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, immune, and reproductive regulatory functions, thus making the WAT the largest endocrine tissue in the body².

Brown adipose tissue (BAT) is a much smaller organ whose main physiological function is non-shivering thermogenesis to prevent hypothermia. In mice and newborn humans, BAT is a well-defined organ located in the interscapular space. Adult humans lack interscapular BAT (iBAT); nevertheless, they develop clusters of brown adipocyte-like cells integrated in depots that otherwise mostly comprise WAT. These "brown-in-white" (brite) adipocytes share morphological and molecular features with classical iBAT adipocytes, but they have a different cellular origin^3,4.

In contrast to white adipocytes, brown adipocytes have multiple small lipid droplets and abundant mitochondria⁵. Uncoupling protein 1 (UCP1, also known as thermogenin) is uniquely expressed by brown and brite adipocytes and mediates proton leakage in the inner mitochondrial membrane (IMM), thus uncoupling electron transport from ATP synthesis and generating heat. Non-shivering thermogenesis in BAT is activated by norepinephrine (NE), which is released from the sympathetic terminals in the BAT in response to cold stimulation⁶. NE binds to beta-adrenoceptors (mostly beta 3) on the surface of brown adipocytes and triggers an intracellular cAMP-mediated signaling cascade. This results in TAG lipolysis, the beta-oxidation of mitochondrial fatty acids, and heat generation upon UCP1 activation³. The close functional relationship between lipid droplets and mitochondria in brown adipocytes has structural parallels, such as the interaction between these organelles in areas that are large and have very tight physical contact^7,8.

iBAT has abundant blood vessels and sympathetic terminals⁹. These structures, along with the preadipocytes, immune cells, fibroblasts, and extracellular matrix molecules, compose the adipose stromal vascular fraction (SVF)¹⁰. Many protocols have been reported to generate mature adipocytes from preadipocytes^{11,12,13,14,15} (Supplementary Table 1); nevertheless, they display extreme variations in tissue processing and the composition of the differentiation culture media. The protocol described herein allows the efficient and reproducible differentiation of brown adipocytes that (1) express the key adipogenic transcription factors peroxisome proliferator-activated receptor gamma (PPARγ) and CCAAT/enhancer-binding protein alpha (C/EBPα), (2) express the mature adipocyte markers perilipin1 (PLIN1), and cluster determinant 36 (CD36), (3) accumulate abundant lipid droplets, (4) have high mitochondrial mass and a high abundance of OXPHOS complexes, (5) have thermogenic potential, as determined by high levels of UCP1, and (6) have mitochondrial morphological changes associated with the phenotype of mature brown adipocytes. This methodology is used for studying the molecular mechanisms underlying generalized lipodystrophy^15,16,17.

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 mid-level of the animal's back using sharp scissors. Remove the skin carefully.
   NOTE: This step is critical because if the skin is not well removed, it might tear and damage the iBAT and preclude the optimal preparation of the SVF.
4. Surgically detach the iBAT from the carcass using small scissors.
   ​NOTE: The iBAT is visualized as two fatty lobules that can be removed independently.
5. Harvest both iBAT lobes by using curved-tip pliers to remove the lobes independently.
6. Place the two lobes in a plastic tube with 1.5 mL of sterile PBS at room temperature. Maintain the tissues in their individual tubes until the tissue extraction is completed for all the pups required.

2. Tissue digestion

1. Aliquot 300 µL of collagenase type II digestion buffer (0.2% type II collagenase in buffer [25 mM KHCO₃, 1.2 mM MgSO₄, 120 mM NaCl, 5 mM glucose, 12 mM KH₂PO₄, 4.8 mM KCl, 1.2 mM CaCl₂, 2.5% BSA, and 1% penicillin/streptomycin, at pH 7.4]) (Table 1) in 1.5 mL tubes in a biosafety cabinet.
   NOTE: Prepare sufficient tubes for the number of individual tissues to be extracted. In the present study, seven tubes were used, corresponding to a litter of seven pups.
2. Move to the bench, and place the iBAT lobes in collagenase type II digestion buffer to degrade the extracellular matrix.
3. Incubate the iBAT tissues for 45 min at 37 °C with continuous gentle shaking at 800 rpm (Figure 1).

3. Tissue processing

NOTE: After the digestion, perform all the following steps in a class II laminar flow tissue culture hood.

