The Mechanism Of SADI-S Improving Type 2 Diabetes Mellitus Based On Multi-omics Analysis

Background and objective:Type 2 diabetes mellitus(T2DM)is a chronic metabolic disease mainly characterized by abnormally elevated blood glucose,and its main pathological feature is insulin resistance.According to the statistics of International Diabetes Federation,the number of people with diabetes worldwide is increasing year by year.Long-term hyperglycaemia can cause chronic damage to organs such as the heart and eyes,leading to a series of serious complications.Therefore,how to effectively treat diabetes is of great significance to ensure the safety of people’s lives.The traditional treatment methods such as diet control and the use of hypoglycemic drugs can’t effectively solve diabetes and its complications because of limited curative effects.Moreover,the traditional treatment methods for diabetes often need to be carried out for a long time.Metabolic and bariatric surgery(MBS)is more effective for T2 DM than traditional treatment methods.However,the mechanism of MBS to relieve T2 DM is not fully understood.At present,most of the researches focus on sleeve gastrectomy(SG)and Roux-enY gastric bypass(RYGB),and there is a lack of researches on the mechanism of single-anastomosis duodenal-ileal bypass with sleeve gastrectomy(SADI-S)VIII improving T2 DM,especially these to explore the mechanism of SADI-S improving T2 DM from the system level.In addition,comprehensively considering the curative effect and safety,the advantages of SADI-S for T2 DM are far greater than those of SG,RYGB and biliopancreatic diversion with duodenal switch.That is to say,SADI-S is a MBS that spawns both efficacy and safety.Based on the above-mentioned points,it is of great significance to explore the potential mechanism of SADI-S improving T2 DM.On the one hand,the pathogenesis of T2 DM is very complex,so it is necessary to adopt a system level research method so as to fully understand the potential mechanism of SADI-S improving T2 DM,and omics technology just meets this requirement.On the other hand,the changes of gut microbita can cause changes in the corresponding metabolites,which are closely related to the occurrence of T2 DM.Gut microbita and its metabolites can mediate the gene and protein expression in liver through the "gut-liver axis".More importantly,as a central metabolic organ,the liver plays an important role in glucose metabolism,such as glycogen breakdown and synthesis,glucose transport,glycolysis and gluconeogenesis.Therefore,this study focuses on rat’s stool and liver,and then explores the potential mechanism of SADI-S improving T2 DM by multi-omics technologies such as gut microbiota,metabolomics,transcriptomics and proteomics.Methods:(1)After 2-week adaptive feeding and 8-week high-fat feeding,rats were intraperitoneally injected with low-dose streptozotocin to establish T2 DM rat model.Next,the T2 DM rats were randomly divided into the experimental group and control group.The rats in experimental group and control group were intervened by SADI-S(SADI-S group,n=10)and sham operation(Sham-PF group,n=9),respectively.The diet plan of rats in the control group was consistent with the SADI-S group to eliminate the possible impact of dietary difference.The body weight,fasting blood glucose(FBG),glycosylated hemoglobin(HBA1c)and area under curve of blood glucose during glucose tolerance test(AUC)were measured to evaluate the weight loss and hypoglycemic effects of SADI-S on T2 DM rats.In addition,histological assessments of rats’ islets were performed using immunofluorescence so as to observe the effects of SADI-S on islet α-cells and β-cells in T2 DM rats.