuz, Switzerland) released into the medium during the incubation period, and expressed as an increase over basal (exocytosis
(stimulated−basal)/basal). The results were statistically evaluated by a two-sample t-test.
At the time of stimulation some cells were fixed with 4% formaldehyde and processed for confocal immunofluorescent microscopy with c-Myc and insulin antibodies.
Blood samples from EAAT2 KO mice and littermates (n
8 of each +/+ and −/−) were taken from the aorta after cervical dislocation and serum was prepared for ELISA measurements. Insulin was analysed using the Mouse Ultrasensitive Insulin ELISA kit (Mercodia, Uppsala, Sweden). Serum samples for each animal were analysed in triplicate and absorbance read at 450 nm in a Photometer for microtitration plate. Blood glucose concentrations from each sample were analysed by Accu-Chek compact (Roche Diagnostics GmbH, Mannheim, Germany).
PCR was conducted on RNA from isolated islets of Langerhans and brain tissue using Qiagen OneStep RT-PCR kit (35 3-step cycles). Primer control and -RT controls were included. Primers used were EAAT2 P1F (GAAAAAACCCATTCTCCTTTTT) and EAAT2 P1R (CCGACTGGGAGGACGAATC). Primers were from Oligold Eurogentec. 20 ng RNA template of each tissue was used per reaction. Electrophoresis was conducted on 2% agarose gels, samples diluted with Fermentas 6× Orange buffer.
Simulation of granule energetics
Details of these simulations are given in the Supplementary Material.