Distinct signaling and transcriptional pathways regulate peri-weaning development and cold-induced recruitment of beige adipocytes Yixuan Wua, Melissa A. Kinnebrewa,b, Vassily I. Kutyavina, and Ajay Chawlaa,c,d,1,2 aCardiovascular Research Institute, University of California, San Francisco, CA 94143-0795; bDepartment of Dermatology, University of California, San Francisco, CA 94115; cDepartment of Physiology, University of California, San Francisco, CA 94143-0795; and dDepartment of Medicine, University of California, San Francisco, CA 94143-0795 Edited by Christian Wolfrum, Swiss Federal Institute of Technology in Zurich, Zurich Schwerzenbach, Switzerland, and accepted by Editorial Board Member David J. Mangelsdorf February 6, 2020 (received for review November 20, 2019) Adipose tissue provides a defense against starvation and environ- Although less well appreciated, iWAT also undergoes beige mental cold. These dichotomous functions are performed by three adipogenesis during the peri-weaning period (7–12). However, distinct cell types: energy-storing white adipocytes, and thermogenic the importance of sympathetic stimulation in acquisition of this beige and brown adipocytes. Previous studies have demonstrated phenotype remains controversial. For example, Contreras et al. that exposure to environmental cold stimulates the recruitment of demonstrated that denervation of iWAT impairs commitment to beige adipocytes in the white adipose tissue (WAT) of mice and the beige adipocyte lineage, whereas Chabowska-Kita et al. + humans, a process that has been extensively investigated. However, reported presence of UCP1 cells in the iWAT of thermoneutral beige adipose tissue also develops during the peri-weaning period in mice that presumably lacked sympathetic stimulation (7, 8). mice, a developmental program that remains poorly understood. Here, we address this gap in our knowledge using genetic, imaging, These findings raise the possibility that a combination of cell physiologic, and genomic approaches. We find that, unlike cold- autonomous and nonautonomous mechanisms might regulate induced recruitment in adult animals, peri-weaning development of beige adipocyte development during the peri-weaning period, a beige adipocytes occurs in a temperature- and sympathetic nerve- gap in our knowledge that we address here. independent manner. Instead, the transcription factor B cell PHYSIOLOGY leukemia/lymphoma 6 (BCL6) acts in a cell-autonomous manner Results and Discussion to regulate the commitment but not the maintenance phase of Beige Fat Develops during the Peri-Weaning Period in a Temperature- beige adipogenesis. Genome-wide RNA-sequencing (seq) studies Independent Manner. To identify mechanisms that control beige adi- reveal that BCL6 regulates a core set of genes involved in fatty acid pogenesis during the peri-weaning period, we employed Adipo-Clear oxidation and mitochondrial uncoupling, which are necessary for development of functional beige adipocytes. Together, our find- Significance ings demonstrate that distinct transcriptional and signaling mech- anisms control peri-weaning development and cold-induced Adipose tissue performs multiple functions in mammals, in- recruitment of beige adipocytes in mammals. cluding insulation, mechanical tissue protection, and energy balance. These functions of adipose tissue are performed by BCL6 | sympathetic nervous system | thermogenesis | development | three distinct cell types: white, beige, and brown adipocytes. metabolism While white adipocytes store energy, beige and brown adi- pocytes dissipate energy through mitochondrial respiration. n mammals, adipose tissue provides a defense against starva- Previous studies have elucidated how environmental cold Ition and environmental cold (1). These distinct functions of stimulates recruitment of beige adipocytes in adult animals. adipose tissue are performed by specialized adipocytes that can However, beige adipogenesis also occurs during the peri- store or dissipate energy. For example, white adipocytes, which weaning period in mice, a process that is poorly understood. contain a single large lipid droplet, are a primary site of energy Here, we demonstrate that distinct mechanisms regulate de- storage in mammals, whereas multilocular beige and brown ad- velopment and recruitment of beige adipocytes. We find that B ipocytes dissipate energy through uncoupled respiration (1–4). cell leukemia/lymphoma 6 (BCL6) controls development of Consistent with their distinct functions in energy metabolism, beige adipocytes during the peri-weaning period, whereas loss of white adipocytes, as in lipodystrophy, leads to ectopic de- sympathetic nerves regulate the recruitment of beige adipo- position of lipids in liver and other organs, whereas