高通量呼吸运动计量法测量棕色脂肪细胞的产热功能评估

Kiana Mahdaviani; Ilan Benador; Orian Shirihai

doi:10.21769/BioProtoc.1641

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Peer-reviewed

Assessment of Brown Adipocyte Thermogenic Function by High-throughput Respirometry

高通量呼吸运动计量法测量棕色脂肪细胞的产热功能评估

KM Kiana Mahdaviani

IB Ilan Benador

OS Orian Shirihai email

发布: 2015年11月05日第5卷第21期 DOI: 10.21769/BioProtoc.1641 浏览次数: 14013

评审: Hong-guang XiaValentine V TrotterAnonymous reviewer(s)

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Cover of The EMBO Journal, featuring study using the protocol.

Mar 2014

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Abstract

Brown adipose tissue (BAT) has the unique ability to dramatically increase mitochondrial uncoupled fuel oxidation for thermogenesis in response to adrenergic stimulation. A key parameter in assessing brown adipocyte thermogenic capacity is mitochondrial uncoupling as determined by respiration. Measuring mitochondrial oxygen consumption rate (OCR) therefore provides valuable information to study the regulation and dysregulation of fuel metabolism and energy expenditure. Adding measurements of mitochondrial membrane potential allows for more in-depth interpretation of the respirometry data. Here we provide protocols for measuring respiration in adherent intact and plasma membrane permeabilized brown adipocytes using the Seahorse XF Analyzer. In the protocol Part I, a combination of norepinephrine and free fatty acids are used to induce uncoupled respiration. The ATP Synthase inhibitor oligomycin, the chemical uncoupler FCCP, and the complex III inhibitor Antimycin A are then used to measure coupled, maximal, and non-mitochondrial oxygen consumption, respectively. In the protocol Part II, the plasma membrane is permeabilized with recombinant perfringolysin O, a cholesterol-dependent cytolysin that oligomerizes into pores exclusively in the plasma membrane. This permits experimental control of metabolite availability without separating mitochondria from the native cell environment.

Part I. Intact brown adipocyte respiration

Materials and Reagents

Note: Rotenone, antimycin, oligomycin, and FCCP are toxic and light sensitive. Wear personal protective equipment when handling and store stocks in the dark.

3-4 weeks-old C57BL6/J mice
Note: Isolate primary pre adipocytes from brown adipose tissue, then differentiate them in an XF-24 well cell culture microplate for 7 days.
XF assay 24‐well cartridge and cell culture microplate (Seahorse Bioscience, catalog numbers: 100850-001 and 100777-004 )
XF assay 24-well cartridge and utility plate
DMEM (Thermo Fisher Scientific, Gibco^TM, catalog number: 12100-046 )
ewborn calf serum (Sigma-Aldrich, catalog number: N4887 )
HEPES (Corning, catalog number: 25-060-Cl )
L-Glutamine (Thermo Fisher Scientific, Gibco^TM, catalog number: 25030-164 )
Penicillin-Streptomycin Solution (100x) (Corning, catalog number: 30-002-Cl )
Sodium L-ascorbate (Sigma-Aldrich, catalog number: A4034 )
Rosiglitazone (used as differentiation agent) (Toronto Research Chemicals, catalog number: R693500 )
Insulin from Porcine Pancreas (Sigma-Aldrich, catalog number: I5523 )
XF-assay/base medium (Seahorse Bioscience, Catalog number: 102365-100 )
D-(+)-Glucose (Sigma-Aldrich, catalog numbers: G8270 )
XF Calibrant solution (Seahorse Bioscience, Catalog number: 100840‐000 )
L-(-)Norepinephrine(+)-bitartrate salt monohydrate (Sigma-Aldrich, Catalog number: N5785 )
Free fatty acids (Palmitic acid or Oleate) (Sigma-Aldrich, catalog number: P0500 )
DMSO (Sigma-Aldrich, catalog number: D2650 )
RPMI medium (no glucose) (Thermo Fisher Scientific, Gibco^TM, catalog number: 11879-020 )
Fatty‐acid‐free BSA (CalBiochem, catalog number: 126575 )
BAT differentiation media (see Recipes)
Seahorse assay media (SH assay media) (see Recipes)
Free fatty acid conjugated to the BSA (see Recipes)
Norepinephrine (see Recipes)
Oligomycin (Sigma-Aldrich, catalog number: 75371 ) (see Recipes)
Antimycin A from Streptomyces sp. (Sigma-Aldrich, catalog number: A8674 ) (see Recipes)
Carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP) (Sigma-Aldrich, catalog number: C2920 ) (see Recipes)
Sodium pyruvate (Sigma-Aldrich, catalog number: P5280 ) (see Recipes)

Equipment

CO₂-free incubator
8% CO₂ incubator
XF24 extracellular flux analyzer (Seahorse Bioscience)
Cell culture hood
pH meter
37 °C Water bath

Procedure

English

中文翻译

文章信息

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如何引用

Readers should cite both the Bio-protocol article and the original research article where this protocol was used:

Mahdaviani, K., Benador, I. and Shirihai, O. (2015). Assessment of Brown Adipocyte Thermogenic Function by High-throughput Respirometry. Bio-protocol 5(21): e1641. DOI: 10.21769/BioProtoc.1641.
Wikstrom, J. D., Mahdaviani, K., Liesa, M., Sereda, S. B., Si, Y., Las, G., Twig, G., Petrovic, N., Zingaretti, C. and Graham, A. (2014). Hormone‐induced mitochondrial fission is utilized by brown adipocytes as an amplification pathway for energy expenditure. EMBO J 33(5): 418-436.