Asprosin Causes Vascular Endothelial Insulin Resistance Through Promoting Mitochondrial Fission Mediated By IKKβ-NF-κBp65 Pathway

ObjectiveAsprosin（ASP）is a newly identified adipokine,which is mainly secreted by white adipose tissue（WAT）.ASP plays important roles in regulating the homeostasis of glycolipid metabolism.Abnormal increases in the ASP levels are observed in the patients with obesity.Also,ASP participates in the pathophysiological process of the obesity complicated with vascular disease and diabetes.However,its effect and mechanism of action on vascular endothelial insulin resistance（IR）are still unclear.The purpose of this study was to explore the effect and mechanism of ASP on vascular endothelial IR induced by high fat from the perspective of vascular endothelial inflammation and mitochondrial dynamics,so as to lay an experimental foundation for the clinical treatment of obesity-related vascular diseases and complications.Methods1.Human umbilical vein endothelial cells（HUVECs）were treated with palmitic acid（PA）to establish the vascular endothelial IR model by detecting the NO levels in medium and the expression levels of e NOS-p1177.2.HUVECs were treated with various concentrations of ASP（50n M）,and the phosphorylation levels of the insulin signaling proteins（PI3K,AKT,e NOS）were examined by western blotting to select the optimal concentration of ASP that leads to IR.Next,the HUVECs were randomly divided into Ctrl,PA（0.25mM）,ASP（50n M）and PA（0.25mM）+ASP（50n M）groups.The phosphorylation levels of PI3K,AKT and e NOS were detected by western blotting to investigate the effects of ASP on the IR of vascular endothelium under hyperlipidemia condition.3.The grouping was the same as described in the method 2,and the expression levels of mitochondrial fission protein DRP1 were detected by western blotting.Mitochondrial morphology was detected by laser confocal microscopy.The effects of ASP on mitochondrial fission under hyperlipidemia were investigated by the above experiments.4.The grouping was the same as described in the method 2,and the levels of inflammatory cytokines including IL-6 and TNF-ɑwere detected by ELISA.The subcellular localization of NF-κBp65 was visualized by immunofluorescence and the cytoplasmic and nuclear levels of NF-κBp65 were assayed by western blotting to evaluate the translocation of NF-κBp65 from the cytoplasm to nucleus.The expression levels of inflammatory sinaling proteins（p-NF-κBp65/NF-κBp65、p-IKKɑ/β/IKKɑ/βand IKBɑ）were measured by western blotting to explore the mechanism of pro-inflammation of ASP.The levels of reactive oxygen species（ROS）were detected by flow cytometry.5.The rats were fed with normal chow in specific pathogen free（SPF）environment（temperature:25°C;humidity:approximately 50%）for 1 week.They were randomly divided into ND group（12 rats）and HFD group（12 rats）and fed with the corresponding diet for the first 24 weeks.In the last 4 weeks,12 rats fed with ND were randomly sub-divided into ND group（6 rats）and ASP group（6 rats）,and 12rats fed with HFD were randomly divided into HFD group（6 rats）and anti-ASP+HFD group（6 rats）.In detail,the rats in ASP group or ND group were intraperitoneally injected with recombinant ASP(0.5μg·kg^-1·day^-1)or equivolume normal saline for 4 weeks and were fed with ND;meanwhile,the rats in anti-ASP+HFD group or HFD group were intraperitoneally injected with a specific neutralization antibody against ASP(anti-ASP,0.5μg·kg^-1·day^-1)or equivolume normal saline for 4 weeks and were fed with HFD.ELISA assay was used to detect serum inflammatory factor levels,immunohistochemistry was used to detect macrophage marker CD68 levels,and Western blotting was used to detect inflammatory pathway proteins（p-IKKɑ/β/IKKɑ/βand IκBɑ）levels.Through the above experiments,we aim to investigate the effects of anti-ASP on HFD induced inflammation of endothelial cells in obese rats.6.To explore the mechanism of ASP on vascular endothelial inflammation and IR and the relationship between mitochondrial fission and vascular endothelial inflammation,an inhibitor of mitochondrial fission（Mdivi-1）and a short hairpin RNA（sh RNA）were used,respectively.The phosphorylation levels of insulin signaling proteins（PI3K-AKT-e NOS）and inflammatory signaling proteins（IKKɑ/β-NF-κBp65）as well as the DRP1 levels were detected by western blotting.Besides,the subcellular localization of NF-κBp65 and the morphology of mitochondria were detected by laser confocal microscopy.Results1.Treatment of HUVECs with 0.25 mM PA for 48 h significantly decreased the levels of NO and p-e NOS,indicating that the high-fat induced IR model was established successfully.2.Treatment of HUVECs with 50 n M ASP alone for 48 h significantly inhibited the activation of the insulin signaling（PI3K-AKT-e NOS）;moreover,the treatment with ASP+PA markedly worsed the decreases in the phosphorylation levels of insulin signaling proteins（PI3K-AKT-e NOS）.3.The Western blotting data showed that the treatment of ASP+PA significantly increaseed the expressions of DRP1,and the immunofluorescence results showed that the fragmented mitochondria were also increased.4.The ELISA results revealed that the treatment of ASP+PA significantly increased the levels of inflammatory factors（IL-6 and TNF-ɑ）in vascular endothelial cells;more importantly,the treatment of ASP+PA promoted the translocation of NF-κBp65 from cytoplasm to nucleus and thus activated the inflammatory signaling pathway（IKKɑ/β-NF-κBp65）.However,ASP treatment had no effect on the level of ROS.5.In the experiments exploring the effects of ASP and anti-ASP on the rats with obesity induced by HFD,both ELISA and immunohistochemistry results showed that both HFD and ASP can cause vascular endothelial inflammatory reactions in rats,such as increased expression of inflammatory factors and increased levels of CD68,while anti-ASP can inhibit the above inflammatory reactions.Western blotting results also showed that anti-ASP can inhibit the activation of the vascular endothelial inflammatory pathway（IKKɑ/β-NF-κBp65）in the rats induced by long-term HFD.6.The Western blotting results displayed that the inhibition of DRP1 with Mdivi-1 markedly normalized the decreased insulin signaling（PI3K-AKT-e NOS）and the elevated inflammatory signaling（IKKɑ/β-NF-κBp65）induced by the ASP+PA treatment.Moreover,the immunofluorescence results also exhibited that the inhibition of DRP1 considerably abolished the nuclear translocation of NF-κBp65induced by the ASP+PA treatment.Besides,after silencing IKKβwith sh RNA,compared with the transfected negative plasmid group（NC sh RNA）,the inhibitory effect of ASP on the insulin signaling（PI3K-AKT-e NOS）was reversed;the DRP1expressions and the fragmented mitochondria were reduced.Conclusion:ASP promotes mitochondrial fission through the IKKβ-NF-κBp65pathway and leads to the vascular endothelial insulin resistance,and there is a mutual promotion relationship between the vascular endothelial inflammation and the mitochondrial fission caused by ASP.