| Insulin resistance(IR)is an abnormal condition in which the body don’t properly respond to circulating Insulin.Reduced insulin sensitivity result in decreased glucose uptake and utilization and the long-term lead to metabolic syndrome and related diseases such as hypertension,type 2 diabetes,atherosclerosis,heart disease,stroke and chronic kidney failure.Obesity,especially the chronic inflammatory state caused by obesity,is one of most important risk factors for IR.The gut contains a broad immune system due to it was exposed to a variety of antigenic stimulus,including microbial antigens and antigens taken from the diet.Recent studies suggest that chronic intestinal inflammation and the changes of immune cells caused by obesity is closely related to insulin resistance.Dipeptidyl peptidase-4(DPP4)is a transmembrane glycoprotein dipeptidase with multiple functions,including activity of serine protease,maintenance of lymphocyte composition and functionality,synergistic stimulatory activity of T cell,and cell adhesion properties and effects.In addition to T cells,it is widely expressed on the surface of a variety of cells including epithelial cells,endothelial cells and other immune cells such as B lymphocytes,NK cells,dendritic cells and macrophages.Research background : A large number of studies have proved that DDP4 inhibition can promote metabolism,control blood glucose,lipids and blood pressure,and improve diabetes and hypertension.Some of its inhibitors are already in clinical use.However,most of these studies have focused on the enzymatic role of DPP4.In epigenetic studies,it was found that methylation levels of DPP4 gene affected its expression and was associated with abnormal lipid metabolism in patients with severe obesity,indicating that DPP4 had many other functions in addition to its enzymatic effects on obesity.Some previous research results of our group also showed that the expression of DPP4 in obese patients,especially the DPP4 expression level on the surface of immune cells such as dendritic cells(DC)is closely related to obesity and obesity-induced insulin resistance/atherosclerosis,but some mechanisms involved are not clear.Objective: This study focused on the DPP4 gene knockout on source of bone marrow cells of mice.To compare the DPP4 gene knockout mice and wild-type mice on obesity and obesity-induced insulin resistance.Then transplanted bone marrow cells from DPP4 knockout mice into wild-type C57 mice.Obesity and glucose tolerance were observed and compared.The most important part of this research topic is inducing mature bone marrow dendritic cells to co-culture with mesenteric lymph nodes in vitro.To investigate the mechanism by which that DPP4 expression on dendritic cells could affect the occurrence of high fat diet-induced obesity and insulin resistance in mice by controlling it to induce preferential expansion of intestinal T cells to regulatory T cells(Treg).Methods: Selected mice between 6 and 8weeks of age and divided these mice into DPP4 gene knockout(DPP4-/-)group and wild-type(WT)control group for further experimental study.Animal experiment: animal experiment is divided into two parts:(1)To clarify the influence of DPP4 deletion in obesity and obesity-induced insulin resistance,all the mice of DPP4-/-(10 Male)and WT group(10 Male)were given a high-fat diet for 14 weeks.Then,we tested and recorded their body weight,oral glucose tolerance test and glucose infusion rate,insulin sensitive index,basal glucose disposal and CLAMP glucose disposal.The effects of DPP4 knockout on blood glucose and insulin were compared in these ways.(2)WT mice were irradiated at a dose of 950 Rad,then they were randomly divided into 2 groups.DPP4 +/+ bone marrow cells and DPP4-/-bone marrow cells were respectively transplanted into 2 groups.After bone marrow reconstruction,fed a normal diet for 2 weeks.And then building models of obesity to fed high-fat diet for 14 weeks to test the same indicators in above(1)Cellular experiment: First,the interaction between DPP4 protein and CYPA was verified by CO-IP experiment in total protein of mesenteric lymph nodes and spleen of WT mice.By flow cytometry,splenic lymph node cells,axillary lymph node cells(ALN),inguinal lymph node cells(ILN),mesenteric lymph node cells(MLN),and lamina