Taking control over intracellular fatty acid levels is essential for the analysis of thermogenic function in cultured primary brown and brite/beige adipocytes

Thermogenesis in brown adipocytes, conferred by mitochondrial uncoupling protein 1 (UCP1), is receiving great attention because metabolically active brown adipose tissue may protect humans from metabolic diseases. In particular, the thermogenic function of brown‐like adipocytes in white adipose tissue, known as brite (or beige) adipocytes, is currently of prime interest. A valid procedure to quantify the specific contribution of UCP1 to thermogenesis is thus of vital importance. Adrenergic stimulation of lipolysis is a common way to activate UCP1. We here report, however, that in this frequently applied setup, taking control over intracellular fatty acid levels is essential for the analysis of thermogenic function in cultured brown and brite adipocytes. By the application of these findings, we demonstrate that UCP1 is functionally thermogenic in intact brite adipocytes and adrenergic UCP1 activation is largely dependent on adipose triglyceride lipase (ATGL) rather than hormone sensitive lipase (HSL). Synopsis This study provides guidelines and protocols for the accurate quantification of the specific contribution of UCP1 to thermogenesis in brown and brite/beige adipocytes. Activation of UCP1 is a prerequisite for quantifying UCP1‐mediated leak respiration in cultured primary adipocytes Free fatty acids released by adrenergic‐stimulated lipolysis mask the specific UCP1‐mediated component of uncoupled respiration by inducing unspecific leak respiration in cultured primary adipocytes Scavenging of free fatty acids with BSA enables specific measurement of UCP1‐mediated uncoupled respiration and reveals thermogenic property of brite adipocytes in a similar fashion as in brown adipocytes, with activation being largely dependent on adipose triglyceride lipase (ATGL). Graphical Abstract This study provides guidelines and protocols for the accurate quantification of the specific contribution of UCP1 to thermogenesis in brown and brite/beige adipocytes.