Short-peptide-based enteral nutrition affects rats MDP translocation and protects against gut-lung injury via the PepT1-NOD2-beclin-1 pathway in vivo.

BACKGROUND: Peptide transporter 1 (PepT1) transports bacterial oligopeptide products and induces inflammation of the bowel. Nutritional peptides compete for the binding of intestinal bacterial products to PepT1. We investigated the mechanism of short-peptide-based enteral nutrition (SPEN) on the damage to the gut caused by the bacterial oligopeptide product muramyl dipeptide (MDP), which is transported by PepT1. The gut-lung axis is a shared mucosal immune system, and immune responses and disorders can affect the gut-respiratory relationship. METHODS AND RESULTS: Sprague-Dawley rats were gavaged with solutions containing MDP, MDP + SPEN, MDP + intact-protein-based enteral nutrition (IPEN), glucose as a control, or glucose with GSK669 (a NOD2 antagonist). Inflammation, mitochondrial damage, autophagy, and apoptosis were explored to determine the role of the PepT1-nucleotide-binding oligomerization domain-containing protein 2 (NOD2)-beclin-1 signaling pathway in the small intestinal mucosa. MDP and proinflammatory factors of lung tissue were explored to determine that MDP can migrate to lung tissue and cause inflammation. Induction of proinflammatory cell accumulation and intestinal damage in MDP gavage rats was associated with increased NOD2 and Beclin-1 mRNA expression. IL-6 and TNF-α expression and apoptosis were increased, and mitochondrial damage was severe, as indicated by increased mtDNA in the MDP group compared with controls. MDP levels and expression of proinflammatory factors in lung tissue increased in the MDP group compared with the control group. SPEN, but not IPEN, eliminated these impacts. CONCLUSIONS: Gavage of MDP to rats resulted in damage to the gut-lung axis. SPEN reverses the adverse effects of MDP. The PepT1-NOD2-beclin-1 pathway plays a role in small intestinal inflammation, mitochondrial damage, autophagy, and apoptosis.

Structure-Based Analysis of Cefaclor Pharmacokinetic Diversity According to Human Peptide Transporter-1 Genetic Polymorphism.

Cefaclor is a substrate of human-peptide-transporter-1 (PEPT1), and the impact of inter-individual pharmacokinetic variation due to genetic polymorphisms of solute-carrier-family-15-member-1 (SLC15A1) has been a topic of great debate. The main objective of this study was to analyze and interpret cefaclor pharmacokinetic variations according to genetic polymorphisms in SLC15A1 exons 5 and 16. The previous cefaclor bioequivalence results were integrated with additional SLC15A1 exons 5 and 16 genotyping results. An analysis of the structure-based functional impact of SLC15A1 exons 5 and 16 genetic polymorphisms was recently performed using a PEPT1 molecular modeling approach. In cefaclor pharmacokinetic analysis results according to SLC15A1 exons 5 and 16 genetic polymorphisms, no significant differences were identified between genotype groups. Furthermore, in the population pharmacokinetic modeling, genetic polymorphisms in SLC15A1 exons 5 and 16 were not established as effective covariates. PEPT1 molecular modeling results also confirmed that SLC15A1 exons 5 and 16 genetic polymorphisms did not have a significant effect on substrate interaction with cefaclor and did not have a major effect in terms of structural stability. This was determined by comprehensively considering the insignificant change in energy values related to cefaclor docking due to point mutations in SLC15A1 exons 5 and 16, the structural change in conformations confirmed to be less than 0.05 Å, and the relative stabilization of molecular dynamic simulation energy values. As a result, molecular structure-based analysis recently suggested that SLC15A1 exons 5 and 16 genetic polymorphisms of PEPT1 were limited to being the main focus in interpreting the pharmacokinetic diversity of cefaclor.

Investigating the Transepithelial Transport and Enzymatic Stability of Lactononadecapeptide (NIPPLTQTPVVVPPFLQPE), a 19-Amino Acid Casein-Derived Peptide in Caco-2 Cells.

