Study Of The Alleviation Of Glucomannan On Insulin Resistance Through Gut Microbiota Via Omics Technology

The number of people with type 2 diabetes mellitus(T2DM)is expected to reach 700 million worldwide by 2045,posing a huge burden to people and society in terms of life and economy.Insulin resistance(IR)is one of the important pathological bases of T2 DM,and the incidence of T2 DM can be effectively reduced by alleviating IR in the prediabetic period.The occurrence of IR is often accompanied by the disruption of gut microbiota,and the use of exogenous substances to target and regulate gut microbiota is important for the treatment and prevention of IR.Natural bioactive polysaccharides have a wide range of regulatory effects on gut microbiota,and it is important to find polysaccharides with targeted regulation of gut microbiota and to investigate their mechanisms of action.In this paper,we identified the gut microbiota(Parabacteroides distasonis)associated with IR alleviation through a case-control study,and determined that Dendrobium offcinale polysaccharides(DOP)has a targeted modulatory effect on it,and further elucidated its mechanism of action in alleviating IR.The main contents and results are as follows:(1)Conducted a case-control study in Chinese adult T2 DM patients,focusing on the differences in the composition of the gut microbiota between patients and healthy subjects.The results showed that compared to healthy controls,the gut microbiota of T2 DM patients had significantly higher abundance of Prevotella,unclassified Enterobacteriae and Megasphaera,while Faecalibacterium,Bacteroides,Roseburia and was significantly lower.Correlation analysis indicated that Parabacteroides(especially P.distasonis)was significantly negatively correlated with the severity of IR,maybe a key species for improving IR.(2)Through in vivo and in vitro experiments,DOP could improve the proliferation of P.distasonis.Three polysaccharides with potential proliferative effects on P.distasonis were selected: DOP,β-glucan and inulin were evaluated in an in vitro fermentation experiment for their effects on the gut microbiota of T2 DM patients and healthy controls.It was shown that all three polysaccharides were efficiently degraded and utilized by gut microbiota and improved the production of beneficial fatty acids such as acetic,propionic,butyric,lactic and succinic acids.The results of sequencing showed that all three polysaccharides had significant effects on the structure of gut microbiota,and all of them could increase the abundance of Parabacteroides,and the best is DOP.Further experimental results showed that DOP mainly promoted the proliferation of P.distasonis,and this promotion effect was also confirmed in animal experiments.In addition,a strain of P.distasonis,named P.distasonis NSP007,was screened from human feces by in vitro culture and screening techniques.(3)The alleviating effects of DOP and P.distasonis NSP007 on IR were investigated in high-fat diet induced-IR mice.The results showed that DOP and P.distasonis NSP007 significantly improved weight gain,fat accumulation,lipid metabolism disorders,insulin sensitivity,degree of glucose tolerance,liver injury,chronic inflammation,and intestinal permeability in IR mice.In addition,DOP altered the structure of gut microbiota and significantly increased the abundance of P.distasonis in the gut of IR mice.Further results from pseudo-germ-free mice showed that DOP was dependent on the gut microbiota to exert its anti-insulin resistance effect;P.distasonis NSP007 exerted its anti-insulin resistance effect independently of others gut microbiota.(4)The characteristic metabolites of P.distasonis NSP007 were investigated based on metabolomics technology and identified the metabolite with the effect of improving IR in mice.Firstly,P.distasonis NSP007 was found to be significantly enriched in nicotinic acid(NA)and nicotinamide(NAM)metabolic pathways during in vitro culture by untargeted metabolomics techniques.Further,the ability of P.distasonis NSP007 to produce NA and NAM was determined by targeted metabolomics techniques.In addition,the results of animal experiments showed that both P.distasonis NSP007 and DOP could effectively increase the level of NA in the feces of mice,not NAM.The results of cellular experiments showed that NA could significantly upregulate the expression of intestinal barrier-related genes in Caco-2cells(human colorectal adenocarcinoma cells)and was dependent on the activation of G protein-coupled receptor 109a(GPR109a,NA receptor).In addition,results from animal experiments showed that NA improved weight gain,insulin sensitivity,degree of glucose tolerance,lipid metabolism disorders,chronic inflammation,liver damage and intestinal barrier function in IR mice and was dependent on the activation of GPR109 a.(5)The negative correlation between P.distasonis and NA and IR severity was again determined by another independent validation cohort.Our results showed that the levels of P.distasonis and NA in the stools of T2 DM patients were significantly lower than those of healthy controls.Correlation analysis showed that P.distasonis and NA were significantly and negatively correlated with IR severity.In addition,a re-analysis of data in the Human Microbiome Project(HMP)database revealed that the abundance of Parabacteroides spp.in the feces of pre-diabetic patients was negatively correlated with body mass index(BMI),venous plasma fasting glucose(FPG)and steady-state glucose(SSPG),which further supported our findings.In summary,this paper found a gut bacterium,P.distasonis,was negatively correlated with the severity of IR through cohort studies;DOP could significantly promote the proliferation of P.distasonis via in vitro and in vivo studies,while a strain of P.distasonis named NSP007 was selected from human feces;further animal experiments revealed that both DOP and P.distasonis NSP007 could effectively improve IR in mice;finally,through multi-omics technology,it was found that P.distasonis NSP007 could produce NA,and confirmed that NA regulates intestinal permeability by activating GPR109 a in the colon of mice,and then plays a role in improving IR.Mechanistically,this study demonstrates that DOP can improve IR through the P.distasonis-NA-GPR109 a axis.The above research results provide theoretical support for the clinical translation of DOP and provide new ideas and evidence for the study of the polysaccharide-gut microbiota-host relationship.