propria mononuclear cells(LPMC)were detected to express higher levels of DPP4 on CD103+DC that come from mesenteric lymph node cells.Therefore,the focus of our research was determined to explore the mechanism of DPP4 regulate the induction of Treg by DC cells through in vitro cell experiments.Based on the mesenteric lymph nodes gathered the number of CD103+DPP4+DC,mature BMDC form WT mice with high expression of CD103+DPP4+ were induced before the co-culture experiment at first,then co-culture experiments were conducted with MLN form WT mice.The proportions of Treg amplification,anti-inflammatory factor IL-10 secreted by Treg were compared between the co-culture group without any treatment,the co-culture group after DC incubation with CYPA recombinant protein in advance,and the co-culture group after continuous CYPA stimulation.Results:(1)DPP4 could regulate blood glucose and insulin resistance in diet-induced obese mice.Compared with wild-type mice that expressing DPP4 normally,DPP4 knockout mice had lower body weight,improved oral glucose tolerance and increased insulin sensitivity in glucose clamp test.(2)DPP4 knockout of bone marrow derived cells can improve insulin resistance induced by chronic high-fat diet.After DPP4 +/+ bone marrow cells and DPP4-/-bone marrow cells were transplanted into WT mice respectively,the oral glucose tolerance test of DPP4-/-receptor group was significantly improved compared with DPP4 +/-receptor group,although the body weight,fasting blood glucose,food intake and tissue weight had no significantly difference between the two groups.(3)DPP4 bind CYPA can affect CD103+DC cells to induce CD4+T cells to preferentially expand Treg cells in MLN.The results of flow cytometry showed the expression of DPP4 in intestinal and exoenteric CD103+DC cells of WT mice,as well as the proportion of Treg between DPP4 knockout mice and WT mice.More CD103+DPP4+DC were enriched in intestinal tract than in other sites,and there are a large number of Treg in intestinal lymph nodes of mice either or not DPP4 knockout.At the same time,the CO-IP experiment of DPP4 and CYPA precipitation by extracting total proteins from the mesenteric lymph nodes and spleen of WT mice showed that CYPA could be precipitated by using anti-DPP4 antibody,and vice versa.In the cell experiment,compared with the co-culture group without any treatment,the co-culture group of BMDC from WT mice and MLN from WT mice,the proportion of Treg in the co-culture group after DC incubation with CYPA recombinant protein in advance and the co-culture group continuously stimulated with CYPA are increased in our flow cytometry results.In addition,the same result was also found in the detection of IL-10 secretion in the co-culture supernatant.In other words,in the co-culture group of WT BMDC,the co-culture group with CYPA recombinant protein incubated DC in advance and the co-culture group with CYPA continuous stimulation had higher secretion than the non-treatment group.The result of RT-PCR showed the expression of Treg-related IL-10 in co-cultured cells are also consistent with that by flow cytometry.(4)DPP4 may affect the expression of TGF-β through binding to CYPA,thus affecting the amplification of intestinal Treg.Intestinal tissue RNA-seq results showed that intestinal inflammation was increased and pathway was down-regulated after DPP4 gene knockout.Total RNA extracted from co-culture cells was analyzed by RT-PCR.Compared with the co-culture group without any treatment the m RNA of TGF-βin co-culture group of WT BMDC the co-culture group after DC incubation with CYPA recombinant protein in advance and the co-culture group treated with CYPA continuous stimulation express increased in our result.There was no significant difference among the three groups of BMDC form DPP4-/-mice.Conclusion: The DPP4 T on the surface of intestinal DC cells(especially CD103 + DC cells)through combined with CYPA regulate the expression of TGF-β,which affect the DC to induce preferential amplification of intestinal regulatory T cells,promotes tolerance to the intestinal immune environment in the diet-induced obesity state,and maintains the intestinal flora in a state that promotes obesity,which ultimately leads to insulin resistance. |
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