TGF‐β effects on adipogenesis of 3T3‐L1 cells differ in 2D and 3D cell culture conditions

The TGF‐β superfamily plays a pivotal role in the regulation of adipogenesis, but little is known about the potential differential role of the three isoforms of TGF‐β, TGF‐β‐1~3. To further elucidate their role, two‐dimensionally (2D) and three‐dimensionally (3D) cultured 3T3‐L1 mouse preadipocytes were subjected to the following analyses: (a) qPCR analysis of adipogenesis‐related factors and major extracellular matrix protein (2D and /or 3D), (b) lipid staining by Oil Red O (2D) or BODIPY (3D), (c) Seahorse cellular metabolic measurement (2D), and (d) size and stiffness measurements of 3D 3T3‐L1 spheroids. In the 2D cultured 3T3‐L1 cells, mRNA expression levels of adipogenesis‐related genes and Oil Red O lipid staining intensity were significantly increased by adipogenesis and they were substantially decreased following treatment with 0.1 nm TGF‐β isoforms, with TGF‐β2 having the greater effects. Consistent with these results, treatment with TGF‐β2 resulted in suppression of mitochondrial and glycolytic functions in 2D cultured 3T3‐L1 cells. However, the inhibitory effect of TGF‐β on adipogenesis decreased under 3D spheroid culture conditions and TGF‐β isoforms did not affect adipogenesis‐induced (a) enlargement and downsizing of 3T3‐L1 spheroids, (b) increase in BODIPY lipid staining intensity, and (c) up‐regulation of the mRNA expression of adipogenesis‐related genes. The findings presented herein suggest that the three TGF‐β isoforms have different suppressive effects on adipogenesis‐related cellular properties of 2D cultured 3T3‐L1 cells and that their effects decrease under 3D spheroid culture conditions.