Lactononadecapeptide (LNDP; NIPPLTQTPVVVPPFLQPE), a casein-derived peptide comprising 19 residues, is known for its capacity to enhance cognitive function. This study aimed to explore the transepithelial transport and stability of LNDP. Results showed that LNDP retained over 90% stability after 2 h of treatment with gastrointestinal enzymes. The stability of LNDP on Caco-2 cell monolayers ranged from 93.4% ± 0.9% to 101.1% ± 1.2% over a period of 15-60 min, with no significant differences at each time point. The permeability of LNDP across an artificial lipid membrane was very low with the effective permeability of 3.6 × 10-11 cm/s. The Caco-2 assay demonstrated that LNDP could traverse the intestinal epithelium, with an apparent permeability of 1.22 × 10-6 cm/s. Its transport was significantly inhibited to 67.9% ± 5.0% of the control by Gly-Pro, a competitor of peptide transporter 1 (PEPT1). Furthermore, PEPT1 knockdown using siRNA significantly inhibited LNDP transport by 77.6% ± 1.9% in Caco-2 cell monolayers. The LNDP uptake in PEPT1-expressing HEK293 cells was significantly higher (54.5% ± 14.6%) than that in mock cells. These findings suggest that PEPT1 plays a crucial role in LNDP transport, and LNDP exhibits good resistance to gastrointestinal enzymes.

Peptide Transporter 1-Mediated Dipeptide Transport Promotes Hepatocellular Carcinoma Metastasis by Activating MAP4K4/G3BP2 Signaling Axis.

Cancer metastasis is the leading cause of mortality in patients with hepatocellular carcinoma (HCC). To meet the rapid malignant growth and transformation, tumor cells dramatically increase the consumption of nutrients, such as amino acids. Peptide transporter 1 (PEPT1), a key transporter for small peptides, has been found to be an effective and energy-saving intracellular source of amino acids that are required for the growth of tumor cells. Here, the role of PEPT1 in HCC metastasis and its underlying mechanisms is explored. PEPT1 is upregulated in HCC cells and tissues, and high PEPT1 expression is associated with poor prognosis in patients with HCC. PEPT1 overexpression dramatically promoted HCC cell migration, invasion, and lung metastasis, whereas its knockdown abolished these effects both in vitro and in vivo. Mechanistic analysis revealed that high PEPT1 expression increased cellular dipeptides in HCC cells that are responsible for activating the MAP4K4/G3BP2 signaling pathway, ultimately facilitating the phosphorylation of G3BP2 at Thr227 and enhancing HCC metastasis. Taken together, these findings suggest that PEPT1 acts as an oncogene in promoting HCC metastasis through dipeptide-induced MAP4K4/G3BP2 signaling and that the PEPT1/MAP4K4/G3BP2 axis can serve as a promising therapeutic target for metastatic HCC.

Irinotecan-induced gastrointestinal damage alters the expression of peptide transporter 1 and absorption of cephalexin in rats.

Irinotecan causes severe gastrointestinal damage, which may affect the expression of intestinal transporters. However, neither the expression of peptide transporter 1 (Pept1) nor the pharmacokinetics of Pept1 substrate drugs has been investigated under irinotecan-induced gastrointestinal damage. Therefore, the present study quantitatively investigated the effects of irinotecan-induced gastrointestinal damage on the intestinal expression of Pept1 and absorption of cephalexin (CEX), a typical Pept1 substrate, in rats. Irinotecan was administered intravenously to rats for 4 days to induce gastrointestinal damage. The expression of Pept1 mRNA and the Pept1 protein in the upper, middle, and lower segments of the small intestine of irinotecan-treated rats was assessed by quantitative real-time polymerase chain reaction (PCR) and western blotting, respectively. The pharmacokinetic profile of CEX was examined after its oral or intravenous administration (10 mg/kg). In irinotecan-treated rats, ∼2-fold increases in Pept1 protein levels were observed in all three segments, whereas mRNA levels remained unchanged. The oral bioavailability of CEX significantly decreased to 76% of that in control rats. The decrease in passive diffusion caused by intestinal damage may have overcome the increase in Pept1-mediated uptake. In conclusion, irinotecan may decrease the intestinal absorption of Pept1 substrate drugs; however, it increased the expression of intestinal Pept1.

