Measurement of Futile Creatine Cycling Using Respirometry.
Measurement of Futile Creatine Cycling Using Respirometry.
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DOI：
10.1007/978-1-0716-2087-8_10
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发表时间：
2022
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产热脂肪组织在调节全身能量消耗和营养平衡方面起着至关重要的作用，因为它能够将化学能作为热量消散，这一过程被称为非寒颤产热。脂肪细胞中肌酸水平的降低会损害产热能力并促进饮食诱导的肥胖。kazak等，Cell 163, 643 - 55,2015；Kazak et al ., Cell Metab 26, 660-671。e3, 2017;Kazak等人，自然科学学报，1,360 - 370,2019)。从机制上讲，产热呼吸可以通过释放过量的ADP来促进，这依赖于肌酸的添加。一个双酶系统的模型，我们称之为无效肌酸循环，已经被假设来支持肌酸的这种产热作用。在纯化的线粒体制剂中可以监测无效的肌酸循环，其中通过测量ADP限制条件下的耗氧量来监测肌酸依赖的ADP释放。目前的模型提出，在产热脂肪细胞中，线粒体靶向肌酸激酶B （CKB）使用线粒体衍生的ATP磷酸化肌酸（Rahbani JF, Nature 590, 480-485, 2021）。肌酸激酶反应产生磷酸肌酸和ADP， ADP刺激呼吸。接下来，线粒体磷酸肌酸池被磷酸酶直接水解，再生肌酸。然后，释放的肌酸可以与线粒体CKB结合，触发另一轮循环，以支持adp依赖性呼吸。在这个模型中，肌酸磷酸化和磷酸肌酸水解的协同作用触发了一个无用的循环，产生了线粒体ADP的克分子过量，以促进产热呼吸（Rahbani JF, Nature 590, 480-485, 2021； Kazak和Cohen， Nat Rev Endocrinol 16, 421-436, 2020）。在这里，我们提供了一种详细的方法来对分离的线粒体进行呼吸测量，并计算肌酸依赖的ADP释放的化学计量。这种方法提供了对无效肌酸循环的直接测量。
Thermogenic adipose tissue plays a vital function in regulating whole-body energy expenditure and nutrient homeostasis due to its capacity to dissipate chemical energy as heat, in a process called non-shivering thermogenesis. A reduction of creatine levels in adipocytes impairs thermogenic capacity and promotes diet-induced obesityKazak et al, Cell 163, 643–55, 2015; Kazak et al, Cell Metab 26, 660–671.e3, 2017; Kazak et al, Nat Metab 1, 360–370, 2019). Mechanistically, thermogenic respiration can be promoted by the liberation of an excess quantity of ADP that is dependent on addition of creatine. A model of a two-enzyme system, which we term the Futile Creatine Cycle, has been posited to support this thermogenic action of creatine. Futile creatine cycling can be monitored in purified mitochondrial preparations wherein creatine-dependent liberation of ADP is monitored through the measurement of oxygen consumption under ADP-limiting conditions. The current model proposes that, in thermogenic fat cells, mitochondria-targeted creatine kinase B (CKB) uses mitochondrial-derived ATP to phosphorylate creatine (Rahbani JF, Nature 590, 480–485, 2021). The creatine kinase reaction generates phosphocreatine and ADP, and ADP stimulates respiration. Next, a pool of mitochondrial phosphocreatine is directly hydrolyzed by a phosphatase, to regenerate creatine. The liberated creatine can then engage mitochondrial CKB to trigger another round of this cycle to support ADP-dependent respiration. In this model, the coordinated action of creatine phosphorylation and phosphocreatine hydrolysis triggers a futile cycle that produces a molar excess of mitochondrial ADP to promote thermogenic respiration (Rahbani JF, Nature 590, 480–485, 2021; Kazak and Cohen, Nat Rev Endocrinol 16, 421–436, 2020). Here, we provide a detailed method to perform respiratory measurements on isolated mitochondria and calculate the stoichiometry of creatine-dependent ADP liberation. This method provides a direct measure of the futile creatine cycle.