Effect Of Acetic Acid On Glucose Metabolism In Macrophages And Its Potential Mechanism In Mice With Sepsis

Objective:Sepsis is a systemic inflammatory response syndrome caused by infection,often accompanied by organ dysfunction,but the exact molecular mechanism is not clear,the lack of effective treatment exists.Proinflammatory factor were mainly released by macrophages in sepsis mice.Its metabolism related to macrophage glycolysis as a therapeutic target.Sepsis in mice were induced by cecal ligation and puncture model.One of the important metabolic intermediate product-acetic acid showed protective effect on sepsis mice.We studied its effect and mechanism on sepsis mice.Methods: 1.Effect of acetic acid on septic mice:Polymicrobial sepsis was induced by cecal ligation and puncture model in mice.For in vivo experiment,500 ul PBS with or without acetate(250 mg/kg or 500 mg/kg)was i.p administrated 0.5h after the establishment of the septic animal models.For the survival assessment,animals were observed for up to 7 days.12 hours after operation,liver and lung tissue injury,Bacteria load in the peritoneal fluid and the effect of proinflammol/latory cytokines were measured.We also used a LPS-induced endotoxemia model to confirm the anti-inflammtory activity of acetate in vivo.To test whether the protective effect of acetate could be extended to other routes of administration,acetate was given to mice by intraperitoneal injection(500mg/kg)0.5h post LPS intraperitoneal administration or by feed in water(150mM)for consecutive 12 days before LPS i.p..2.Effects of acetic acid on proinflamtory cytokines in macrophages:To examine the mechanism by which acetate protect mice from sepsis,BMDMs,human THP-1 cells,mononuclear cells isolated from healthy human peripheral blood(PBMC)and human CD14 monocytes were used to assess the effect of acetate on production of proinflammatory cytokines upon LPS exposure.Pretreatment with acetic acid(5mM~100mM)exhibited a dose-dependent inhibitory effect on LPS-induced TNF-? and IL-6 in BMDMs.Notably,post-treatment of acetate at 0.5h after LPS injection also showed potent anti-inflammatory effect.3.The mechanism of acetic acid in macrophage:We investigated the possible effect of acetate on the phosphorylation of NF-kB and MAPKs.BMDMs and RAW 264.7 macrophages cell line were pretreated with acetate(10mM)for 15 min before LPS stimulation.The phosphor-and total protein levels of NF-?B p65 were determined using phosphor-specific or total protein antibodies at 15 min,30min and 60 min.Western blotting method was used to observe the effects of acetic acid on the phosphorylation of NF-?B p65,JNK,ERK and p38 MAPK and the expression of total protein.BMDMs were knockdown by GPR43 and GPR41 by siRNA for 48 h before 30min-pretreated with acetate(20mM),followed by LPS for additional 6 hours,supernatant TNF-? and IL-6 level were detected.Secondly,we evaluate glycolysis effect on acetic acid through determination of lactic acid concentration in culture medium of BMDMs,analysis of 2-NBDG uptake.We estimate whether acetic acid change LPS induced GLUT1,PFKFB3,HK2,MCT4 mRNA expression and further analysis of changes in the rate of ECAR and OCR.Whether glycosis involving in the effect of acetic acid were studied by pre-incubating of IFN-? or GM-CSF.BMDMs were activated with or without recombinant 100ng/ml IFN-? or 100ng/ml GM-CSF for 3 h before pretreatment of acetate,followed by LPS exposure for 6h.Supernatant was collected to measure TNF-? and IL-6 production.Relative mRNA expression levels of Glut1,HK2,MCT4 and PFKFB3 in BMDMs were examined.Glut1 expression of cell surface was analyzed by flow cytometry.Change of lactate in IFN-? or GM-CSF activated BMDMs were measured.We analysed total p65 and phosphorylated p65,total mTOR and phosphorylated mTOR in acetate pretreated BMDMs followed by LPS stimulated for 0,15 or 30 min in the presence of IFN-? or GM-CSF by Immunoblot.4.How acetic acid inhibits glycolysis in vivo and vitro:Firstly,the effect of acetic acid on HIF-1?were observed.HIF-1? mRNA in BMDMs pretreated with or without 10 mM acetate for 0.5h followed by LPS stimulated.TNF-? and IL-6 mRNA were measured by qPCR.We analysed HIF-1? and actin