[Role of cholinergic anti-inflammatory pathway in Ghrelin regulation of peptide transporter 1 expression in small intestinal epithelium of septic rats].

OBJECTIVE: To investigate the role of cholinergic anti-inflammatory pathway in the regulation of peptide transporter 1 (PepT1) expression in small intestinal epithelium of septic rats by Ghrelin. METHODS: One hundred adult male Sprague-Dawley (SD) rats were randomly divided into sham operation group, sepsis group, sepsis+vagotomy group, sepsis+Ghrelin group, and sepsis+vagotomy+Ghrelin group, with 20 rats in each group. In the sham operation group, the cecum was separated after laparotomy, without ligation and perforation. In the sepsis group, the rats received cecal ligation puncture (CLP). In the sepsis+vagotomy group, the rats received CLP and vagotomy after laparotomy. In the sepsis+Ghrelin group, 100 µmol/L Ghrelin was intravenously injected after CLP immediately. The rats in the sepsis+vagotomy+Ghrelin group received CLP and vagotomy at the same time, then the Ghrelin was intravenously injected immediately with the same dose as the sepsis+Ghrelin group. Ten rats in each group were taken to observe their survival within 7 days. The remaining 10 rats were sacrificed 20 hours after the operation to obtain venous blood and small intestinal tissue. The condition of the abdominal intestine was observed. The injury of intestinal epithelial cells was observed with transmission electron microscopy. The contents of tumor necrosis factor-α (TNF-α) and interleukin-1ß (IL-1ß) in serum and small intestinal tissue were detected by enzyme-linked immunosorbent assay (ELISA). The brush border membrane vesicle (BBMV) was prepared, the levels of mRNA and protein expression of PepT1 in the small intestinal epithelium were detected by real-time fluorescence quantitative polymerase chain reaction (RT-qPCR) and Western blotting. RESULTS: All rats in the sham operation group survived at 7 days after operation. The 7-day cumulative survival rate of rats in the sepsis group was significantly lower than that in the sham operation group (20% vs. 100%, P < 0.05). The cumulative survival rate of rats after Ghrelin intervention was improved (compared with sepsis group: 40% vs. 20%, P < 0.05), but the protective effect of Ghrelin was weakened after vagotomy (compared with sepsis+Ghrelin group: 10% vs. 40%, P < 0.05). Compared with the sham operation group, in the sepsis group, the small intestine and cecum were dull red, the intestinal tubules were swollen and filled with gas, the intestinal epithelial cells were seriously injured under transmission electron microscopy, the levels of TNF-α and IL-1ß in serum and small intestinal were significantly increased, and the expression levels of PepT1 mRNA and protein in the small intestinal epithelium were significantly decreased. It indicated that the sepsis rat model was successfully prepared. After vagotomy, the intestinal swelling and gas accumulation became worse in septic rats, leading to the death of all rats. Compared with the sepsis group, the abdominal situation in the sepsis+Ghrelin group was improved, the injury of intestinal epithelial cells was alleviated, the serum and small intestinal TNF-α and IL-1ß were significantly decreased [serum TNF-α (ng/L): 253.27±23.32 vs. 287.90±19.48, small intestinal TNF-α (ng/L): 95.27±11.47 vs. 153.89±18.15, serum IL-1ß (ng/L): 39.16±4.47 vs. 54.26±7.27, small intestinal IL-1ß (ng/L): 28.47±4.13 vs. 42.26±2.59, all P < 0.05], and the expressions of PepT1 mRNA and protein in the small intestinal epithelium were significantly increased [PepT1 mRNA (2-ΔΔCt): 0.66±0.05 vs. 0.53±0.06, PepT1 protein (PepT1/GAPDH): 0.80±0.04 vs. 0.60±0.05, both P < 0.05]. Compared with the sepsis+Ghrelin group, after vagotomy in the sepsis+vagotomy+Ghrelin group, the effect of Ghrelin on reducing the release of inflammatory factors in sepsis rats was significantly reduced [serum TNF-α (ng/L): 276.58±19.88 vs. 253.27±23.32, small intestinal TNF-α (ng/L): 144.28±12.99 vs. 95.27±11.47, serum IL-1ß (ng/L): 48.15±3.21 vs. 39.16±4.47, small intestinal IL-1ß (ng/L): 38.75±4.49 vs. 28.47±4.13, all P < 0.05], the up-regulated effect on the expression of PepT1 in small intestinal epithelium was lost [PepT1 mRNA (2-ΔΔCt): 0.58±0.03 vs. 0.66±0.05, PepT1 protein (PepT1/GAPDH): 0.70±0.02 vs. 0.80±0.04, both P < 0.05], and the injury of small intestinal epithelial cells was worse. CONCLUSIONS: Ghrelin plays a protective role in sepsis by promoting cholinergic neurons to inhibit the release of inflammatory factors, thereby promoting the transcription and translation of PepT1.

