Cellular and Biochemical Pathways of Adipose Metabolism and Thermogenesis

脂肪代谢和产热的细胞和生化途径

基本信息

批准号：
10540420
负责人：
BRUCE M. SPIEGELMAN
金额：
$ 53.23万
依托单位：
DANA-FARBER CANCER INST
依托单位国家：
美国
项目类别：
财政年份：
2019
资助国家：
美国
起止时间：
2019-12-10 至 2024-11-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10540420
关键词：
Ablation Address Adipocytes Adipose tissue Adrenergic Agents Affect Alkaline Phosphatase Anabolism Animal Model Animals Biochemical Biochemical Pathway Biology Biophysics Calories Cardiovascular Diseases Carrier Proteins Cells Cellular biology Chemicals Circulation Creatine Data Development Diabetes Mellitus Diet Electrodes Energy Metabolism Enterobacteria phage P1 Cre recombinase Epidemic Exposure to Fatty Liver Fatty acid glycerol esters Futile Cycling Generations Genes Genetic Genetic Models Grant High Fat Diet Homeostasis Human Hydrolase Hydrolysis Individual Isotope Labeling Knockout Mice Knowledge Label Light Malignant Neoplasms Mass Spectrum Analysis Membrane Messenger RNA Metabolic Metabolic Diseases Metabolism Mitochondria Mitochondrial Proteins Modeling Molecular Mus Mutant Strains Mice Mutation Nature Non-Insulin-Dependent Diabetes Mellitus Obesity Oxygen Pathway interactions Phosphocreatine Phosphoric Monoester Hydrolases Physiological Physiology Play Post-Translational Protein Processing Preparation Process Protein Dephosphorylation Proteins Protocols documentation Reaction Regulation Research Resolution Respiration Role Site Stimulus Structure Temperature Therapeutic Thermogenesis Tissues Visceral adiponectin creatine transporter experimental study fighting gain of function human subject in vivo inhibitor inorganic phosphate interest liquid chromatography mass spectrometry metabolomics natural hypothermia pharmacologic promoter protein structure response stoichiometry

项目摘要

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.

项目概要/摘要人们对脂肪生物学产生了很大的兴趣，特别是考虑到肥胖和肥胖症在世界范围内的流行代谢疾病，包括2型糖尿病、心血管疾病和癌症。虽然脂肪组织是某些脂肪组织作为卡路里的主要储存场所而闻名，在适应性方面发挥着关键作用生热作用，化学能响应外部压力以热的形式耗散的过程刺激。产热脂肪组织（棕色和米色）可保护身体免受体温过低、肥胖和其他代谢紊乱。未满足的关键需求包括了解详细的分子途径化学能转化为热量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生热物质棕色和米色脂肪中的途径，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破坏会导致肥胖水平，而通过消融则无法观察到。任何先前描述的生热机制，包括 UCP1；针对这些观察结果，我这笔资金完全集中于对这种无效肌酸途径的进一步生化和生理学研究。一目标将重点关注肌酸转运蛋白 (CrT) 在脂肪组织中的作用，其中 adipo-CrT 的初步数据 KO 小鼠表明，这种肌酸积累的外源途径对全身有显着贡献能量稳态。 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。该突变也将与我们之前的遗传模型（adipo-GATM-KO）无法从头合成肌酸，因此无法创建完全缺乏脂肪肌酸积累的动物模型。一个相关的目标是研究监管 CrT mRNA 和蛋白；初步数据显示肥胖人脂肪细胞中 mRNA 下调科目。重要的是，我们还将使用代谢组学研究 (LC/MS) 来追踪磷酸肌酸 (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。我们的最后一个目标将集中在一个主要目标上未解答的生化问题：CrP 上的高能磷酸盐到底是如何消散的？徒劳的循环。在这方面，我们使用 31P NMR 获得了令人兴奋的初步数据：来自生热脂肪含有可以直接水解肌酸磷酸的活性。我们已经净化了这个活动并有鉴定其为 TNAP，一种碱性磷酸酶。虽然没有注释为线粒体蛋白，但我们发现该蛋白的大部分位于线粒体相关膜 (MAM) 部分。我们将表演 TNAP 的遗传和药理学操作以确定其在生热作用中的作用，但徒劳无功肌酸循环。我们还将使用蛋白质质谱来确定如何修饰该蛋白质实现与线粒体的关联。这些研究将共同推进适应性的基础知识生热作用并为人类代谢疾病治疗提供潜在的新途径。