Anti-adipogenic effects of KD025 (SLx-2119), a ROCK2-specific inhibitor, in 3T3-L1 cells

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.

Effects of the Selective EP2 Receptor Agonist Omidenepag on Adipocyte Differentiation in 3T3-L1 Cells

“…25,26 Further, it is reported that differentiation starts with the induction of Cebpb at an early stage, during which cells begin to express Pparg and Cebpa. 28,29 Our results are consistent with those of previous reports (Fig. 5).…”

“…Overall, 3T3-L1 cells were originally developed by clonal expansion from murine Swiss 3T3 cells 27 and have been widely used as preadipocytes in studies of adipocyte differentiation. [27][28][29] In our hands, LAT-A and PGF 2a prevented the accumulation of lipid droplets in 3T3-L1 cells and inhibited adipogenesis ( Figs. 3 and 4).…”

“…5). 28,29 These observations suggest that FP agonists prevent the accumulation of lipid droplets initially by inhibiting adipogenic Cebpb expression, and subsequently by limiting the associated increases in Pparg and Cebpa expression.…”

ROCK2 inhibition enhances the thermogenic program in white and brown fat tissue in mice

“…Indeed, its therapeutic potential has been explored in fibrotic disease, 15 focal cerebral ischemia, 16,61,62 and auto-immune disease. 67 However, we have noticed some differences between our study in SV cells with the reported study in 3T3-L1 cells: (a) KD025 treatment reduced total ROCK activity reflected by reduced p-MLC and p-cofilin levels in SV cells, but not 3T3-L1 cells, (b) KD025 treatment disrupted actin cytoskeleton in SV cells, but not 3T3-L1 cells, (c) KD025 reduced adipogenesis on terminally differentiated SV cells, but not on terminally differentiated 3T3-L1 cells, and (d) the effects on beige adipogenesis were not examined in 3T3-L1 cells. Importantly, both dose-and time-dependent analyses ( Figure 7E; Figure S8A) helped to dissociate the pro-beige adipogenic (mediated by ROCK2 inhibition) and anti-adipogenic (targets are not clear) actions of KD025.…”

Determination of KD025 (SLx-2119), a Selective ROCK2 Inhibitor, in Rat Plasma by High-Performance Liquid Chromatography-Tandem Mass Spectrometry and Its Pharmacokinetic Application

“…Upon administration, KD025 binds to and thereby inhibits the serine/threonine kinase activity of ROCK2. Furthermore, KD025 has been reported in various studies to be a potential candidate for the treatment of obesity, insulin resistance [23][24][25][26], rheumatoid arthritis, systemic lupus erythematosus [27], inflammatory bowel disease [28], chronic autoimmune disorders, chronic graft-versus-host disease [29], and fibrosis [30], as well as protection of the blood-brain barrier during thrombolysis [31]. In several phase 1 clinical trials in healthy volunteers, oral administration of KD025 was well tolerated and produced no significant adverse events, including cardiovascular side effects [19,32].…”