Secretory Granule-mediated Co-secretion ofl-Glutamate and Glucagon Triggers Glutamatergic Signal Transmission in Islets of Langerhans

Mitsuko Hayashi,Hiroshi Yamada,Shunsuke Uehara,Riyo Morimoto,Akiko Muroyama,Shouki Yatsushiro,Jun Takeda,Akitsugu Yamamoto,Yoshinori Moriyama

doi:10.1074/jbc.m206758200

Abstract

L-Glutamate is believed to function as an intercellular transmitter in the islets of Langerhans. However, critical issues, i.e. where, when and how L-glutamate appears, and what happens upon stimulation of glutamate receptors in the islets, remain unresolved. Vesicular glutamate transporter 2 (VGLUT2), an isoform of the vesicular glutamate transporter essential for neuronal storage of L-glutamate, is expressed in alpha cells (Hayashi, M., Otsuka, M., Morimoto, R., Hirota, S., Yatsushiro, S., Takeda, J., Yamamoto, A., and Moriyama, Y. (2001) J. Biol. Chem. 276, 43400-43406). Here we show that VGLUT2 is specifically localized in glucagon-containing secretory granules but not in synaptic-like microvesicles in alpha TC6 cells, clonal alpha cells, and islet alpha cells. VGLUT1, another VGLUT isoform, is also expressed and localized in secretory granules in alpha cells. Low glucose conditions triggered co-secretion of stoichiometric amounts of L-glutamate and glucagon from alpha TC6 cells and isolated islets, which is dependent on temperature and Ca(2+) and inhibited by phentolamine. Similar co-secretion of L-glutamate and glucagon from islets was observed upon stimulation of beta-adrenergic receptors with isoproterenol. Under low glucose conditions, stimulation of glutamate receptors facilitates secretion of gamma-aminobutyric acid from MIN6 m9, clonal beta cells, and isolated islets. These results indicate that co-secretion of L-glutamate and glucagon from alpha cells under low glucose conditions triggers GABA secretion from beta cells and defines the mode of action of L-glutamate as a regulatory molecule for the endocrine function. To our knowledge, this is the first example of secretory granule-mediated glutamatergic signal transmission.

Highlights

L-Glutamate is believed to function as an intercellular transmitter in the islets of Langerhans
Indirect immunofluorescence microscopy indicated that Vesicular glutamate transporter 2 (VGLUT2) is colocalized with glucagon, a marker of secretory granules, but not with synaptophysin, a marker of synaptic vesicles or synaptic-like microvesicles (SLMVs), in ␣TC6 cells (Fig. 1, A and B)
In the islets of Langerhans, triple labeling for immunoelectronmicroscopy indicated that 15 nm of gold particles for VGLUT2 were associated with the membranes of secretory granules (Fig. 1, D and E, and insets, arrowheads) that had been labeled with 5 nm of gold particles for glucagon (Fig. 1, D and E, and insets, arrows)

Summary

Introduction

L-Glutamate is believed to function as an intercellular transmitter in the islets of Langerhans. Low glucose conditions triggered co-secretion of stoichiometric amounts of L-glutamate and glucagon from ␣TC6 cells and isolated islets, which is dependent on temperature and Ca2؉ and inhibited by phentolamine. Stimulation of glutamate receptors facilitates secretion of ␥-aminobutyric acid from MIN6 m9, clonal ␤ cells, and isolated islets.

Results

Conclusion