(2)Eight weeks after operation,rats’ stools were collected for 16 S r DNA high-throughput sequencing and non-targeted metabolomics testing to identify the differential bacteria and differential metabolites.Spearman correlation analysis was carried out between differential bacteria and FBG,HBA1 c and AUC to identify the key bacteria that SADI-S affected glucose metabolism.Based on the Kyoto Encyclopedia of Genes and Genomes(KEGG)database,differential metabolites were enriched for pathway analysis to find potential metabolic pathways that SADI-S affected glucose metabolism.Moreover,Spearman correlation analysis was performed between differential metabolites involved in metabolic pathways and glucose metabolism indicators to find the key metabolites that SADI-S affected glucose metabolism.X(3)Eight weeks after operation,the rats’ liver tissues were collected for transcriptomics and proteomics testing to identify differentially expressed genes and proteins in the liver,and these differentially expressed genes/proteins were annotated using the KEGG database so as to find the key genes and signaling pathways that SADI-S improved glucose metabolism in T2 DM rats.A conjoint analysis of transcriptome and proteome were performed to further screen out genes/proteins showing the same trend at m RNA and protein levels.According to the changes of genes at the m RNA level,the corresponding proteins were selected for parallel reaction monitoring(PRM)to verify the accuracy of proteomics results.Results:(1)Six rats and eight rats survived to the end of the experiment in the SADI-S group and Sham-PF group,respectively.There was no statistical difference between the SADI-S group and Sham-PF group in terms of preoperative body weight,FBG and AUC(P>0.05).At each time point after operation,the body weight and FBG of SADI-S group were significantly lower than those of Sham-PF group(P<0.05).At 8 weeks after operation,the HBA1 c and AUC of SADI-S group were also significantly lower than those of Sham-PF group(P<0.05).At 8 weeks after operation,the rats’ pancreatic islet α-cells andβ-cells in the Sham-PF group were distributed disorderly and the pancreatic isletβ-cells were injured severely with marked vacuolation degeneration.In contrast,the SADI-S group showed complete pancreatic islets and compact and uniform morphology of pancreatic islet β-cells without significant vacuolar degeneration.(2)A total of 690,893 OTUs were obtained from 14 stool samples and identified as 14 phyla.Bacteroidota was the most abundant phyla.At 8 weeks after operation,the Ace,Chao and Shannon indexes in the SADI-S group were significantly lower than those of the Sham-PF group,and the Simpson index was significantly larger than that of the Sham-PF group(P<0.05).At the phylum level,SADI-S decreased the relative abundance of Bacteroidetes and increased the relative abundance of Proteobacteria,Verrucomicrobiota and Actinomycetota(P<0.05).At the genera level,SADI-S significantly changed the abundances of33 bacteria,such as the increase of anti-inflammatory bacteria(Akkermansia and Bifidobacterium),the decrease of pro-inflammatory bacteria(Bacteroides).The increased Bifidobacterium also belonged to short-chain fatty acid(SCFA)-producing bacteria.Among the 33 differential bacteria,the three indicators of FBG,HBA1 c,and AUC positively correlated with abundances of Anaerofilum,Quinella,Anaerovibrio,Anaerotruncus,Oscillibacter,Intestinimonas,Lactobacillus,Bacteroides,Peptococcus,Gelria,Ruminococcus and Parabacteroides,and negatively correlated with abundances of Streptococcus,Parasutterella,Bifidobacterium,Veillonella,Eubacterium,Escherichia and Enterococcus.(3)Compared with the Sham-PF group,81 differential metabolites(43 upregulation and 38 down-regulation)were identified under the positive ion model,and 164 differential metabolites(138 up-regulation and 26 down-regulation)were identified under the negative ion model in the SADI-S group,of which 8XII metabolites were detectable in both models.Therefore,a total of 237 differential metabolites were identified between the SADI-S group and Sham-PF group.According to the KEGG enrichment analysis,a total of 30 metabolic pathways(impact > 0)were obtained which mainly involved in unsaturated fatty acid metabolism,riboflavin metabolism,amino acid metabolism,fatty acid synthesis,bile acid synthesis,steroid hormone biosynthesis and tricarboxylic acid cycle.Among them,the linoleic acid metabolic pathway has the highest impact value.A total of 52 metabolites were involved in the above-mentioned 30 metabolic pathways,of which 10 metabolites such as taurochenodesoxycholic acid and 27 metabolites such as linoleic acid were positively and negatively correlated with the three indicators of FBG,HBA1 c,and AUC,respectively.