ATP2B1 Silencing Increase Insulin Sensitivity Through Facilitating Akt Activation Via Ca²⁺/Calmodulin Signaling Pathway In HUVECs

Objective: To study the effect of silencing ATP2B1 on insulin sensitivity in human umbilical vein endothelial cells?HUVEC?,explore the basic mechanism involved in and provide some experimental datas to reduce the risk of cardiovascular disease in patients with insulin resistance.Methods: HUVEC cells in logarithmic growth phase were digested with 0.25% trypsin and then plated in a 6-well plate at a density of 0.5×106cells/well.The negative SiRNA?Si-Neg?and ATP2B1 gene specific SiRNA?Si-ATP2B1?were separately transfected into HUVECs by riboFECTTMCP transfection kit.Then the experiment was conducted in three parts.In part 1,after 48 hours of transfection,HUVECs were harvested,the expression of PMCA1 was tested by Western Blotting and the concentration of Ca²⁺ was measured using Fluo-3-acetoxymethyl ester?Fluo-3 AM?.In part 2,after 32 hours of transfection,the transfeced HUVECs were starved of serum for overnight followed with pretreatments with or without 10?M BAPTA-AM for 90 min,and then the HUVECs were stimulated with 100 n M insulin for 30 minutes.Otherwise,the transfeced HUVECs were starved of serum for overnight followed with treatment with or without 500 nM A23187 for 30 min.Then the protein expressions of p-Akt and Akt were detected by Western Blotting in HUVECs.In part 3,after 32 hours of transfection,the transfeced HUVECs were starved of serum for overnight followed with pretreatment with or without 30?M W7 for 30 min,and then the HUVECs were stimulated with 100 nM insulin for 30 minutes.Then the protein expressions of p-Akt and Akt were detected by Western Blotting in HUVECs.Otherwise,the transfeced HUVECs were starved of serum for overnight followed with treatment with 100 nM insulin for 30 min and the levels of Akt co-immunoprecipited with antibody against calmodulin were analyzed by Western Blotting.Results:1.The protein expression of PMCA1 was significantly decreased in the Si-ATP2B1 transfected HUVECs than that in the SiRNA transfected cells?P<0.05?.2.The intracellular Ca²⁺ concentration was higher in the Si-ATP2B1 transfected HUVECs than that in the Si-Neg transfected cells?P<0.05?.3.Compared with transfected cells without insulin stimulation,the p-Akt/Akt ratios were significantly increased in Si-Neg and Si-ATP2B1 transfected cells with 100 n M insulin stimulation?P<0.05?.Insulin could induce phosphoration of Akt on serine 473 to phosphor-Akt?ser473?.Otherwise,compared with Si-Neg transfected cells with 100 nM insulin stimulation,the p-Akt/Akt ratio was significantly increased in Si-ATP2B1 transfected cells with 100 nM insulin stimulation?P<0.05?.ATP2B1 silencing could promote insulin-induced phosphoration of Akt on serine 473 and increase insulin sensitivity in HUVECs.4.Compared with Si-ATP2B1 transfected cells without pretreatment with BAPTA-AM,the ratio of p-Akt/Akt was significantly decreased in Si-ATP2B1 transfected cells with pretreatment with BAPTA-AM?P<0.05?.ATP2B1 silencing increase insulin sensitivity through facilitating Akt activation in Ca²⁺-dependent manner.5.Calcium ionopore A23187 can transport Ca²⁺ into cytoplasm to increase the intracellular Ca²⁺ concentration.Compared with transfected cells without A23187 stimulation,the p-Akt/Akt ratios were significantly increased in Si-Neg and Si-ATP2B1 transfected cells with A23187 stimulation?P<0.05?.The same as insulin,A23187 could induce phosphoration of Akt on serine 473 to phosphor-Akt?ser473?.Compared with Si-Neg transfected cells with A23187,the ratio of p-Akt/Akt was significantly increased in Si-ATP2B1 transfected cells with A23187?P<0.05?.ATP2B1 silencing increase insulin sensitivity through facilitating Akt activation in Ca²⁺-dependent manner.6.W7 was a specific calmodulin antagonist.Compared with Si-ATP2B1 transfected cells without pretreatment with W7,the ratio of p-Akt/Akt was significantly decreased in Si-ATP2B1 transfected cells with pretreatment with W7?P<0.05?.ATP2B1 silencing increase insulin sensitivity through facilitating Akt activation in calmodulin-dependent manner.7.The co-immunoprecipitation experiment data showed that the Akt associated with calmodulin was higher in Si-ATP2B1 transfected cells than that in Si-Neg transfected cells.The increased insulin sensitivity in the ATP2B1 silencing HUVECs was calmodulin-dependent through higher levels of interaction between calmodulin and Akt.Conclusion:ATP2B1 silencing increase insulin sensitivity through facilitating Akt activation via Ca²⁺/calmodulin signaling pathway in HUVECs.