Vesicular Inhibitory Amino Acid Transporter Is Present in Glucagon-Containing Secretory Granules in αTC6 Cells, Mouse Clonal α-Cells, and α-Cells of Islets of Langerhans

Abstract

Islets of Langerhans contain ␥-aminobutyrate (GABA) and may use it as an intercellular transmitter. In ␤-cells, GABA is stored in synaptic-like microvesicles and secreted through Ca 2؉ -dependent exocytosis. Vesicular inhibitory amino acid transporter (VIAAT), which is responsible for the storage of GABA and glycine in neuronal synaptic vesicles, is believed to be responsible for the storage and secretion of GABA in ␤-cells. . Islets of Langerhans contain GABA, the enzymes that catalyze its biosynthesis (GAD) and metabolism (GABA transaminase), and GABA receptors at concentrations comparable with levels in the central nervous system (3-7). In particular, ␤-cells contain a much higher concentration of GABA than other types of islet cells. In ␤-cells, GABA is stored in synaptic-like microvesicles (SLMVs), endocrine counterparts of neuronal synaptic vesicles, but not in insulin granules (8). ATP-dependent vesicular GABA transport activity has been detected in isolated SLMVs (9). Furthermore, ␤-cells and MIN6 cells (clonal ␤-cells) secrete GABA through Ca 2ϩ -dependent and -independent pathways (10 -14). Exogenous GABA has been shown to inhibit glucagon secretion by way of GABAA receptors on ␣-cells (15-17). These results indicate that ␤-cells are the major sites of GABA signal appearance in the islets and that GABA functions as a paracrine-like intercellular chemical transmitter that regulates islet function.Vesicular inhibitory amino acid transporter (VIAAT) is responsible for the storage of GABA and glycine in synaptic vesicles through active transport at the expense of ATP hydrolysis by a vacuolar proton pump (18,19). The cDNA encoding VIAAT has been cloned, and its primary amino acid sequence has been determined (19). Because VIAAT is a potential probe for GABA signal appearance in neurons, 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, immunohistochemical evidence obtained with antibodies against either the COOH-terminal or NH 2 -terminal region of VIAAT indicated that VIAAT immunoreactivity is predominantly present in nonϪ␤-cells and probably ␣-cells, although the precise localization of VIAAT at the cellular or subcellular level has not yet been determined (20).L-glutamate may also function as an intercellular messenger in the islets of Langerhans (21). We have shown that vesicular glutamate transporter (VGLUT), which is responsible for the vesicular storage of L-glutamate and thus a potential probe for L-glutamate signal appearance, is expressed in ␣-cells and is specifically associated with GABA, ␥-aminobutyrate; SLMV, synaptic-like microvesicle; VGLUT, vesicular glutamate transporter; VIAAT, vesicular inhibitory amino acid transporter.

Identification and characterization of a novel isoform of the vesicular γ-aminobutyric acid transporter with glucose-regulated expression in rat islets

“…Results published subsequently have been conflicting (Hayashi et al 2003a, Gammelsaeter et al 2004. The immunostaining results shown herein provide further evidence of cell expression of VIAAT.…”

“…Lower levels of VIAAT were also detected in the rat islet cells, which contain GAD65 (Chessler et al 2002). Hayashi et al (2003a) subsequently confirmed the presence of VIAAT in the rat islet mantle, detecting none in the islet cells. Unexpectedly, VIAAT was found to localize to the glucagon-containing secretory granules rather than to the synaptic-like microvesicles also present in islet cells.…”

“…Conflicting results have been published concerning whether VIAAT is expressed in cells (Chessler et al 2002, Hayashi et al 2003a, Gammelsaeter et al 2004. Immunoblot analysis showed that the rat cell line Figure 5 Effects of glucose concentration on islet VIAAT content.…”

“…This form was predominant in islet cell extracts and has an apparent molecular weight of 52·5 kDa (V52). Hayashi et al (2003a), using a novel antibody that may have a relatively lower affinity for V57, observed only an 52 kDa VIAAT band in rat islet extracts and TC6 cells, while both V57 and V52 were detected in brain. The 52 kDa form of VIAAT is present in low abundance in rat and human brain tissue extracts and is detected, as well, in purified synaptic vesicles and rat pineal gland extracts (Takamori et al 2000, Chessler et al 2002, Jin et al 2003, Echigo & Moriyama 2004.…”

Physiology of the pancreatic α-cell and glucagon secretion: role in glucose homeostasis and diabetes

“…Similar conclusions were obtained in mouse islets and clonal aTC1-9 cells (Xu et al 2006, Bailey et al 2007). The neurotransmitter L-glutamate also accumulates in the a-cell secretory granules because of vesicular glutamate transporters 1 and 2 found in these cells (Hayashi et al 2003a). In low-glucose conditions, L-glutamate is cosecreted with glucagon, triggering GABA release from neighbouring b-cells and, subsequently, inhibiting the a-cell function as previously described (Hayashi et al 2003b).…”