Clonal analyses and gene profiling identify genetic biomarkers of the thermogenic potential of human brown and white preadipocytes


Targeting brown adipose tissue (BAT) content or activity has therapeutic potential for treating obesity and the metabolic syndrome by increasing energy expenditure. However, both inter- and intra-individual differences contribute to heterogeneity in human BAT and potentially to differential thermogenic capacity in human populations. Here we generated clones of brown and white preadipocytes from human neck fat and characterized their adipogenic and thermogenic differentiation. We combined an uncoupling protein 1 (UCP1) reporter system and expression profiling to define novel sets of gene signatures in human preadipocytes that could predict the thermogenic potential of the cells once they were maturated. Knocking out the positive UCP1 regulators, PREX1 and EDNRB, in brown preadipocytes using CRISPR-Cas9 markedly abolished the high level of UCP1 in brown adipocytes differentiated from the preadipocytes. Finally, we were able to prospectively isolate adipose progenitors with great thermogenic potential using the cell surface marker CD29. These data provide new insights into the cellular heterogeneity in human fat and offer potential biomarkers for identifying thermogenically competent preadipocytes.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

Figure 1: Generation and characterization of immortalized human brown and white fat progenitors.
Figure 2: Use of a UCP1 reporter system for in vitro and in vivo monitoring of UCP1 expression.
Figure 3: Clonal analysis of human brown and white fat progenitors.
Figure 4: Gene expression profiles in adipose progenitors predict the thermogenic capacity of mature adipocytes.
Figure 5: PREX1 and EDNRB are required for determining thermogenic competency.
Figure 6: Isolation of progenitors possessing thermogenic potential using a cell surface marker.

Accession codes

Primary accessions

Gene Expression Omnibus


  1. 1

    Cannon, B. & Nedergaard, J. Brown adipose tissue: function and physiological significance. Physiol. Rev. 84, 277–359 (2004).

  2. 2

    Schulz, T.J. & Tseng, Y.H. Brown adipose tissue: development, metabolism and beyond. Biochem. J. 453, 167–178 (2013).

  3. 3

    Guerra, C., Koza, R.A., Yamashita, H., Walsh, K. & Kozak, L.P. Emergence of brown adipocytes in white fat in mice is under genetic control. Effects on body weight and adiposity. J. Clin. Invest. 102, 412–420 (1998).

  4. 4

    Petrovic, N. et al. Chronic peroxisome proliferator-activated receptor gamma (PPARγ) activation of epididymally derived white adipocyte cultures reveals a population of thermogenically competent, UCP1-containing adipocytes molecularly distinct from classic brown adipocytes. J. Biol. Chem. 285, 7153–7164 (2010).

  5. 5

    Harms, M. & Seale, P. Brown and beige fat: development, function and therapeutic potential. Nat. Med. 19, 1252–1263 (2013).

  6. 6

    Nedergaard, J. & Cannon, B. The browning of white adipose tissue: some burning issues. Cell Metab. 20, 396–407 (2014).

  7. 7

    Bartelt, A. et al. Brown adipose tissue activity controls triglyceride clearance. Nat. Med. 17, 200–205 (2011).

  8. 8

    Stanford, K.I. et al. Brown adipose tissue regulates glucose homeostasis and insulin sensitivity. J. Clin. Invest. 123, 215–223 (2013).

  9. 9

    Nedergaard, J., Bengtsson, T. & Cannon, B. Unexpected evidence for active brown adipose tissue in adult humans. Am. J. Physiol. Endocrinol. Metab. 293, E444–E452 (2007).

  10. 10

    Cypess, A.M. et al. Identification and importance of brown adipose tissue in adult humans. N. Engl. J. Med. 360, 1509–1517 (2009).

  11. 11

    van Marken Lichtenbelt, W.D. et al. Cold-activated brown adipose tissue in healthy men. N. Engl. J. Med. 360, 1500–1508 (2009).

  12. 12

    Virtanen, K.A. et al. Functional brown adipose tissue in healthy adults. N. Engl. J. Med. 360, 1518–1525 (2009).

  13. 13

    Saito, M. et al. High incidence of metabolically active brown adipose tissue in healthy adult humans: effects of cold exposure and adiposity. Diabetes 58, 1526–1531 (2009).

  14. 14

    Zingaretti, M.C. et al. The presence of UCP1 demonstrates that metabolically active adipose tissue in the neck of adult humans truly represents brown adipose tissue. FASEB J. 23, 3113–3120 (2009).

  15. 15

    Himms-Hagen, J. et al. Effect of CL-316,243, a thermogenic β3-agonist, on energy balance and brown and white adipose tissues in rats. Am. J. Physiol. 266, R1371–R1382 (1994).

  16. 16

    Yoneshiro, T. et al. Recruited brown adipose tissue as an antiobesity agent in humans. J. Clin. Invest. 123, 3404–3408 (2013).

