Effects And Mechanisms Of Metformin Combined With Pioglitazone On Glucose And Lipid Metabolism In Obese Mic

Objectives:The prevalence of diabetes has been increasing rapidly and China has the highest number of diabetes patients in the world.More than 90%of patients are type 2 diabetes mellitus(T2DM),posing a serious health threat to the patients and bringing heavy socioeconomic burden.However,the current proportions of diabetes treatment and control are both below 50%,and glycemic control with combination therapy becomes essential for most diabetes patients.The pathogenesis of T2DM is complex,and insulin resistance(IR)is the most important core pathological mechanism.Both pioglitazone and metformin have been proven to improve IR,and clinical research showed that the combination of the two medications can improve glycemic and lipid metabolism and IR.Previous studies have confirmed that T2DM and obesity are closely related to dysbiosis of gut microbiota,alteration of intestinal metabolites,non-alcoholic fatty liver disease(NAFLD),and abnormal cardiac metabolic profiling.However,the combined effects of pioglitazone and metformin on these underlying mechanisms are still unclear.Therefore,this study aims to assess the protective effects of metformin and pioglitazone combination therapy in highfat diet(HFD)-fed mice.Based on multi-omics detection and analysis techniques,we explored the potential mechanisms from various aspects such as gut microbiota,liver lipid metabolism,and cardiac metabolic profiling,to comprehensively understand the effectiveness of the combination of the two drugs and provide a more comprehensive theoretical basis for clinical practice.Materials and Methods:Part 1:Male C57BL/6 mice aged 7 weeks adapted for one week and then they were randomly divided them into 5 groups:the control group,fed a normal control diet;the HFD group,fed an HFD;metformin monotherapy group,orally administered with metformin(150 mg/kg/day body weight);pioglitazone monotherapy group,orally administered with pioglitazone(5mg/kg/day body weight);and combination therapy group,orally administered with metformin and pioglitazone.All intervention groups were given HFD.The experimental period of these mice was 18 weeks,with only dietary intervention for the first 10 weeks and drug intervention with HFD was given for the next 8 weeks.Body weight and food intake were monitored during the experimental period.Glucose and insulin tolerance tests at the end of the experiment were conducted and all mice were sacrificed.Adipose tissue and liver weight were recorded.Serum samples were collected for the biochemical tests for insulin,blood lipids,and transaminases.Part 2:Based on the mice models in the part 1,cecal contents of the mice were collected for 16S rDNA amplicon sequencing and analysis.Untargeted metabolomics of the cecal contents was performed to access the alterations of gut microbiota and intestinal metabolites.At the same time,the phenotype data of glucose and lipid metabolism were jointly analyzed with the differential microbiota and differential metabolites.Part 3:Based on the mice models in the part 1,liver tissues were obtained.Hematoxylin-eosin(HE)staining and oil red O staining were performed to examine the histological changes.Quantitative lipidomics analysis of liver tissue was carried out to explore the changes of liver lipid profile.The hepatic gene expressions were detected by RNA-seq,with a special focus on the changes in lipid metabolism-related genes and metabolic pathways.The vital genes will be validated by real-time fluorescence quantitative polymerase chain reaction(RT-qPCR).Part 4:C57BL/6 male mice were randomly divided into normal control group,HFD group,and combined intervention of metformin and pioglitazone group.The present batch of mice used in this study is the second batch of mice.The experimental period lasted for 28 weeks,with 16 weeks of dietary intervention followed by 12 weeks of drug intervention to verify the effect of metformin combined with pioglitazone on glucose and lipid metabolism and NAFLD phenotype in the first batch of mice.At the same time,shortchain fatty acids(SCFAs)in colon were detected.Cardiac tissue HE staining,various free fatty acids(FFA)molecular levels and targeted metabolomics was performed and analyzed.Results:Part 1:Mice fed with HFD showed significant weight gain and IR phenotype.Metformin or pioglitazone monotherapy have partial metabolic improvement effects.The improvement effects in the combination therapy group were strong than monotherapy groups,significantly reducing of body weight,white adipose tissue weight and liver weight,improving glucose tolerance and insulin tolerance,as well as decreasing of insulin,homeostasis model assessment of insulin resistance,total cholesterol,low-density lipoprotein cholesterol,and alanine aminotransferase.Part 2:The 16s rDNA sequencing showed that metformin combined with pioglitazone could significantly improve the abundance of various beneficial bacterial genera in HFD mice,including Bifidobacterium,Roseburia,Christensenellaceae_R-7_group,and Faecalibacterium.Meanwhile,the abundance of several potentially pathogenic bacterial genera increased in the HFD group but significantly decreased in the combination therapy group,such as Oscillibacter,Eubacterium_xylanophilum_group,and Muribaculaceae.Untargeted metabolomics analysis of cecal contents revealed that multiple amino