Differentiation and Imaging of Brown Adipocytes from the Stromal Vascular Fraction of Interscapular Adipose Tissue from Newborn Mice

Summary

Preadipocytes are isolated from the stromal vascular fraction of interscapular brown adipose tissue from newborn mice and differentiated into cells that accumulate lipid droplets, express molecular markers, and show the mitochondrial morphology of mature brown adipocytes. These cells are further analyzed by immunofluorescence and transmission electron microscopy.

Abstract

Brown adipose tissue (BAT) is only present in mammals and has a thermogenic function. Brown adipocytes are characterized by a multilocular cytoplasm with multiple lipid droplets, a central nucleus, a high mitochondrial content, and the expression of uncoupling protein 1 (UCP1). BAT has been proposed as a potential therapeutic target for obesity and its associated metabolic disorders due to its ability to dissipate metabolic energy as heat. To investigate BAT function and regulation, brown adipocyte culturing is indispensable. The present protocol optimizes tissue processing and cell differentiation for culturing brown adipocytes from newborn mice. Additionally, procedures for the imaging of differentiated adipocytes with both confocal immunofluorescence and transmission electron microscopy are shown. In the brown adipocytes differentiated with the techniques described herein, the major defining features of classical BAT are preserved, including high UCP1 levels, increased mitochondrial mass, and very close physical contact between the lipid droplets and mitochondria, making this method a valuable tool for BAT studies.

Introduction

White and brown adipose tissue differ in their anatomical location, cellular origin, function, morphology, and total mass. White adipose tissue (WAT) is the major physiological energy reservoir of the body and stores large amounts of triacylglycerol (TAG) in highly specialized cells that have a single giant lipid droplet occupying most of their cellular volume¹. TAG lipolysis releases free fatty acids, which enter the systemic circulation to meet energy demands during fasting or other states of negative energy balance. Additionally, the WAT secretes protein and lipid products, called adipokines and lipokines, respectively, that have metabolic, ....

Protocol

The animal procedures were approved by the Institutional Animal Care and Use Committee at Pontificia Universidad Católica de Chile. P0.5 newborn mice of both sexes, derived from a mixed background of C57BL/6J and 129J strains, were used for this study.

1. Tissue extraction

1. Clean and disinfect the workbench with a 70% ethanol solution, and use sterile surgical material.
2. Euthanize P0.5 newborn pups by decapitation with surgical scissors following institutionally approved protocol.
3. Place the mouse in a prone position (facing downward), and make a 1 cm long incision in the skin at the m....

Results

Adipogenesis is regulated by a network of transcription factors that are responsible for both the expression of key proteins that induce brown adipocyte formation and functioning²², including classical adipogenic regulators such as PPARγ and C/EBPα^23,24,25, as well as markers of mature adipocytes^26,27. Through testing the different concentrations .......

Discussion

The present protocol is a simple and replicable two-phase differentiation procedure (Figure 2) for generating cells with the molecular and morphological characteristics of mature brown adipocytes. The surgical harvesting of the iBAT is the first critical step because tissue tearing severely limits the viability of the starting material. Tissue processing is also key because a homogeneous cell suspension that is free of debris greatly increases the amount of SVF that can be cultured. In the c.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
10x Tris/Glycine Buffer	BioRad	1610734
16% Paraformaldehyde Aqueous Solution	Electron Microscopy Sciences	15710
35 mm TC-treated Easy-Grip Style Cell Culture Dish	Falcon	353001
3-Isobutyl-1-methylxanthine (IBMX)	Calbiochem	410957
40% Acrylamide/Bis Solution, 37.5:1	BioRad	1610148
6-well plate	SPL Life Science	-
96 well optical black w/lid cell culture sterile	Thermo Scientific	165305
AccuRuler RGB Plus Pre-stained Protein Ladder	Maestrogen	02102-250
ACK lysing buffer	Gibco	A10492-01
Ammonium Persulfate	BioRad	1610700
Antibiotic-antimycotic	Gibco	15240062
Anti-mouse IgG, HRP-linked Antibody	Cell Signaling	7076
Anti-rabbit IgG, HRP-linked Antibody	Cell Signaling	7074
Blotting-grade blocker	BioRad	170-6404
BODIPY 493/503	Invitrogen	D3922
BSA	Sigma	A1470
C/EBPα antibody	Cell Signaling	2295
CaCl2	Calbiochem	208291
CD36 antibody	Invitrogen	PA1-16813
Cell Strainer 100 µm, nylon	Falcon	352360
Cell Strainer 40 µm, nylon	Falcon	352340
Collagenase type II	Gibco	17101-015
Cytation 5 Cell Imaging Multimode Reader	Biotek
Dexamethasone	Sigma	D4902
DMEM/F-12, powder	Gibco	12500062
EMBed-812 EMBEDDING KIT (Epon)	Electron Microscopy Sciences	14120
Ethanol absolute	Merck	100983
Fetal bovine serum	Gibco	16000-044
Gelatin from cold water fish skin	Sigma	G7041
Glucose	Gibco	15023-021
Glutaraldehyde 25% Aqueous Solution	Electron Microscopy Sciences	16210
Goat anti-Rabbit IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 594	Life Technologies	A11012
Halt Phosphatase Inhibitor Cocktail 100X	Thermo Scientific	78427
Halt Protease Inhibitor Cocktail 100X	Thermo Scientific	78429
Hoechst 33258	Invitrogen	H1398
Immun-Blot PVDF Membrane	BioRad	1620177
Indomethacin	Sigma	I7378
Insulin	Sigma	I3536
KCl	Calbiochem	529552
KH2PO4	Calbiochem	529568
KHCO3	Sigma	60339
Lane Marker Reducing Sample Buffer	Thermo Scientific	39000
MgSO4	Sigma	M2643
MilliQ water sterile	-	-
Mitoprofile Total OXPHOS Rodent WB antibody Cocktail	Abcam	MS604
NaCl	Merck	1064041000
OmniPur 10x PBS Liquid Concentrate	Calbiochem	6505-OP
Osmium Tetroxide	Electron Microscopy Sciences	19100
Perilipin 1 antibody	Cell Signaling	9349
PPARγ (81B8) antibody	Cell Signaling	2443
RIPA buffer lysis	Thermo Scientific	89901
Rosiglitazone	Merck	557366
Sodium bicarbonate	Sigma	S5761
Sodium Cacpdylate	Electron Microscopy Sciences	12300
Sodium Dodecyl Sulfate	BioRad	1610301
T3	Sigma	T6397
Talos F200C G2	Thermo Scientific
TEMED	BioRad	1610800
TOM20 antibody	Cell Signaling	42406
Tris Buffered Saline (TBS-10X)	Cell Signaling	12498
Triton X-100	Sigma	93443
Trypsin-EDTA (0,25%)	Gibco	25200056
Tween 20, Molecular Biology Grade	Promega	H5152
UCP1 antibody	Cell Signaling	14670
Ultracut R	Leica
Uranyl Acetate	Electron Microscopy Sciences	22400
Westar Sun	Cyanagen	XLS063
Westar Supernova	Cyanagen	XLS3