Sign In

A subscription to JoVE is required to view this content. Sign in or start your free trial.

In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

This study describes a new method of isolating murine brown adipocytes for gene and protein expression analysis.

Abstract

Brown adipose tissue (BAT) is responsible for non-shivering thermogenesis in mammals, and brown adipocytes (BAs) are the functional units of BAT. BAs contain both multilocular lipid droplets and abundant mitochondria, and they express uncoupling protein 1 (UCP1). BAs are categorized into two sub-types based on their origin: embryo derived classical BAs (cBAs) and white adipocytes derived BAs. Due to their relatively low density, BAs cannot be isolated from BAT with traditional centrifugation method. In this study, a new method was developed to isolate BAs from mice for gene and protein expression analysis. In this protocol, interscapular BAT from adult mice was digested with Collagenase and Dispase solution, and the dissociated BAs were enriched with 6% iodixanol solution. Isolated BAs were then lysed with Trizol reagent for simultaneous isolation of RNA, DNA, and protein. After RNA isolation, the organic phase of the lysate was used for protein extraction. Our data showed that 6% iodixanol solution efficiently enriched BAs without interfering with follow-up gene and protein expression studies. Platelet-derived growth factor (PDGF) is a growth factor that regulates the growth and proliferation of mesenchymal cells. Compared to the brown adipose tissue, isolated BAs had significantly higher expression of Pdgfa. In summary, this new method provides a platform for studying the biology of brown adipocytes at a single cell-type level.

Introduction

Both mice and humans have two types of adipose tissues: white adipose tissue (WAT) and brown adipose tissue (BAT)1. WAT stores energy in the form of triglycerides in white adipocytes, and the brown adipocytes (BAs) of BAT dissipate chemical energy as heat2. Based on their developmental origin, BAs are further categorized into classical BAs (cBAs) that formed during embryo development and white adipocytes derived BAs (beige/brite cells, converted from white adipocytes under stress conditions)3. BAs are multilocular and express the thermogenic protein uncoupling protein 1 (UCP1)4

Protocol

All mice were maintained in pathogen-free conditions, and all procedures were approved by Masonic Medical research Institutional Animal Care and Use Committee (IACUC). UCP1::Cre9 and Rosa 26tdTomato mice lines13 were reported previously. All mice were kept at room temperature with a 12 h light/dark cycle.

1. Preparing the Solutions and brown adipose tissue (BAT)

  1. Prepare digestion solution and separation solution in 15 mL ce.......

Representative Results

Preparation of interacapular BAT for brown adipocytes isolation
The brown adipocytes (BAs) isolation process is depicted in Figure 1A. The whole process, from preparing BAT and digestion/separation solutions to obtaining isolated BAs will take around 4 h.

In adult mice, abundant BAT exists in the interscapular region. This interscapular BAT (iBAT) is covered by muscle layers and WAT (Figure 1B). Before starting .......

Discussion

In this study, a new method of isolating BAs for gene and protein expression analysis was developed.

In a published BAs isolation protocol, 3% BSA solution was used to enrich BAs12. Nevertheless, the enriched BAs achieved by this published protocol could not be directly used for protein expression analysis. This is because the concentrated BSA existing in the BAs solution interferes with following-up protein extraction. When the BAs enriched in the 3% BSA solution were .......

Acknowledgements

Z. Lin was supported by National Institutes of Health HL138454-01 and Masonic Medical Research Institute funds.

....

Materials

NameCompanyCatalog NumberComments
Antibodies
AntigenCompanyCatalog
PPARγLSBioLs-C368478
PDGFRaSanta Cruzsc-398206
UCP1R&D systemIC6158P
Chemical and solutions
Collagenase, Type IIThermo Fisher Scientific17101015
1-Bromo-3-chloropropaneSigma-AldrichB62404
Bovine Serum Albumin (BSA) GoldbioA-421-10
Calcium chlorideBio BasicCT1330
ChloroformIBI ScientificIB05040
Dispase II, proteaseSigma-AldrichD5693
EDTABio BasicEB0107
EthanolIBI ScientificIB15724
LiQuant Universal Green qPCR Master MixLifeSctLS01131905Y
Magnesium Chloride HexahydrateBoston BioProductsP-855
OneScrip Plus cDNA Synthesis SuperMixABMG454
OptiPrep (Iodixanol)Cosmo Bio USAAXS-1114542
PBS (10x)Caisson LabsPBL07
PBS (1x)Caisson LabsPBL06
Pierce BCA Protein Assay KitThermo Fisher Scientific23227
Potassium ChlorideBoston BioProductsP-1435
SimplyBlue safe StainInvitrogenLC6060
Sodium dodecyl sulfate (SDS)Sigma-Aldrich75746
Trizol reagentLife technoologies15596018
Primers
Gene name (Species) ForwardReverse
Pdgfra (Mouse)CTCAGCTGTCTCCTCACAgGCAACGCATCTCAGAGAAAAGG
Pdgfa (Mouse)TGTGCCCATTCGCAGGAAGAGTTGGCCACCTTGACACTGCG
36B4(Mouse)TGCTGAACATCTCCCCCTTCTCTCTCCACAGACAATGCCAGGAC
Ucp1ACTGCCACACCTCCAGTCATTCTTTGCCTCACTCAGGATTGG
Equipment
NameCompanyApplication
Keyence BZ-X700KeyenceImaging brown adipocytes
Magnetic stirrerVWRDissociate BAT
QuantStudio 6 Flex Real-Time PCR SystemApplied BiosystemQuantitative PCR
The Odyssey Fc Imaging systemLI-CORWestern blot immaging

References

  1. Zwick, R. K., Guerrero-Juarez, C. F., Horsley, V., Plikus, M. V. Anatomical, physiological, and functional diversity of adipose tissue. Cell Metabolism. 27, 68-83 (2018).
  2. Symonds, M. E. Brown adipose tissue growth and development.

Explore More Articles

Brown AdipocytesInterscapular Brown Adipose TissueGene ExpressionProtein ExpressionAdipocyte IsolationAdipose Tissue PurificationIodixanol GradientTRIzolRNA IsolationProtein Isolation

This article has been published

Video Coming Soon

JoVE Logo

Privacy

Terms of Use

Policies

Research

Education

ABOUT JoVE

Copyright © 2024 MyJoVE Corporation. All rights reserved