Adipogenesis

Cell Biology Laboratory PCB 4023
Laboratory 7 and 8: Adipogenesis

Serbus and Mayoral, Adapted from Mustafi et al 2014 lab manual
THE DEVELOPMENT OF FAT CELLS
Adipogenesis is the development of fat cells

(adipocytes) from pre-adipocytes, originally derived
from mesenchymal stem cells. Adipogenesis has been
one of the most extensively studied models of cellular
differentiation partly because of our ability to
recapitulate most of the key features of fat cell
formation in vitro. More recently, interest in
adipogenesis has increased due to its association with
health hazards like obesity and a number of pathological
disorders, including diabetes, hypertension, cancer, gall
bladder disease, and atherosclerosis.
Adipocyte differentiation. Image derived from Gregoire
FM, Smas CM, Sui HS (1998). Physiol Rev 78:783-809.
GROWTH AND DIFFERENTIATION OF FAT CELLS:
BAT and WAT tissue
Adipose tissue growth involves an increase in adipocyte size and differentiation of new
adipocytes from precursor cells. There are two types of adipose tissue, namely white adipose
tissue (WAT) and brown adipose tissue (BAT). WAT and BAT adipocytes are derived from
distinct precursor cells and have distinct morphological characteristics. White adipocytes
can be derived from mesenchymal precursor cells and brown adipocytes are derived from
Myf5-expressing precursor cells (see figure to the right).
WAT is the major site of energy

storage in periods of energy
excess and its mobilization in
response to hormones and
nutrient deprivation. WAT is
characterized by one large fat
droplet (unilocular). In humans,
WAT is the predominant fatty
tissue and accounts for 20% and
25% of body weight in men and
women, respectively.
In contrast, BAT serves to

dissipate energy as heat instead of Source: Sargeant & Stephens, Cold Spring Harb Perspect Biol 2012;4:a008417
storing it. Brown adipose cells
contain numerous fat droplets (multilocular) and notably large numbers of mitochondria.
Mitochondria use energy from fat metabolism to drive the electron transport chain, which
1
creates a proton gradient that drives ATP synthesis. In BAT cells, the mitochondria have
further included an “uncoupling protein” Thermogenin in the inner mitochondrial
membrane that weakens the proton gradient. This allows mitochondria to run the electron
transport chain and generate heat without creating excess amounts of ATP. This uncoupling
mechanism is particularly important for hibernating animals and newborn babies, which
require the heat from fat metabolism to stay warm.
ADIPOGENISIS IS INITIATED BY TRANSCRIPTION FACTORS
In order to achieve a successful transformation into mature adipocytes, preadipocytes

undergo marked changes in morphology and gene expression. The process of adipogenesis
consists of three stages: growth arrest, clonal expansion and differentiation (See Figure 1
above). These stages are orchestrated by a sequential activation of multiple transcriptions
factors, being the most important ones the CCAAT/enhancer-binding protein (C/EBP) gene
family and the peroxisome proliferator activated receptor- (PPAR-). C/EBP-δ and C/EBP-
β are increased early during differentiation to induce PPAR-γ and C/EBP-α for adipocyte
differentiation. These transcription factors in turn decide the expression of many genes.
Clonal expansion Increasing Lipid accumulation
Source: Modifed from Sargeant & Stephens, Cold Spring Harb Perspect Biol 2012;4:a008417
Other stimulator of adipogenesis includes the transcriptional factor sterol-regulatory-

