Effects Of BRL37344on Differentiation Of3T3-L1Preadipocytes And The Mechanism Study

ObjectiveObesity is closely correlated with the prevalence of diabetes and cardiovascular disease. Obesity is caused not only by hypertrophy of adipose tissue but also by adipose tissue hyperplasia, which triggers transformation of pre-adipocytes into adipocytes. The program of adipocyte differentiation is a complex process that involves coordinated expression of specific genes and proteins associated with each stage of differentiation. This process is regulated by several signaling pathways. The MEK/ERK signaling pathway is the most concerned. β3-adrenergic receptors have been proved localised to tissues involved in fat mobilisation and thermogenesis. BRL37344, a specific β3-adrenergic receptor agonist could promote lipolysis, against arrhythmia, which have potential weight loss effects in rodents through stimulating the lipolysis of white adipose tissue and non-shivering thermogenesis of brown adipose tissue, offer perspective on anti-obesity drug research. In this study, we aimed to examine the effect of BRL37344on3T3-L1preadipocyte different and further to clear the role of ERK1/2pathway phosphorylation in this process.Methords3T3-L1preadipocytes were cultured then induced differentiation after they were adherent and reached confluence24hours. Preadipocytes were cultured in vitro and differentiated by four groups of inducers:control, DMSO group, BRL group, PD group and PD+BRL group, On5d, the lipid acccumulation in the cells was evaluated by oil Red O staining, the lysates were analyzed for PPARy, ERK1/2and activated ERK1/2. To further assess the involvement of ERK signaling in the effects of BRL37344on adipocyte differention, until24hours postconfluent3T3-L1cells were serum deprived for4h and then treated the3T3-L1cells with Insulin, BRL37344, Insulin+BRL37344, BRL37344+PD98059for60min, and the lysates were analyzed for activated ERK1/2in four points(0min、5min、10min、60min)during adipocytes differentiation were detected.Data and figures in the text were expressed as means±SE(x±s). Statistical analysis was undertaken using t-text for comparison of control versus treatment groups. The threshold for significance was P<0.05.Results1. Successfully induced of3T3-L1preadipocyte differentiation into mature fat cells.2. BRL37344could inhibit adipocyte differentiation in3T3-L1preadipocytes.3.3T3-L1cells were cultured in the differentiation medium for5d, compared with NC, the protein experessions of PPARy in BRL group and PD group were significantly declined36.07%、45.08%(P<0.05), there is no significant differenc-es in DMSO group and PD+BRL group (P>0.05); pERKl/2in BRL group showed strong expression, was2.99fold as much as control group (P<0.05), the other three groups had no difference (P>0.05), compared with NC.4. In addition, cells were treated insulin for60min, a rapid increase in ERK1/2phosphorylation occurred at5min, was2.99fold as much as0min intervention group (P<0.05), however, the increase in the phosphorylation of ERK1/2lasted for up to60min when the cells were treated with BRL37344, were1.60(P<0.05)、1.46、1.69(P<0.05) fold as much as0min intervention group, or co-treated with insulin and BRL37344, were1.71(P<0.05)、1.33、1.59(P<0.05) fold as much as0min intervention group; Contreatment of BRL37344with PD98059, an upstream kinase for ERK, reduced ERK phosphorylation, a rapid increase in ERK1/2phosphorylation occurred at5min, was2.12fold as much as0min intervention group (P<0.05).Conclusions1. We successfully established the differentiation model of3T3-L1preadipocyt-es.2. BRL37344could inhibit adipocyte differentiation in3T3-L1preadipocytes.3. The long time and high levels phosphorylation of ERK1/2signaling pathway trigged by BRL37344could down-regulate the the protein experessions of PPARy2, plays an important role in the process of inhibition of3T3-L1preadipocyte differention.4. Cotreatment of evodiamine with PD98059, a specific inhibitor of MAPK kinase (an upstream kinase for ERK), reduced ERK phosphorylation and restored the protein expressions of PPARy and adipocyte differentiation.