Effect Of CDK5 On Telmisartan’s PPARγ Phosphorylation In 3T3-L1 Adipocytes

Objective 1.Clearing inflammatory factor TNF-α whether have an impact on 3T3-L1 adipocytes PPARγ phosphorylation role.2.Clear telmisartan can antagonize the impact of TNF-α on PPARγ.3.Clear the role of telmisartan on fat cells is indeed dependent on the activity of protein kinase CDK5 and telmisartan effect site.Methods The 3T3-L1 preadipocytes were induced to differentiate and mature by three-step method.TNF-α stimulated the maturation of 3T3-L1 and treated with telmisartan at different doses(T0.1 group,0.1 μmol /L telmisartan;T5 group,5 μmol/L telmisartan,T10 group,10 μmol/L telmisartan).Western blot was used to detect peroxisome peroxisome proliferator activated receptor γ(PPARγ)and its phosphorylation levels.The 3T3-L1 PPARγ gene was silenced by sh RNA.The 3T3-L1 cell line stably expressing PPARγ serine was constructed by retroviral infection and the adipocyte differentiation ability was evaluated by oil red O-stained isopropanol extraction.WB was used to detect phosphorylation sites and their upstream kinases.sh RNA silenced cyclin-dependent kinase 5(CDK5)in mature 3T3-L1 and interfere with telmisartan,PPARγ and phosphorylation levels was detected by WB,Adiponectin Release volumewere detected by ELISA.Result 1.Three-step method successfully established 3T3-L1 preadipocyte differentiation model,the screening of PPARγ mutant cell lines and wild-type compared to the same differentiation ability.2.Telmisartan affect the phosphorylation of PPARγ in 3T3-L1,and the expression of p PPARγ in each group was decreased after intervention with telmisartan at different doses,especially in T5 and T10 groups.The content of adiponectin significantly increased.3.Western blot results showed that phosphorylation level of PPARγ in S273 A cells no4.Longer responded to telmisartan intervention compared with WT,that is,no decrease in p PPARγ expression was observed,but p PPARγ expression in S112 A and S186 A decreased.5.CDK5 mediated the inhibition of PPARγS273 phosphorylation by CDK5,silenced CDK5 gene and screened ΔCDK5-3T3-L1 preadipocyte line.The results of oil red O staining showed that CDK5 gene silenced after 3T3-L1 differentiation ability was not significantly affected,but compared with the control group,the ability of telmisartan to reduce PPARγphosphorylation in ΔCDK5-3T3-L1 mature adipocytes was significantly inhibited,meanwhile the expression of adiponectin was significantly increased.Conclusion 1.The decrease of adiponectin release by 3T3-L1 adipocytes induced by TNF-α was achieved by up-regulating the phosphorylation level of PPARγ and then affecting the transcriptional activity rather than increasing the expression level of PPARγ.Telmisartan can antagonize the above TNF-α effect.2.The use of retrovirus gene silencing and site-directed mutagenesis successfully constructed a series of PPARγ phosphorylation site mutation 3T3-L1 cell line.The cell line was used to determine the site of telmisartan serine 273.3.The ability of 3T3-L1 differentiation was not significantly affected by CDK5 silencing,but the ability of telmisartan to reduce the phosphorylation level of PPARγ was significantly inhibited by silencing CDK5 gene as above and selecting for ΔCDK5-3T3-L1 preadipocyte line.4.Telmisartan can inhibit CDK5-mediated serine phosphorylation at position 273 of PPARγ,thereby upregulating the expression of adiponectin,improving insulin resistance,and clarifying the molecular mechanism of telmisartan in improving inflammatory response.Early application of drugs in the clinic to provide a theoretical basis to fill the gaps in the current field of T2 DM drug prevention.