The Association Of Tryptophan Metabolism And Related Gut Microbiota With Fatty Liver Disease: A Cross-Sectional Study

ObjectiveFatty liver disease（FLD）is a condition characterized by the accumulation of fats in the liver cells,with more than 5%of liver weight.FLD approximately affects a 30%of the world population,which is the leading cause of liver-related mortality.FLD will become a global public health problem.Tryptophan is one of the essential amino acids in the body,and is a precursor of microorganisms and host metabolites.In addition to its participation in protein synthesis,most of tryptophan is metabolized through the kynurenine pathway,serotonin pathway,and indole pathway.Existing studies have shown that kynurenine can increase the risk of metabolic diseases,while indole-3-propionic acid can decrease the risk of metabolic diseases.A few studies have reported an abnormal tryptophan metabolism in patients with FLD,however,the findings are still controversial.Among the tryptophan metabolic pathways,indole pathway metabolites are derived from tryptophan by gut microorganisms.However,it is not clear which intestinal bacteria are involved in the indole pathway,and the association of indole pathway-related intestinal bacteria with FLD remains poorly understood.Previous studies have found that intestinal bacteria can be involved in the development of FLD through producing several metabolites.Therefore,we hypothesized that abnormal tryptophan metabolism was associated with FLD,and the intestinal bacteria that were involved in the indole pathway were associated with FLD.We utilized a community-based cross-sectional study to assess the association between tryptophan metabolites and FLD.Furthermore,we aimed to identify which intestinal bacteria were associated with indole pathway metabolites,and to construct gut microbiota score（GMBS）based on indole pathway-related intestinal bacteria,and to assess the association of indole pathway-related intestinal bacteria and GMBS with FLD.MethodsThis study was a community-based cross-sectional study using a multi-stage sampling in Huoshan County.A structured questionnaire was use to collect information on demographic characteristics,food intake,and other lifestyle behaviors.Blood specimens were collected,and nineteen tryptophan metabolites were tested using liquid chromatography-mass spectrometry,including 2 tryptophan and its derivatives（tryptophan and N-acetyltryptophan）,6 metabolites of kynurenine pathway（kynurenine,kynurenic acid,3-hydroxyanthranilic acid,quinolinic acid,picolinic acid,and nicotinic acid）,3 metabolites of serotonin pathway（5-hydroxy-tryptophan,N-acetylserotonin,and melatonin）,and 8 metabolites of indole pathway（indole-3-acetamide,indole-3-acetic acid,indole-3-methyl acetate,indole-3-acrylic acid,indole-3-propionic acid,tryptamine,indole-3-carboxaldehyde,and indole-3-carboxylic acid）.In addition,the activity of two rate-limiting enzymes including tryptophan 2,3-dioxygenase（TDO）in kynurenine pathway and tryptophan hydroxylase（TPH）in serotonin pathway was calculated.Fecal specimens were collected and amplicon sequencing of the 16S ribosomal RNA（r RNA）gene was used for the taxonomic classification of bacteria.The participant underwent a liver test via vibration-controlled transient elastography,and the controlled attenuation parameter≥274 d B/m was defined as FLD.Logistic regression was used to evaluate the association of tryptophan metabolites（quartiles）and intestinal bacteria（tertiles）with FLD.Based on the operational taxonomic units（OTU）,the alpha diversity and beta diversity（PCo A analysis）were calculated.We compared the difference in beta diversity using ANOSIM test and PERMANOVA test.Multiple linear regression was used to evaluate the association of intestinal bacteria（tertiles）with