followed by LPS stimulated for 0,15 or 30 min by Immunoblot.Subsequently,it is discussed whether HIF-1? is necessary for acetic acid.BMDMs incubated with DMSO or DMOG(0.5mmol/l)for 2.5h before treatment with 10 mM acetic acid for 0.5 hour,followed by 100ug/ml LPS for 6 h.TNF-? and IL-6 in supernatant were determined by ELISA.mRNA expression of Glut1,HK2,MCT4 and PFKFB3 in BMDMs 1 h after LPS were determined by PCR.Expression of Glut1 in BMDMs examined by flow cytometry at 15 min,30min,1h and 3h after LPS.Lactate production and glucose consumption in BMDMs were also measured.We also analysed total NF-?B p65,phosphorylated NF-?B p65,HIF-1? and actin in acetate pretreated BMDMs followed by LPS stimulated for 0,15 or 30 min in the presence of IFN-?or GM-CSF.RAW264.7 cells were transduced with vector specific for HIF-1? and control vector for 24 h and treated with 10mmol/l acetate for 0.5 hour,then stimulated with LPS for 6 h,and the effect of overexpression HIF-1? on the effect of acetic acid was observed.Finally,we investigated the relationship among HIF-1? and glycolysis and the effect of acetic acid in septic mice.The 40 wild type(wild type,WT)C57BL/6 mice were randomly divided into five groups as sham operation group(sham,n=8),CLP model group(CLP,n=8),DMOG+CLP Group(DMOG+CLP,n=8),acetic acid(CLP+A,n=8 group)and acetic acid+DMOG group(DMOG+CLP+A,n=8).DMOG(320mg/kg)by intraperitoneal injection of 2.5h followed by CLP operation,and then use acetic acid(500mg/kg,i.p)0.5h after operation.For the survival assessment,animals were observed for up to 7 days.12 hours after operation,liver and lung tissue injury,Bacteria load in the peritoneal fluid and the effect of proinflammol/latory cytokines were measured.Results: 1.Effect of acetic acid on septic mice:survival of CLP with acetate treatment was firstly measured.Acetate(250mg/kg or 500mg/kg)reduced CLP mortality from 62.5% to 25% or 15%,respectively.The sepsis induced organ dysfunction was also assessed by histological analysis of liver and lung.Acetate treated mice displayed significant ameliorated pathological changes,including necrosis,inflammatory cell infiltration,and degeneration.Moreover,acetate intervention enhanced pathogen clearance capacity,as indicated by the peritoneal bacteria counts.Furthermore,acetate decreased the level of TNF-?,IL-6 and IL-1? in plasma compared with sham group(P <0.05).We also used a LPS-induced endotoxemia model to confirm the anti-inflammatory activity of acetate in vivo.We observed an increased resistance to LPS challenge in acetate treated mice,as compared to that in PBS controls,evidenced by a significantly decreased production of TNF-? and IL-6 in both routes of administration.These results suggest that acetate protect mice from CLP.2.Effects of acetic acid on macrophages:Pretreatment with acetate(5mM~100mM)exhibited a dose-dependent inhibitory effect on LPS-induced TNF-? and IL-6 in BMDMs.Notably,post-treatment of acetic acid at 0.5h after LPS ligation also showed potent anti-inflammatory effect.Acetate also blunted LPS-induced TNF-? and IL-6 gene expression at all the time points we examined.Similar result was observed in human THP-1 cell line and peripheral blood mononuclear cells(PBMCs)obtained from healthy human.TNF-? and IL-6 production induced by LPS decreased dramatically in the acetate-treated group at both mRNA and at protein levels.To further confirm the role of acetate in human monocytes,we purified human CD14+ monocytes and obtained the similar results.To exclude the possible cell toxicity of acetate,we measured LDH release after LPS stimulation in the presence of different dose of acetate and found that acetate had no significant effect on LDH release even at the highest dose(100mM).These findings indicate that acetate could blunt TLR4-driven production of proinflamatory cytokines.3.The role of glycolysis to the effect of acetic acid in macrophage:BMDMs exposed to LPS exhibited a significant increase in the phosphorylation of NF-kB compared with control,whereas acetate pretreatment resulted in significant attenuated activation of NF-k B,which is consistant with a previous report in RAW 264.7 macrophages cell line.However,we did not observe any inhibitory