1. Prepare and label two sets of 1.5 mL plastic tubes for each individual sample.
2. Mechanically disrupt the tissue suspension in collagenase type II digestion buffer by pipetting up and down using a P1,000 micropipette and filtered tips. Use a new tip for each sample.
   NOTE: This is a critical step. Ensure to mechanically disaggregate the tissue with the tip and pipette up and down until macroscopic homogenization is complete.
3. Pass the disaggregated tissues through individual 100 µm cell strainers (see the Table of Materials) into new 1.5 mL tubes.
4. Add 500 µL of ACK buffer (see the Table of Materials) to the filtered tissue samples, invert the tubes 4-5 times, and incubate at room temperature for 4 min.
   NOTE: Do not exceed the incubation time, as this may damage the cells of interest.
5. Centrifuge the tissue samples at 300 x g for 5 min at room temperature.
6. Discard the supernatant by inverting the tube, and resuspend the pellet in 1 mL of culture medium (DMEM-F12 with 10% FBS, 1% antibiotic antimycotic, pH 7.2) (see the Table of Materials).
7. Pass the suspensions through a 40 µm cell strainer into new 1.5 mL tubes using a P1,000 micropipette and filtered tips.
8. Centrifuge the samples at 300 x g for 5 min at room temperature.
   NOTE: The pellet corresponds to the preadipocyte-containing SVF.
9. Discard the supernatant by inverting the tube, and resuspend the pellet in 1 mL of culture medium by pipetting.
10. Prepare one 15 mL tube for each sample by adding 5 mL of culture medium.
11. Add each resuspended pellet to its corresponding 15 mL tube containing 5 mL of culture medium. Invert the tube 4-5 times to mix the cell content.
12. Seed each sample into six wells of a 24-well culture plate by adding 1 mL of the suspension to each well (Figure 1).

4. Stromal vascular fraction (SVF) culture

1. Change the culture medium every other day until the cells reach 100% confluence.
   NOTE: During this period, non-plastic adherent cells are removed, resulting in a primary culture enriched with a preadipocyte-containing SVF.
2. When the cells become 100% confluent, remove the culture medium, and wash the cells with 800 µL of sterile PBS.
3. Add 150 µL of 0.25% trypsin-EDTA (see the Table of Materials) to every well, and place the culture plate for 3 min in the incubator for the detachment of the cells.
   NOTE: Verify that the cells are detached using a phase contrast light microscope.
4. Inactivate the trypsin by adding 850 µL of culture medium to each well.
5. Collect the cells from the six wells seeded in step 3.12, and transfer them into a 15 mL tube.
6. Make the total volume up to 12 mL by adding 6 mL of culture medium. Invert the tube 4-5 times to mix the content.
7. Seed 1 mL of the sample into each well of a 12-well plate. Change the culture medium every other day until the cells reach confluence.
8. When the cells become 100% confluent, detach them with 150 µL of 0.25% trypsin-EDTA following the same incubation conditions as in step 4.3, and seed them according to the surface area of the well required for the experiment.
9. Perform high-resolution imaging after fixing the cells following step 8.
   NOTE: A density of 35,000 cells/cm² is recommended for further experiments (protein and total RNA extraction, 330,000 cells [one well of a 6-well plate]; immunofluorescence [IF], 10,500 cells [one well of a 96-well plate]).

5. Adipogenic differentiation

1. Incubate the cells with induction medium (DMEM-F12 with 10% FBS, 1% antibiotic antimycotic, pH 7.2, supplemented with 500 nM dexamethasone, 125 nM indomethacin, 0.5 mM 3-isobutyl-1-methylxanthine [IBMX], 5 µM rosiglitazone, 1 nM T3, and 20 nM insulin) (Table 1) for 72 h (day 0 to day 2) (Figure 2).
   NOTE: The culture medium must be changed every other day; thus, the first medium change must occur on day 2 after the initial induction.
2. Change to maintenance medium (DMEM-F12 with 10% FBS, 1% antibiotic antimycotic, pH 7.2, supplemented with 5 µM rosiglitazone, 1 nM T3, and 20 nM insulin) (Table 1) at day 3, and maintain this maintenance medium until day 7 (Figure 2).
   ​NOTE: The maintenance medium must be changed on day 5 and day 7. Small lipid droplets can be visualized by phase contrast microscopy from day 3 of differentiation. Large lipid droplets are evident on day 7 of differentiation. Differentiation procedures longer than 7 days result in cell loss by detachment, likely due to the high buoyancy of lipid-laden cells.

6. Cellular homogenate preparation

1. Remove the culture medium at day 0 and day 7 of differentiation, and wash the cells 3 times with sterile PBS.
2. After the last cleaning, add 1.3 mL of sterile PBS to each well.
   NOTE: Maintain the cells in ice until the cleaning of the last plate.
3. Harvest the cells mechanically from one well of a 6-well plate (~330,000 cells) with a cell scraper, and put the cellular suspension in a 1.5 mL tube.
4. Centrifuge the samples at 2,800 x g for 5 min at 4 °C.
5. Remove the PBS with a micropipette, and maintain the tubes on ice.
6. Resuspend the cellular pellet in RIPA buffer with protease and phosphatase inhibitors (see the Table of Materials), and incubate for 30 min on ice.
   NOTE: To lysate the cellular pellet, 70-80 µL are sufficient.
7. Centrifuge the samples at 21,000 x g for 15 min at 4 °C.
8. Carefully remove the supernatant to a 1.5 mL tube, and discard the pellet.
   NOTE: The supernatant represents the cellular homogenate.
9. Determine the protein concentration of the cellular homogenate using bicinchoninic acid (BCA) for the colorimetric detection and quantitation of the total protein^18,19.

7. Western blotting

1. Prepare 30 µg of the cellular homogenate (from step 6.8) and add commercially available SDS-PAGE sample loading buffer (see the Table of Materials).
2. Resolve in polyacrylamide gel electrophoresis-SDS²⁰ (PAGE-SDS) according to the molecular weight of every protein.
   NOTE: Use 10% PAGE-SDS for PPARγ, C/EBPα, PLIN1, CD36, and UCP1, 12% PAGE-SDS for OXPHOS, and 15% PAGE-SDS for TOM20 (see the Table of Materials).
3. Electrotransfer the proteins onto the PVDF membrane (see the Table of Materials) at 350 mA for 2 h.
   NOTE: The ice container must be changed after 1 h. During the entire process, maintain the chamber on ice.
4. Block the membranes with 5% BSA or fat-free milk in Tris-buffered saline (TBS) 0.1% Tween 20 (TBS-T) for 2 h following the recommendations from the antibodies' datasheets.
5. Incubate with the primary antibodies (the antibodies used in this study are detailed in the Table of Materials) overnight at 4 °C.
   NOTE: All the antibodies used in this study were diluted to 1:1,000 in blocking solution.
6. Wash the membranes 3 times with 0.1% TBS-T, and incubate them with horseradish peroxidase (HRP) conjugated secondary antibody (see the Table of Materials) for 2 h at room temperature.
7. Wash the membranes 3 times with 0.1%. TBS-T. Perform one last wash with 1x TBS. Store the membranes in 1x TBS until visualization. Storing for a maximum of 1 h is recommended.
8. Visualize the membranes by chemiluminescence²⁰ or any other preferred method.

8. High-resolution imaging

1. Perform the immunofluorescence assay following the steps below.
   1. Seed and differentiate the primary cultures of brown preadipocytes from P0.5 newborn mice (following step 4.8) in 96-well plates with an optical bottom (see the Table of Materials).
   2. Fix the cells at day 0 and day 7 of differentiation with 4% PFA for 20 min, and wash 3 times with sterile PBS.
   3. Permeabilize the cells with 0.1% Triton X-100 for 15 min, and block them with 3% fish gelatin (see the Table of Materials) for 1 h at room temperature.
   4. Wash 3 times with sterile PBS, and incubate with PLIN1 antibody (1:400, see the Table of Materials) in 1% BSA/PBS overnight at 4 °C in a humid chamber.
   5. Wash 3 times with sterile PBS, and incubate with fluorophore-conjugated secondary antibody at 1:1,000 (1% BSA/PBS) (see the Table of Materials) for 1 h at room temperature.
   6. Stain the nuclei with 1 µg/mL Hoechst 33342 and the lipid droplets with 1 µg/mL BODIPY for 30 min at room temperature.
      NOTE: The fluorescence images were acquired using a cell imaging system (see the Table of Materials). Overall, 9 or 16 photos per well were acquired with two to three channels with a magnification of 20x.
2. Perform transmission electron microscopy following the steps below.
   1. Grow and differentiate the brown preadipocytes in 35 mm x 10 mm cell culture dishes.
   2. On day 0 and day 7 of differentiation, remove the medium, and fix the cells for 1 h with 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer, pH 7.0 (see the Table of Materials).
   3. Wash the cells 3 times with 0.05 M sodium cacodylate buffer for 10 min, and treat them with 1% aqueous osmium tetroxide (see the Table of Materials) for 15 min.
   4. Wash the cells 3 times with bi-distilled water for 5 min, and treat them with 1% aqueous uranyl acetate (see the Table of Materials) for 15 min.
   5. Dehydrate the cells with an ethanol gradient of 30%, 50%, 70%, 95%, and 100% for 5 min each time.
   6. Pre-embed the cells in 1:1 epon/ethanol for 30 min and then in epon for 1 h (see the Table of Materials).
      NOTE: The inclusion of cells is performed directly in the dish by adding fresh epon to cover the entire cell culture monolayer. Let the epon polymerize in an oven at 60 °C for 24 h.
   7. Immerse the cell culture dish in liquid nitrogen for 2 min, and then fracture it using pliers to loosen the plastic inclusion.
   8. Glue the inclusion segments with cells to a block of resin to obtain fine cuts (80 nm) parallel to the surface in an ultramicrotome (see the Table of Materials).
   9. Stain the cuts with 1% aqueous uranyl acetate for 4 min and lead citrate for 5 min, according to Reynolds et al.²¹.
   10. Visualize the grids in an electron microscope (see the Table of Materials).

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 conce...

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

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