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（UCP）是内部线粒体膜蛋白，可将线粒体膜电位的耗散作为热而不是ATP合成。为了阐明UCPS依赖性代谢控制的细胞和分子机制，尤其是与细胞ATP水平有关，我们首先研究了VIVO中UCP1-KNOCKOUT（KO）小鼠的2-脱氧葡萄糖（2-DG）的组织吸收。在野生型（WT）小鼠中，施用去甲肾上腺素（NE）加速了血浆2-DG的消失，并增加了2-DG摄取到棕色脂肪组织（BAT）中，专门表达UCP1和心脏，而没有血浆胰岛素水平上升。在UCP1-KO小鼠中，NE对2-DG摄取BAT的刺激作用，而不是进入心脏，完全消失了。在WT和UCP1 -KO小鼠中，胰岛素给药增加了2 -DG摄取BAT，心脏也同样增加了心脏。 NE还增加了AMP/ATP的比率和在WT蝙蝠中的AMP激活蛋白激酶（AMPK）的活性，而不是UCP1-KO，小鼠。这些结果表明，BAT中刺激的葡萄糖利用归因于UCP1和AMPK的序列激活。接下来，我们通过使用Stratagene的Lacswitch II诱导型哺乳动物表达系统和HEHP3B人肝细胞癌来建立在线粒体分数中表达功能性UCP1的哺乳动物细胞，该哺乳动物不表达任何内源性UCP亚型。在开发表达功能性UCP2和UCP3的各自的细胞之后，它们对于以同工型特异性方式研究了UCP依赖性代谢控制机制的研究。