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

解偶联蛋白（Uncoupling proteins, UCPs）是线粒体内膜蛋白，它介导线粒体膜电位以热形式消散，而不是ATP合成。为了阐明UCP1依赖性代谢控制的细胞和分子机制，特别是与细胞ATP水平的关系，我们首先研究了UCP1敲除（KO）小鼠体内2-脱氧葡萄糖（2-DG）的组织摄取。在野生型（WT）小鼠中，给药去甲肾上腺素（NE）加速了血浆2-DG的消失，增加了2-DG进入棕色脂肪组织（BAT）的摄取，而只表达UCP1和心脏，而血浆胰岛素水平没有升高。在UCP1-KO小鼠中，NE对2-DG进入BAT而非进入心脏的刺激作用完全消失。在WT和UCP1 -KO小鼠中，胰岛素给药增加了BAT和心脏对2-DG的摄取。NE还增加了WT小鼠BAT中AMP/ATP的比值和AMP活化蛋白激酶（AMPK）的活性，但对UCP1-KO小鼠没有影响。这些结果表明，BAT中交感刺激的葡萄糖利用是由于UCP1和AMPK的连续激活。接下来，我们利用Stratagene的LacSwitch II诱导哺乳动物表达系统和不表达任何内源性UCP亚型的Hep3B人肝细胞癌，建立了在线粒体部分表达功能性UCP1的哺乳动物细胞。在分别培养出表达功能性UCP2和UCP3的细胞后，它们将有助于以异构体特异性的方式研究ucp依赖性代谢控制的机制。

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.