Metagenomic Study On The Relevance Of Gut Microbiota With The Susceptibility To High-fat Diet-induced Obesity In Mice

Obesity is a kind of nutrition disorder that people consume more energy than they expend for a long time,showing an excessive body fat accumulation,body weight exceeds a certian range.Unlike simple obesity,obesity would become incentives to cause a variety of metabolic diseases,such as diabetes,cardiovascular disease,chronic kidney disease,etc.Obesity was declared a global health epidemic in 2003 by the World Health Organization and its prevalence has been projected to grow by 40% in the next decade,which has become one of the major diseases that endanger human health.In modern time,irregular diet and irrational diet structure,excessive calories ingestion are main factors which lead to obesity.Long-term high-calorie diet(high fat,high protein,high sugar)may rise obesity,causing human metabolic disorders,promoting all sorts of dangerous complications.In real life,it was found that: some people never get fat,while some other people who don't eat more but get fat easier than former.It is clear that there are differences in individual fat metabolism and genetics in obesity.Besides study of human group induced by high-fat diet,mice are often used as the common laboratory animals in scientific research.In the early 1980 s,Levin et al.gave high-fat diet to the same batch of Sprague-Dawley rats and found that some rats became obese,known as diet-induced obesity(DIO),and the other part of the rats did not develop obesity,called diet-induced obesity resistance(DIOR).We can see that there are differences in susceptibility to high-fat diet-induced obesity in different individuals.Recent studies have shown that high-fat diet would affect the composition and function of human intestinal flora,and intestinal flora is called the body's "second genome",which can regulate a variety of metabolic pathways to maintain the balance of intestinal microecology.Among which,lipid metabolism,carbohydrate metabolism,etc.They have a close relationship with the occurrence of obesity,but the relationship between intestinal flora and obesity susceptibility is far less clearly understood.Benefiting from the development of metagenomics,we have a further understanding of the structure and function of the intestinal flora by using some bioinformatics analysis tools,which can help us understand the relationship between intestinal flora and the differences of individual susceptibility in obesity induced by high-fat diet,and the role of intestinal flora in obesity.In this study,we used C57BL/6J mice induced by high-fat diet to establish obesity and obesity resistance models.The differences in the constituent and function of intestinal flora between the obesity group and the obesity resistance group were compared by metagenomic analysis,which can help us to investigate the relationship between intestinal flora and obesity.The main contents and results are as following:1.Establishment and determination of high-fat diet mouse model: 30 C57BL/6J mice,10 as Control,fed normal diet;20 as model group,fed high-fat diet.After the end of modeling period,mice which growed over twice average rate of growth of the Control group mice were called "high-fat diet-induced obesity mice"(HFDIO group,8 mice),the other which did not meet the obesity standard were called "high-fat diet-induced obesity-resistant mice"(HFDIOR group,12 mice).2.Collection of mouse feces and genomic DNA extraction,metagenomic sequencing: using the abdominal massage method to collect the fresh mouse feces after the end of the model building;DNA extraction in accordance with the QIAamp? Fast DNA Stool Mini kit handbook(purchased from Germany QIAGEN company),detecting genomic concentration,purity,genomic integrity was deteced by agarose gel electrophoresis;metagenomic sequencing commissioned by BGI's Shenzhen Technology Service Co.,Ltd.A total of 18 samples(Control 6,HFDIO 7,HFDIOR 5)were selected for the construction of genomic DNA libraries and metagenomic sequencing,and data quality control,filtration,assembly and gene catalog construction.3.High-fat diet-induced mouse intestinal flora species annotation and species abundance analysis: comparing the collected gene catalog with the NCBI non-redundant protein database nr.The results showed that comparing with Control group,HFDIO group and HFDIOR group had a significant decrease in Bacteroidetes;yet,the contents of Firmicutes,Fusobacteria and Proteobacteria were increased significantly.HFDIO group and HFDIOR group mice' Proteobacteria,Spirochaetes and Verrucomicrobia had some differences,there were significant differences between HFDIO group and HFDIOR group in Firmicutes,Fusobacteria and Proteobacteria(P<0.05).4.Metagenomic diversity analysis: diversity analysis was divided into ? diversity analysis and ? diversity analysis.The results of ? diversity analysis showed that the diversity of intestinal flora of the Control group was much higher than that of the high-fat group,and the difference between HFDIO group and HFDIOR group was not obvious.The results of ? analysis showed that there were significant differences in Control group,HFDIO group and HFDIOR group in terms of species composition,gene function and metabolic pathway.In the species composition,high-fat diet-induced two groups of mice which tended to be similar,and had some overlaps,but these two groups had a greater gap with the Control group,cluster analysis suggested that there was gender differences;and in the gene function and metabolic pathways,three groups of mice were significantly different,HFDIOR group was closer to the Control group,suggesting that the key function of intestinal flora in HFDIOR group mice was not significantly damaged,and HFDIO group mice may have been in a state of metabolic disorders.5.Metagenomic key biomarkers screening: through the Linear discriminant Effect size(LEfSe)analysis,we found that the key species of the Control group were the Bacteroides and the Prevotella,belonging to the Bacteroides;the key species of the HFDIO group was only Lachnospiraceae bacterium28-4,which belongs to Firmicutes;the most key species of the HFDIOR group was Lachnospiraceae bacterium10-1,belonging to Firmicutes.6.GO function significant difference analysis: comparing annotated genes by Gene ontology(Gene Ontology,GO)analysis,we found that there were significant differences between HFDIO group and HFDIOR group in cellular component,molecular function and biological process.a total of 23 were detected with statistically significant difference(P<0.05),and significant differences included oxidoreductase activity,transferase activity and nucleic acid binding.7.KEGG Pathway difference analysis: comparing annotated genes through the KEGG(Kyoto Encyclopedia of Genes and Genomes)Pathway Database,we found that HFDIO and HFDIOR mice had great differences,a total of 17 were detected with statistically significant difference(P<0.05),the key differences were focused on carbohydrate metabolism.8.High-fat diet-induced mouse intestinal virus species annotation and species abundance analysis: comparing the collected gene catalog with the NCBI non-redundant protein database nr and virus.The results showed that comparing with the Control group,HFDIO group and HFDIOR group both had a decrease in Myoviridae and Microviridae,and the content of Podoviridae was decreased significantly in HFDIOR group.HFDIO group and HFDIOR group mice' characteristics of intestinal virus were not significantly different(P>0.05).9.Related network analysis between virus and intestinal bacteria: 18 types of viruses and 46 bacteria species produced 183 related network lines,of which 82 were negative related,and the remaining 101 were positive related.A large number of crAssphages were found in the mouse intestine which infects bacteroides mainly for the first time.In conclusion,by comparing the community structure,key species,gene function and metabolic pathways of the intestinal flora in obese mice and obesity-resistant mice induced by high-fat diet through metagenomic analysis,we found that there was a difference in individual susceptibility to obesity certainly between high-fat diet-induced obesity mice and high-fat diet-induced obesity-resistant mice,suggesting that the genetic function of key flora was important for the occurrence or resistance of obesity,and further study of these key genes and crAssphage helps to assess the relationship between intestinal flora and obesity correctly,and provide a theoretical basis for the mechanism of obesity and control strategies.