In-Vitro Adipocytes Differentiation
|Author: Shalini Jain and Hariom Yadav|
|Affiliation: Animal Biochemistry Division, National Dairy Research Institute, Karnal-132001, Haryana, India|
|Date Added: Mon Feb 02 2009|
|Date Modified: Mon Feb 18 2013|
|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|
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.
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:
Preparation of solutions
Preadipocyte maintenance and passage:
Plate the cells in 10% CS/ FBS-DMEM on culture dishes and incubate them at 37øC 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