Addition of ROCK Inhibitors Alleviates Prostaglandin-Induced Inhibition of Adipogenesis in 3T3L-1 Spheroids

Abstract

To elucidate the additive effects of the ROCK inhibitors (ROCK-i), ripasudil (Rip) and Y27632 on bimatoprost acid (BIM-A), a prostaglandin analog (PG), on adipose tissue, two- and three-dimensional (2D or 3D) cultures of 3T3-L1 cells, the most well characterized cells in the field of lipid research, were used. The cells were subjected to a variety of analyses including lipid staining, real-time cellular metabolic analysis, the mRNA expressions of genes related to adipogenesis and extracellular matrices (ECMs) as well as the sizes and physical properties of the 3D spheroids by a micro-squeezer. BIM-A induced strong inhibitory effects on most of the adipogenesis-related changes in the 2D and 3D cultured 3T3-L1 cells, including (1) the enlargement and softening of the 3D spheroids, (2) a dramatic enhancement in lipid staining and the expression of adipogenesis-related genes, and (3) a decrease in mitochondrial and glycolytic metabolic function. By adding ROCK-i to the BIM-A, most of these BIM-A-induced effects were cancelled. The collective findings reported herein suggest that ROCK-i eliminated the PG-induced suppression of adipogenesis in the 3T3-L1 cells, accompanied by the formation of enlarged 3D spheroids. Such effects of adding ROCK-i to a PG in preadipocytes on cellular properties appear to be associated with the suppression of PG-induced adverse effects, and provide additional insight into our understanding of lipid-related research.