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Cellular and molecular mechanisms of the UCPs-dependent metabolic control and its patho-physiological relevance

UCPs依赖性代谢控制的细胞和分子机制及其病理生理学相关性

基本信息

批准号：
15580252
负责人：
KIMURA Kazuhiro
金额：
$ 2.37万
依托单位：
HOKKAIDO UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2003
资助国家：
日本
起止时间：
2003 至 2004
项目状态：
已结题

项目摘要

Uncoupling proteins (UCPs) are inner mitochondrial membrane proteins that mediate dissipation of the mitochondrial membrane potential as heat rather than ATP synthesis. To clarify the cellular and molecular mechanisms of the UCPs-dependent metabolic control, especially in relation with cellular ATP levels, we firstly investigated tissue uptake of 2-deoxyglucose (2-DG) in UCP1 -knockout (KO) mice in vivo. In wild-type (WT) mice, administration of norepinephrine (NE) accelerated the disappearance of plasma 2-DG and increased 2-DG uptake into brown adipose tissue (BAT) exclusively expressing UCP1 and heart without any rise of plasma insulin level. In UCP1-KO mice, the stimulatory effect of NE on 2-DG uptake into BAT, but not into heart, disappeared completely. Insulin administration increased 2-DG uptake into BAT and also heart similarly in WT and UCP1 -KO mice. NE also increased the ratio of AMP/ATP and the activity of AMP-activated protein kinase (AMPK) in BAT of WT, but not of UCP1-KO, mice. These results suggest that the sympathetically stimulated glucose utilization in BAT is due to the serial activation of UCP1 and AMPK. We next established mammalian cells expressing functional UCP1 in a mitochondrial fraction by using Stratagene's LacSwitch II Inducible Mammalian Expression System and Hep3B human hepatocellular carcinoma that dose not express any endogenous UCP isoforms. After developing respective cells expressing functional UCP2 and UCP3, they would be useful for the studies on the mechanisms of the UCP-dependent metabolic control in an isoform-specific manner.

解偶联蛋白（UCPs）是线粒体内膜蛋白，介导线粒体膜电位的耗散作为热量，而不是ATP的合成。为了阐明UCPs依赖性代谢调控的细胞和分子机制，特别是与细胞ATP水平的关系，我们首先研究了UCP 1基因敲除（KO）小鼠体内组织摄取2-脱氧葡萄糖（2-DG）的情况。在野生型（WT）小鼠，去甲肾上腺素（NE）的管理加速血浆2-DG的消失，并增加2-DG摄取到棕色脂肪组织（BAT）只表达UCP 1和心脏没有任何血浆胰岛素水平的上升。在UCP 1-KO小鼠中，NE对BAT摄取2-DG的刺激作用完全消失，但对心脏摄取2-DG的刺激作用不明显。在WT和UCP 1-KO小鼠中，胰岛素给药增加了BAT和心脏的2-DG摄取。NE还增加WT小鼠BAT中AMP/ATP比值和AMP活化蛋白激酶（AMPK）活性，但不增加UCP 1-KO小鼠BAT中AMP/ATP比值和AMPK活性。这些结果表明，交感神经刺激BAT的葡萄糖利用是由于UCP 1和AMPK的串行激活。我们接下来通过使用Stratagene的LacSwitch II诱导性哺乳动物表达系统和不表达任何内源性UCP同种型的Hep 3B人肝细胞癌建立了在线粒体部分中表达功能性UCP 1的哺乳动物细胞。在开发表达功能性UCP 2和UCP 3的相应细胞后，它们将用于以异构体特异性方式研究UCP-dependent代谢控制机制。