Effects of thymol on 3T3‐L1 differentiation and lipase activity

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The antibacterial properties of thyme extracts have been well documented, however the effects of thyme on mammalian cells are less well studied and of great importance. Evidence suggests that the antibacterial activities are in part due to the effects of thymol on the membrane structure, effectively altering permeability. As varied permeability and fluidity in the membrane are commonly linked to insulin sensitivity, the exploration of its effects on cell morphology and differentiation in adipose tissue is essential. As a cell model system to explore obesity and diabetes, 3T3‐L1 pre‐adipocytes are well suited due to the ability to examine both adipogenesis and differentiation. Pre‐adipocytes were grown in monolayer in expansion media containing DMEM supplemented with 10% bovine calf serum, as suggested by the supplier. Differentiation was chemically induced following trypsinization of 70% confluent cells with DMEM, 10% fetal bovine serum (FBS), 1 μM Dexamethasone, 0.5 mM methylisobutylxanthine, and 1 μg/ml insulin. Pre‐adipocytes were also treated with thymol in DMEM with 10% FBS minus the chemical induction components dexamethasone, methylisobutylxanthine, and insulin for comparison. Differentiation was monitored at 12, 18, 24, 36, and 48 hours by fixing and staining with oil red O. Some cells were allowed to grow to maturity at 2 weeks and stained for comparison. Cultures were replicated multiple times, allowing testing for cell death using the trypan blue exclusion assay as well as an assay of lipase activity in live unfixed cultures. Lipid droplet volume was estimated based on droplet size and count relative to the cell size. Lipid volume was also assayed using dye extraction and quantitation. Results suggest that, like other phenolic compounds, thymol enhances differentiation from pre‐adipocyte to adipocyte at certain concentrations. However, like other phenolic compounds, this effect is highly sensitive to concentration. The lipase activity was expected to increase where differentiation had occurred, but more data is needed to determine significant changes in lipase activity. A moderate change in cell size was also observed, which can more appropriately monitored further in three‐dimensional culture.