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Abstract

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Protocol

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Biology

Isolation and Differentiation of Stromal Vascular Cells to Beige/Brite Cells

Published: March 28th, 2013

DOI:

10.3791/50191

1UCSF Diabetes Center and Department of Cell and Tissue Biology, University of California, San Francisco , 2Department of Biology, University of Copenhagen, Denmark, 3National Institute of Nutrition and Seafood Research, Bergen, Norway

Primary white preadipocytes isolated from white adipose tissues in mice can be differentiated into beige/brite cells. Presented here is a reliable cellular model system to study the molecular regulation of "browning" of white fat.

Brown adipocytes have the ability to uncouple the respiratory chain in mitochondria and dissipate chemical energy as heat. Development of UCP1-positive brown adipocytes in white adipose tissues (so called beige or brite cells) is highly induced by a variety of environmental cues such as chronic cold exposure or by PPARγ agonists, therefore, this cell type has potential as a therapeutic target for obesity treatment. Although most immortalized adipocyte lines cannot recapitulate the process of "browning" of white fat in culture, primary adipocytes isolated from stromal vascular fraction in subcutaneous white adipose tissue (WAT) provide a reliable cellular system to study the molecular control of beige/brite cell development. Here we describe a protocol for effective isolation of primary preadipocytes and for inducing differentiation to beige/brite cells in culture. The browning effect can be assessed by the expression of brown fat-selective markers such as UCP1.

Obesity is dramatically increasing worldwide and is now considered one of the most serious concerns to public health 1. This condition is related to a misbalance in energy intake relative to expenditure and results in excess energy stored as lipid in white adipose tissue (WAT). Enlarged WAT is associated with increased body mass and weight, while brown adipose tissue has the ability to dissipate excess energy to produce heat. Hence BAT can function as protection against both cold and obesity 2,3 . This is achieved by uncoupling of the electron transport in mitochondria by uncoupling protein 1 (UCP1). This protein is considered a hallmark for nons....

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1. Prepare Digestion Medium

Make 5 ml per 5 mice per tissue (approximately 1 ml/1 g adipose tissue).

  1. Weigh in digestion enzymes:
    - Collaginase D: 1.5 u/ml (1108874103, 1 g, Roche, 70334223)
    - Dispase II: 2.4 u/ml (04942078001, 0,980 mg/lyo, Roche, 11466200)
  2. Add 25 ml PBS and mix well to dissolve
  3. Add CaCl2 just prior to digestion of the tissue at a final concentration of 10 mM

2. Dissect Adipose .......

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Browning of primary adipocytes can be accessed by measuring mRNA expression of Ucp1 and other brown fat-specific or selective genes by qRT-PCR. Presented in Figure 1 is gene expression data in inguinal WAT-derived primary adipocytes. The cells were induced to differentiate in the presence of two different doses of rosiglitazone at 50 nM and 500 nM, respectively. A subset of cells was treated with forskolin at 10 μM for 4 hr prior to harvest. This will induce cyclic-AMP (cAMP) in the cells and.......

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Here we present a reliable cellular system to study the development of beige/brite cells in primary cultured adipocytes in mice. As compared to several available immortalized cell lines, this system is likely to offer enhanced relevance to the browning of white fat in vivo.

Even though the study of these primary adipocytes offers some advantages, there also exist some limitations and concerns that are important to consider. First, this system is highly reliant on differentiation poten.......

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We thank Haruya Ohno, Kosaku Shinoda, Louis Sharp, Emi Tomoda, and Lauren Ruiz for discussion, technical help and editorial assistance on the manuscript. This work was supported by grants from the NIH (DK087853), from the Program for Breakthrough Biomedical Research and from Asubio Pharm Inc. to S.K. U.L.A. was supported by a SHARE PhD fellowships from The University of Copenhagen and the EU FP7 project DIABAT (HEALTH-F2-2011-278373) to Lise Madsen and Karsten Kristiansen. We also acknowledge the DERC center grant (NIH P30 DK063720).

....

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Name Company Catalog Number Comments
Reagent
Collaginase D Roche 11088874103
Dispase II Roche 04942078001
CaCl2
DMEM medium Fisher 10017-CV With 2,5 g/l glucose & L-glutamine without sodium pyruvate
Insulin
T3 (3,3',5-Triiodo-L-thyronine) Sigma T-2877
Indomethacin Sigma I-7378
Dexamethasone Sigma D-1756
IBMX Sigma I-5879
Rosiglitazone Sigma R-2408
Equipment
Collagen coated dishes BD 354450 10 cm plates
70 μm filter BD Falcon 352350 Cell strainer,70 μm nylon 1/ea

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