Quantitative assessment of adipocyte differentiation in cell culture

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Abstract

Adipocyte cell culture is an important tool for mechanistic studies of energy metabolism. Many factors affect the differentiation of adipocytes in culture. Oil red O staining can be used to assess the degree of differentiation. However, the validity of this method for quantitative analysis has not yet been established. Here we show that a protocol with arbitrarily chosen parameters does not measure in the linear range and is not suitable for quantitative analysis (R 2 = 0.077, p = 0.382), and develop and validate an optimized protocol for quantitative oil red O staining of cultured adipocytes. 3T3-L1 preadipocytes and adipocytes are fixed with 4% formaldehyde and stained with 0.2% oil red O solution in 40% 2-propanol for 30 minutes. Dye is eluted with 2-propanol, and absorption of the eluate is measured photometrically at 510 nm. This optimized protocol achieves excellent correlation between defined amounts of differentiated adipocytes on constant-size culture plates and photometric absorption (R 2 = 0.972, p = 6.585E-14). The performance of the method is independent of the culture plates used. Thus, the optimized oil red O staining protocol can be universally employed to quantitatively assess adipocyte differentiation.

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An established preadipose cell line and its differentiation in culture. II. Factors affecting the adipose conversion.

Howard Green, Olaniyi Kehinde (1975)

When cells of the established preadipose line 3T3-L1 enter a resting state, they accumulate triglyceride and convert to adipose cells. The adipose conversion is brought about by a large increase in the rate of triglyceride synthesis, as measured by the incorporation rate of labeled palmitate, acetate, and glucose. In a resting 3T3 subline which dose not undergo the adipose conversion, the rate of triglyceride synthesis from these precursors is very low, and similar to that of growing 3T3-L1 cells, before their adipose conversion begins. If 3T3-L1 cells incorporate bromodeoxyuridine during growth, triglyceride synthesis does not increase when the cells reach a stationary state, and triglycerides do not accumulate. As would be expected from their known actions on tissue adipose cells, lipogenic and lipolytic hormones and drugs affect the rate of synthesis and accumulation of triglyceride by 3T3-L1 cells, but in contrast to bromodeoxyuridine, these modulating agents do not seem to affect the proportion of cells which undergoes the adipose conversion. Insulin markedly increases the rate of synthesis and accumulation of triglyceride by fatty 3T3-L1 cells, and produces a related increase in cell protein content. Of 20 randomly selected clones isolated from the original 3T3 stock, 19 are able to convert to adipose cells. The probability of such a conversion varies greatly among the different clones, in most cases being much lower than for 3T3-L1; but once the conversion takes place, the adipose cells produced from all of the 19 clones appear similar. The adipose conversion would seem to depend on an on-off switch, which is on with a different probability in different clones. This probability is quasistably inherited by the clonal progeny.

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Regulation of Wnt signaling during adipogenesis.

Christina Bennett, Sarah Ross, Kenneth Longo … (2002)

We have identified Wnt10b as a potent inhibitor of adipogenesis that must be suppressed for preadipocytes to differentiate in vitro. Here, we demonstrate that a specific inhibitor of glycogen synthase kinase 3, CHIR 99021, mimics Wnt signaling in preadipocytes. CHIR 99021 stabilizes free cytosolic beta-catenin and inhibits adipogenesis by blocking induction of CCAAT/enhancer-binding protein alpha and peroxisome proliferator-activated receptor gamma. Preadipocyte differentiation is inhibited when 3T3-L1 cells are exposed to CHIR 99021 for any 24 h period during the first 3 days of adipogenesis. Consistent with this time frame of inhibition, expression of Wnt10b mRNA is suppressed upon induction of differentiation, with a 50% decline by 6 h and complete inhibition by 36 h. Of the agents used to induce differentiation, exposure of 3T3-L1 cells to methyl-isobutylxanthine or cAMP is sufficient to suppress expression of Wnt10b mRNA. Inhibition of adipogenesis by Wnt10b is likely mediated by Wnt receptors, Frizzled 1, 2, and/or 5, and co-receptors low density lipoprotein receptor-related proteins 5 and 6. These receptors, like Wnt10b, are highly expressed in preadipocytes and stromal vascular cells. Finally, we demonstrate that disruption of extracellular Wnt signaling by expression of secreted Frizzled related proteins causes spontaneous adipocyte conversion.