Mechanism Of Glutamate Sensing By MTorc1 Signaling Network In Promoting Intestinal Development Of Piglets

Mechanistic target of rapamycin complex 1(mTORC1)is a highly evolutionarily conserved serine/threonine kinase that regulates cell growth and metabolism in response to multiple environmental cues,such as nutritional factors,hormones,and energy.Glutamate(Glu)is a major metabolic fuel for the intestinal epithelium.Furthermore,as a functional amino acid,Glu extensively participates in a variety of metabolic and physiological processes.Undoubtedly,normal intestinal epithelial renewal is the basis for the maintenance of intestinal function and health.The renewal ability of this tissue originates from intestinal stem cells(ISCs)at the bases of crypts,and the rapid turnover of the intestinal epithelium requires a continuous energy supply.Moreover,ISCs are able to self-renew and differentiate into various functional cells(enterocytes,goblet cells,enteroendocrine cells,Paneth cells,etc.).However,the effects and mechanism of glutamate on the expansion of porcine ISCs and intestinal epithelial renewal(or development)are poorly studied.Therefore,this study focuses on whether glutamate regulates the expansion of ISCs and affects intestinal development,whether this process is related to mTORC1 signal and the possible molecular mechanism.The main research contents and experimental results are as follows:In Experiment 1,research was conducted to study the effect of dietary Glu on the development of intestinal epithelium of piglets.A total of 14 weaned piglets(Duroc ×Landrace × Large White)with similar body weights(7.82 ± 0.11 kg)were randomly allocated into 2 groups.A nitrogen-free diet was used to exclude the effects of other amino acids.The first group was fed a nitrogen-free diet(the NFD group,n = 7),and the second group was fed a nitrogen-free diet plus 1.00% Glu(the NFDG group,n = 7),and monoculture cages were raised for 21 days.Compared with the control group,dietary Glu significantly increased the jejunum weight and mucosal weight of weaned piglets(P < 0.05),increased tendency of the average daily feed intake(ADFI)of weaned piglets(P = 0.084),and significantly increased jejunal villus height and villus height/crypt depth ratio(P < 0.05),and scanning electron microscope(SEM)observation revealed that the jejunal villus development in the Glu group was more complete.Dietary Glu had no effect on the organ index(heart,liver,spleen,lung,kidney,stomach)(P > 0.05).The content of serum Glu,citrulline,lysine,tyrosine,and proline was increased significantly in dietary Glu group compared with the control group(P < 0.05),and serum leucine(P = 0.051)and valine acid(P = 0.055),histidine(P = 0.069),phenylalanine(P = 0.057),alanine(P = 0.054)have an increasing trend.The serum total protein content,globular protein content,serum aspartate aminotransferase(AST)activity,and urea content all significantly increased(P < 0.05).The results of Western blotting and quantitative real-time PCR and IHC showed that dietary Glu upregulated proliferating cell nuclear antigen(PCNA),Leucine-rich-repeat-containing G-protein-coupled receptor 5(Lgr5),and B cell specific moloney murine leukemia virus insertion site 1(Bmi1)expression.In addition,the m RNA abundance of differentiation and functional cell marker genes were also increased.Dietary Glu stimulated the proliferation and differentiation of intestinal epithelial cells and promoted intestinal development.Experiment 2: In order to analyze the molecular mechanism of Glu affects ISCs to regulate intestinal development,three control samples and three Glu group jejunum tissue samples were selected in the first experiment.i TRAQ proteomics and bioinformatics were used to analyze the key signaling pathway of Glu regulation in intestinal development.A total of 5181 proteins were detected and 48 proteins were differentially expressed between the groups,of which 24 were up-regulated and 24 were down-regulated compared with the control group.Biological processes of differential protein enrichment analysis include epithelial development,regulation of intestinal absorption,digestive tract development,columnar epithelial cell differentiation,the stem cell population maintenance,stem cell differentiation,and so on.The molecular function of differential protein enrichment analysis includes G-protein coupled peptide receptor