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

Summary

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.

Abstract

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.

Introduction

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 nonshivering thermogenesis in BAT³. Several studies in recent years revealed that adult humans have functional BAT ^4-8 and, consequently, manipulation of BAT in humans can be a potential therapeutic intervention in the battle against obesity and its related diseases.

Current evidence indicates that two types of brown adipocytes exist in rodents; "classical" or "pre-existing" brown fat develops during the prenatal stage and forms dedicated brown adipose depots in interscapular region and other peripheral tissues. On the other hand, an "inducible" form of brown fat (so called brite or beige cells) develops during post-natal stage and appears interspersed in white adipose tissues. The two types of brown adipocytes are also separated by different developmental origins. While the pre-existing brown adipocytes arise from myoblastsic-like Myf5 precursors, the inducible brite/beige cells interspersed in WAT arise from a non-Myf5 lineage^9,10 . In addition, regulatory pathways of this cell type is likely to be different from the Myf5-derived brown adipocytes ¹¹. The development of the beige cells (i.e. "browning" of white fat) can be activated in response to chronic cold exposure and to β3-adrenoceptor-agonists or PPARγ agonists in adults^12-14. The beige/brite cells are likely to be a promising therapeutic target for manipulation of overall energy balance and can potentially become part of obesity treatment; hence, it is important to understand precise molecular mechanisms and signaling pathways by which environmental cues control the development of beige cells.

To understand the molecular control of the browning of white fat, in vitro experiments are best suited as differentiation of preadipocytes takes place rather asynchronously and it is difficult to detect the cells in situ ¹⁵. Although studies on adipocyte development have thus far been performed primarily on cell lines such as 3T3-L1, 3T3-F442A or HIB1, these cell lines appear to lack the molecular signature of beige cells. On the other hand, primary adipocytes isolated from subcutaneous WAT are most likely to recapitulate the process of browning of white fat in a cell autonomous fashion. Here we provide a protocol for effective isolation of the stromal-vascular fraction from adipose tissues and for inducing the browning of white fat in response to PPARγ agonists. Rosiglitazone has been shown to be an especially effective mediator of browning in these cells. As previously suggested ¹⁶, this cellular system can be used to serve a reliable cellular system to study the development of beige/brite cells.

Protocol

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 Tissue from Mice

1. Dissection should be performed carefully, and immediately after the mice (6-8 weeks old) are sacrificed. Work sterile with ethanol sprayed on the mouse fur before opening it. Place the tissue directly in clean PBS, separate interscapular BAT and subcutaneous inguinal WAT.
2. For interscapular BAT: have the mouse placed with the back faced up, cut open along the back and all the way to the neck. The brown adipose tissue can be found right under the skin between the shoulders (interscapular). BAT can be seen as two lobes, butterfly shaped. A thin layer of white fat covering the BAT should to be carefully removed.
3. For subcutaneous WAT (inguinal WAT): remove skin. Inguinal WAT is found directly under the skin on both sides, starting almost on the back and going alongside, inside of the thighs and down toward the testicle.

3. Cut and Digest Adipose Tissue

1. Quickly remove all contamination, like hair, skeletal muscle and connective tissue from the tissue sections.
2. Dry tissue quickly on paper to not dilute dissection medium with PBS and place it on a dry plate.
3. Add digestion medium (added CaCl₂ prior to digestion) and mince the tissue into small pieces. Mix very well by pipetting up and down with a 5 ml pipette. Transfer the minced tissues in 50 ml tubes with the rest of the digestion medium, mix even more by pipetting up and down.
4. Digest in 37 °C with constant agitation at 150 rpm for 40-50 min. Check every 10-15 min to make sure the digestion is going well and to prevent over-digestion.

Note: Correct digestion medium and time are important. The tissue needs to be well digested, but excess digestion can damage the cells.

4. Filter Cell Suspension

1. Stop digestion by adding 5 ml complete medium (DMEM/F12 containing 10% FPS and P/S). The cells should almost be completely homogenous. Mix well by pipetting.
2. Centrifuge at 700 x g for 10 min.
3. SVF can now be seen as a brownish pellet on the bottom of the tube. Aspirate the oily mature adipocyte layer on top and most of the liquid layer - keep the pellet and dissolve it in 10 ml complete medium. Mix well.
4. Place a cell strainer (50-70 μm diameter) over a new 50 ml tube and filter the cell suspension.

5. Plate Cells

1. Transfer cell suspension to a 15 ml tube and centrifuge at 700 x g for 10 min.
2. Aspirate medium and re-suspend pellet in 10 ml complete medium. Pipette and mix well.
3. Estimate how many plates are needed. This may vary and depends on the pellet size. A general rule is 2 of 10 cm plates from 5 mice for inguinal WAT and one 10 cm plate for BAT.
4. Plate cells on collagen coated dishes (R&D) in complete medium
5. 1-2 hr after plating the cells, aspirate medium, wash with PBS twice and add fresh medium. This step is important as this can remove red blood cells, immune cells and other contaminants.

6. Differentiate Cells

1. Make maintenance and induction mediums.

Maintenance medium

Complete medium with added:

Insulin, final conc. 5 μg/ml (5 mg/ml stock, ***100 μl of acetic acid in 10 ml H₂O to prepare acidified water pH 2.5. dissolve insulin in the acidified water. Store stock at -20 °C)

3,3',5-Triiodo-L-thyronine (T3), final conc. 1 nM (10 μM stock, ***dissolve T3 in 1N NaOH and add medium to make 10 μM stock. Sigma cat# T-2877)

Store at 4 °C, good for one week

Induction medium

Maintenance medium containing following compounds:

Indomethacin, final conc. 125 μM (0.125 M stock in ethanol, Sigma cat#I-7378). Indomethacin must be heated to 60 °C to be dissolved.

Dexamethasone, final conc. 2 μg/ml (2 mg/ml sock in ethanol, Sigma cat# D-1756)
3-Isobutyl-1-methylxanthine (IBMX), final conc. 0.5 mM (0.25 M stock in DMSO, Sigma cat# I-5879)
Rosiglitazone, final conc. 0.5 μM (10 mM stock in DMSO, Sigma cat# R-2408)

Note: Make fresh induction medium each time.

2. Grow primary adipocytes to 95-97% confluence in complete medium (confluent but not too packed)
3. Change regular complete medium with induction medium (day 0)
4. After 48 hr (day 2), change medium to maintenance medium with rosiglitazone (0.5 μM)
5. After additional 48 hr (day 4), change to fresh maintenance medium with rosiglitazone (1 μM) for additional 2-3 days. 6-7 days after adding the induction medium, cells are fully differentiated to mature fat cells and are filled with oil droplets. Droplets will appear around 3-4 days after the induction.

Note: Change medium every 2-3 days until the cells are fully differentiated.

Cells can now be harvested and mRNA expression of Ucp1 and other brown adipocyte-specific genes can be measured by qRT-PCR. Western blot will be used to detect proteins.

Results

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 ac...

Discussion

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...

Materials

List of materials used in this article

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