Abstract: This protocol is very useful for researchers working in the field of obesity, diabetes, CVD especially in adipocyte biology. Here it has been descirbed with its importance and basic principle of the assay

Introduction

Obesity is a significant clinical problem that contributes to life-threatening diseases such as diabetes and atherosclerosis. With an increasing incidence of obesity worldwide, rational strategies are needed to control adipogenesis. Cells that undergo determination to the adipose lineage called adipoblasts and this process called adipogenesis. Adipogenesis has been extensively studied using in vitro model systems consisting either of established adipogenic cell lines (clonal lines 3T3-L1, 3T3-F442A, Ob17, BFC-1, ST13, A31T, ...) and primary culture of adipocyte precursors and pre-adipocytes. Growth arrest of adipoblasts at the G1/S phase of the cell cycle appears to be required for the commitment of adipoblasts to preadipose cells which is accompanied by the emergence of early markers of adipgenesis. Using cell culture systems cultivated either in the presence of serum or in defined medium, it has been possible to identify factors regulating either positively or negatively adipose differentiation. The positive regulators include several hormones such as insulin, growth hormone, and triiodothronine. In addition, serum, partially purified serum factors, and conditioned medium from several adipogenic cell lines have been shown to stimulate adipose differentiation in vitro and assumed to contain adipogenic regulators distinct from the hormones. Mammalian 3T3-L1 cells differentiate into adipocytes after continuous exposure to pharmacological doses of insulin or physiological doses of insulin-like growth factor I.

Principle

When adipoblasts treated with a combination of dexamethasone, isobutylmethylxanthine (IBMX or MIX) and insulin, cells start to adopt a rounded phenotype and within 5-8 days begin to accumulate lipids intracellularly in the form of lipid droplets. The induction condtions and media vary according to the cell lines. Treatment of cells with dexamethasone activates the transcription factor CCAAT/enhancer-binding protein b (C/EBPb). IBMX inhibits soluble cyclic nucleotide phosphodiesterases and results in increased intracellular cAMP levels. At the nuclear level, treatment with IBMX results in activation of the related transcription factor C/EBPd. C/EBPb and d in turn induce transcription of C/EBPa and PPAR . Within 3 days of exposure to inducers, the cells undergo two rounds of mitosis, termed mitotic clonal expansion, which are required for differentiation. Insulin or insulin-like growth factor-1 promote adipocyte differentiation by activating PI3-kinase and Akt activity. Modulation of the activity of the forkhead transcription factor Foxo1 appears to be necessary for insulin to promote adipocyte differentiation. C/EBPa and PPAR direct the final phase of adipogenesis by activating expression of adipocyte-specific genes, such as fatty acid synthetase, fatty acid binding protein, leptin and adiponectin.

Reagents and requirements

Material and sources:

• DMEM (GibcoBRL-Cat# 11965-084)
• Calf Serum (GibcoBRL-Cat#16170-078)
• Fetal Bovine Serum (GibcoBRL-Cat# 10437-028)
• Isobutylmethylxanthine (IBMX; Sigma I-7018)
• Dexamethasone (Sigma D-4902)
• Insulin (Bovine; Sigma I-5500)
• MEM Sodium Pyruvate (100mM; GibcoBRL Cat#11360-070)
• Pen/Strep/Glutamine (100x P/S/G; GibcoBRL Cat#10378-016)
• Sterilized petriplates
• Leminar air hood
• CO2 incubator
• Sterilized pipettes

Preparation of solutions

1. 10% Calf Serum/DMEM
   60 mL : Calf Serum
   6 mL : 100 mM MEM Sodium Pyruvate
   6 mL : 100x P/S/G
   500 mL : DMEM
2. 10% FBS/DMEM
   60 mL : Fetal Bovine Serum (Filter Sterilized)
   6 mL : 100 mM MEM Sodium Pyruvate
   6 mL : 100x P/S/G
   500 mL : DMEM
3. IBMX Solution (make fresh)
   Dissolve IBMX in a solution made of 0.5N KOH to a final concentration of
   0.0115 g/mL.
   Filter sterilize through a 0.22 mm syringe filter.
4. Insulin Stock Solution
   167 mM (1 mg/mL) in 0.02 M HCl
   Filter sterilized through 0.22 mm filter
   Can store at -20°C for long term, 4°C short term.
5. Dexamethasone Stock Solutions
   Freezer Stock: 10 mM of Dex in 100% ethanol (store at -20°C)
   Working Stock: Dilute Freezer stock to 1mM in PBS
   Filter sterilize and store at 4°C.
6. MDI Induction Media (10 mL/ 10cm plate; 5 mL/ 6 cm plate)
   To required volume of 10% FBS/DMEM add:
   1:100 IBMX
   1:1000 Insulin
   1:1000 Dexamethasone working stock
7. Insulin Media (10 mL/ 10 cm plate; 5 mL /6 cm plate)
   To required volume of 10% FBS/DMEM add:
   1:1000 Insulin
8. Oil red O stock solution (0.5 g/ 100 ml isopropanol)
   Just before staining: mix 60 ml of stock with 40 ml of H₂0, let it sit for 1 hr at RT
   and filter through whatman paper 3MM.

Procedures

Preadipocyte maintenance and passage:

Plate the cells in 10% CS/ FBS-DMEM on culture dishes and incubate them at 37°C in 10% CO₂. It is important to feed the preadipocytes every couple of days and avoid letting them get too confluent (>70%), if you want to continue to passage them and differentiate them at a later date. So, take care to split them appropriately. They can be split as far as 1:15, though we usually do 1:10 or less depending on need.

Adipocyte Differentiation Protocol

1. Grow preadipocytes/ fibrobalst to confluency in 10% FBS-DMEM.
2. After two days of post confluency (which is counted as day 0), stimulate the cells with MDI induction media. You will notice a distinct change in the morphology of the cells (become more spindly) in the next 2 days.
3. After two days of MDI an induction medium (which is called as day 2) replace the MDI induction media with Insulin Media. The media will begin to get more viscous as free fatty acids are produced by the cells and secreted into the media.
4. Two days later (which is called as day 4) change media to 10% FBS-DMEM. Feed cells with 10% FBS-DMEM every two days. Full differentiation is usually achieved by day 8.

Staining procedure:

1. Aspirate media, add formaldehyde slowly and let sit for 30 min.
2. Aspirate formaldehyde and add Oil red O solution to cover the well, leave 1 hr at RT.
3. Remove the stain and wash with distilled water twice.
4. Let it dry for pictures.

Precautions:

1. Dexamethasone Solution, IBMX Solution and Insulin Solution should be stored at -20ºC.
2. Oil Red O Solution, Wash Solution, and Dye Extraction Solution should be stored at room temperature. Storage of Oil Red O Solution and Dye Extraction Solution at -20ºC may result in formation of insoluble precipitates and is not recommended. If Oil Red O solution forms a precipitate, remove particulates by passage through a 0.22 or 0.45 micron filter. Oil Red O stains skin and clothing.
3. IBMX and dexamethasone are irritants and potentially toxic. DMSO is readily absorbed through the skin. Wear a lab coat and gloves when handling these solutions.
4. Isopropanol is flammable. Keep solutions containing isopropanol (Oil Red O Solution, Wash Solution and Dye Extraction Solution) away from open flames.