Abstract
Adipose tissue is a specialized organ that synthesizes and stores fat. During adipogenesis, Rho and Rho-associated kinase (ROCK) 2 are inactivated, which enhances the expression of pro-adipogenic genes and induces the loss of actin stress fibers. Furthermore, pan ROCK inhibitors enhance adipogenesis in 3T3-L1 cells. Here, we show that KD025 (formerly known as SLx-2119), a ROCK2-specific inhibitor, suppresses adipogenesis in 3T3-L1 cells partially through a ROCK2-independent mechanism. KD025 downregulated the expression of key adipogenic transcription factors PPARγ and C/EBPα during adipogenesis in addition to lipogenic factors FABP4 and Glut4. Interestingly, adipogenesis was blocked by KD025 during days 1~3 of differentiation; after differentiation terminated, lipid accumulation was unaffected. Clonal expansion occurred normally in KD025-treated cells. These results suggest that KD025 could function during the intermediate stage after clonal expansion. Data from depletion of ROCKs showed that KD025 suppressed cell differentiation partially independent of ROCK’s activity. Furthermore, no further loss of actin stress fibers emerged in KD025-treated cells during and after differentiation compared to control cells. These results indicate that in contrast to the pro-adipogenic effect of pan-inhibitors, KD025 suppresses adipogenesis in 3T3-L1 cells by regulating key pro-adipogenic factors. This outcome further implies that KD025 could be a potential anti-adipogenic/obesity agent.
Highlights
Fats, or triacylglycerols, are highly efficient sources of energy in the body, and mammals have developed intricate mechanisms to store fats in adipocytes to minimize the loss of energy
The Rho-Rho-associated kinase (ROCK) signaling pathway plays an important role in adipocyte differentiation, and ROCK2 has been suggested as the messenger and transducer of the anti-adipogenic activity of Rho
When KD025 was administered with differentiation media (DMI) from day 0 to day 7, the amount of fat significantly decreased in a dose-dependent manner (Fig. 1B,C)
Summary
Triacylglycerols, are highly efficient sources of energy in the body, and mammals have developed intricate mechanisms to store fats in adipocytes to minimize the loss of energy. PPARγ and C/EBPα induce the expression of various metabolic genes that are required to maintain adipocyte phenotypes, such as fatty acid-binding protein 4 (FABP4; aP2) and glucose transporter 4 (GLUT4; SLC2A4)[2]. Rho-associated coiled-coil-containing protein kinases (ROCKs) were first introduced as RhoA-binding proteins that regulate actin cytoskeleton remodeling in cells[7,8]. ROCK isoforms play pivotal roles in the regulation of actin cytoskeleton organization, cytokinesis, differentiation, apoptosis, glucose metabolism, cell adhesion/motility, and inflammation[2,9,10,11]. Active Rho promotes the expression of YAP (Yes-associated protein) and TAZ (transcriptional co-activator with PDZ-binding motif), transcription factors that suppress adipocyte differentiation[17]. We provide important evidence that KD025 suppresses adipocyte differentiation in 3T3-L1 cells by inhibiting the expression of pro-adipogenic factors such as PPARγ and C/EBPα. We suggest that KD025 could suppress adipogenesis by targeting an unknown adipogenic factor other than ROCK2
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