Measurement of Futile Creatine Cycling Using Respirometry

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.