Skip to main content
SpringerLink
Log in

Flow Cytometric Isolation and Differentiation of Adipogenic Progenitor Cells into Brown and Brite/Beige Adipocytes

  • Protocol
  • First Online:
Thermogenic Fat

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1566))

Abstract

Aside from mature adipocytes, adipose tissue harbors several distinct cell populations including immune cells, endothelial cells, and adipogenic progenitor cells (AdPCs). AdPCs represent the reservoir of regenerative cells that replenishes adipocytes during normal cellular turnover and during times of increased demand for triglyceride-storage capacity. The worldwide increase in pathologies associated with the metabolic syndrome, such as obesity and type-2 diabetes, has heightened public and scientific interest in adipose tissues and the cell biological processes of adipose tissue formation and function. Two distinct types of fat cells are known: White and brown adipocytes. Especially brown adipose tissue (BAT) has received considerable attention due to its unique capacity for thermogenic energy expenditure and potential role in the treatment of adiposity. Accordingly, the cold-induced conversion of white into brown-like adipocytes has become a feasible approach in humans and a study-subject in rodents to better understand the underlying molecular processes. Fluorescence-activated cell sorting (FACS) provides a method to isolate AdPCs and other cell populations from adipose tissue by using antibodies detecting unique surface markers. We here describe an approach to isolate cells committed to the adipogenic lineage and summarize established protocols to differentiate FACS-purified primary AdPCs into UCP1-expressing brown adipocytes under in vitro conditions.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 119.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 159.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 159.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. World Health Organization (2014) Obesity and overweight fact sheet N°311.http://www.who.int/mediacentre/factsheets/fs311/en/

  2. Bluher M (2014) Adipokines—removing road blocks to obesity and diabetes therapy. Mol Metab 3:230–240

    Article  PubMed  PubMed Central  Google Scholar 

  3. Kershaw EE, Flier JS (2004) Adipose tissue as an endocrine organ. J Clin Endocrinol Metab 89:2548–2556

    Article  CAS  PubMed  Google Scholar 

  4. Zhang Y, Proenca R, Maffei M et al (1994) Positional cloning of the mouse obese gene and its human homologue. Nature 372:425–432

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  6. Cypess AM, Lehman S, Williams G et al (2009) Identification and importance of brown adipose tissue in adult humans. N Engl J Med 360:1509–1517

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. van Marken Lichtenbelt WD, Vanhommerig JW, Smulders NM et al (2009) Cold-activated brown adipose tissue in healthy men. N Engl J Med 360:1500–1508

    Article  PubMed  Google Scholar 

  9. Virtanen KA, Lidell ME, Orava J et al (2009) Functional brown adipose tissue in healthy adults. N Engl J Med 360:1518–1525

    Article  CAS  PubMed  Google Scholar 

  10. Zingaretti MC, Crosta F, Vitali A et al (2009) 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

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Matsushita M, Yoneshiro T, Aita S et al (2013) Impact of brown adipose tissue on body fatness and glucose metabolism in healthy humans. Int J Obes (Lond) 38:812–817

    Article  Google Scholar 

  13. Bakker LEH, Boon MR, van der Linden RAD et al (2014) Brown adipose tissue volume in healthy lean south Asian adults compared with white Caucasians: a prospective, case-controlled observational study. Lancet Diabetes Endocrinol 2:210–217

    Article  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  15. Lichtenbelt WV, Kingma B, van der Lans A et al (2014) Cold exposure—an approach to increasing energy expenditure in humans. Trends Endocrinol Metab 25:165–167

    Article  CAS  PubMed  Google Scholar 

  16. Schulz TJ, Tseng YH (2013) Brown adipose tissue: development, metabolism and beyond. Biochem J 453:167–178

    Article  CAS  PubMed  Google Scholar 

  17. Rosen ED, Spiegelman BM (2014) What we talk about when we talk about fat. Cell 156:20–44

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Ishibashi J, Seale P (2010) Medicine. Beige can be slimming. Science 328:1113–1114

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Petrovic N, Walden TB, Shabalina IG et al (2010) Chronic peroxisome proliferator-activated receptor gamma (PPAR gamma) 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

    Article  CAS  PubMed  Google Scholar 

  20. Shabalina IG, Petrovic N, de Jong JM et al (2013) UCP1 in brite/beige adipose tissue mitochondria is functionally thermogenic. Cell Rep 5:1196–1203

    Article  CAS  PubMed  Google Scholar 

  21. Kajimura S, Seale P, Spiegelman BM (2010) Transcriptional control of brown fat development. Cell Metab 11:257–262

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Kajimura S, Seale P, Kubota K et al (2009) Initiation of myoblast to brown fat switch by a PRDM16-C/EBP-beta transcriptional complex. Nature 460:1154–1158

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Seale P, Bjork B, Yang W et al (2008) PRDM16 controls a brown fat/skeletal muscle switch. Nature 454:961–967

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Seale P, Conroe HM, Estall J et al (2011) Prdm16 determines the thermogenic program of subcutaneous white adipose tissue in mice. J Clin Invest 121:96–105

    Article  CAS  PubMed  Google Scholar 

  25. Vitali A, Murano I, Zingaretti MC et al (2012) The adipose organ of obesity-prone C57BL/6J mice is composed of mixed white and brown adipocytes. J Lipid Res 53:619–629