The sugar transporter ZmSUGCAR1 of the nitrate transporter 1/peptide transporter family is critical for maize grain filling.

Maternal-to-filial nutrition transfer is central to grain development and yield. nitrate transporter 1/peptide transporter (NRT1-PTR)-type transporters typically transport nitrate, peptides, and ions. Here, we report the identification of a maize (Zea mays) NRT1-PTR-type transporter that transports sucrose and glucose. The activity of this sugar transporter, named Sucrose and Glucose Carrier 1 (SUGCAR1), was systematically verified by tracer-labeled sugar uptake and serial electrophysiological studies including two-electrode voltage-clamp, non-invasive microelectrode ion flux estimation assays in Xenopus laevis oocytes and patch clamping in HEK293T cells. ZmSUGCAR1 is specifically expressed in the basal endosperm transfer layer and loss-of-function mutation of ZmSUGCAR1 caused significantly decreased sucrose and glucose contents and subsequent shrinkage of maize kernels. Notably, the ZmSUGCAR1 orthologs SbSUGCAR1 (from Sorghum bicolor) and TaSUGCAR1 (from Triticum aestivum) displayed similar sugar transport activities in oocytes, supporting the functional conservation of SUGCAR1 in closely related cereal species. Thus, the discovery of ZmSUGCAR1 uncovers a type of sugar transporter essential for grain development and opens potential avenues for genetic improvement of seed-filling and yield in maize and other grain crops.

Susceptibility Loci in SLC15A1, UGT1A3, and CWC27 Genes Associated with Bladder Cancer in the Northeast Chinese Population.

Bladder cancer (BCa) is an increasingly severe clinical and public health issue. Therefore, we aim to investigate BCa susceptibility loci in the Chinese population. In this study, 487 BCa patients and 563 controls were recruited from the First Affiliated Hospital of China Medical University from July 2015 to September 2020. A total of ten single-nucleotide polymorphisms (SNPs) in solute carrier family 15 member 1 (SLC15A1), CWC27 spliceosome associated cyclophilin (CWC27), or UDP glucuronosyltransferase family 1 member A3 (UGT1A3) genes were genotyped. The associations between the candidate SNPs and BCa were analyzed using genotype and haplotype analysis. The results demonstrated that Rs4646227 of SLC15A1 has a significant association with BCa. The patients with CG (OR =2.513, p < 0.05) and GG (OR =2.859, p < 0.05) genotypes had an increasing risk of BCa compared with the CC genotype. For the CWC27 gene, genotypic frequency analysis revealed that the GT or TT genotype of rs2042329 and the CT or TT genotype of rs1870437 were more frequent in BCa patients than those in the control group, indicating that these genotypes were associated with a higher risk of BCa (all p < 0.05). Haplotypes of SLC15A1, UGT1A3, and CWC27 genes found that the C-C-C haplotype of SLC15A1 was associated with a lower risk of BCa while the C-G-C haplotype was associated with a higher risk. For the UGT1A3 gene, a moderate protective effect was observed with the most frequent T-T-C haplotype, and for the CWC27 gene, most of the haplotypes showed no association with BCa, except the G-G-C-T haplotype (order of SNPs: rs2042329-rs7735338-rs1870437-rs2278351, OR =0.81, p =0.038). In sum, this study indicated that rs2042329 and rs1870437 in the CWC27 gene and rs4646227 in the SLC15A1 gene are independent indicators for BCa risk in Chinese people. Further large-scale studies are required to validate these findings. Also, this study provided the theoretical basis for developing new therapeutic drug targeting of BCa.

