The Anti-obesity-associated Metabolic Disorders Role Of Major Vault Protein And Underlying Mechanisms

Objectives:Obesity is becoming a leading concern of insulin resistance,hepatic steatosis and other metabolic disorders.Macrophage-orchestrated,low-grade chronic inflammation is a key contributor to obesity related metabolic disorders.However,the underlying regulatory mechanisms remain incompletely understood.The major vault protein(MVP)is the main component of unique cellular ribonucleoprotein particles known as vaults,which mediates cellular multi-drug resistance and participates in multiple signaling pathways and apoptosis.Recently,it has been found that MVP is also implicated in immunoregulation against bacterial and viral infections.However,MVP has been shown conflicting effects on immunoregulation under different stimuli.This suggests that MVP specific role in inflammation should be identified carefully.Therefore,this study aims to investigate the role and mechanism of MVP in metaflammation and pave the way for clinical intervention strategies for metabolic diseases.Methods:In order to determine the role of MVP in obesity,C57BL/6J mice were fed with a chow diet(CD)or high-fat diet(HFD).We then detected the expression MVP in the epididymal white adipose tissue(epi WAT).The localization of MVP in epi WAT was conducted with immunoflourence.We also measured the expressional level of MVP in the visceral adipose tissue of human beings with different BMI.Both global and myeloid-specific MVP gene knockout mice and their control mice were fed with an HFD to induce the animal models of obesity.Body weight and fasting glucose level of mice were detected once a week.We performed glucose induced-insulin secreting test to determine the insulin level of mice.The effects of MVP deficiency on insulin sensitity were explored by glucose tolerance test(GTT)and insulin tolerance test(ITT).The phosphorylation of AKT in epi WAT,liver,and skeletal muscle of mice were determined by Western Blot.We obtained the plasma and various metabolic tissue depots of mice at the end of modeling.Levels of triglycerides(TG),total cholesterol(TCH)and other metabolic index in plasma and liver were assayed.Lipid accumulation in liver was examined by H&E staining and Oil Red O staining.We then studied the effect of MVP deficiency on macrophage inflammatory response in adipose tissue.The expressions of inflammatory mediators in adipose tissue and liver were determined by RT-q PCR.F4/80~+macrophages in epi WAT were sorted by fluorescence-activated cell sorting(FACS)and the expression of inflammatory mediators was detected by RT-q PCR.The inflammatory mediators in plasma were assayed by ELISA.Immunohistochemistry(IHC)staining and flow cytometry were used to analyze the content of macrophages in the epi WAT.The effects of MVP on macrophage inflammatory response and IKK-NF-k B signaling pathway in vitro were observed.Macrophages were treated with the inhibitor of NF-k B pathway to determine whether the effect of MVP on inflammatory response was dependent on NF-k B signaling pathway.Co-immunoprecipitation(Co-IP)was used to find the target molecule directly interacting with MVP,and we then examined the effects of MVP and its fragments on the target molecule.We further studied the functional fragment of MVP regulating the activity of the target molecule.Finally,we overexpressed MVP gene and its functional fragment via lentivirus to determine whether overexpression of MVP and its functional fragment could inhibit inflammatory response in macrophages.Results:In the epi WAT of obese mice,MVP expression was significantly increased,particularly in the macrophages of stromal vascular fraction cells(SVFs)rather than the adipocytes of epi WAT.MVP expression was also upregulated in macrophages of visceral adipose tissue in overweight or obese individuals(BMI≥24).Both global and myeloid-specific MVP gene knockout mice gained more weight and displayed a higher weight of multiple adipose depots than their control mice.MVP deficiency exacerbated HFD-induced high fasting blood glucose and glucose-induced hyperinsulinemia.Furthermore,MVP deficiency impaired glucose tolerance and insulin resistance in mice by GTT and ITT.MVP deficiency increased levels of TG and TCH in the plasma and liver significantly.H&E staining and Oil Red O staining showed a dramatic change in the overall liver morphology with accumulation of large droplet-like structures in MVP deficiency mice.IHC analysis and flow cytometry measurements revealed macrophage content in epi WAT was significantly increased in HFD-fed MVP deficiency mice.The m RNA levels of pro-inflammatory mediators were obviously increased in epi WAT,sub WAT,BAT and liver of HFD-fed MVP deficiency mice.The pro-inflammatory mediators were significantly increased in F4/80~+macrophages isolated from epi WAT of HFD-fed MVP deficiency mice.The plasma level of TNF-αin HFD-fed MVP deficiency mice was also increased consistently.Further mechanism studies indicated that the deficiency of MVP increased the expressions of LPS-mediated inflammatory mediators TNF-αand CCL2 and the activated of the NF-k B signaling pathway.When macrophages were treated with Bay11-7082,the inhibitor of NF-k B signaling pathway,MVP deficiency induced over-production of TNF-αand CCL2 was effectively reversed.Co-IP revealed that MVP interacted with TRAF6.MVP depletion significantly enhanced LPS-induced polyubiquitination of TRAF6 in murine bone marrow derived macrophages(BMDMs).MVP depletion can also promote TRAF6 recruitment to IRAK1 and subsequently oligomerization of TRAF6.TRAF6 interacted with the MVP-(1-480),MVP-(481-685)and MVP-(686-870).However,only MVP and itsα-helical domain(686-870),inhibited the oligomerization and polyubiquitination of TRAF6 functionally.Transfection of macrophages with lentivirus to overexpress MVP or itsα-helical domain suppressed LPS-induced macrophage inflammatory response.Conclusion:MVP is up-regulated in adipose tissue in human and mice during obesity,especially in macrophages.Both global and myeloid-specific MVP gene knockout aggravate high-fat diet induced obesity,insulin resistance,hepatic steatosis and inflammatory response in mice.In vitro studies reveal that MVP deficiency promotes LPS-mediated macrophage inflammatory response and activates NF-κB signaling in macrophages.MVP interacts with TRAF6 and prevents its recruitment to IRAK1 and subsequent oligomerization and polyubiquitination,which inhibits NF-k B signaling pathway.Overexpression of MVP and itsα-helical domain inhibits the activity of TRAF6 and suppresses macrophage inflammation.Our results demonstrate that macrophage MVP constitutes a key constraint of NF-κB signaling thereby suppressing metabolic diseases.