Inflammatory Reaction Induced By TSE-MVs And Mechanisms On Inhibiting Insulin Signaling Pathway In Adipocytes

Objective Growing studies have been indicating that tobacco smoking is associated with glucose intolerance,insulin resistance,dyslipidemia and unhealthy body fat distribution which are the risk factors of metabolic syndrome(MS).Microvesicles(MVs),which also are known as microparticles,are small membranous structures that are released from cells during activation or apoptosis.The release of MVs is a mechanism of self modulation and involved in the signaling activation,transduction,immunoregulation and renovation in physiological and pathological process of diseases.Our purpose of this study is to confirm that MVs,which were induced by the exposure of tobacco smoke extract(TSE)to human macrophages,would be contribute to induce the early stage of inflammatory reaction and inhibit insulin signaling pathways.Meanwhile,in this study we explored that high mobility group box-1(HMGB-1)which was carried on the TSE-MVs may be the mechanism of the effects of TSE-MVs and would be given some new theoretical supports to the relationship between tobacco smoking and metablic syndrome.Methods 1.The extraction of TSE-MVs.Human THP-1 monocytic cells were differentiated to human monocyte/marophages by PMA.Primary human monocyte-derived macrophages(h MDMs)were obtained from fresh anticoagulant blood.Research-grade cigarettes were obtained from the Reference Cigarette Program at the University of Kentucky(Lexington,KY).Tobacco smoke extract(TSE)was prepared by extracting mainstream smoke through 10 m L of phenol red-free RPMI 1640 medium containing 0.2% bovine serum albumin(BSA).TSE-MVs were isolated by ultracentrifugation from the conditional medium of TSE-exposed human macrophage which differentiated by human THP-1 monocytic cells or primary human monocyte-derived macrophages(h MDMs).TSE-MVs were resuspended by RPMI 1640 medium.2.TSE-MVs induce the early stage of inflammatory reaction.Cultured 3T3-L1 adipocytes were stimulated to mature adipocyte by dexamethasone,IBMX,rosiglitazone and insulin.TSE-MVs were added into cultured 3T3-L1 adipocytes for 24 hours.20 ?m cell trackers were used for marking the monocytes to green fluorescence.We observed the activation of the early inflammation,which indicated monocytes could transmigrate and adhesion to the adipocyte,was followed by the assays of chemoattraction of monocytes across a 8.0 ?m boyden chamber to the adipocytes.3.TSE-MVs inhibit the phosphorylation of insulin signaling pathway.TSE-MVs were added into cultured 3T3-L1 adipocytes for 24 hours followed by 10 n M insulin for 20 minutes.To examine biological effects on insulin signaling pathways,we observed the phosphorylation of IRS1(Y612)and Akt(S473)by western blotting after TSE-MVs stimulated adipocytes.Furthermore,TSE-MVs and/or monocyte could inhibit the the phosphorylation of insulin signaling pathway IRS-612 and AKT-473 in adipocytes.4.HMGB-1 is the mechanism of the effects of TSE-MVs.Western blot was applied for finding out high mobility group box 1(HMGB-1)maybe carried by MVs.Recombinant HMGB-1(r HMGB-1)had the same effects with TSE-MVs on activating the early inflammatory reaction and inhibiting the phosphorylation of insulin signaling pathway followed by the methods aboved.Pretreatment of TSE-MVs or recombinant HMGB-1(r HMGB-1)with glycyrrhizin,which binds the active sites of HMGB-1,attenuated the ability of TSE-MVs to induce MCP-1 secretion,early inflammation and phosphorylation of IRS-612 and AKT-473 in adipocytes.Results 1.TSE-MVs were generated by TSE-exposed human macrophage which differentiated by human THP-1 monocytic cells or h MDMs.2.TSE-MVs induced the secretion of MCP-1 and increased the transmigration and adherence of monocytes to adipocytes.Anti-MCP-1 neutralizing antibody or the CCR2 antagonist RS504393 significantly inhibited monocytes' transmigration and adherence to adipocytes under the stimulation of TSE-MVs.3.TSE-MVs inhibited insulin-induced phosphorylation of IRS-612 and AKT-473.4.HMGB-1 was secreted by TSE in human THP-1 monocytic cells and h MDMs.HMGB-1 carried by TSE-MVs was released as a soluble form from activated or dying macrophage.r HMGB-1 had the same effect with TSE-MVs on monocytes' transmigration/adherence to adipocytes and the inhibition of phosphorylation of IRS-612 and AKT-473.5.Glycyrrhizin,which binds the active sites of HMGB-1,attenuated the ability of TSE-MVs and r HMGB-1 which could induce MCP-1 secretion and enhanced monocytes' transmigration/adherence to adipocytes.Furthermore,glycyrrhizin could recovere the injury of phosphorylation of IRS-612 and AKT-473 stimulated by TSE-MVs and r HMGB-1.Conclusion 1.TSE-MVs were released by TSE-induced human macrophage which differentiated by human THP-1 monocytic cells or h MDMs.2.TSE-MVs could induce the early inflammatory reaction including monocytes' transmigration and adherence to adipocytes by stimulating the secretion of MCP-1.TSE-MVs could inhibit insulin-induced phosphorylation of IRS-612 and AKT-473 to induce the development of insulin resisitance.3.Owing to HMGB-1 carried by TSE-MVs,HMGB-1 could be the mechanism of TSE-MVs to induce the early inflammation and inhibited phosphorylation of insulin signaling pathways.Glycyrrhizin,which binds the active sites of HMGB-1,attenuated the ability of TSE-MVs in early inflammation and insulin resistant.