Development of a functional beige fat cell line uncovers independent subclasses of cells expressing UCP1 and the futile creatine cycle

Highlights•Development of a beige fat cell line containing both UCP1 and the futile creatine cycle•Clonal analysis reveals different subclasses of beige adipocytes•Identification of UCP1high and TNAPhigh adipocytes in different fat cells in vivo•Detection of TNAPhigh adipocyte population in human deep neck brown fatSummaryAlthough uncoupling protein 1 (UCP1) is established as a major contributor to adipose thermogenesis, recent data have illustrated an important role for alternative pathways, particularly the futile creatine cycle (FCC). How these pathways co-exist in cells and tissues has not been explored. Beige cell adipogenesis occurs in vivo but has been difficult to model in vitro; here, we describe the development of a murine beige cell line that executes a robust respiratory response, including uncoupled respiration and the FCC. The key FCC enzyme, tissue-nonspecific alkaline phosphatase (TNAP), is localized almost exclusively to mitochondria in these cells. Surprisingly, single-cell cloning from this cell line shows that cells with the highest levels of UCP1 express little TNAP, and cells with the highest expression of TNAP express little UCP1. Immunofluorescence analysis of subcutaneous fat from cold-exposed mice confirms that the highest levels of these critical thermogenic components are expressed in distinct fat cell populations.Graphical abstract