Piston D W, Knobel S M, Postic C, Shelton K D, Magnuson M A
Department of Molecular Physiology and Biophysics, Vanderbilt University, Nashville, Tennessee 37232, USA.
J Biol Chem. 1999 Jan 8;274(2):1000-4. doi: 10.1074/jbc.274.2.1000.
The relationship between glucokinase (GK) and glucose-stimulated metabolism, and the potential for metabolic coupling between beta cells, was examined in isolated mouse islets by using a recombinant adenovirus that expresses Cre recombinase (AdenoCre) to inactivate a conditional GK gene allele (gklox). Analysis of AdenoCre-treated islets indicated that the gklox allele in approximately 30% of islet cells was converted to a nonexpressing variant (gkdel). This resulted in a heterogeneous population of beta cells where GK was absent in some cells. Quantitative two-photon excitation imaging of NAD(P)H autofluorescence was then used to measure glucose-stimulated metabolic responses of individual islet beta cells from gklox/lox mice. In AdenoCre-infected islets, approximately one-third of the beta cells showed markedly lower NAD(P)H responses. These cells also exhibited glucose dose responses consistent with the loss of GK. Glucose dose responses of the low-responding cells were not sigmoidal and reached a maximum at approximately 5 mM glucose. In contrast, the normal response cells showed a sigmoidal response with an KcatS0.5 of approximately 8 mM. These data provide direct evidence that GK is essential for glucose-stimulated metabolic responses in beta cells within intact islets and that intercellular coupling within the islet plays little or no role in glucose-stimulated metabolic responses.
通过使用表达Cre重组酶的重组腺病毒（腺病毒Cre）来使条件性葡萄糖激酶（GK）基因等位基因（gklox）失活，在分离的小鼠胰岛中研究了GK与葡萄糖刺激的代谢之间的关系以及β细胞之间代谢偶联的可能性。对经腺病毒Cre处理的胰岛的分析表明，约30%的胰岛细胞中的gklox等位基因转变为不表达变体（gkdel）。这导致了β细胞群体的异质性，其中一些细胞中不存在GK。然后，利用NAD(P)H自发荧光的定量双光子激发成像来测量来自gklox/lox小鼠的单个胰岛β细胞的葡萄糖刺激的代谢反应。在感染腺病毒Cre的胰岛中，约三分之一的β细胞显示出明显较低的NAD(P)H反应。这些细胞还表现出与GK缺失一致的葡萄糖剂量反应。低反应性细胞的葡萄糖剂量反应不是S形的，在约5 mM葡萄糖时达到最大值。相比之下，正常反应细胞显示出S形反应，KcatS0.5约为8 mM。这些数据提供了直接证据，表明GK对于完整胰岛内β细胞中葡萄糖刺激的代谢反应至关重要，并且胰岛内的细胞间偶联在葡萄糖刺激的代谢反应中几乎不起作用。
