Submitted on December 13, 2002
Accepted on March 10, 2003
Glucose Metabolism and Glutamate Analogue Acutely Alkalinize pH of Insulin Secretory Vesicles of Pancreatic 
Cells
Kazuhiro Eto1, Tokuyuki Yamashita2, Kenzo Hirose3, Yoshiharu Tsubamoto2, Edward K. Ainscow4, Guy A. Rutter4, Satoshi Kimura2, Mitsuhiko Noda5, Masamitsu Iino3, and Takashi Kadowaki1*
1 Department of Metabolic Diseases, University of Tokyo, Graduate School of Medicine, Tokyo, Japan; CREST of Japan Science and Technology Corporation, Kawaguchi, Japan
2 Department of Metabolic Diseases, University of Tokyo, Graduate School of Medicine, Tokyo, Japan
3 Department of Pharmacology, University of Tokyo, Graduate School of Medicine, Tokyo, Japan
4 Department of Biochemistry, University of Bristol, School of Medical Sciences, Bristol, United Kingdom
5 CREST of Japan Science and Technology Corporation, Kawaguchi, Japan; Asahi Life Foundation, Institute for Diabetes Care and Research, Tokyo, Japan
* To whom correspondence should be addressed. E-mail: kadowaki-3im{at}h.u-tokyo.ac.jp.
We studied acute changes of secretory vesicle pH in pancreatic 
cells with a fluorescent pH indicator lysosensor green DND-189. The fluorescence was decreased by 0.66 ± 0.10% at 149 ± 16 sec with 22.2 mM glucose stimulation, indicating that vesicular pH was alkalinized by ~0.016 unit. Glucose-responsive pH increase was observed when cytosolic Ca2+ influx was blocked but disappeared when an inhibitor of glycolysis or mitochondrial ATP synthase was present. Glutamate dimethylester (GME), a plasma membrane-permeable analogue of glutamate, potentiated glucose-stimulated insulin secretion at 5 mM, without changing cellular ATP content or cytosolic [Ca2+]. Application of GME at basal glucose concentration decreased DND-189 fluorescence by 0.83 ± 0.19% at 38 ± 2 sec. These results indicated that the acutely alkalinizing effect of glucose on cell secretory vesicle pH was dependent on glucose metabolism but independent of modulations of cytosolic [Ca2+]. Moreover, glutamate derived from glucose may be one of the mediators of this alkalinizing effect of glucose, which may potentially be relevant to the alteration of secretory function by glutamate.
