BMP7 Increases UCP1-Dependent and Independent Thermogenesis with a Unique Gene Expression Program in Human Neck Area Derived Adipocytes

Abstract

White adipocytes contribute to energy storage, accumulating lipid droplets, whereas brown and beige adipocytes mainly function in dissipating energy as heat primarily via the action of uncoupling protein 1 (UCP1). Bone morphogenic protein 7 (BMP7) was shown to drive brown adipocyte differentiation in murine interscapular adipose tissue. Here, we performed global RNA-sequencing and functional assays on adipocytes obtained from subcutaneous (SC) and deep-neck (DN) depots of human neck and differentiated with or without BMP7. We found that BMP7 did not influence differentiation but upregulated browning markers, including UCP1 mRNA and protein in SC and DN derived adipocytes. BMP7 also enhanced mitochondrial DNA content, levels of oxidative phosphorylation complex subunits, along with PGC1α and p-CREB upregulation, and fragmentation of mitochondria. Furthermore, both UCP1-dependent proton leak and UCP1-independent, creatine-driven substrate cycle coupled thermogenesis were augmented upon BMP7 addition. The gene expression analysis also shed light on the possible role of genes unrelated to thermogenesis thus far, including ACAN, CRYAB, and ID1, which were among the highest upregulated ones by BMP7 treatment in both types of adipocytes. Together, our study shows that BMP7 strongly upregulates thermogenesis in human neck area derived adipocytes, along with genes, which might have a supporting role in energy expenditure.

Mitophagy Mediates the Beige to White Transition of Human Primary Subcutaneous Adipocytes Ex Vivo

“…However, the droplets merged and enlarged when the beige protocol was replaced by the white ( Figure 2 A). The quantification of this phenomenon was performed by texture sum variance, which separated the white and beige adipocyte populations well in several previous cellular models [ 40 , 41 , 45 , 46 ], is shown in Figure 2 B. To investigate the mitochondrial morphology, we performed immunostaining for translocase of outer mitochondrial membrane (TOM) 20 [ 37 , 47 ] ( Figure 2 A; see the secondary antibody control in Figure S1 ).…”

“…A list of all the probes is provided in Table S1 . Quantification of mtDNA was performed by qPCR as previously described [ 37 , 46 ].…”

“…Propidium iodide (PI, 1.5 μg/mL, 1 h) was used for nuclei labeling. Images were obtained with an Olympus FluoView 1000 (Olympus Scientific Solutions, Tokyo, Japan) confocal microscope and FluoView10-ASW (Olympus Scientific Solutions) software version 3.0, as previously described [ 37 , 46 ]. For the excitation of Alexa Fluor 488, the 488 nm line of an argon ion laser was used, while for Alexa Fluor 647, a 633-nm He–Ne laser was used; for PI, a 543-nm He–Ne laser was used.…”

“…The optimum size values for the LC3 punctae and fragmented mitochondria were determined based on an analysis of all immunostaining images and manual verification of the counting accuracy by checking the outlines of counts. Both LC3 punctae and fragmented mitochondria content were normalized to per nucleus for individual images [ 37 , 46 ]. Colocalization of LC3 and TOM20 was evaluated by calculation of the PCC [ 37 ].…”

Mitophagy mediates beige to white transition of human primary subcutaneous adipocytes ex vivo

“…Propidium Iodide (1.5 μg/mL, 1 h) was used for nuclei labelling. Images were obtained by Olympus FluoView 1000 confocal microscope as previously described [39,46]. LC3 punctae and fragmented mitochondria were quantified using FIJI as previously described [39,46].…”