element-binding-protein-1 (SREBP1), fatty acids, prostaglandins and glucocorticoids. On the
other hand, the pre-adipocyte factor-l (PREF-1), glycoproteins, transforming growth factor-
 (TGF-), inflammatory cytokines and growth hormones have a negative (or inhibitory
effect) on the differentiation process. For example, PREF-1 is highly expressed in
preadipocytes but it is absent after adipocyte differentiation. Overall, PREF-1 may function
as a soluble factor, maintaining proliferating cells in an undifferentiated state during
development.
IN VITRO STUDIES OF ADIPOGENESIS
2
The in vitro differentiation of fat cells from preadipocytes is an excellent model to study
adipogenesis in mammals because it captures most of the characteristic features of in vivo
adipogenesis. Also, a cell line derived from cloning guaranties a homogeneous population of
cells synchronized all at the same stage of differentiation, allowing for a coordinated
response to treatments. For in vitro studies, adipogenesis is achieved by exposing cultured
cells to a combination of small molecules: the hormone dexamethasone (DEX), which
modifies transcriptional patterns of the cell; isobutylmethylxanthine (IBMX), which modifies
the signaling environment of the cell; and insulin, which activates specific signaling
processes in the cell. IBMX can inhibit phosphodiesterases, which causes an elevation of
intracellular cAMP. High levels of cAMP leads to the alteration in transcription factors
through protein kinase A. At the same time, IBMX can directly induce C/EBP-β expression as
well. Similarly, DEX activates C/EBP-δ expression through binding to intracellular
glucocorticoid receptor. As growth factors are generally considered to be inhibitors of
adipocyte differentiation, growth factors are not administered to the cells in this exercise.
PROTOCOL:
In this exercise, students will evaluate the effects of insulin concentration on adipogenesis
using 3T3-L1 cell line, originally derived from mouse embryo fibroblasts. Lipid accumulation
and changes in adipocyte cell number as a result of differentiation will be visualized by
staining adipocytes with Oil-Red-O stain.
MATERIALS:
• Insulin stock 1mg/mL
• IBMX stock 500 mM
• DEX stock 10 mM
• 4% Formaldehyde (in PBS)
• Differentiation Medium
90% DMEM (Dulbecco's Modified Eagle Medium) - High Glucose
10% FBS (Fetal Bovine Serum)
1:100 L-Glutamine stock
1:100 Penicillin – Streptomycin stock
1:1000 Insulin stock
0.5 mM IBMX (isobutylmethylxanthine)
0.25 µM DEX (Dexamethasone)
• Growth Medium
90% DMEM (Dulbecco's Modified Eagle Medium) – High Glucose
10% FBS (Fetal Bovine Serum)
1:100 L-Glutamine stock
1:100 Penicillin – Streptomycin
• Oil Red O (ORO) Protocol
➢ Oil Red O stock solution:
0.5 g Oil Red O
3
100 mL Isopropanol
Mix the ORO and Isopropanol with magnetic stirrer. Then filter solution with 0.20
µm filter. Store at 4°C.
➢ Oil Red O working solution:
3 parts Oil Red-O stock
2 parts ddH20
Mix together. Let the solution sit for 20 min. Pass the solution through a 0.20 µm
filter.
*Note: Working solution must be prepared right before it will be used.
ESTABLISH 3T3-L1 CELL CULTURE FROM A FROZEN STOCK

(Set up in advance by the lab coordinator)
1. After quickly thawing the frozen cells (stored in liquid nitrogen) in a 37°C water
bath, immediately transfer the thawed cell suspension into a 50 mL centrifuge tube
containing 10 mL of culture medium.
2. Centrifuge the tube for 5 min at 1300 to 1500 rpm to get rid of DMSO (used in
freezing the cells).
3. Discard the supernatant and break the cell pellet by finger tapping.
4. Add a few drops of culture medium with gentle shaking and finger tap the tube a few
times.
5. Add 2 mL of culture medium to the tube and gently pipet the cell suspension up and
down twice.
6. Transfer the cell suspension to a 10 cm culture dish containing 8 mL of culture
medium.
7. Swirl the culture plate well to mix the cells, and then incubate the cells for two to
three days before expansion.
8. Grow preadipocytes to 70% confluency in growth medium.
9. Maintain cells at confluency for 2 days.
FIRST LAB DAY: ADIPOCYTE DIFFERENTIATION
1. On “Day 0” of the experiment (your first lab day working through this procedure),
first observe the cell morphology through the microscope.
2. Draw a sketch in your notebook or take a picture of what you see.
3. Next, remove media from the culture dish.
4. Before the cells dry out, gently add 2 mL of differentiation media, containing a pre-
specified insulin concentration. (Each group will use a different concentration of
insulin, which will be one of the following: 0, 0.25, 1, or 4 micrograms/mL.
5. Change the differentiation medium after two days (Day 2). The cells will have an
altered morphology by this point. The media will also begin to become more viscous
as free fatty acids are produced by the cells and secreted into the media.
6. Two days later (Day 4), replace the differentiation medium with growth medium.
4
Spherical organelles will begin to become visible in the cytoplasm, indicating