effect of acetate on the activation of LPS driven JNK,ERK and p38 MAPK.Unexpectedly,neither knockdown of GPR43,GPR41 respectively nor combinational knockdown of both molecules reverse the effect of acetate on LPS-induced production of 2 cytokines,indicating GPR43 and GPR41 may not involve in the anti-inflammatory effect of acetate.LPS induced a slightly increased level of lactate,while acetate treatment resulted in a slightly but notably lowered extracellular lactate level.We sought acetate acid altered the expression of these molecules LPS induced a rapid increase in mRNA expression of Glut1,PFKFB3,HK2,MCT4,whereas acetate impaired the upregulation of all these molecules at 1 h and 3 h after LPS exposure,suggesting acetate dampen the expression of key glycolytic enzymes upon LPS stimulation.LPS stim? lation resulted in a markedly increase in the ECAR and decrease in the OCR,whereas in the presence of the acetate,both of ECAR and OCR were reduced.Pre-incubation with IFN-? or GM-CSF resulted in nearly completely abrogated the effect of acetate on LPS-induced production of cytokines,though both IFN-? and GM-CSF showed substantially augmented production of cytokine in the absence of LPS.As expected,decreased LPS-driven mRNA expression of Glut1,PFKFB3,HK2,and MCT4 by acetate were also reversed by IFN-? or by GM-CSF.Flow cytometry analysis of GLUT1 on cell surface showed the similar results,indicating that the enhancement of glycolysis could reverse the anti-inflammatory effect of acetate.4.How acetic acid inhibits glycolysis in vivo and vitro:LPS enhanced m RNA expression of HIF-1?,whereas acetate dampened this upregulation.At protein level,acetate also markedly decreased HIF-1?expression after LPS stimulation,indicating that acetate inhibited the upregulation of HIF-1? both at mRNA level and at protein level in response to LPS.DMOG abolished nearly completely the inhibitory effect of acetate on decreased cytokines production upon LPS exposure.Overexpression of HIF-1? in RAW264.7 showed the similar results,demonstrating that HIF-1? is needed for acetate to inhibit LPS-driven release of cytokines.We examined the mRNA expression of glycolytic enzymes in DMOG incubated BMDMs.The blunted upregulated expression of these glycolytic pathway-related genes by acetate in response to LPS were all abolished.The decreased Glut1 expression in cell surface was also reversed by DMOG after LPS exposure.Accordingly,DMOG abolished the decreased lactate production by acetate in response to LPS and restored the glucose level in extracell? lar culture medium.We also found the attenuated activation of NF-?B by acetate was reversed completely by DMOG,indicating that in addition to directly heighten glycolysis,HIF-1? may regulate glycolysis indirectly through activation of NF-?B in macrophage.To further establish the role of HIF-1? in vivo,we found that DMOG significantly impaired prolonged survival in acetate treatment CLP mice.HE stain of liver and lung also did not display mitigated damages in DMOG injected CLP mice compared to control mice in response to acetate treatment.Similarly,DMOG reversed the decreased TNF-?,IL-6 and IL-1? in serum by acetate compared with control mice.These data approve the essential role of HIF-1? in the anti-inflammatory effect of acetate on septic model.Conclusion:We here report that acetate,a small molecule fatty acid,protect mice from experimental sepsis produced by cecal ligation and puncture.Mechanistically,acetate impaired LPS-driven glycolysis of macrophages by decreased the expression of glucose transporter and key glycolytic enzymes.Enhancement of glycolysis by IFN-? or GM-CSF reversed the inhibitory effect of acetate both in vitro and in septic models.Further investigations showed that acetate blunted LPS driven hypoxia-inducible factor 1?(HIF-1?)expression.Stabilization or overexpression of HIF-1? reversed the decreased glycolysis and production of proinflammatory cytokines in macrophages and abrogated the protective effect of acetate on sepsis mice.Our results reveal acetate is a potential therapeutic molecule for sepsis and our study enhance the scope of the limited drugs aimed at regulating glucose metabolism for sepsis therapy.