YAMASAKI Tomoyuki Osaka University Medical School, Assistant Proffessor, 医学部, 助手 (00303975)
KITOJIMA Koichi Osaka University Graduate School of Medicine, Assistant Proffessor, 医学系研究科, 助手 (00314310)
NAKAJIMA Hiromu Osaka University Graduate School of Medicine, Assistant Proffessor, 医学系研究科, 助手 (50252680)
|Budget Amount *help
¥1,000,000 (Direct Cost : ¥1,000,000)
Fiscal Year 1999 : ¥1,000,000 (Direct Cost : ¥1,000,000)
Glucose-6-phosphatase (G-6-Pasc) hydrolyzes glucose-6-phosphate to glucose, reciprocal to 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 the rat G-6-Pase catalytic subunit cDNA and have established permanent clones with 3-, 7-, and 24-fold G-6-Pase activity of mouse beta cell line MIN6. In these clones, glucose usage and ATP production in the presence of 5.5 or 25 mM glucose were reduced, and glucose-stimulated insulin secretion was decreased proportionally to the increased G-6-Pase activity. In addition, insulin secretory capacity in response to D-fructose and pyruvate was unchanged, however, 25 mM glucose-stimulated insulin secretion and intracellular calcium response were completely inhibited. In the clone with 24-fold G-6-Pasc activity, changes in intracellular NAD(P)H autofluorescence in response to 25 mM glucose were reduced, but the changes with 20 mM f
ructose and 20 mM pyruvatc were not altered. Stable overexpression of G-6-Pasc in beta cells resulted in the 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 the glucose cycling at the GK/G-6-Pase step.
Moreover, to analyze the molecular mechanisms of lipotoxicity, we here studied the impact of FFA on glucose and lipid metabolism in pancreatic beta cells with special reference to insulin secretion. Pancreatic beta cell line MIN6 was exposed to various concentrations of palmitate for 3 days. Glucose-stimulated insulin secretion and insulin content were decreased corresponding to the concentration of the palmitate exposed. Glycolytic flux and ATP synthesis was unchanged, but pyruvate flux into mitochondria was decreased. Pyruvate carboxylase was decreased at the protein level, which was restored by the removal of palmitate or the inhibition of beta oxidation.
Intracellular content of triglyceride and FFA were elevated, beta oxidation was increased, and de novo lipogenesis from glucose was decreased. Though NADPH content was decreased, malic enzyme activity was unaffected. The malic enzyme inhibitor alone inhibited insulin response to glucose. From these results, we conclude that the decreased pyruvate flux into mitochondria would play a critical role in the inhibition of glucose stimulated insulin secretion by long term exposure of FFA. Less