CLARIFICATION OF THE MECHANISM OF ANIT-DIABETIC EFFET BY THE ADMINISTRATION OF BIOTIN

生物素是一种水溶性维生素，是参与哺乳动物脂质、碳水化合物和氨基酸代谢的四种羧化酶的辅助因子。本课题组发现生物素具有改善糖尿病患者碳水化合物代谢的新功能。本研究旨在阐明生物素这种新作用的机制。首先，我们试图确定生物素在刺激胰腺B细胞分泌胰岛素中的作用部位。从大鼠胰腺中分离出朗格汉胰岛，并进行培养。用生物素或不加生物素刺激葡萄糖后，测定培养基中分泌的胰岛素。注射生物素后胰岛素分泌呈剂量依赖性增加（可达50μM）。当丙酮酸代替葡萄糖作为兴奋剂时观察到这种现象，因此生物素被认为参与了线粒体内糖酵解途径的反应。然后，利用不同标记位点的葡萄糖放射性同位素测量了TCA循环中磷酸化的能力。结果表明，以[U-^<14>C]葡萄糖为刺激物时，生物素可使CO_2生成量增加2倍，而以[6-^<14>C]葡萄糖为刺激物时，CO_2生成量没有变化。这些结果表明，生物素可以在不改变TCA循环旋转能力的情况下，通过显著增加葡萄糖氧化来刺激ATP合成。接下来，我们分析了生物素在肝脏磷酸烯醇丙酮酸羧激酶（PEPCK）基因表达调控中的作用。链脲佐菌素诱导的糖尿病大鼠的PEPCK mRNA较对照组降低了40%。因此，生物素具有胰岛素样作用，可以改善糖尿病动物的状况。我们试图利用具有胰岛素敏感性的大鼠肝癌细胞H4IIE来确定PEPCK基因的生物素调控位点。生物素处理3小时后，H4IIE细胞与胰岛素培养液孵育后，PEPCK mRNA升高。这表明生物素可以抑制该细胞系的胰岛素作用。少

Biotin is one of water-soluble vitamin that functions as a cofactor of four types of carboxylase involved in lipid, carbohydrate and amino acids metabolism in mammalian. Our group has shown that biotin has a novel function that improves carbohydrate metabolism in diabetes mellitus. This research aimed clarification of mechanism of this novel action of biotin.First of all, we tried to identify site to action of biotin in the stimulation of insulin secretion from the B cells of pancreas. The Langerhan's islet was isolated from rat pancreas, and cultured. Secreted insulin in cultured medium was measured after stimulation of glucose with or without biotin. The secretion of insulin has been increased by biotin administration dose-dependent manner (up to 50μM). This phenomenon was observed when pyruvate was used as a stimulant instead of glucose, therefore biotin was presumed to be participation in the reaction within mitochondria on the glycolytic pathway. Then, the ability of oxidative pho … More sphorylation at the TCA cycle was measured by using radioisotope of glucose with a different labeling site. As a result, the amount of ^<14>CO_2 formations was increased twice by biotin administration when [U-^<14>C] glucose was used as a stimulant, while it was not changed in case of [6-^<14>C] glucose administration. These results indicate that biotin could stimulate ATP synthesis through significant increase of glucose oxidation without the change of rotation ability in TCA cycle.Next, we analyzed the involvement of biotin in the regulation of liver phosphoenolpyruvate carboxykinase (PEPCK) gene expression. PEPCK mRNA was decreased by 40% compared with the control when the biotin was administered to the streptozotocin induced diabetic rat. Therefore biotin has insulin-like action and could improve the condition of diabetic animal. We tried to identify the site of biotin regulation of PEPCK gene using rat hepatoma cell H4IIE that has the insulin susceptibility. PEPCK mRNA increased in H4IIE cells 3 hours after treatment of biotin when cells were incubated with insulin medium. This result indicated that biotin could suppress the insulin action in this cell line. Less