indole pathway metabolites.Based on the indole pathway-related intestinal bacteria,the weighted quantile sum（WQS）regression was used to construct GMBS with considering FLD as an outcome.ResultsA total of 333 participants,with median age 51（39,62）and 132（39.64%）men,were included in this study.The prevalence of FLD was 24.02%（80 cases）.Compared with non-FLD,patients with FLD tended to be male and obese,to have a history of smoking,alcohol consumption,hypertension and diabetes,and to have a higher level of homeostatic model assessment for insulin resistance and c-reactive protein.Among kynurenine pathway,we found a positive association of TDO enzyme activity(OR_{Q4 vs.Q1}=3.22,95%CI:1.23～8.90,P_trend=0.013)and kynurenine(OR_{Q4 vs.Q1}=2.79,95%CI:1.12～7.36,P_trend=0.011)with FLD.Among serotonin pathway,N-acetylserotonin was shown to be positively associated with FLD(OR_{Q4 vs.Q1}=2.39,95%CI:1.04～5.70,P_trend=0.113).Among indole pathway,indole-3-propionic acid(OR_{Q4 vs.Q1}=0.35,95%CI:0.14～0.83,P_trend=0.011)and indole-3-carboxylic acid(OR_{Q4 vs.Q1}=0.41,95%CI:0.18～0.91,P_trend=0.034)were negatively associated with FLD.There was no association of TPH and other tryptophan metabolites with FLD.After annotation and filtration of OTU,a total of 7 phyla,12 classes,16 orders,30families,74 genera and 130 species were found.Patients with FLD had a lower alpha diversity than non-FLD（ACE index:P=0.005,Chao1 index:P=0.006,Observed index:P=0.005）,and ANOSIM test（R=0.091,P=0.011）and PERMANOVA test（R²=0.008,P=0.012）showed a difference in beta diversity between participants with FLD and non-FLD.Among indole pathway,there was a strong association between indole-3-propionic acid（IPA）and intestinal bacteria.A total of 34 genera were associated with IPA obtained from multiple linear regression.After FDR correction,21 genera（FDR<0.05）were positively associated with IPA.Based on the identified 21 genera,the GMBS was constructed using WQS regression,the genus Odoribacter,Sporobacter and Akkermansia were the three strongest contributors for the GMBS,with weight of 0.57,0.14 and 0.11,accounting for 82%of the total weight.As the GMBS increasing from the lowest quartile to the highest quartile（the lowest quartile as reference）,the serum IPA increased by 131.53%（95%CI:55.38%～244.98%）,190.26%（95%CI:94.64%～332.87%）,and 249.38%（95%CI:132.9%～424.12%）,while the odds of FLD decreased by 9%（OR=0.91,95%CI:0.43～1.93）,46%（OR=0.54,95%CI:0.24～1.20）,and 84%（OR=0.16,95%CI:0.05～0.43）,respectively.Among the association between IPA-related intestinal bacteria and FLD,six genera were found to be associated with FLD.Barnesiella(OR_{T3 vs.T1}=0.35,95%CI:0.17～0.69),Odoribacter(OR_{T3 vs.T1}=0.30,95%CI:0.13～0.64),Oscillibacter(OR_{T3 vs.T1}=0.40,95%CI:0.18～0.84),Prevotella(OR_{T3 vs.T1}=0.45,95%CI:0.21～0.93),and Sporobacter(OR_{T3 vs.T1}=0.39,95%CI:0.18～0.80)were negatively associated with FLD.However,Escherichia were positively associated with FLD(OR_{T2 vs.T1}=2.41,95%CI:1.14～5.24;OR _{T3 vs.T1}=1.66,95%CI:0.78～3.60).ConclusionThe association between tryptophan metabolites and FLD depended on the metabolic pathway.There was a positive association of TDO enzyme activity and kynurenine with FLD,while indole-3-propionic acid and indole-3-carboxylic acid were shown to be negatively associated with FLD.The alpha diversity of gut microbiota decreased among FLD.GMBS was positively associated with serum IPA and negatively associated with FLD.This study suggests that tryptophan metabolites exhibit pathogenic and protective effect in the development of FLD.Furthermore,we may attenuate the risk of FLD through promotion of IPA by improving the composition of gut microbiota.The findings provide evidence for the development of treatment for FLD based on regulating tryptophan metabolism and gut microbiota.