Additional data for flavonoids inhibit 3T3-L1 adipocyte differentiation through reducing ROS generation

The data are mainly to explore the dosage effect and intervention stage of flavonoids including chrysin (CH), apigenin (AP), luteolin (LU), kaempferol (KA), quercetin (QU), and myricetin (MY) on 3T3-L1 preadipocyte viability, differentiation, reactive oxygen species (ROS) regulated adipogenesis.
MTT assay was used to investigate the cytotoxicity of the six flavonoids on 3T3-L1 preadipocytes. 5, 10, 20, 30, 40, or 50 μM LU, QU, MY, AP, KA, or CH were added to 3T3-L1 for 4 h, respectively. 40 μM LU and QU, 50 μM LU, QU, MY, AP, and CH significantly inhibited cell viability in 3T3-L1 preadipocytes. All the flavonoids did not damage cell viability until 30 μM.
To measure the anti-adipogenic activity of six dietary flavonoids, we added non-toxic 10 or 30 μM flavonoids to 3T3-L1 adipocytes culture media during the full stage of 8 days differentiation. Oil red O staining showed that 10 μM LU, QU, MY, AP, KA, or CH did not affect lipid storage in 3T3-L1 adipocytes. However, 30 μM LU, QU, MY, AP, KA, or CH prominently decreased lipid droplet content in 3T3-L1 adipocytes.
Adipogenesis during adipocyte differentiation can be divided into growth arrest, post-confluent mitosis and clonal expansion, and lipid accumulation. 3T3-L1 preadipocytes were induced to mature adipocytes for 8 days. To explore which stage of 3T3-L1 adipocyte differentiation was inhibited by the dietary flavonoids, 20 μM flavonoids were added to 3T3-L1 differentiated media at intervals of 2 days, as shown in. Oil red staining showed that, during the different stages of 3T3-L1 differentiation, 20 µM QU, MY, AP, KA, or CH effectively inhibited the lipid droplet content of 3T3-L1 when the flavonoids were added during 0-2, 0-4, 0-6 or 0-8 days, but not during 2-8, 4-8 or 6-8 days.
The level of intracellular ROS increases during adipocyte differentiation, and exogenous hydrogen peroxide causes lipid storage increasing in adipocytes. N-acetylcysteine is a cell-permeable cysteine precursor and removes intracellular ROS. To test whether LU inhibits 3T3-L1 adipocyte adipogenesis through lowering ROS generation, we used 20 μM LU and 10 mM NAC to treat 3T3-L1 cells with or without 100 μM exogenous hydrogen peroxide, respectively. Exogenous hydrogen peroxide significantly increased adipogenic gene expression including peroxisome proliferator-activated receptor γ (PPARγ), CCAAT/ enhancer binding protein α (C/EBPα), fatty acid binding protein 4 (AP2), CD36, fatty acid synthase (FAS) and stearoyl-CoA desaturase 1 (SCD1) in 3T3-L1 cells. LU and NAC treatments not only directly resisted insulin cocktail-induced adipogenic gene expression, but also reversed the action of exogenous hydrogen peroxide on adipocyte differentiation.