The Molecular Mechanism Of Melatonin Modulating Lipid Metabolism Through Shaping The Pattern Of Gut Microbiome And Adipose-derived Exosomes

In China,increased living standard-induced requirement for high-level of meat quality has been a key problem in animal agriculture.The meat quality is easily affected by various factors,and intramuscular fat is directly related to the quality of meat quality,taste and flavor.Lipid metabolism is closely related to the formation and distribution of adipose in livestock and poultry,lipid dysmetabolism-induced obesity prejudices health of livestock.Therefore,it is very important to identify the key regulatory pathways which affects lipid metabolism in vivo and in vitro as well as to analyze specific mechanisms of the improvement in livestock and poultry genetics and breeding.Previous investigations have indicated melatonin(MT,N-acetyl-5-methoxytryptamine)to alleviate lipid dysmetabolism in multiple physiological sites(including adipose tissue,liver,and skeletal muscles)via various regulatory pathways.Admittedly,melatonin-mediated positive effects on various tissues/organs have been respectively reported,but regulatory mechanisms of melatonin on the crosstalk among different organs/tissues have been investigated rarely.Moreover,since MT has been used clinically as an oral agent,the possibility of MT-mediated improvements in physiology may be secondary to MT actions on microbiota.Overall,this study identified the target gut strains and key molecular in MT-mediated regulation under various physiological models through multi-omics sequencing.Further,the MT-mediated modulations on key physiological sties(including ileum,liver,and adipose tissue)were investigated by RT-q PCR,immunoblotting,enzyme linked immunosorbent assay(ELISA),immunofluorescence,and histological section.Finally,the mechanisms of MT have been revealed by the technique of gene interference,antibiotics-mediated depletion of gut microbiota,fecal microbiome transportation,mono-colonization,agonist/inhibitor,and luciferase test.The results include:1.Melatonin ameliorates intestinal and adipose lipid dysmetabolism through reducing Escherichia coli-derived lipopolysaccharideHerein,we found that jet-lagged(JL)mice have a higher level of ileal lipid uptake,fat accumulation in epididymal white adipose tissue(e WAT)and lower level of circulating angiopoietin-like 4(ANGPTL4)in comparison with the control(P<0.05).JL mice also showed a significantly higher abundance of Escherichia coli(E.coli)and lipopolysaccharide(LPS)than the control(P<0.05).Conversely,oral melatonin supplementation remarkably reversed these phenotypes(P<0.05).The test of depletion of gut microbiota further demonstrated that oral melatonin-mediated improvements on lipometabolism in JL mice were depended on the presence of gut microbiota.By mono-colonization of E.coli,LPS has been determined to trigger these changes similar to JL treatment.Furthermore,we found that LPS served as a pivotal link which contributed to activating Toll-like receptor 4(TLR4)/Signal transducer and activator of transcription 3(STAT3)/REV-ERB?signaling to up-regulate Nuclear factor interleukin-3-regulated protein(NFIL3)expression,resulting in increased lipid uptake in ileum.In MODE-K cells,the activation of NFIL3 has further been shown to inhibit ANGPTL4 transcription,which is closely associated with lipid uptake and transport in peripheral tissues.Finally,we confirmed that melatonin inhibited LPS via repressing the expression of LpxC in E.coli.Overall,oral melatonin decreased the quantity of E.coli-generated LPS,which alleviated NFIL3-induced transcriptional inhibition of ANGPTL4 through TLR4/STAT3 signaling in ileum,thereby resulting in the amelioration of ileal lipid intake and lower fat accumulation in e WAT.2.Reduced delivery of epididymal adipocyte-derived exosomal resistin is essential for melatonin ameliorating hepatic steatosisHere,we first found the amount of adipocyte-derived exosomal resistin to be significantly decreased by melatonin supplementation(P<0.05).Compared to mice with Exos _HFDor Exos_resistintreatment,Exos _MTremarkably ameliorated hepatic steatosis(P<0.05).Further test demonstrated that resistin was a pivotal cytokine which repressed phosphorylation of 5?adenosine monophosphate-activated protein kinase?(p AMPK?Thr172)to trigger endoplasmic reticulum(ER)stress,resulting in hepatic steatosis,whereas Exos _MTreversed these risks in hepatocytes.In adipocytes,we identified melatonin to reduce the production of resistin through the brain and muscle arnt-like protein 1(Bmal1)transcriptional inhibition.Notably,we also confirmed that melatonin enhanced N6-Methyladenosine(m⁶A)RNA demethylation to degrade resistin m RNA in adipocytes.Overall,melatonin decreases traffic volume of adipocyte-generated exosomal resistin from adipocytes to hepatocytes,which further alleviates ER stress-induced hepatic steatosis.3.The improvement of the pattern of secondary bile acids is essential for oral melatonin inhibiting trimethylamino oxide production and improving hepatic lipid dysmetabolism through gut microbiota in aged miceIn this study,compared to the control,aging significantly shaped the pattern of gut microbiota and bile acid,whereas MT treatment reversed these phenotypes(P<0.05),which was identified to depend on the existence of gut microbiota.Further,aging obviously triggered activation of ileac farnesoid X receptor(FXR)signaling and downstream fibroblast growth factor 15(Fgf15)expression,whereas MT remarkedly inhibited ileac FXR signaling via shaping the pattern of gut microbiome in aged mice(P<0.05).Moreover,by the mono-colonization,aging-triggered high-level expression of ileac FXR was significantly decreased through MT-mediated inhibition on Campylobacter jejuni-induced deconjugation of TUDCA and GUDCA(P<0.05).In liver,the aging-induced high-level of serum TCDCA activated trimethylamine-N-oxide(TMAO)-triggered protein kinase R—like ER kinase(PERK)/activating transcriptional factor 4(ATF4)signaling via hepatic FXR,which further promoted SREBP1 transcription and regulated hepatic bile acid metabolism,whereas TUDCA inhibited senescence-triggered high-level expression of hepatic AFT4.Overall,melatonin reduces Campylobacter jejuni-mediated deconjugation of TUDCA to inhibit senescence-triggered high-level expression of hepatic FXR/flavin-containing monooxygenase 3(FMO3),which further decreases hepatic TMAO production and bile acid dysmetabolism.Taken together,these results systematically address the mechanism of MT on modulating lipid dysmetabolism in ileum,liver,and adipose tissue via inhibiting E.coli-LPS and Campylobacter jejuni-mediated deconjugation of TUDCA,meanwhile the mechanism of MT on hepatic steatosis via shaping adipocyte-derived exosomes has also been identified.These data provide a novel foundation of research for gut microbiota and adipocyte-derived exosomes-mediated regulation on animal lipometabolism and selective breeding,which also suggests that melatonin is a clinically potential therapies for nutritional obesity and related metabolic syndrome.