Obesity increases the risk of mortality because of metabolic sequelae such as type 2 diabetes and cardiovascular disease¹. Thermogenesis by adipocytes can counteract obesity and metabolic diseases^2,3. In thermogenic fat, creatine liberates a molar excess of mitochondrial ADP-purportedly via a phosphorylation cycle⁴-to drive thermogenic respiration. However, the proteins that control this futile creatine cycle are unknown. Here we show that creatine kinase B (CKB) is indispensable for thermogenesis resulting from the futile creatine cycle, during which it traffics to mitochondria using an internal mitochondrial targeting sequence. CKB is powerfully induced by thermogenic stimuli in both mouse and human adipocytes. Adipocyte-selective inactivation of Ckb in mice diminishes thermogenic capacity, increases predisposition to obesity, and disrupts glucose homeostasis. CKB is therefore a key effector of the futile creatine cycle.

Brown and beige adipocytes are specialized cells that express uncoupling protein 1 (UCP1) and dissipate chemical energy as heat. These cells likely possess alternative UCP1-independent thermogenic mechanisms. Here, we identify a secreted enzyme, peptidase M20 domain containing 1 (PM20D1), that is enriched in UCP1(+) versus UCP1(-) adipocytes. We demonstrate that PM20D1 is a bidirectional enzyme in vitro, catalyzing both the condensation of fatty acids and amino acids to generate N-acyl amino acids and also the reverse hydrolytic reaction. N-acyl amino acids directly bind mitochondria and function as endogenous uncouplers of UCP1-independent respiration. Mice with increased circulating PM20D1 have augmented respiration and increased N-acyl amino acids in blood. Lastly, administration of N-acyl amino acids to mice improves glucose homeostasis and increases energy expenditure. These data identify an enzymatic node and a family of metabolites that regulate energy homeostasis. This pathway might be useful for treating obesity and associated disorders.

The mitochondrial ADP/ATP carrier (AAC) is a major transport protein of the inner mitochondrial membrane. It exchanges mitochondrial ATP for cytosolic ADP and controls cellular production of ATP. In addition, it has been proposed that AAC mediates mitochondrial uncoupling, but it has proven difficult to demonstrate this function or to elucidate its mechanisms. Here we record AAC currents directly from inner mitochondrial membranes from various mouse tissues and identify two distinct transport modes: ADP/ATP exchange and H⁺ transport. The AAC-mediated H⁺ current requires free fatty acids and resembles the H⁺ leak via the thermogenic uncoupling protein 1 found in brown fat. The ADP/ATP exchange via AAC negatively regulates the H⁺ leak, but does not completely inhibit it. This suggests that the H⁺ leak and mitochondrial uncoupling could be dynamically controlled by cellular ATP demand and the rate of ADP/ATP exchange. By mediating two distinct transport modes, ADP/ATP exchange and H⁺ leak, AAC connects coupled (ATP production) and uncoupled (thermogenesis) energy conversion in mitochondria.

Adipose tissues dynamically remodel their cellular composition in response to external cues by stimulating beige adipocyte biogenesis; however, the developmental origin and pathways regulating this process remain insufficiently understood owing to adipose tissue heterogeneity. Here, we employed single-cell RNA-seq and identified a unique subset of adipocyte progenitor cells (APCs) that possessed the cell-intrinsic plasticity to give rise to beige fat. This beige APC population is proliferative and marked by cell-surface proteins, including PDGFRα, Sca1, and CD81. Notably, CD81 is not only a beige APC marker but also required for de novo beige fat biogenesis following cold exposure. CD81 forms a complex with αV/β1 and αV/β5 integrins and mediates the activation of integrin-FAK signaling in response to irisin. Importantly, CD81 loss causes diet-induced obesity, insulin resistance, and adipose tissue inflammation. These results suggest that CD81 functions as a key sensor of external inputs and controls beige APC proliferation and whole-body energy homeostasis.

With the increasing prevalence of type 2 diabetes and fatty liver disease, there is still an unmet need to better treat hyperglycemia and hyperlipidemia. Here, we identify isthmin-1 (Ism1) as an adipokine and one that has a dual role in increasing adipose glucose uptake while suppressing hepatic lipid synthesis. Ism1 ablation results in impaired glucose tolerance, reduced adipose glucose uptake, and reduced insulin sensitivity, demonstrating an endogenous function for Ism1 in glucose regulation. Mechanistically, Ism1 activates a PI3K-AKT signaling pathway independently of the insulin and insulin-like growth factor receptors. Notably, while the glucoregulatory function is shared with insulin, Ism1 counteracts lipid accumulation in the liver by switching hepatocytes from a lipogenic to a protein synthesis state. Furthermore, therapeutic dosing of recombinant Ism1 improves diabetes in diet-induced obese mice and ameliorates hepatic steatosis in a diet-induced fatty liver mouse model. These findings uncover an unexpected, bioactive protein hormone that might have simultaneous therapeutic potential for diabetes and fatty liver disease.

Brown adipose tissue (BAT) regulates metabolic physiology. However, nearly all mechanistic studies of BAT protein function occur in a single inbred mouse strain, which has limited the understanding of generalizable mechanisms of BAT regulation over physiology. Here, we perform deep quantitative proteomics of BAT across a cohort of 163 genetically defined diversity outbred mice, a model that parallels the genetic and phenotypic variation found in humans. We leverage this diversity to define the functional architecture of the outbred BAT proteome, comprising 10,479 proteins. We assign co-operative functions to 2,578 proteins, enabling systematic discovery of regulators of BAT. We also identify 638 proteins that correlate with protection from, or sensitivity to, at least one parameter of metabolic disease. We use these findings to uncover SFXN5, LETMD1, and ATP1A2 as modulators of BAT thermogenesis or adiposity, and provide OPABAT as a resource for understanding the conserved mechanisms of BAT regulation over metabolic physiology.