Extracellular domain of PepT1 interacts with TM1 to facilitate substrate transport.

Mammalian peptide transporters, PepT1 and PepT2, mediate uptake of small peptides and are essential for their absorption. PepT also mediates absorption of many drugs and prodrugs to enhance their bioavailability. PepT has twelve transmembrane (TM) helices that fold into an N-terminal domain (NTD, TM1-6) and a C-terminal domain (CTD, TM7-12) and has a large extracellular domain (ECD) between TM9-10. It is well recognized that peptide transport requires movements of the NTD and CTD, but the role of the ECD in PepT1 remains unclear. Here we report the structure of horse PepT1 encircled in lipid nanodiscs and captured in the inward-open apo conformation. The structure shows that the ECD bridges the NTD and CTD by interacting with TM1. Deletion of ECD or mutations to the ECD-TM1 interface impairs the transport activity. These results demonstrate an important role of ECD in PepT1 and enhance our understanding of the transport mechanism in PepT1.

Hypertension alters the function and expression profile of the peptide cotransporters PEPT1 and PEPT2 in the rodent renal proximal tubule.

Hypertension is a major risk factor for kidney and cardiovascular disease. The treatment of hypertensive individuals by selected ACE inhibitors and certain di-and tripeptides halts the progression of renal deterioration and extends life-span. Renal reabsorption of these low molecular weight substrates are mediated by the PEPT1 and PEPT2 cotransporters. This study aims to investigate whether hypertension and ageing affects renal PEPT cotransporters at gene, protein expression and distribution as well as function in the superficial cortex and the outer medulla of the kidney. Membrane vesicles from the brush border (BBMV) and outer medulla (OMMV) were isolated from the kidneys of young Wistar Kyoto (Y-WKY), young spontaneously hypertensive (Y-SHR), and middle aged SHR (M-SHR) rats. Transport activity was measured using the substrate, ß-Ala-Lys (AMCA). Gene expression levels of PEPT genes were assessed with qRT-PCR while renal localisation of PEPT cotransporters was examined by immunohistochemistry with Western Blot validation. The Km and Vmax of renal PEPT1 were decreased significantly in SHR compared to WKY BBMV, whilst the Vmax of PEPT2 showed differences between SHR and WKY. By contrast to the reported cortical distribution of PEPT1, PEPT1-staining was detected in the outer medulla, whilst PEPT2 was expressed primarily in the cortex of all SHR; PEPT1 was significantly upregulated in the cortex of Y-SHR. These outcomes are indicative of a redistribution of PEPT1 and PEPT2 in the kidney proximal tubule under hypertensive conditions that has potential repercussions for nutrient handling and the therapeutic use of ACE inhibitors in hypertensive individuals.

Expression, purification and characterization of human proton-coupled oligopeptide transporter 1 hPEPT1.

The human peptide transporter hPEPT1 (SLC15A1) is responsible for uptake of dietary di- and tripeptides and a number of drugs from the small intestine by utilizing the proton electrochemical gradient, and hence an important target for peptide-like drug design and drug delivery. hPEPT1 belongs to the ubiquitous major facilitator superfamily that all contain a 12TM core structure, with global conformational changes occurring during the transport cycle. Several bacterial homologues of these transporters have been characterized, providing valuable insight into the transport mechanism of this family. Here we report the overexpression and purification of recombinant hPEPT1 in a detergent-solubilized state. Thermostability profiling of hPEPT1 at different pH values revealed that hPEPT1 is more stable at pH 6 as compared to pH 7 and 8. Micro-scale thermophoresis (MST) confirmed that the purified hPEPT1 was able to bind di- and tripeptides respectively. To assess the in-solution oligomeric state of hPEPT1, negative stain electron microscopy was performed, demonstrating a predominantly monomeric state.

Indoxyl Sulfate Elevated Lnc-SLC15A1-1 Upregulating CXCL10/CXCL8 Expression in High-Glucose Endothelial Cells by Sponging MicroRNAs.

