Cellular and Biochemical Pathways of Adipose Metabolism and Thermogenesis

项目摘要/摘要 脂肪生物学引起了人们的极大兴趣，特别是在肥胖和肥胖的世界性流行的情况下 代谢性疾病，包括2型糖尿病、心血管疾病和癌症。而脂肪组织 作为卡路里的主要储存场所，某些脂肪组织在适应 生热作用：化学能以热的形式散失以响应外界环境的过程。 刺激物。产热脂肪组织，棕色和米色，保护身体免受体温过低、肥胖和 其他代谢紊乱。未得到满足的关键需求包括了解详细的分子途径 哪种化学能被转化为热，以及人类疗法的发现可能会增加 生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物质 棕色和米色脂肪中的一种途径，在能量消耗和抑制肥胖方面发挥着重要作用 动物模型。这种无用的肌酸循环的中断会导致肥胖水平，而消融术中观察不到 任何以前描述的生热机制，包括UCP1；作为对这些观察的回应，我 将这笔拨款完全集中在对这一徒劳的肌酸途径的进一步生化和生理学研究上。一 AIM将侧重于肌酸转运蛋白(CRT)在脂肪组织中的作用，在脂肪组织中，ADIPO-CRT的初步数据 KO小鼠表明，这种外源性肌酸积累途径对全身有很大贡献 能量平衡。CRT在脂肪中的生理作用将通过代谢笼进行分析，以 研究几种不同生理扰动下的突变小鼠。这种突变还将与 我们之前的遗传模型(ADIPO-GATM-KO)无法合成从头开始的肌酸，以创造一种 完全缺乏肌酸脂肪蓄积的动物模型。一个相关的目标将是研究对 CRT的mRNA和蛋白；初步数据显示肥胖者脂肪细胞中的mRNA下调 研究对象。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， 因为它是在产热脂肪细胞的线粒体中加工/水解的。我们的最终目标将集中在一个主要的 悬而未决的生化问题：作为这一过程的一部分，CRP上的高能磷酸到底是如何消散的 徒劳的循环。在这方面，我们有令人兴奋的初步数据使用31P核磁共振：线粒体制剂来自 生热脂肪具有直接水解C反应蛋白的活性。我们已经提纯了这一活性，并已 经鉴定，它是一种碱性磷酸酶TNAP。虽然没有被注释为线粒体蛋白质，但我们发现了一个 这种蛋白质的很大一部分存在于线粒体相关膜(MAM)部分。我们将表演 TnAP的遗传和药理学操作以确定其在产热中的作用和无用 肌酸循环。我们还将使用蛋白质质谱学来确定这种蛋白质可能如何被修饰以 实现其与线粒体的关联。总之，这些研究将增进自适应的基本知识 生热作用，为人类治疗代谢性疾病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.