REGULATION OF BROWN ADIPOSE TISSUE THERMOGENESIS

棕色脂肪组织产热的调节

• 批准号: 3231067
• 负责人: EARL S SHRAGO
• 金额: $ 7.03万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 1983
• 资助国家: 美国
• 起止时间: 1983-08-01 至 1991-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3231067
• 关键词: acyl coA; affinity chromatography; binding proteins; bioenergetics; body temperature regulation; brown fat; cold temperature; electron spin resonance spectroscopy; free fatty acids; gel electrophoresis; guanosine diphosphate; hamsters; high performance liquid chromatography; laboratory mouse; lipid metabolism; liposomes; membrane lipids; membrane potentials; membrane reconstitution /synthesis; mitochondria; mitochondrial membrane; obesity; oxygen consumption; protein reconstitution; proteins; purine nucleotides; thermogenesis

The hypothesis that nonshivering thermogenesis (NST) mediated by brown adipose tissue mitochondria (BATM) is linked to obesity and the regulation of body weight has been recently proposed. The molecular basis for this weight regulating ability is the occurrence of a nucleotide binding protein called uncoupling protein (UP) within the BATM inner membrane which imparts the unique capability of controlled uncoupling of respiration from normal rgulatory mechanisms thus permitting loss of energy as heat. The metabolic regulation of the UP and thus NST has not been ascertained. It is known that purine nucleotides will bind and inhibit the function of the UP. However, no activator of the UP function has been clearly established. We have recently purified and reconstituted the UP into a liposome system and observed faty acid activation of proton transport. In future studies we will attempt to purify the protein to homogeneity for use in the reconstituted system to determine its kinetic properties and mechanism of action by fatty acids. The UP will be extracted with Triton X-100 from mitochondria, purifed on hydroxylapatite and the Triton exchanged with octylglucoside for final reconstitution into a liposome system. Proton conductance will be assayed using the pH system as well as incorporation of cytochrome oxidase with the UP. Measurements of O2 consumption which would be more physiological could then be carried out. The mechanism of fatty acid activation of the UP will be studied using spin labeled fatty acids and measurement by electron spin resonance. Individual intracellular free fatty acids and their long chain thioesters will be determined by HPLC and thier concentrations related to activation of the UP. Hamsters will be the main source of BATM for purification and biochemical studies relating to mechanism of the UP. However, more physiological experiments will be carried out by reconstituting the UP from cold adapted rats and the ob/ob mouse which will provide the information as to whether the UP is non functional under certain conditions or merely decreased in concentration. The long term objectives of the proposal are to determine the mechanism of action of the UP in BATM and how certain aspects of the biochemical and molecular action of the protein might be extended to understanding basal energy expenditure in humans.

棕色介导的非颤抖性产热假说 脂肪组织线粒体（BATM）与肥胖有关， 体重最近被提出。 其分子基础是 体重调节能力是核苷酸结合蛋白的出现 称为解偶联蛋白（UP），位于BATM内膜内， 控制呼吸与正常呼吸分离的独特能力 调节机制，从而允许能量作为热量损失。 代谢 因此，对UP和NST的监管尚未确定。 已知 嘌呤核苷酸会结合并抑制UP的功能。 然而，没有明确确定UP功能的激活剂。 我们 最近将UP纯化并重组到脂质体系统中， 观察到脂肪酸激活质子运输。 在未来的研究中，我们 将尝试将蛋白质纯化至同质，以用于 重组系统，以确定其动力学性质和机制， 脂肪酸的作用。 UP将用Triton X-100从 线粒体，纯化羟基磷灰石和Triton交换与 辛基葡糖苷用于最终重构到脂质体系统中。 质子 将使用pH系统以及掺入 细胞色素氧化酶与UP。 测量O2消耗量， 更符合生理的方式来进行。 脂肪的作用机制 将使用自旋标记的脂肪酸研究UP的酸活化 并通过电子自旋共振进行测量。 单个细胞内游离 脂肪酸及其长链硫酯将通过HPLC测定， 它们的浓度与UP的激活有关。 仓鼠将是 BATM的主要来源，用于纯化和生物化学研究， UP的机制。 然而，更多的生理实验将 通过重建来自冷适应大鼠的UP和ob/ob 鼠标，它将提供关于UP是否为非 在某些条件下起作用或仅仅是浓度降低。 该提案的长期目标是确定 行动的UP在BATM和如何某些方面的生化和 蛋白质的分子作用可能会扩展到理解基础 人类的能量消耗。