Characterization Of Gut Microbiota In Adults With Type 1 Diabetes And Its Relationship With Serum Metabolites

BackgroundT1D is a polygenic genetic disease,with approximately 70%of T1D cases carrying a high allele causing genetic susceptibility and only about 10%of individuals at genetic risk eventually developing T1D.Recent research evidence attributes the increasing incidence of T1D to a combination of genetic and environmental factors.One of the most common environmental factors is altered gut microbiota,and imbalances in intestinal flora can cause the development of T1D by disrupting the immune system and intestinal barrier.The close relationship between gut microbiota and T1D has been recognized,but it is mostly focused on children with T1D,and there are few studies based on adult population.In addition,a comprehensive analysis of gut microbiota changes and metabolic dysregulation in adults with T1D is lacking.ObjectiveCharacterization of the structure and composition of the adult T1D gut microbiota using macrogenomics techniques.Screening for differences in serum metabolites between T1D and healthy populations using untargeted metabolomics techniques.The correlation between T1D gut microbiota and serum metabolites was analysed by combined multi-omics to identify potential disease markers and possible therapeutic targets,providing an experimental basis for the prevention,diagnosis and treatment of T1D.MethodsData were collected from 123 patients who were hospitalized at the Department of Endocrinology,Qilu Hospital of Shandong University from October 2020 to October 2022 and who were eligible for the diagnosis of T1D,and their serum and stool specimens were collected.Seventeen patients were randomly screened for inclusion in the T1D group,while an age-and sex-matched concurrent health screening population was included as a healthy control group.A multi-omics study was then performed.Macrogenomic testing of faecal specimens from two cohorts was used to characterise the structure of the gut microbiota of patients with T1D.Nontargeted metabolomic assays were performed on serum specimens from the two populations to analyze differential serum metabolites between the two groups.Bioinformatics methods were applied to the joint analysis of differential gut microbiota and differential metabolites to obtain correlations between changes in differential gut microbiota and differential metabolites between T1D patients and healthy populations.Results1.The structural characteristics of the T1D intestinal flora were characterized by macrogenomics,and the species composition of the T1D group and the healthy control group at the phylum level did not differ significantly,and the intestinal flora of both groups belonged to four major phyla,namely,Bacteroidetes,Firmicutes,Proteobacteria and Antinobacteria,with the largest proportion of Bacteroidetes and Firmicutes belonging to these groups.Species that differed significantly at the species level between the T1D group and the healthy controls included s_Prevotella_copri,s_Lachnospira_pectinoschiza,s_Klebsiella_pneumoniae,s_Megamonas_funiformis,s_Prevotella_marseillensis,s_Prevotella_sp.₁09,s_Prevotella_sp._CA G:255,s_Prevotella_sp._AM42-24,s_Prevotella_sp.₈85,s_Succinatimonas_sp._CAG:777,s_Dialister_sp.₅BBH33,s_Eubacterium_ventriosum,s_Dialister_sp._CAG:357,s_Dialister_sp._Marseille-P5638,s_Prevotella_stercorea_CA G:629,and s_Fusobacterium_ulcerans;Further screening of strains that could be used as biomarkers for the identification of T1D intestinal flora by LEFSe analysis revealed that s_Escherichia_coli and s Klebsiella_pmeumoniae.were enriched in the T1D group,while s_Prevotella_sp_CAG₇32,s_Lachnospira_pectinoschiza,s_Prevotella_sp_AM42₂4,s_Megamonas_funiformis,s_Prevotella_sp_AM42₂4,s_Megamonas_funiformis,s_Prevotella_sp_AM42₂4,s_Prevotella_sp_CAG₂55,Prevotella marseillensis,s_Prevotella_sp₁09,s_Prevotella_copri were enriched in the normal control group.The KEGG pathway function prediction was performed on the differential genes obtained from the macrogenome,and the enriched pathways were mainly focused on glycine,serine and threonine metabolism pathway,microbial metabolism pathway,biosynthesis of secondary metabolites pathway,biosynthesis of amino acid pathway,biosynthesis of cofactor pathway,two-component system pathway,cysteine and methionine metabolism pathway.2.A total of 1161 differential metabolite ions were identified by the non-targeted metabolome for T1D serum metabolite changes,of which 990 were up-regulated and 171 were down-regulated compared to healthy controls.A total of 72 known metabolites were identified by comparing the differential metabolic ions with the HMDB database.Compared to healthy controls,17 metabolites were up-regulated and 55 metabolites were down-regulated.Statistical analysis of the differential metabolites identified 30 metabolite changes that were statistically significant,including（R）-3-Hydroxybutyric acid,Threonic acid,D-Lactic acid,2,4-Octadiene,2,6-Dimethoxy-4-propylphenol,3-Hydroxydodecanoic acid,2-Ethyl-1,3,3-trimethyl-2norbornanol,3-Hydroxymyristic acid,1,2-Dimethyl-4-（6-methyl-4-heptenyl）-1,3cyclohexadiene,3,5-di-tert-butyl-2-hydroxybenzaldehyde,Oleic acid,Stearic acid,3Oxooctadecanoic acid,2-Hydroxydesipramine,2-Methyl-5-（8,11-hydroxybenzaldehyde）（8,11-pentadecadienyl）-1,3-benzenediol,2-Methyl-5-（8-pentadecenyl）-1,3-benzenediol,LysoPA 16:1,LysoPA 16:0,LysoPA 18:3,LysoPA 18:2,LysoPA 20:5,LysoPA 22:6,2（3H）Benzothiazolethione,Acylcarnitine 22:0,Acylcarnitine 24:0 were significantly upregulated in the T1D group was significantly up-regulated and 12,13-Dihydroxy-9Z-octadecenoic acid,Glycoursodeoxycholic acid,Chenodeoxyglycocholic acid,LysoPE 18:3 and LysoPC 12:0 were significantly down-regulated in the T1D group.3.Correlation analysis between the differential flora obtained from the macrogenome and the differential metabolites obtained from the metabolome revealed a broad association between the two.In particular,the significantly reduced metabolite Chenodeoxyglycocholic acid in the serum of patients in the T1D group was positively correlated with the significantly reduced s_Selenomonas_sp._FC4001 strain in the gut and the significantly reduced metabolite Glycoursodeoxycholic acid was positively correlated with the s_Enterococcus_phage_IMEEFm5 strain that was significantly elevated in the gut was negatively correlate,the significantly elevated metabolite Threonic acid was positively correlated with s_Enterobacter_cloacae_complex_sp._CH23B strain that was significantly elevated in the gut,the significantly elevated metabolite D-Lactic acid was positively correlated with significantly elevated s_Serratia_sp._BIGb0156 and s_Blautia_sp._AF13-16 strains in the gut,and the significantly decreased metabolite 2,4-Octadiene was negatively correlated with significantly decreased s_Prevotella_sp.₁09 strains in the gut.ConclusionsIn this study,structural characteristics of gut microbiota species in adult T1D patients were found to be different compared with healthy population using macrogenomics techniques,while differential serum metabolites between adult T1D patients and healthy population were screened using non-targeted metabolomics techniques,and further combined multi-omics analysis revealed significant correlations between differential gut microbiota species and differential serum metabolites in adult T1D patients and healthy population,and the screened differential gut microbiota and related metabolites could be used as potential disease markers for future research on the pathophysiological mechanisms of T1D.