Obesity increases mortality risk because of metabolic sequelae such as type 2 diabetes and cardiovascular disease 1 . Thermogenesis by adipocytes can counter obesity and metabolic diseases 2,3 . In thermogenic fat, creatine liberates a molar excess of mitochondrial ADP to drive thermogenic respiration, hypothesized to be caused by a phosphorylation cycle 4 . However, the proteins that control this futile creatine cycle are unknown. Here we show that creatine kinase B (CKB) is indispensable for futile creatine cycling-based thermogenesis by trafficking to mitochondria through the utilization of an internal mitochondrial targeting sequence. CKB abundance is powerfully induced by thermogenic stimuli in both mouse and human adipocytes. Adipocyte-selective inactivation of Ckb in mice diminishes thermogenic capacity, increases the predisposition to obesity, and disrupts glucose homeostasis. Thus, CKB is a key effector of the futile creatine cycle.The obesity epidemic is a major public health concern 1 . Thermogenesis by brown and beige adipocytes can combat obesity and cardiometabolic diseases 2,5-7 . Adipocyteselective creatine depletion impairs thermogenesis and promotes diet-induced obesity 8,9 , recapitulating the higher fat accretion and lower energy expenditure that occurs with *

Obesity results from energy imbalance, when energy intake exceeds energy expenditure. Brown adipose tissue (BAT) drives non-shivering thermogenesis which represents a powerful mechanism of enhancing the energy expenditure side of the energy balance equation. The best understood thermogenic system in BAT that evolved to protect the body from hypothermia is based on the uncoupling of protonmotive force from oxidative phosphorylation through the actions of uncoupling protein 1 (UCP1), a key regulator of cold-mediated thermogenesis. Similarly, energy expenditure is triggered in response to caloric excess, and animals with reduced thermogenic fat function can succumb to diet-induced obesity. Thus, it was surprising when inactivation of Ucp1 did not potentiate diet-induced obesity. In recent years, it has become clear that multiple thermogenic mechanisms exist, based on ATP sinks centered on creatine, lipid, or calcium cycling, along with Fatty acid-mediated UCP1-independent leak pathways driven by the ADP/ATP carrier (AAC). With a key difference between cold- and diet-induced thermogenesis being the dynamic changes in purine nucleotide (primarily ATP) levels, ATP-dependent thermogenic pathways may play a key role in diet-induced thermogenesis. Additionally, the ubiquitous expression of AAC may facilitate increased energy expenditure in many cell types, in the face of over feeding. Interest in UCP1-independent energy expenditure has begun to showcase the therapeutic potential that lies in refining our understanding of the diversity of biochemical pathways controlling thermogenic respiration.

Highlights d COA6 is a coiled-coil-helix-coiled-coil-helix domain containing protein d COA6 preferentially interacts with SCO1 over SCO2 d COA6 acts as a disulfide reductase of SCO1 and COX2 d COA6 function can be bypassed under hypoxic conditions