Vesicular Inhibitory Amino Acid Transporter Is Present in Glucagon-Containing Secretory Granules in ␣TC6 Cells, Mouse Clonal ␣-Cells, and ␣-Cells of Islets of Langerhans Mitsuko Hayashi,1 Masato Otsuka,1 Riyo Morimoto,1 Akiko Muroyama,1 Shunsuke Uehara,1 Akitsugu Yamamoto,2 and Yoshinori Moriyama1 Islets of Langerhans contain ␥-aminobutyrate (GABA) and may use it as an intercellular transmitter. In ␤-cells, GABA is stored in synaptic-like microvesicles and se- -Aminobutyrate (GABA) is an inhibitory chemical creted through Ca2؉-dependent exocytosis. Vesicular ␥transmitter in the central and peripheral nervous inhibitory amino acid transporter (VIAAT), which is systems (1,2). Islets of Langerhans contain GABA, responsible for the storage of GABA and glycine in the enzymes that catalyze its biosynthesis (GAD) neuronal synaptic vesicles, is believed to be responsible and metabolism (GABA transaminase), and GABA recep- for the storage and secretion of GABA in ␤-cells. How- tors at concentrations comparable with levels in the ever, a recent study by Chessler et al. indicated that central nervous system (3–7). In particular, ␤-cells contain VIAAT is expressed in the mantle region of islets. In the a much higher concentration of GABA than other types of present study, we investigated the precise localization islet cells. In ␤-cells, GABA is stored in synaptic-like of VIAAT in rat islets of Langerhans and clonal islet microvesicles (SLMVs), endocrine counterparts of neuro- cells and found that it is present in ␣-cells, a minor nal synaptic vesicles, but not in insulin granules (8). population of F-cells and ␣TC6 cells, and clonal ␣-cells ␤ ␦ ATP-dependent vesicular GABA transport activity has but not in -cells, -cells, or MIN6 m9-cells (clonal ␤ ␤-cells). Combined biochemical, immunohistochemical, been detected in isolated SLMVs (9). Furthermore, -cells and electronmicroscopical evidence indicated that and MIN6 cells (clonal ␤-cells) secrete GABA through 2ϩ VIAAT is specifically localized with glucagon-containing Ca -dependent and -independent pathways (10–14). Ex- secretory granules in ␣-cells. ATP-dependent uptake of ogenous GABA has been shown to inhibit glucagon secre- radiolabeled GABA, which is energetically coupled with tion by way of GABAA receptors on ␣-cells (15–17). These a vacuolar proton pump, was detected in digitonin- results indicate that ␤-cells are the major sites of GABA permeabilized ␣TC6 cells as well as in MIN6 m9 cells. signal appearance in the islets and that GABA functions as These results demonstrate that functional neuronal a paracrine-like intercellular chemical transmitter that VIAAT is present in glucagon-containing secretory gran- regulates islet function. ules in ␣-cells and suggest that the ATP-dependent Vesicular inhibitory amino acid transporter (VIAAT) is ␤ GABA transporter in -cells is at least immunologically responsible for the storage of GABA and glycine in synap- distinct from VIAAT. Because glucagon-containing se- tic vesicles through active transport at the expense of ATP cretory granules also contain vesicular glutamate trans- hydrolysis by a vacuolar proton pump (18,19). The cDNA porter and store L-glutamate, as demonstrated by Hayashi et al., the present results suggest more complex encoding VIAAT has been cloned, and its primary amino features of the GABAergic phenotype of islets than acid sequence has been determined (19). Because VIAAT previously supposed. Diabetes 52:2066–2074, 2003 is a potential probe for GABA signal appearance in neu- rons, it can be supposed that islet cells, especially ␤-cells, express VIAAT. Very recently, it was found that islet cells actually contain VIAAT (20). Unexpectedly, however, im- munohistochemical evidence obtained with antibodies against either the COOH-terminal or NH2-terminal region of VIAAT indicated that VIAAT immunoreactivity is pre- From the 1Department of Biochemistry, Faculty of Pharmaceutical Sciences, dominantly present in nonϪ␤-cells and probably ␣-cells, Okayama University, Okayama, Japan; and the Department of Physiology, although the precise localization of VIAAT at the cellular Kansai Medical University, Osaka, Japan. Address correspondence and reprint requests to Yoshinori Moriyama, or subcellular level has not yet been determined (20). Okayama University, Department of Biochemistry, Faculty of Pharmaceutical L-glutamate may also function as an intercellular mes- Sciences, Okayama 700-8350, Japan. E-mail: [email protected]. okayama-u.ac.jp. senger in the islets of Langerhans (21). We have shown Received for publication 27 November 2002 and accepted in revised form 16 that vesicular glutamate transporter (VGLUT), which is May 2003. responsible for the vesicular storage of L-glutamate and GABA, ␥-aminobutyrate; SLMV, synaptic-like microvesicle; VGLUT, vesicu- lar glutamate transporter; VIAAT, vesicular inhibitory amino acid transporter. thus a potential probe for L-glutamate signal appearance, is © 2003 by the American Diabetes Association. expressed in ␣-cells and is specifically