Study On The Transport Mechanism Of The Dietary Dipeptide-Bound Pyrraline Across Intestinal Epithelial Cells

Dietary advanced glycation end-products(d AGEs)are a class of products formed in foods through a non-enzymatic glycation reaction between protein and reducing sugar,their structures are complex and diverse.Up to now,the limited studies on the digestion,absorption,metabolism and distribution of well-defined d AGEs make their biohazard assessment difficult.If the transport characteristics of d AGEs in the small intestine could be clarified,it will help to provide a theoretical basis for biohazard assessment of d AGEs.Hence,the study focuses on pyrraline,a typical representative of d AGEs.Specifically,the transport assay of well-defined dipeptide-bound pyrralines was performed by adopting the Caco-2 cells monolayer.Firstly,the transmembrane transport assay of dipeptide-bound pyrralines was conducted to indicate their transport ability,transport form and transport mechanism.Secondly,during the transmembrane transport,the interactions between the dipeptide-bound pyrralines and brush border peptidase(APN)were investigated to clarify the hydrolysis of dipeptide-bound pyrralines by APN.Lastly,to elucidate the biological changes of intestinal epithelial cells(IECs)induced by dietary dipeptide-bound pyrraline,the expression levels of genes and proteins in IECs treated with dipeptide-bound pyrraline were fully analyzed using transcriptomic and proteomic techniques.The main research contents and results were as follows:(1)Since the biological effects of dietary dipeptide-bound pyrralines depend on their bioavailability,their transmembrane transport assay was investigated.Using the intestinal epithelial cell model constructed by Caco-2 cells,the transmembrane transport form and transport ability of dipeptide-bound pyrralines with different chemical structures(Pyrr-Ile、Pyrr-Leu、Pyrr-Thr、Pyrr-Ala)were analyzed,and a relationship between the structure and transport ability of dipeptide-bound pyrralines was established.Results showed that the transmembrane transport ability,transport form,intracellular retention of dipeptide-bound pyrralines,and their distributions on both sides of the Caco-2 cells monolayer were highly correlated with their own structures.The transmembrane transport ability of Pyrr-Ile(log P =-0.73)and Pyrr-Leu(log P =-0.85)with higher hydrophobicity was stronger than that of PyrrAla(log P =-1.36)and Pyrr-Thr(log P =-1.49).Pyrr-Ile and Pyrr-Leu could be transported across the Caco-2 cells monolayer not only in the form of free pyrraline but also in the intact form,while Pyrr-Thr and Pyrr-Ala were translocated across the Caco-2 cells monolayer only in the free form.These results demonstrated that the C-terminal amino acid residue,in which the structure difference of the four dipeptide-bound pyrralines lies,has an important influence on the transmembrane transport of dipeptide-bound pyrralines.(2)The study on the interactions between Aminopeptidase N(APN)and dipeptide-bound pyrralines could help to clarify the hydrolysis mechanism of dipeptide-bound pyrralines during transport,because APN is one of the main peptidases secreted by intestinal brush border.Results showed that dipeptide-bound pyrralines could be specifically recognized by APN,the hydrolysis of dipeptide-bound pyrralines by APN could cause the release of free pyrraline,and the release rate relied on the structures of dipeptide-bound pyrralines.For example,the hydrolysis effect of APN on Pyrr-Ala was better than that of Pyrr-Ile.Since the free pyrraline released from dipeptide-bound pyrraline is an inhibitor of APN,the occurrence of product inhibition in hydrolysis of dipeptide-bound pyrraline was possible.The interaction mechanism between dipeptide-bound pyrralines and APN was further studied from the perspective of spectroscopy and thermodynamics.When APN interacted with dipeptide-bound pyrraline,the position and shape of characteristic peaks of APN at 220 nm and 208 nm did not change significantly but a slight red shift of the peak at 197 nm was observed.These results demonstrated that the interaction between APN and dipeptide-bound pyrraline may not cause significant changes in the conformation of APN,but it may reduce the hydrophobicity of APN.Thermodynamic analysis showed that the interaction between APN and dipeptide-bound pyrraline was thermodynamically favorable,and was highly enthalpy driven.(3)Using the intestinal epithelial cell model constructed by Caco-2 cells,the biological changes of IECs caused by dietary dipeptide-bound pyrraline(Pyrr-Leu)were analyzed at the m RNA level to reveal the transport mechanism of dietary Pyrr-Leu.The results of transcriptomics showed that dietary Pyrr-Leu did not regulate the expression of key transporter SLC15A1-encoding gene,but it significantly and positively regulated the expressions of encoding genes of proteins(ABCC2,ABCG2,etc.)responsible for transporting exogenous substances from the cytoplasm to extracellular space.Besides,dietary Pyrr-Leu had no significant effect on the expressions of protease-encoding genes(DPPIV,GGH,XPNPEP1,MEP1 A,ACE and ANPEP)in IECs.Moreover,the expression levels of genes encoding tight junction proteins and integrin proteins were mostly up-regulated after dietary Pyrr-Leu treatment,indicating that dietary Pyrr-Leu does not damage the integrity of intestinal barrier by regulating the transcription of genes that encode tight junction proteins and integrin proteins.In addition,the expressions of encoding genes of key proteases that regulate the glycolysis pathway were significantly regulated by dietary Pyrr-Leu,suggesting the possible effects on the energy metabolism of IECs caused by dietary Pyrr-Leu.(4)Using the intestinal epithelial cell model constructed by Caco-2 cells,the effects of dietary dipeptide-bound pyrraline(Pyrr-Leu)on energy homeostasis,redox homeostasis,and protein homeostasis of IECs were analyzed at m RNA and protein levels.Associated analysis of multiple omics showed that dietary Pyrr-Leu significantly regulated the expressions of mitochondrial-related genes and proteins in IECs,suggesting that mitochondria are the main target organelles of dietary Pyrr-Leu.Moreover,the expressions of metabolism-related genes and proteins were significantly regulated by dietary Pyrr-Leu,it might activate an energy reprogramming in IECs by inducing the transformation between glycolysis and oxidative phosphorylation pathway.Besides,the general up-regulation of 26 S proteasome abundance and the significant up-regulation of Nrf2 at m RNA level were observed in IECs,suggesting the potential influence of dietary Pyrr-Leu on protein homeostasis and redox homeostasis.In summary,the main contents of this study included synthesis of four well-defined dipeptide-bound pyrralines,calculation of parameters reflecting the chemical reactivity of dipeptide-bound pyrralines,analysis of transmembrane transports of dipeptide-bound pyrralines,study of interactions between APN and dipeptide-bound pyrralines,and evaluation of the biological changes of IECs caused by dipeptide-bound pyrraline at the m RNA and protein levels.These works can provide basic theoretical support for the biohazard assessment of dietary pyrraline and guidance for the rational dietary design of patients with diabetes and nephropathy.