formation of lipid droplets.
7. Feed cells by changing the growth medium again 2 days later (Day 6), preparing the
cells to be examined at the next lab (Day 7). Full differentiation is usually achieved by
day 8 or 9 but in our course the differentiation is ended on Day 7 so that the
experiment can continue in the appropriate lab time slot.
SECOND LAB DAY: Oil Red O STAINING PROCEDURE
1. Remove your cell culture dish from incubator.

2. Examine cell morphology at the microscope.
3. Draw a sketch of what you see in your notebook or take a picture of what you see.
4. Bring the dish over to the fume hood for Oil Red O (ORO) staining.
5. Remove growth medium from the culture dish using the vacuum trap.
6. In the FUME HOOD, pipette 1 mL of fixing solution into your dish.
7. Allow your dish to incubate for 1 hour in the 4°C (refrigerator).
8. Prepare the ORO working solution in the fume hood.
9. Carefully bring your dish of cells back to the fume hood.
10. Remove ALL of the fixing solution, transfer it to the hazardous waste bottle (DO NOT
USE the vacuum trap).
11. Add 0.5 mL of the ORO working solution to your dish. Try not to touch the sides of
the well as you do this.
12. Allow your plate to incubate for maximum 15 min at room temperature, NO
LONGER.
13. Remove ORO working solution, transfer to the ORO WASTE CONTAINER (don’t use
the vacuum trap).
14. Gently rinse your dish twice with 1 mL ddH20. (Try not to blast cells off the surface.)
15. Take pictures of your cells? (Optional).
16. Remove water from the dish, transfer to dye waste bottle.
17. Allow the dish to incubate with 1.5 mL of isopropanol for 10 min in the 37°C
incubator.
18. Carefully bring your dish of cells back to the fume hood.
19. Transfer the isopropanol from the dish into an Eppendorf tube.
20. Centrifuge the test tube for 3 min at 12,000 rpm to remove any cells collected (with
the help of your instructor, make sure the centrifuge is balanced).
21. Gently transfer the supernatant into a cuvette (avoid making air bubbles).
22. In a spectrophotometer, measure the optical density of the liquid in response to 540
nm light (i.e. “measure the OD540”).
23. Record your findings in your notebook and also in the class data table on the board.
24. Sketch out a graph that plots OD value against insulin concentration in your lab
notebook, and interpret the results.
5
QUESTIONS: Testing and applying your understanding.
1.) How does insulin concentration affect adipogenesis? i.e. what do you interpret from
the overall picture of the data?
2.) Can patients injected with insulin for their diabetes be affected in the way shown by the
results you got today? Why or why not?
3.) If you would have to inhibit adipogenesis most efficiently, what transcription factor
will you target from the figure in the section “ADIPOGENISIS IS INITIATED BY
TRANSCRIPTION FACTORS” above? Explain your answer.

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Cell Biology Laboratory PCB 4023

Laboratory 7 and 8: Adipogenesis

THE DEVELOPMENT OF FAT CELLS

Adipogenesis is the development of fat cells

WAT is the major site of energy

In contrast, BAT serves to

ADIPOGENISIS IS INITIATED BY TRANSCRIPTION FACTORS

In order to achieve a successful transformation into mature adipocytes, preadipocytes

Clonal expansion Increasing Lipid accumulation

Other stimulator of adipogenesis includes the transcriptional factor sterol-regulatory-

IN VITRO STUDIES OF ADIPOGENESIS

ESTABLISH 3T3-L1 CELL CULTURE FROM A FROZEN STOCK

FIRST LAB DAY: ADIPOCYTE DIFFERENTIATION

Spherical organelles will begin to become visible in the cytoplasm, indicating

SECOND LAB DAY: Oil Red O STAINING PROCEDURE

1. Remove your cell culture dish from incubator.

QUESTIONS: Testing and applying your understanding.

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