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Promoting brown adipose tissue (BAT) activity has been recognized as innovative therapeutic approach to improve obesity and metabolic disease. Whilst the molecular circuitry underlying thermogenic activation of BAT is well understood, the processes underlying rheostatic regulation of BAT to maintain homeostasis and avoid excessive energy dissipation remain ill-defined. Increasing cyclic AMP (cAMP) biosynthesis is key for BAT activation. Here, we demonstrate that ADCY3, an adenylyl cyclase whose expression is induced during cold exposure and regulates cAMP homeostasis in thermogenic fat, is dispensable for BAT function in lean mice, but becomes critical during obesity. Furthermore, by combining RNA-seq with epigenomic H3K4me3 profiling, we detected a novel, cold-inducible promoter that generates a 5’ truncated Adcy3-at mRNA isoform, Adcy3-at. Mice lacking only Adcy3-at, but not full-length Adcy3, displayed increased energy expenditure already under lean conditions and were protected against obesity and ensuing metabolic imbalances. Subcellularly, translated ADCY3-AT proteins are retained in the endoplasmic reticulum (ER), did not translocate to the cell membrane, and lacked enzymatic activity. By interacting with ADCY3, ADCY3-AT retained ADCY3 in the ER and, thereby, reduced the plasma membrane pool of ADCYs available for G-protein mediated cAMP synthesis. Thereby, ADCY3-AT acts as a signaling rheostat in BAT, limiting adverse consequences of uncurbed cAMP activity after long-term BAT activation. Adcy3-at induction was driven by a cold-induced, truncated isoform of the transcriptional cofactor PPARGC1A (PPARG Coactivator 1 Alpha, PPARGC1A-AT). Expression of Ppargc1a-at and Adcy3-at are evolutionary conserved, indicating that transcriptional rewiring by commissioning of alternative promoters is key for thermogenic fat function.

The different adipose tissues can be distinguished according to their function. For example, white adipose tissue (WAT) stores energy in form of lipids, whereas brown adipose tissue (BAT) dissipates energy in the form of heat. These functional differences are represented in the respective adipocyte morphology: whereas white adipocytes contain large, unilocular lipid droplets, brown adipocytes contain smaller, multilocular lipid droplets. However, an automated, image-analysis pipeline to comprehensively analyze adipocytes in vitro in cell culture as well as ex vivo in tissue sections is missing. We here present AdipoQ, an open-source software implemented as ImageJ plugins that allows to analyze adipocytes in tissue sections and in vitro after histological and/or immunofluorescent labelling. AdipoQ is compatible with different imaging modalities and staining methods, allows batch processing of large datasets and simple post-hoc analysis, provides a broad band of parameters, and allows combining multiple fluorescent read-outs. Thereby, AdipoQ is of immediate use not only for basic research but also for clinical diagnosis.