acid and protein-related metabolic pathways were down-regulated in the combination therapy group.Several lipid metabolic pathways were up-regulated,and metabolites including leukotriene A4,linolenic acid A and some glycerophospholipid metabolites were up-regulated in the combination therapy group.Correlation analysis found that the improvement of glucose and lipid metabolism phenotype was significantly correlated with differential intestinal microbiota and differential metabolites.Part 3:Hepatic HE staining showed that metformin or pioglitazone monotherapy partly reduced liver lipid droplets,but the combined therapy group had the most significant reduced effect on liver lipid droplets and Oil Red O staining area.A total of 664 lipid metabolites were detected by quantitative lipidomics in liver,including triglycerides,diacylglycerols,FFA,and various other metabolites in glycerophospholipid and sphingolipid classes.The lipidomics analyzed results showed that the combination effects of metformin and pioglitazone significantly improved the liver lipid profiles,including significant decreases in triglycerides,diacylglycerols and FFA levels,and increases in the levels of some certain glycerophospholipids and sphingolipid metabolites.RNA-seq analysis showed that the gene expression in the liver of the combination group was closest to that of the normal control group.The key genes related to FFA uptake Cd36 and de novo lipogenesis were significantly upregulated in the HFD group,but were significantly reversed in the combination group,including Elvol5,Fads1,Fads2,Fasn,Pklr,and Scd1.RT-qPCR verification of some genes showed the same trend.In addition,genes related to methylation,including Mat1a,Got1,and Ahcy,involved in the metabolism pathway of cysteine and methionine were significantly upregulated in the combination treatment group.Part 4:In this part,we used mice models with a longer intervention period of HFD.We first validated the improvement of the glucose and lipid metabolism,insulin resistance,and hepatic steatosis phenotypes.Furthermore,significant increases of SCFAs such as propionic acid,butyric acid,and valeric acid were observed in the combination treatment group,which indirectly confirmed the positive effect of the combination treatment on the gut microbiota.The HE staining of cardiac tissue showed no significant abnormality in the arrangement of myocardial cells,while edema of myocardial cells were observed in the HFD group,which was slightly attenuated in the combined treatment group.FFA detection of cardiac tissue revealed significant alterations of various FFA molecules in the HFD group,including increased levels of all-cis-5,8,11,14-Eicosatetraenoic acid,and decreased levels of several beneficial fatty acids such as cis,cis,cis-9,12,15-Linolenic acid,cis,cis9,12-Linoleic acid,and cis,cis,cis-6,9,12-Linolenic acid.The combination of metformin and pioglitazone reduced the level of all-cis-5,8,11,14-Eicosatetraenoic acid,a molecule associated with inflammation.However,the levels of beneficial fatty acids such as cis,cis,cis-9,12,15-Linolenic acid,cis,cis-9,12-Linoleic acid,and cis,cis,cis-6,9,12Linolenic acid were not significantly improved in the combined treatment group.Targeted metabolomics analysis of cardiac tissue identified nearly 300 functional small molecules,among which metabolites such as p-cresol,glucose-1-phosphate,and thymidine showed an increasing trend in the HFD group,while metabolites such as bilirubin and betaine showed a decrease trend in the HFD group.The combined treatment significantly reversed these changes.However,still plenty of metabolites were not reversed in the combined treatment group.Conclusions:This thesis explored for the first time the molecular changes and potential mechanisms of the benefits of metformin combined with pioglitazone on glucose and lipid metabolism in obese mice.Based on multi-omics detection and analysis techniques from multiple perspectives,we comprehensively understand the effect of the combination of the two drugs and provide more theoretical basis for clinical medication.The main conclusions of the thesis are as follows:1.Combined therapy effect of metformin and pioglitazone had synergistic effects or at least have an additive effect on improvement of the glucose and lipid metabolism phenotype in HFD-induced obesity and IR in mice.2.Combination therapy markedly altered gut microbiota by increasing beneficial bacteria and reducing the abundance of potentially pathogenic bacteria,along with the increased level of beneficial lipid metabolites such as Lipoxin A4,Lucidenic acid A,propionic acid and butyric acid.Improvement of gut microbiota and intestinal metabolites might be involved in the mediation of metabolic benefits achieved by the combination therapy.3.Combined therapy of metformin pioglitazone could synergistically alleviate NAFLD in HFD mice,significantly improving the hepatic lipid profiles.And the decreased expressions of genes associated with liver FFA uptake and de novo synthesis might be the key to the relief of NAFLD in the combined therapy group.4.The results of cardiac FFA and targeted metabonomics analysis indicated that metformin and pioglitazone combined intervention might have a certain role in improving cardiac metabolic profiling.However,it was not the vital mechanism for the significant improvement of glucolipid metabolism of HFD-induced mice in the combined therapy group.