Cellular and Biochemical Pathways of Adipose Metabolism and Thermogenesis

项目总结/摘要 人们对脂肪生物学非常感兴趣，特别是鉴于肥胖症在世界范围内的流行， 代谢性疾病，包括2型糖尿病、心血管疾病和癌症。虽然脂肪组织是 作为卡路里的主要储存场所，某些脂肪组织在适应性饮食中起着关键作用。 生热，化学能以热的形式耗散的过程，以响应外部环境的变化。 刺激。棕色和米色的产热脂肪组织可以保护身体免受体温过低、肥胖和 其他代谢紊乱。关键的未满足的需求包括了解详细的分子途径， 化学能转化为热能，以及人类治疗方法的发现， 产热脂肪的数量和功能。四年前，我们描述了一种以前未知的产热性 棕色和米色脂肪中的一种途径，在能量消耗和抑制肥胖中起着重要作用， 动物模型这种无用的肌酸循环的中断导致肥胖水平，而这些肥胖水平是在消融肌酸时观察不到的。 任何先前描述的产热机制，包括UCP 1;作为对这些观察结果的回应，我 把这个资助完全集中在对这种无用的肌酸途径的进一步的生化和生理研究上。一 目的将集中在肌酸转运蛋白（CrT）在脂肪组织中的作用， KO小鼠表明，肌酸积累的这种外源性途径显著有助于全身 能量平衡将使用代谢笼分析CrT在脂肪中的生理作用， 在几种不同的生理扰动下研究突变小鼠。这种突变也将与 我们先前的遗传模型（adipo-GATM-KO）不能从头合成肌酸， 完全缺乏肌酸的脂肪积累的动物模型。一个相关的目标将是研究 CrT mRNA和蛋白;初步数据显示mRNA在肥胖人脂肪细胞中下调 科目重要的是，我们还将使用代谢组学研究（LC/MS）来跟踪磷酸肌酸（CrP）的命运， 因为它在线粒体中从产热脂肪细胞加工/水解。我们最后的目标将集中在一个主要的 未回答的生物化学问题：究竟是如何高能量磷酸盐对磷酸肌酸消散的一部分，这是 徒劳的循环在这方面，我们使用31 P NMR获得了令人兴奋的初步数据： 产热脂肪具有直接水解磷酸肌酸活性。我们已经净化了这种活动， 鉴定为TNAP，一种碱性磷酸酶。虽然没有被注释为线粒体蛋白，但我们发现了一种 在线粒体相关膜（MAM）部分中的该蛋白质的相当大的部分。我们将执行 TNAP的遗传学和药理学操作，以确定其在产热和无效性中的作用。 肌酸循环我们还将使用蛋白质质谱法来确定这种蛋白质如何被修饰， 实现与线粒体的结合。总之，这些研究将推进适应性的基础知识， 产热作用，并为代谢性疾病的人类治疗提供了潜在的新途径。

PROJECT SUMMARY/ABSTRACT There is a great deal of interest in adipose biology, particularly in light of the world-wide epidemic in obesity and metabolic diseases, including type 2 diabetes, cardiovascular disease and cancer. While adipose tissues are best known as the major storage site for calories, certain fat tissues play a critical role in adaptive thermogenesis, the process whereby chemical energy is dissipated in the form of heat in response to external stimuli. Thermogenic adipose tissues, brown and beige, defend the body against hypothermia, obesity and other metabolic disorders. Critical unmet needs include understanding the detailed molecular pathways by which chemical energy is converted into heat and the discovery of human therapeutics that might increase amounts and function of thermogenic fat. Four years ago, we described a previously unknown thermogenic pathway in brown and beige fat that plays a major role in both energy expenditure and suppression of obesity in animal models. Disruptions of this futile creatine cycle causes levels of obesity not observed with ablations of any previously described thermogenic mechanisms, including UCP1; in response to these observations, I am focusing this grant entirely on further biochemical and physiological studies of this futile creatine pathway. One Aim will focus on the role of the creatine transporter (CrT) in fat tissues, where preliminary data with adipo-CrT KO mice shows that this exogenous pathway for creatine accumulation contributes significantly to whole body energy homeostasis. The physiological role of the CrT specifically in fat will be analyzed with metabolic cages to study mutant mice under several different physiological perturbations. This mutation will also be combined with our previous genetic model (adipo-GATM-KO), which is unable to synthesize creatine de novo, to create an animal model totally lacking adipose accumulation of creatine. A related Aim will be to study regulation of the CrT mRNA and protein; preliminary data shows mRNA to be down-regulated in fat cells from obese human subjects. Importantly, we will also use metabolomic studies (LC/MS) to follow the fate of phosphocreatine (CrP), as it is processed/hydrolyzed in mitochondria from thermogenic fat cells. Our last Aim will focus on a major unanswered biochemical question: exactly how is the high energy phosphate on CrP dissipated as part of this futile cycle. In this regard, we have exciting preliminary data using 31P NMR: mitochondrial preparations from thermogenic fat contain an activity that can hydrolyze CrP directly. We have purified this activity and have identified it as TNAP, an alkaline phosphatase. While not annotated as a mitochondrial protein, we find a substantial portion of this protein in the mitochondrial associated membrane (MAM) fraction. We will perform genetic and pharmacological manipulations of TNAP to determine its role in thermogenesis and the futile creatine cycle. We will also use protein Mass Spectrometry to determine how this protein may be modified to achieve its association with mitochondria. Together, these studies will advance basic knowledge of adaptive thermogenesis and provide potential new avenues to human therapeutics in metabolic diseases.