Cardiovascular disease (CVD) is the leading cause of mortality in diabetes mellitus (DM). Immunomodulatory dysfunction is a primary feature of DM, and the emergence of chronic kidney disease (CKD) in DM abruptly increases CVD mortality compared with DM alone. Endothelial injury and the accumulation of uremic toxins in the blood of DM/CKD patients are known mechanisms for the pathogenesis of CVD. However, the molecular factors that cause this disproportional increase in CVD in the DM/CKD population are still unknown. Since long non-protein-coding RNAs (lncRNAs) play an important role in regulating multiple cellular functions, we used human endothelial cells treated with high glucose to mimic DM and with the uremic toxin indoxyl sulfate (IS) to mimic the endothelial injury associated with CKD. Differentially expressed lncRNAs in these conditions were analyzed by RNA sequencing. We discovered that lnc-SLC15A1-1 expression was significantly increased upon IS treatment in comparison with high glucose alone, and then cascaded the signal of chemokines CXCL10 and CXCL8 via sponging miR-27b, miR-297, and miR-150b. This novel pathway might be responsible for the endothelial inflammation implicated in augmenting CVD in DM/CKD and could be a therapeutic target with future clinical applications.

PEPT1 is essential for the growth of pancreatic cancer cells: a viable drug target.

PEPT1 is a proton-coupled peptide transporter that is up-regulated in PDAC cell lines and PDXs, with little expression in the normal pancreas. However, the relevance of this up-regulation to cancer progression and the mechanism of up-regulation have not been investigated. Herein, we show that PEPT1 is not just up-regulated in a large panel of PDAC cell lines and PDXs but is also functional and transport-competent. PEPT2, another proton-coupled peptide transporter, is also overexpressed in PDAC cell lines and PDXs, but is not functional due to its intracellular localization. Using glibenclamide as a pharmacological inhibitor of PEPT1, we demonstrate in cell lines in vitro and mouse xenografts in vivo that inhibition of PEPT1 reduces the proliferation of the cancer cells. These findings are supported by genetic knockdown of PEPT1 with shRNA, wherein the absence of the transporter significantly attenuates the growth of cancer cells, both in vitro and in vivo, suggesting that PEPT1 is critical for the survival of cancer cells. We also establish that the tumor-derived lactic acid (Warburg effect) in the tumor microenvironment supports the transport function of PEPT1 in the maintenance of amino acid nutrition in cancer cells by inducing MMPs and DPPIV to generate peptide substrates for PEPT1 and by generating a H+ gradient across the plasma membrane to energize PEPT1. Taken collectively, these studies demonstrate a functional link between PEPT1 and extracellular protein breakdown in the tumor microenvironment as a key determinant of pancreatic cancer growth, thus identifying PEPT1 as a potential therapeutic target for PDAC.

Onset of nutrient consumption during early life stage digestive system development of two tuna species (Thunnus orientalis and Thunnus albacares).

To specify the timing of exogenous nutrient consumption in the larvae of two commercially important tuna species, the Pacific bluefin tuna (PBF) Thunnus orientalis and the yellowfin tuna (YFT) Thunnus albacares, the gene expressions of peptide transporter 1 (PEPT1) were examined. The mRNA expressions of PEPT1 first occurred at 2 days post hatching (dph) in PBF larvae and 3 dph for the YFT, and PEPT1 was found to only be expressed in the intestinal tract. The histological changes of the digestive tract of the YFT larvae were observed and compared to PBF larvae from a previous study. The intestines were developed at the hatching day for both species. It was found that the developmental timing of internal organs differed between the species, with the YFT showing an approximately one-day delay. The major organs such as liver, pancreas and gall bladder that excrete digestive enzymes appeared at 1 dph for PBF and 2 dph for YFT. The development of external morphological features was similar to organ development timings, with mouth-opening and first feeding starting at 2 dph for PBF, and 3 dph for YFT. Growth during the first month is rapid and variable for both species, ranging from 1.06 to 1.56 mm/d. Our findings provide new information about the early onset of feeding and larval development for the two species which would contribute to future aquaculture.