activity.KEGG analysis results suggest that the mechanistic target of rapamycin(m TOR),and its upstream insulin signaling pathway,phosphoinositide 3-kinase(PI3K)/protein kinase B(Akt)signaling pathway,and extracellular regulated protein kinase(ERK)signaling pathway may participate in the process of Glu regulation of intestinal development.the phosphorylation levels of insulin receptor(IR),insulin receptor substrate(IRS),PI3 K,Akt,epidermal growth factor receptor(EGFR),mitogen-activated protein kinase/extracellular signal-regulated kinase kinase(MEK),ERK,m TOR,ribosomal S6 protein kinase 1(S6K1),and S6 were significantly upregulated by Western blotting assay(P < 0.05).The above results suggest that Glu may activate mTORC1 signal through IR/ PI3K/Akt and EGFR/MEK/ERK pathways,improve the expansion of pig ISCs,and then promote the intestinal development of piglets.Experiment 3: Study the effect of Glu on the expansion of porcine ISCs and its molecular mechanism.On the one hand,the fresh jejunal samples of piglets in the control group and Glu group were used to isolate porcine intestinal crypt cells(including ISCs)and threedimensional culture;on the other hand,and the crypts were isolated from the jejunum of 7-day-old piglets.Different concentrations of Glu were added from seeding to carry out threedimensional culture in vitro.The results showed that dietary 1.00% Glu supplementation significantly increased the organoid forming efficiency and budding efficiency in vivo.Compared with the control group(glutamate deficiency),ISCs treated with 2 mmol/L and 5mmol/L Glu significantly increased the organoid forming efficiency and budding efficiency,and the budding efficiency of 5 mmol/L Glu treatment was significantly higher than that of2 mmol/L Glu treatment.MTT assay,Ed U staining,real-time PCR,and automatic protein expression quantitative analysis system(WES)assay showed that 5 mmol/L Glu significantly promoted the viability,proliferation,and differentiation of ISCs.The WES results further showed that 5 mmol/L Glu activated IR/PI3K/Akt pathway,EGFR/MEK/ERK pathway and mTORC1 signaling.In addition,the inhibition of the IR and MEK/ERK pathway significantly decreased the efficiency of organoid forming and budding efficiency.What’s more,the inhibition of the EGFR pathway blocked the forming of organoids.These results suggest that 5 mmol/L Glu promotes the expansion of ISCs,which may be related to the activation of IR/PI3K/Akt pathway,EGFR/MEK/ERK pathway and mTORC1 signaling.Experiment 4: Intestinal porcine enterocyte cell line(IPEC-J2)was used as a model to further analyze the mechanism of Glu action.Compared with the control group(without Glu and glutamine),MTT and Ed U staining showed that 5 mmol/L Glu significantly increased the viability and proliferation of IPEC-J2 cells for 24 h.5 mmol/L Glu significantly activated mTORC1 signaling and activated both IR/PI3K/Akt pathway and EGFR/MEK/ERK pathway in IPEC-J2 cells treated with Glu for 60 min.It was also found that inhibition of IR/PI3K/Akt pathway and EGFR/MEK/ERK pathway significantly down-regulated the Glu-induced mTORC1 activation.Glu may be the ligand of IR and EGFR as evaluated by molecular docking.At the same time,Western blotting detection showed that there was no difference in the expression of other key proteins(Mios,NPRL2,Rag A,ATP6V1B2)except Rag C downregulation in Glu-treated GTPase-activating protein activity toward Rags 2(GATOR2)/GATOR1/Rag GTPase pathway,and Rag C overexpression and Rag A knockdown did not affect Glu-induced mTORC1 activation.In addition,Glu treatment for60 min did not change the expression of excitatory amino acid transporter-3(EAAT3)and taste receptor type 1-member-1/3(T1R1/3)and metabotropic glutamate receptor-2/3(m Glu R2/3)in IPEC-J2 cells.These results suggest that Glu activates mTORC1 signaling through IR/PI3K/Akt and EGFR/MEK/ERK pathway rather than the GATOR2/GATOR1/Rag GTPase pathway.These results indicate that extracellular Glu activates mTORC1 signaling through the IR/PI3K/Akt pathway and EGFR/MEK/ERK pathway,increasing the viability of pig ISCs and promoting their proliferation,thus promoting intestinal epithelial renewal and intestinal development.The results provide new perspective for regulating the growth and health of the intestinal epithelium and enrich the theoretical system of Glu nutrition to regulate intestinal development.