(4)Compared with the Sham-PF group,235 up-regulated genes and 380down-regulated genes were identified in the SADI-S group,respectively.The up-regulated genes were mainly enriched in signaling pathways such as PPAR signaling pathway,bile secretion,biosynthesis of unsaturated fatty acids,ABC transporter,retinol metabolism,AMPK signaling pathway,fatty acid degradation and pyruvate metabolism,of which the PPAR signaling pathway was most significant and enriched the largest number of differential genes.The downregulated genes were mainly enriched in signaling pathways such as steroid hormone biosynthesis,chemical carcinogenesis,axon guidance,amino acid metabolism,linoleic acid metabolism,retinol metabolism and bile secretion,of which the steroid hormone biosynthesis was most significant and enriched the largest number of differential genes.(5)Compared with the Sham-PF group,198 up-regulated proteins and 114down-regulated proteins were identified in the SADI-S group,respectively.The up-regulated proteins were mainly enriched in ribosome biogenesis in eukaryotes,glutathione metabolism,insulin signaling pathway,aminoacylt RNA biosynthesis,chemical carcinogenesis-DNA adducts,metabolism of xenobiotics by cytochrome P450,cysteine and methionine metabolism,and pyruvate metabolism.The down-regulated proteins were mainly enriched in chemical carcinogenesis-reactive oxygen species,steroid hormone biosynthesis,chemical carcinogenesis-DNA adducts,Alanine,aspartate and glutamate metabolism,linoleic acid metabolism,drug metabolism-cytochrome P450,PPAR signaling pathway,arginine biosynthesis,arachidonic acid metabolism,and retinol metabolism.(6)A conjoint analysis between the differential genes screened in transcriptome and the differential proteins screened in proteome revealed that a total of 120 genes/proteins including the rate-limiting enzyme of gluconeogenesis(Pck1)and the rate-limiting enzyme of glycolysis(Pklr)were screened with the consistent expression trends,of which 64 genes/proteins showed upregulation and 56 genes/proteins showed down-regulation at both m RNA and protein level.Among the signaling pathways enriched in up-regulated genes/proteins,the pathways involved in glucose metabolism included pyruvate metabolism,insulin signaling pathway,glycolysis/gluconeogenesis pathway,XIV glucagon signaling pathway,and AMPK signaling pathway.In addition,the upregulated genes/proteins were also enriched in the PPAR signaling pathway related to the inflammatory response and glutathione metabolic pathway related to the antioxidant effect.Down-regulated genes/proteins were mainly enriched in amino acid catabolic pathways.In addition,the down-regulated genes/proteins were also enriched in glutathione metabolic pathway related to antioxidant effect,PPAR signaling pathway related to inflammatory response and pyruvate metabolic pathway involved in glucose metabolism.The trends of the screened 16 proteins in PRM were completely consistent with those of proteomics,indicating that the results of proteomics were reliable.Conclusions:(1)SADI-S had a significant weight loss and hypoglycemic effects on T2 DM rats.(2)SADI-S significantly altered the structure composition of gut microbiota in T2 DM rats,such as the increase of anti-inflammatory bacteria(Akkermansia and Bifidobacterium)and the decrease of pro-inflammatory bacteria(Bacteroides).The postoperative blood glucose level of rats was positively correlated with the relative abundances of 12 bacteria including Bacteroides,and negatively correlated with the relative abundances of 7 bacteria including Bifidobacterium.SADI-S significantly changed the composition of metabolites in T2 DM rats,such as branched-chain amino acids,bile acids and SCFA.The postoperative blood glucose level of rats was positively correlated with 10 metabolites including taurochenodeoxycholic acid and negatively correlated with 27 metabolites including linoleic acid.The above-mentioned changes in gut microbita and metabolites may play a role in SADI-S improving T2 DM in rats.(3)SADI-S significantly altered the expression of liver genes/proteins in T2 DM rats,such as the rate-limiting enzyme of gluconeogenesis(Pck1)and the rate-limiting enzyme of glycolysis(Pklr).Among the signaling pathways enriched by differential genes/proteins caused by SADI-S,the pyruvate metabolism pathway,insulin signaling pathway,glycolysis/gluconeogenesis pathway,glucagon signaling pathway and AMPK signaling pathway were related to glucose metabolism.This may be the mechanism by which SADI-S improved T2 DM.