loss of beige cytes in cold environments. and brown adipocytes compromises organismal defense against environmental cold (1). Author contributions: Y.W., M.A.K., and A.C. designed research; Y.W., M.A.K., and V.I.K. performed research; V.I.K. contributed new reagents/analytic tools; Y.W., M.A.K., V.I.K., Beige and brown adipocytes are considered to be two distinct and A.C. analyzed data; and Y.W. and A.C. wrote the paper. cell types based on their developmental origin, transcriptome, The authors declare no competing interest. and epigenome (1, 4, 5). Myogenic precursors give rise to brown This article is a PNAS Direct Submission. C.W. is a guest editor invited by the adipocytes during embryogenesis in a temperature-independent Editorial Board. manner, whereas environmental cold is the primary stimulus for This open access article is distributed under Creative Commons Attribution-NonCommercial- the recruitment of mesenchyme-derived beige adipocytes in the NoDerivatives License 4.0 (CC BY-NC-ND). white adipose tissue (WAT) of adult animals. A focus on cold- Data deposition: All RNA-seq data are available at the Gene Expression Omnibus (GEO), induced recruitment of beige adipocytes has identified a large https://www.ncbi.nlm.nih.gov/geo/ (accession no. GSE140259). number of signaling and transcriptional pathways that control its 1Present address: Discovery Biology, Merck Research Labs, South San Francisco, CA 94080. biogenesis in adult animals (4). In nearly all of these cases, beige- 2To whom correspondence may be addressed. Email: [email protected]. promoting factors directly or indirectly modulate the adrenergic This article contains supporting information online at https://www.pnas.org/lookup/suppl/ activity of sympathetic nerves, which densely innervate the in- doi:10.1073/pnas.1920419117/-/DCSupplemental. guinal WAT (iWAT) of mice (6). First published March 5, 2020. www.pnas.org/cgi/doi/10.1073/pnas.1920419117 PNAS | March 24, 2020 | vol. 117 | no. 12 | 6883–6889 Downloaded by guest on September 28, 2021 together with light sheet fluorescence microscopy to investigate at P14 between mice bred at 22 °C and 30 °C but differed in older how the ambient temperature (Ta) regulates developmental ex- (P21–P35) animals (Fig. 1C). Hematoxylin and eosin staining of pression of UCP1 in the iWAT (13). In C57BL/6J mice that were tissue sections independently confirmed the presence of multi- bred at Ta = 22 °C, UCP1 expression began in the inguinal region locular beige adipocytes in P28 iWAT of thermoneutral mice (Fig. (left) of iWAT as early as postnatal day (P)14, spread to the 1D). Taken together with previous observations that neonatal dorsolumbar region (right) by P21, peaked around P28, and mice are poikilothermic (body temperature mirrors ambient tem- began to regress by P35 (Fig. 1A, SI Appendix, Fig. S1 A and B, perature) and only develop full thermoregulatory capacity around and Movie S1). To our surprise, this spatiotemporal pattern of P19 to P28 (14), our data suggest that beige adipogenesis is initiated UCP1 expression was preserved in iWAT of C57BL/6J mice that prior to acquisition of homeothermy in a temperature-independent were bred at thermoneutrality (Ta = 30 °C), suggesting that the manner. commitment to the beige adipocyte lineage occurs in a temperature- We next asked whether the pattern of UCP1 expression in independent manner (Fig. 1B and Movie S2). In support of this iWAT of cold-exposed adult animals was similar to its develop- hypothesis, quantitation of UCP1 protein expression during the peri- mental expression during the peri-weaning period. We found weaning period (P14–P35) revealed that while beige adipocyte de- that cold-induced beige fat recruitment in adult animals followed velopment occurred in a temperature-independent manner, the a similar spatiotemporal pattern as young mice; i.e., it began in amplitude of this response was gated by environmental temper- the inguinal region and progressively spread to the dorsolumbar + ature (Fig. 1C). For example, the UCP1 volume was comparable region of iWAT, as previously reported (Fig. 1 A and E) (13). C57BL/6J 22 C C57BL/6J 30 C A P14 UCP1 P21 UCP1 P28 UCP1 P35 UCP1 CD 5×109 ) 3 9 m 4×10 P 9 3×10 p<0.000001 p=0.00283 9 p=0.0058 B P14 UCP1 P21 UCP1 P28 UCP1 P35 UCP1 2×10 1×109 UCP1 volume ( 0 14 21 28 35 14 21 28 35 Age (days) E UCP1 RT UCP1 UCP1 UCP1 F 20 wk 20 wk G Ai14 Ai14 x Ucp1Cre J I 2 Biological Process -logP 1 Upregulated (210 genes) Sc5D 0 Ptges2 Muscle contraction 18.4 -1 Cpt2 Ehhadh Sarcomere organization 4.6 -2 Hsd17B12 Abhd5 Cardiac muscle tissue morphogenesis 4.1 Hmgcs1 Lss Regulation of ATPase activity 3.4 Hadha Cardiac muscle contraction 2.7 Fam57B Hadhb Insig1 (111 genes) (111 Aspg K Downregulated Hsd17B4 Acaa1B Beige White Thrsp