Stable overexpression of the glucose-6-phosphatase catalytic subunit attenuates glucose sensitivity of insulin secretion from a mouse pancreatic beta-cell line K Iizuka, H Nakajima, A Ono, K Okita, J-i Miyazaki 1 , J-i Miyagawa, M Namba, T Hanafusa and Y Matsuzawa Department of Internal Medicine and Molecular Science, Graduate School of Medicine (B5), Osaka University, 2–2 Yamada-oka, Suita, Osaka 565–0871, Japan 1 Department of Nutrition and Physiological Chemistry, Graduate School of Medicine (B5), Osaka University, 2–2 Yamada-oka, Suita, Osaka 565–0871, Japan (Requests for offprints should be addressed to H Nakajima; Email: hinakaji@med2.med.osaka-u.ac.jp) Abstract Glucose-6-phosphatase (G-6-Pase) hydrolyzes glucose-6- phosphate to glucose, reciprocal with the so-called glucose sensor, glucokinase, in pancreatic beta cells. To study the role of G-6-Pase in glucose-stimulated insulin secretion from beta cells, we have introduced rat G-6-Pase catalytic subunit cDNA and have established permanent clones with 3-, 7- and 24-fold G-6-Pase activity of the mouse beta-cell line, MIN6. In these clones, glucose usage and ATP production in the presence of 5·5 or 25mM glucose were reduced, and glucose-stimulated insulin secretion was decreased in proportion to the increased G-6-Pase activity. In addition, insulin secretory capacity in response to -fructose and pyruvate was unchanged; however, 25 mM glucose-stimulated insulin secretion and intra- cellular calcium response were completely inhibited. In the clone with 24-fold G-6-Pase activity, changes in intracellular NAD(P)H autofluorescence in response to 25 mM glucose were reduced, but the changes with 20 mM fructose and 20 mM pyruvate were not altered. Stable overexpression of G-6-Pase in beta cells resulted in attenuation of the overall glucose-stimulated metabolic responses corresponding to the degree of overexpression. This particular experimental manipulation shows that the possibility exists of modulating glucose-stimulated insulin release by thoroughly altering glucose cycling at the glucokinase/G-6-Pase step. Journal of Endocrinology (2000) 164, 307–314 Introduction Glucose-6-phosphatase (G-6-Pase; -glucose-6-phos- phate phosphohydrolase; EC 3·1·3·9) hydrolyzes glucose- 6-phosphate to glucose, which serves as a key enzyme of gluconeogenesis and glucose output from tissue. The physiological significance of G-6-Pase has been widely studied in liver (Nordlie et al. 1999). It has been questioned whether the existence of G-6- Pase activity is significant in normal and diabetic beta cells. Several investigations have addressed the existence of the enzymatic activity of G-6-Pase and/or mRNA expression of its catalytic subunit in the islets of Langerhans (Ashcroft & Randle 1968, Taljedal 1969, Tokuyama et al. 1995). If they are present, the glucokinase (GK)/G-6-Pase system can facilitate glucose cycling and its dysregulation may contribute to metabolic abnormality in diabetic islets. In some animal models of diabetes mellitus, attenuation of glucose-stimulated insulin secretion is associated with enhanced glucose cycling and increased G-6-Pase activity (Khan et al. 1989, 1990). In Zucker diabetic fatty (ZDF) rats, diabetic animals exhibit increased G-6-Pase mRNA expression by approximately 43% of the islets of pre- diabetic littermates. In the islets of diabetic male ZDF animals, fivefold G-6-Pase activity is shown to be relative to that of Zucker lean controls (Tokuyama et al. 1995). In normal mouse islets, G-6-Pase activity is reported to be comparable to that of the reciprocal glucose phosphor- ylation. It has also been reported that pancreatic islets from normal rats contain three times as much specific activity of G-6-Pase as liver in terms of microsomal protein (Waddel & Burchell 1988). The question here arises as to whether or not abnormal regulation of G-6-Pase activity of the islets per se may be responsible and sufficient to establish glucose insensitivity. The glucose-sensing ability of pancreatic beta cells has been considered to be represented predominantly by GK activity, which also helps to determine the overall glucose utilization rate. Thus, glucose flux can be a metabolic signal for insulin secretion from beta cells. As recently reported, the physiological amount of G-6-Pase activity does not play a significant role in glucose metabolism and 307 Journal of Endocrinology (2000) 164, 307–314 0022–0795/00/0164–307  2000 Society for Endocrinology Printed in Great Britain Online version via http://www.endocrinology.org Downloaded from Bioscientifica.com at 06/13/2020 11:13:17AM via free access