  17. 17

    van der Lans, A.A. et al. Cold acclimation recruits human brown fat and increases nonshivering thermogenesis. J. Clin. Invest. 123, 3395–3403 (2013).

  18. 18

    Ouellet, V. et al. Brown adipose tissue oxidative metabolism contributes to energy expenditure during acute cold exposure in humans. J. Clin. Invest. 122, 545–552 (2012).

  19. 19

    Wu, J. et al. Beige adipocytes are a distinct type of thermogenic fat cell in mouse and human. Cell 150, 366–376 (2012).

  20. 20

    Cypess, A.M. et al. Anatomical localization, gene expression profiling and functional characterization of adult human neck brown fat. Nat. Med. 19, 635–639 (2013).

  21. 21

    Lidell, M.E. et al. Evidence for two types of brown adipose tissue in humans. Nat. Med. 19, 631–634 (2013).

  22. 22

    Jespersen, N.Z. et al. A classical brown adipose tissue mRNA signature partly overlaps with brite in the supraclavicular region of adult humans. Cell Metab. 17, 798–805 (2013).

  23. 23

    Schulz, T.J. et al. Identification of inducible brown adipocyte progenitors residing in skeletal muscle and white fat. Proc. Natl. Acad. Sci. USA 108, 143–148 (2011).

  24. 24

    Lee, Y.H., Petkova, A.P., Mottillo, E.P. & Granneman, J.G. In vivo identification of bipotential adipocyte progenitors recruited by β3-adrenoceptor activation and high-fat feeding. Cell Metab. 15, 480–491 (2012).

  25. 25

    Berry, R. & Rodeheffer, M.S. Characterization of the adipocyte cellular lineage in vivo. Nat. Cell Biol. 15, 302–308 (2013).

  26. 26

    Wang, W. et al. Ebf2 is a selective marker of brown and beige adipogenic precursor cells. Proc. Natl. Acad. Sci. USA 111, 14466–14471 (2014).

  27. 27

    Tchkonia, T. et al. Fat depot–specific characteristics are retained in strains derived from single human preadipocytes. Diabetes 55, 2571–2578 (2006).

  28. 28

    Whittle, A.J. et al. BMP8B increases brown adipose tissue thermogenesis through both central and peripheral actions. Cell 149, 871–885 (2012).

  29. 29

    Tseng, Y.H. et al. New role of bone morphogenetic protein 7 in brown adipogenesis and energy expenditure. Nature 454, 1000–1004 (2008).

  30. 30

    Seale, P. et al. Transcriptional control of brown fat determination by PRDM16. Cell Metab. 6, 38–54 (2007).

  31. 31

    Timmons, J.A. et al. Myogenic gene expression signature establishes that brown and white adipocytes originate from distinct cell lineages. Proc. Natl. Acad. Sci. USA 104, 4401–4406 (2007).

  32. 32

    Welch, H.C. et al. P-Rex1, a PtdIns(3,4,5)P3- and Gβγ-regulated guanine-nucleotide exchange factor for Rac. Cell 108, 809–821 (2002).

  33. 33

    Cheung, J. et al. Identification of the human cortactin-binding protein-2 gene from the autism candidate region at 7q31. Genomics 78, 7–11 (2001).

  34. 34

    Zhang, S.X. et al. Identification of direct serum-response factor gene targets during Me2SO-induced P19 cardiac cell differentiation. J. Biol. Chem. 280, 19115–19126 (2005).

  35. 35

    Yamada, Y. et al. Cloning and functional characterization of a family of human and mouse somatostatin receptors expressed in brain, gastrointestinal tract and kidney. Proc. Natl. Acad. Sci. USA 89, 251–255 (1992).

  36. 36

    Kikkawa, T. et al. Dmrta1 regulates proneural gene expression downstream of Pax6 in the mammalian telencephalon. Genes Cells 18, 636–649 (2013).

  37. 37

    Garciafigueroa, D.Y., Klei, L.R., Ambrosio, F. & Barchowsky, A. Arsenic-stimulated lipolysis and adipose remodeling is mediated by G-protein–coupled receptors. Toxicol. Sci. 134, 335–344 (2013).

  38. 38

    Chen, T.Y. et al. Endogenous n-3 polyunsaturated fatty acids (PUFAs) mitigate ovariectomy-induced bone loss by attenuating bone marrow adipogenesis in FAT1 transgenic mice. Drug Des. Devel. Ther. 7, 545–552 (2013).

  39. 39

    Behjati, S. et al. Recurrent PTPRB and PLCG1 mutations in angiosarcoma. Nat. Genet. 46, 376–379 (2014).

  40. 40

    Takada, Y., Ye, X. & Simon, S. The integrins. Genome Biol. 8, 215 (2007).

  41. 41

    Margadant, C., Monsuur, H.N., Norman, J.C. & Sonnenberg, A. Mechanisms of integrin activation and trafficking. Curr. Opin. Cell Biol. 23, 607–614 (2011).