associated with 2066 DIABETES, VOL. 52, AUGUST 2003 M. HAYASHI AND ASSOCIATES glucagon-containing secretory granules (21,22). In fact, RT-PCR. Total RNAs extracted from isolated islets and ␣TC6 cells (1 ␮g) were transcribed into cDNA in a final volume of 20 ␮l reaction buffer ␣-cells cosecrete stoichiometric amounts of L-glutamate ␤ comprising 0.2 mmol/l each dNTP, 10 mmol/l dithiothreitol, 25 pmol random and glucagon upon stimulation by either -aderenergic octamers, and 200 units Molony murine leukemia virus reverse transcriptase receptors or low-glucose conditions (22). Thus, it is of (Amersham). After a 1-h incubation at 42°C, the reaction was terminated by interest to determine whether ␣-cells express VIAAT; if so, heating at 90°C for 5 min. For PCR amplification, the 10-fold diluted synthe- the subcellular localization of VIAAT is of special interest sized cDNA solution was added to the reaction buffer comprising 0.6 mmol/l total dNTP (150 ␮mol/l each dNTP), 25 pmol primers, and 1.5 units Ampli with reference to the localization of VGLUT. Taq-Gold DNA polymerase (Perkin Elmer). Thirty-five temperature cycles In this study, we found that functional VIAAT is ex- were conducted as follows: denaturation at 94°C for 30 s, annealing at 54°C for pressed and localized with glucagon-containing secretory 30 s, and extension at 72°C for 1 min. The amplification products were granules in ␣- and ␣TC6 cells but not in ␤-cells, ␦-cells, or analyzed by polyacrylamide gel electrophoresis. The sequences of the oligo- nucleotides used as primers were based on the published sequences of a rat MIN6 cells. VIAAT and VGLUT are coexpressed and colo- VIAAT-specific sense primer, 5Ј-GAGACATTCATTATCAGCGCGG-3Ј (bases calized in ␣-cells. It is suggested that vesicular GABA 328–349) and antisense primer, 5Ј-CCATCTTCGTTCTCCTCGTACA-3Ј (bases transporter immunologically distinct from VIAAT is 595–616) (19), or a mouse VIAAT specific sense primer, 5Ј-CTGCCCTCTCTC present in ␤-cells, and therefore the mode of action of GAAGGCAAC-3Ј (bases 1,008–1,028) and antisense primer, 5Ј-GGATAGGA CAGCAGCGCCTTG-3Ј (25). DNA sequencing was performed by the chain- GABA in the islets of Langerhans is more complex than termination method. previously supposed. ATP-dependent uptake of radiolabeled GABA. The previously published (21) method was used with slight modification. Cultured cells (4.0 ϫ 105 cells/35-mm dish) were washed with 1 ml buffer comprising 20 mmol/l MOPS-Tris (pH. 7.0), 0.3 mol/l sucrose, 0.1 mol/l KCl, and 2 mmol/l Mg acetate. RESEARCH DESIGN AND METHODS Then, the cells were permeabilized with digitonin at 10 ␮mol/l for 10 min at Animals and cell cultures. Wistar male rats at 6–7 postnatal weeks were 37°C. The medium was then replaced with fresh buffer containing ATP-Tris used in this study. ␣TC6, MIN6 m9, PC12, and COS7 cells were cultured as (pH. 7.0) at 2 mmol/l. In some experiments, reagents at the indicated described (21–24). concentrations were also included in the assay medium (see the legend to Fig. Preparation of anti-VIAAT antibodies. The DNA fragment encoding the 3). Then, GABA uptake was started by the addition of radiolabeled GABA (2.5 seventh transmembrane-loop region of VIAAT (236 bp), which corresponds to ␮Ci at 0.1 mmol/l; specific activity, 60 Ci/mmol; New England Nuclear) at D364-C437 (19), was amplified by PCR and then cloned into the EcoRI and 37°C. After a 15-min incubation, uptake was terminated by washing the cells XhoI sites of expression vector pGEX4T-2 (Amersham Pharmacia Biotech) to twice with 1 ml ice-cold 20 mmol/l MOPS-Tris (pH. 7.0) containing 0.3 mol/l obtain a GST fusion-expressing plasmid. The plasmid was transformed to sucrose. The radioactivity in the culture medium and cell lysate was then Escherichia coli BL21, and the resultant transformant was cultured and counted with a liquid scintillation counter. In some experiments, an extract of harvested after induction with 1 mmol/l isopropyl 1-thio-␤-D-galactoside for cell lysate was applied on a CAPCELL PAK C18 column (2.0 ϫ 250 mm; 3h.TheE. coli cells were suspended in 50 mmol/l Tris-HCl (pH 8.0) 50 mmol/l Siseido), and then the content of GABA was determined by high-performance NaCl, 1 mmol/l EDTA, and 1 mmol/l dithiothreitol and were sonicated. The liquid chromatography and amperometric detection, as previously described lysate was centrifuged, and the resultant supernatant was applied toa1ml (12). Degradation of GABA was negligible (Ͻ3%) under the assay conditions glutathione-Sepharose 4B column. The GST fusion proteins were eluted with used. 16 mmol/l reduced glutathione in buffer A. The GST fusion protein was injected twice at 2-week intervals into a rabbit with complete adjuvant and then with incomplete adjuvant. RESULTS Western blotting. Samples were denatured with SDS sample buffer contain- ing 1% SDS and 10% ␤-mercaptoethanol, and then Western analysis was VIAAT is localized in glucagon-containing secretory conducted as described (22). granules. As the first step of this study, we raised Immunohistochemistry.