EPAC1 enhances brown fat growth and beige adipogenesis.

Brown adipose tissue (BAT) is a central thermogenic organ that enhances energy expenditure and cardiometabolic health. However, regulators that specifically increase the number of thermogenic adipocytes are still an unmet need. Here, we show that the cAMP-binding protein EPAC1 is a central regulator of adaptive BAT growth. In vivo, selective pharmacological activation of EPAC1 increases BAT mass and browning of white fat, leading to higher energy expenditure and reduced diet-induced obesity. Mechanistically, EPAC1 coordinates a network of regulators for proliferation specifically in thermogenic adipocytes, but not in white adipocytes. We pinpoint the effects of EPAC1 to PDGFRα-positive preadipocytes, and the loss of EPAC1 in these cells impedes BAT growth and worsens diet-induced obesity. Importantly, EPAC1 activation enhances the proliferation and differentiation of human brown adipocytes and human brown fat organoids. Notably, a coding variant of RAPGEF3 (encoding EPAC1) that is positively correlated with body mass index abolishes noradrenaline-induced proliferation of brown adipocytes. Thus, EPAC1 might be an attractive target to enhance thermogenic adipocyte number and energy expenditure to combat metabolic diseases.

Triglyceride cycling enables modification of stored fatty acids.

Triglyceride cycling is the process of continuous degradation and re-synthesis of triglyceride in cellular stores. We show in 3T3-L1 adipocytes that triglycerides are subject to rapid turnover and re-arrangement of fatty acids with an estimated half-life of 2-4 h. We develop a tracing technology that can simultaneously and quantitatively follow the metabolism of multiple fatty acids to study the triglyceride futile substrate cycle directly and with molecular species resolution. Our approach is based on alkyne fatty acid tracers and mass spectrometry. The triglyceride cycling is connected to modification of released fatty acids by elongation and desaturation. Through cycling and modification, saturated fatty acids are slowly converted to monounsaturated fatty acids, and linoleic acid to arachidonic acid. We conclude that triglyceride cycling renders stored fatty acids accessible for metabolic alteration. The overall process facilitates cellular adjustments to the stored fatty acid pool to meet changing needs of the cell.

Apoptotic brown adipocytes enhance energy expenditure via extracellular inosine.

Brown adipose tissue (BAT) dissipates energy1,2 and promotes cardiometabolic health3. Loss of BAT during obesity and ageing is a principal hurdle for BAT-centred obesity therapies, but not much is known about BAT apoptosis. Here, untargeted metabolomics demonstrated that apoptotic brown adipocytes release a specific pattern of metabolites with purine metabolites being highly enriched. This apoptotic secretome enhances expression of the thermogenic programme in healthy adipocytes. This effect is mediated by the purine inosine that stimulates energy expenditure in brown adipocytes by the cyclic adenosine monophosphate-protein kinase A signalling pathway. Treatment of mice with inosine increased BAT-dependent energy expenditure and induced 'browning' of white adipose tissue. Mechanistically, the equilibrative nucleoside transporter 1 (ENT1, SLC29A1) regulates inosine levels in BAT: ENT1-deficiency increases extracellular inosine levels and consequently enhances thermogenic adipocyte differentiation. In mice, pharmacological inhibition of ENT1 as well as global and adipose-specific ablation enhanced BAT activity and counteracted diet-induced obesity, respectively. In human brown adipocytes, knockdown or blockade of ENT1 increased extracellular inosine, which enhanced thermogenic capacity. Conversely, high ENT1 levels correlated with lower expression of the thermogenic marker UCP1 in human adipose tissues. Finally, the Ile216Thr loss of function mutation in human ENT1 was associated with significantly lower body mass index and 59% lower odds of obesity for individuals carrying the Thr variant. Our data identify inosine as a metabolite released during apoptosis with a 'replace me' signalling function that regulates thermogenic fat and counteracts obesity.