  42. 42

    Balamatsias, D. et al. Identification of P-Rex1 as a novel Rac1-guanine nucleotide exchange factor (GEF) that promotes actin remodeling and GLUT4 protein trafficking in adipocytes. J. Biol. Chem. 286, 43229–43240 (2011).

  43. 43

    Lewis, J.P. et al. Analysis of candidate genes on chromosome 20q12–13.1 reveals evidence for BMI-mediated association of PREX1 with type 2 diabetes in European Americans. Genomics 96, 211–219 (2010).

  44. 44

    Wu-Wong, J.R., Berg, C.E. & Dayton, B.D. Endothelin-stimulated glucose uptake: effects of intracellular Ca2+, cAMP and glucosamine. Clin. Sci. 103 (suppl. 48), 418S–423S (2002).

  45. 45

    Juan, C.C. et al. Effect of endothelin-1 on lipolysis in rat adipocytes. Obesity (Silver Spring) 14, 398–404 (2006).

  46. 46

    Shinoda, K. et al. Genetic and functional characterization of clonally derived adult human brown adipocytes. Nat. Med. 21, 389–394 (2015).

  47. 47

    Gierloff, M. et al. Adipogenic differentiation potential of rat adipose tissue–derived subpopulations of stromal cells. J. Plast. Reconstr. Aesthet. Surg. 67, 1427–1435 (2014).

  48. 48

    Farnier, C. et al. The signaling pathway for β1-integrin/ERKs is involved in the adaptation of adipocyte functions to cell size. Ann. NY Acad. Sci. 973, 594–597 (2002).

  49. 49

    Kawaguchi, N. et al. ADAM12 induces actin cytoskeleton and extracellular matrix reorganization during early adipocyte differentiation by regulating β1 integrin function. J. Cell Sci. 116, 3893–3904 (2003).

  50. 50

    Irizarry, R.A. et al. Exploration, normalization, and summaries of high-density oligonucleotide array probe level data. Biostatistics 4, 249–264 (2003).

  51. 51

    Benjamin, Y. et al. Controlling the false discovery rate: a practical and powerful approach to multiple testing. J. R. Statist. Soc. B 57, 289–300 (1995).

Download references


This work was supported in part by US National Institutes of Health (NIH) grants R01DK077097 (to Y.-H.T.), R01DK099511 (to L.J.G.), K23DK081604 (to A.M.C.) and P30DK036836 (to Joslin Diabetes Center's Diabetes Research Center, DRC) from the National Institute of Diabetes and Digestive and Kidney Diseases, a sponsored research grant from Chugai Pharmaceutical Co. (to Y.-H.T. and A.M.C.), a research grant from the American Diabetes Association (ADA 7-12-BS-191, to Y.-H.T.), the Intramural Research Program of the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK), and by funding from the Harvard Stem Cell Institute (to Y.-H.T.). M.D.L. was supported by NIH fellowships (T32DK007260 and F32DK102320). We thank M.-E. Patti and K. Hughes of the Advanced Genomics and Genetics Core of Joslin's DRC for advice and expert technical assistance. The authors thank Stryker Regenerative Medicine (Hopkinton, Massachusetts) for the generous gift of recombinant BMP7.

Author information

The study was designed by Y.-H.T., R.X., M.D.L. and A.M.C. The manuscript was written by Y.-H.T., M.D.L., R.X. and J.M.D. R.X. performed the majority of the experiments. M.D.L. did the time-lapse imaging, IVIS scanning and FACS. J.M.D. analyzed microarray data. F.S. performed bioenergetics analyses in knockout cells. T.J.S. and H.Z. established the method of isolation, immortalization and differentiation of human fat progenitors. T.L.H. did the human cell implantation and gene expression microarrays. K.L.T. provided assistance with the Seahorse bioanalyzer. Y.L. provided research assistance. H.T. and L.J.G. helped with fuel utilization experiments. A.M.C., L.S.W. and A.P.W. collected human fat samples. M.S.L. and L.L.R. helped with the time-lapse imaging. All authors contributed to editing the manuscript.

Correspondence to Yu-Hua Tseng.

Ethics declarations

Competing interests

A.M.C. and Y.-H.T. are recipients of a sponsored research grant and licensing payments from Chugai Pharmaceutical Co., Ltd through Joslin Diabetes Center.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–9 & Supplementary Tables 1–5 (PDF 1480 kb)

Time lapse imaging of hBAT-SVF differentiation (MOV 31877 kb)

Time lapse imaging of hBAT-SVF differentiation (MOV 31877 kb)

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Xue, R., Lynes, M., Dreyfuss, J. et al. Clonal analyses and gene profiling identify genetic biomarkers of the thermogenic potential of human brown and white preadipocytes. Nat Med 21, 760–768 (2015).

Download citation

Further reading