Becker T C, Noel R J, Johnson J H, Lynch R M, Hirose H, Tokuyama Y, Bell G I, Newgard C B
Gifford Laboratories for Diabetes Research, University of Texas Southwestern Medical Center, Dallas 75235, USA.
J Biol Chem. 1996 Jan 5;271(1):390-4. doi: 10.1074/jbc.271.1.390.
Glucose-stimulated insulin secretion is believed to require metabolism of the sugar via a high Km pathway in which glucokinase (hexokinase IV) is rate-limiting. In this study, we have used recombinant adenoviruses to overexpress the liver and islet isoforms of glucokinase as well as low Km hexokinase I in isolated rat islets of Langerhans. Glucose phosphorylating activity increased by up to 20-fold in extracts from islets treated with adenoviruses containing the cDNAs encoding either tissue isoform of glucokinase, but such cells exhibited no increase in 2- or 5-[3H]glucose usage, lactate production, glycogen content, or glucose oxidation. Furthermore, glucokinase overexpression enhanced insulin secretion in response to stimulatory glucose or glucose plus arginine by only 36-53% relative to control islets. In contrast to the minimal effects of overexpressed glucokinases, overexpression of hexokinase I caused a 2.5-4-fold enhancement in all metabolic parameters except glycogen content when measured at a basal glucose concentration (3 mM). Based on measurement of glucose phosphorylation in intact cells, overexpressed glucokinase is clearly active in a non-islet cell line (CV-1) but not within islet cells. That this result cannot be ascribed to the levels of glucokinase regulatory protein in islets is shown by direct measurement of its activity and mRNA. These data provide evidence for functional partitioning of glucokinase and hexokinase and suggest that overexpressed glucokinase must interact with factors found in limiting concentration in the islet cell in order to become activated and engage in productive metabolic signaling.
葡萄糖刺激的胰岛素分泌被认为需要通过一条高Km途径对糖进行代谢，其中葡萄糖激酶（己糖激酶IV）是限速酶。在本研究中，我们使用重组腺病毒在分离的大鼠胰岛中过表达葡萄糖激酶的肝脏和胰岛同工型以及低Km的己糖激酶I。在用含有编码葡萄糖激酶两种组织同工型cDNA的腺病毒处理的胰岛提取物中，葡萄糖磷酸化活性增加了高达20倍，但这些细胞在2-或5-[3H]葡萄糖利用、乳酸产生、糖原含量或葡萄糖氧化方面没有增加。此外，相对于对照胰岛，葡萄糖激酶过表达仅使对刺激性葡萄糖或葡萄糖加精氨酸的胰岛素分泌增强了36%-53%。与过表达葡萄糖激酶的最小作用相反，在基础葡萄糖浓度（3 mM）下测量时，己糖激酶I的过表达导致除糖原含量外的所有代谢参数提高了2.5-4倍。基于对完整细胞中葡萄糖磷酸化的测量，过表达的葡萄糖激酶在非胰岛细胞系（CV-1）中明显有活性，但在胰岛细胞内无活性。通过直接测量其活性和mRNA表明，该结果不能归因于胰岛中葡萄糖激酶调节蛋白的水平。这些数据为葡萄糖激酶和己糖激酶的功能分区提供了证据，并表明过表达的葡萄糖激酶必须与胰岛细胞中以限制浓度存在的因子相互作用才能被激活并参与有效的代谢信号传导。
