Positional cloning of carnitine transporter gene and its contribution on hypoglycemia.

肉碱转运蛋白基因的定位克隆及其对低血糖的贡献。

基本信息

批准号：
10470232
负责人：
KUWAJIMA Masamichi
金额：
$ 8.19万
依托单位：
The University of Tokushima
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
1998
资助国家：
日本
起止时间：
1998 至 1999
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-10470232/
关键词：
carnitine carnitine transporter hypoglycemia JVS mouse octn2 lactate pyruvate NaィイD1+ィエD1 dependency 心筋細胞 OCTN^2 ポジショナルクローニング β-酸化 JVS マウスカルニチントランスポーター

项目摘要

Juvenile Visceral Steatosis (JVS) mouse, which we reported in 1991, serves as an animal model of primary carnitine deficiency. Because this mouse is suffered from severe hypoglycemia, we analyzed the cause and mechanism The summary of the results is as follows ;1. Assay of carnitine transport activityWe have conducted a kinetic analysis using fibroblasts derived from normal, heterozygous, and homozygous JVS mice and found that the high-affinity carnitine transporter, which shows NaィイD1+ィエD1 dependency, is defective in homozygous JVS mice. Moreover, a gene dose-dependent decrease of carnitine transport activity was found in heterozygous JVS mice.2. Analysis of candidate geneAs a human OCTN2 gene encoding a sodium-dependent carnitine cotranspoter was isolated, we isolated the mouse octn2 gene and screened for its mutation in the JVS mouse. DNA sequencing analysis disclosed a missense mutation from CTG (Leu) to CGG (Arg) at codon 352 located within the sixth transmembrane domain of octn2. This amino acid replacement possibly causes the conformational change of the protein that leads to dysfunction of the gene product.3. Analysis of hypoglycemiaThe glucose level of JVS mouse at ad lib feeding was lower than that of normal control. After the prolonged starvation, the glucose level of JVS mouse was significantly decreased at 60 hours in comparison with normal control. The insulin level was not changed between JVS and normal control mouse. The levels of pyruvate and lactate were decreased. This decreased was thought as a cause of hypoglycemia.

我们于1991年报道了幼年内脏脂肪变性(JVS)小鼠作为原发性肉碱缺乏的动物模型，由于该小鼠患有严重的低血糖，我们分析了其原因和机制，结果总结如下： 1．肉碱转运活性的测定我们使用来自正常、杂合和纯合 JVS 小鼠的成纤维细胞进行了动力学分析，发现高亲和力肉碱纯合子JVS小鼠中显示NaiiD1+IeD1依赖性的转运蛋白存在缺陷，并且在杂合子JVS小鼠中发现了肉碱转运活性的基因剂量依赖性降低。2．候选基因分析作为编码钠依赖性的人OCTN2基因。分离出肉碱共转运蛋白后，我们分离了小鼠 octn2 基因并在 JVS 小鼠中筛选了其突变，DNA 测序分析发现了 CTG 的错义突变。 octn2 第六跨膜结构域内的密码子 352 处的 (Leu) 变为 CGG (Arg)。这种氨基酸替换可能导致蛋白质构象发生变化，从而导致基因产物功能障碍。 3. 低血糖分析 JVS 的血糖水平。自由进食的小鼠低于正常对照。长期饥饿后，60小时时JVS小鼠的血糖水平与正常对照相比显着降低，而胰岛素水平则没有变化。 JVS 和正常对照小鼠的丙酮酸和乳酸水平降低，这被认为是低血糖的原因。