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Himms-Hagen J, Melnyk A, Zingaretti MC et al (2000) Multilocular fat cells in WAT of CL-316243-treated rats derive directly from white adipocytes. Am J Physiol Cell Physiol 279:C670–C681

    CAS  PubMed  Google Scholar 

  27. Barbatelli G, Murano I, Madsen L et al (2010) The emergence of cold-induced brown adipocytes in mouse white fat depots is determined predominantly by white to brown adipocyte transdifferentiation. Am J Physiol Endocrinol Metab 298:E1244–E1253

    Article  CAS  PubMed  Google Scholar 

  28. Schulz TJ, Huang P, Huang TL et al (2013) Brown-fat paucity due to impaired BMP signalling induces compensatory browning of white fat. Nature 495:379–383

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Weisberg SP, McCann D, Desai M et al (2003) Obesity is associated with macrophage accumulation in adipose tissue. J Clin Invest 112:1796–1808

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Rodeheffer MS, Birsoy K, Friedman JM (2008) Identification of white adipocyte progenitor cells in vivo. Cell 135:240–249

    Article  CAS  PubMed  Google Scholar 

  31. Schulz TJ, Huang TL, Tran TT et al (2011) Identification of inducible brown adipocyte progenitors residing in skeletal muscle and white fat. Proc Natl Acad Sci USA 108:143–148

    Article  CAS  PubMed  Google Scholar 

  32. Tang W, Zeve D, Suh JM et al (2008) White fat progenitor cells reside in the adipose vasculature. Science 322:583–586

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Berry DC, Stenesen D, Zeve D et al (2013) The developmental origins of adipose tissue. Development 140:3939–3949

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Berry R, Rodeheffer MS (2013) Characterization of the adipocyte cellular lineage in vivo. Nat Cell Biol 15:302–308

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Tseng YH, Kokkotou E, Schulz TJ et al (2008) New role of bone morphogenetic protein 7 in brown adipogenesis and energy expenditure. Nature 454:1000–1004

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Vegiopoulos A, Muller-Decker K, Strzoda D et al (2010) Cyclooxygenase-2 controls energy homeostasis in mice by de novo recruitment of brown adipocytes. Science 328:1158–1161

    Article  CAS  PubMed  Google Scholar 

  37. Steenhuis P, Pettway GJ, Ignelzi MA Jr (2008) Cell surface expression of stem cell antigen-1 (Sca-1) distinguishes osteo-, chondro-, and adipoprogenitors in fetal mouse calvaria. Calcif Tissue Int 82:44–56

    Article  CAS  PubMed  Google Scholar 

  38. Himms-Hagen J, Cui J, Danforth E Jr et al (1994) Effect of CL-316,243, a thermogenic beta 3-agonist, on energy balance and brown and white adipose tissues in rats. Am J Physiol 266:R1371–R1382

    CAS  PubMed  Google Scholar 

  39. Klaus S, Choy L, Champigny O et al (1994) Characterization of the novel brown adipocyte cell line HIB 1B. Adrenergic pathways involved in regulation of uncoupling protein gene expression. J Cell Sci 107:313–319

    CAS  PubMed  Google Scholar 

  40. Lehr L, Canola K, Asensio C et al (2006) The control of UCP1 is dissociated from that of PGC-1alpha or of mitochondriogenesis as revealed by a study using beta-less mouse brown adipocytes in culture. FEBS Lett 580:4661–4666

    Article  CAS  PubMed  Google Scholar 

  41. Orr JS, Kennedy AJ, Hasty AH (2013) Isolation of adipose tissue immune cells. J Vis Exp 22(75):e50707

    Google Scholar 

Download references

Acknowledgments

This work was supported by grants from the German Research Foundation (DFG; grant # SCHU 2445/2-1) and the European Research Council (grant # ERC-StG-2012-311082) to T.J.S. The authors gratefully acknowledge support from the German Center for Diabetes Research (DZD).

Author information

Authors and Affiliations

  1. Research Group Adipocyte Development, German Institute of Human Nutrition (DlfE), Potsdam-Rehbrucke 114-116, Arthur-Scheunert-Allee, D-14558, Nuthetal, Germany

    Jochen Steinbring, Antonia Graja, Anne-Marie Jank & Tim J. Schulz

  2. German Center for Diabetes Research (DZD), 85764, München-Neuherberg, Germany

    Tim J. Schulz

Authors
  1. Jochen Steinbring
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Antonia Graja
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Anne-Marie Jank
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Tim J. Schulz
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Tim J. Schulz .

Editor information

Editors and Affiliations

  1. Department of Molecular and Integrative Physiology, Life Sciences Institute, University of Michigan, Ann Arbor, Michigan, USA

    Jun Wu

Rights and permissions

Reprints and permissions

Copyright information

© 2017 Springer Science+Business Media LLC

About this protocol

Cite this protocol

Steinbring, J., Graja, A., Jank, AM., Schulz, T.J. (2017). Flow Cytometric Isolation and Differentiation of Adipogenic Progenitor Cells into Brown and Brite/Beige Adipocytes. In: Wu, J. (eds) Thermogenic Fat. Methods in Molecular Biology, vol 1566. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6820-6_4

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-6820-6_4

  • Published:

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-6819-0

  • Online ISBN: 978-1-4939-6820-6

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation