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 origin3,4.
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 contact7,8.
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 preadipocytes11,12,13,14,15 (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 lipodystrophy15,16,17.
Protocol
Representative Results
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…
Disclosures
The authors have nothing to disclose.
Acknowledgements
Funding was provided by FONDECYT (1181214 and 1221146) and Anillos (ACT210039) to VC and doctoral scholarships ANID 21171743 to AMF and ANID 21150665 to FS. We thank Alejandro Munizaga for help in the processing of the samples and technical advice for the transmission electron microscopy. The illustrations were produced using BioRender.
Materials
| 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 |
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