Effect of dipeptide on intestinal peptide transporter 1 gene expression: An evaluation using primary cultured chicken intestinal epithelial cells.

Peptide transporter 1 (PepT1) is a transporter responsible for absorbing dipeptide and tripeptide in enterocytes and is upregulated by dipeptide in mammals. It has not been certain whether intestinal PepT1 expression is responsive to dipeptides in chickens because of the lack of in vitro study using the cultured enterocytes. This study established a primary culture model of chicken intestinal epithelial cells (IECs) in two-dimensional monolayer culture using collagen gel by which the response of chicken PepT1 gene expression to dipeptide stimuli was evaluated. The cultured chicken IECs showed the epithelial-like morphology attached in a patch-manner and exhibited positive expression of cytokeratin and epithelial cadherin, specific marker proteins of epithelial cells. Moreover, the chicken IECs exhibited the gene expression of intestinal cell type-specific marker, villin1, mucin 2, and chromogranin A, suggesting that the cultured IECs were composed of enterocytes as well as goblet and enteroendocrine cells. PepT1 gene expression was significantly upregulated by synthetic dipeptide, glycyl-l-glutamine, in the cultured IECs. From the results, we herein suggested that dipeptide is a factor upregulating PepT1 gene expression in chicken IECs.

Epigenetic regulation of intestinal peptide transporter PEPT1 as a potential strategy for colorectal cancer sensitization.

Human intestinal peptide transporter PEPT1 is commonly repressed in human colorectal cancer (CRC), yet its relationship with sensitivity to the common CRC treatment ubenimex has not previously been elucidated. In this study, we confirmed PEPT1 suppression in CRC using real-time quantitative polymerase chain reaction and western blotting and then investigated the underlying epigenetic pathways involved using bisulfite sequencing, chromatin immunoprecipitation, siRNA knockdown, and reporter gene assays. We found that PEPT1 transcriptional repression was due to both DNMT1-mediated DNA methylation of the proximal promoter region and HDAC1-mediated histone deacetylation, which blocked P300-mediated H3K18/27Ac at the PEPT1 distal promoter. Finally, the effects of the epigenetic activation of PEPT1 on the CRC response to ubenimex were evaluated using sequential combination therapy of decitabine and ubenimex both in vitro and in xenografts. In conclusion, epigenetic silencing of PEPT1 due to increased DNMT1 and HDAC1 expression plays a vital role in the poor response of CRC to ubenimex.

Protonation State of a Histidine Residue in Human Oligopeptide Transporter 1 (hPEPT1) Regulates hPEPT1-Mediated Efflux Activity.

To clarify the role of an amino acid residue in the pH-dependent efflux process in Chinese hamster ovary (CHO) cells expressing the human oligopeptide transporter hPEPT1 (CHO/hPEPT1), we determined the effect of extracellular pH on the hPEPT1-mediated efflux process. The efflux of glycylsarcosine (Gly-Sar), a typical substrate for hPEPT1, was determined using an infinite dilution method after cells were preloaded with [3H]-Gly-Sar. The efflux of [3H]-Gly-Sar was stimulated by 5 mM unlabeled hPEPT1 substrates in the medium. This trans-stimulation phenomenon showed that hPEPT1 mediated the efflux of [3H]-Gly-Sar from CHO/hPEPT1 and that hPEPT1 is a bi-directional transporter. We then determined the effect of extracellular pH (varying from 8.0 to 3.5) on the efflux activity. The efflux activity by hPEPT1 decreased with the decrease in extracellular pH. The Henderson-Hasselbälch-type equation, which fitted well to the pH-profile of efflux activity, indicated that a single amino acid residue with a pKa value of approximately 5.7 regulates the efflux activity. The pH-profile of the efflux activity remained almost unchanged irrespective of the proton gradient across the plasma membrane. In addition, the chemical modification of the histidine residue with diethylpyrocarbonate completely abolished the efflux activity from cells, which could be prevented by the presence of 10 mM Gly-Sar. These data indicate that the efflux process of hPEPT1 is also regulated in a pH-dependent manner by the protonation state of a histidine residue located at or near the substrate recognition site facing the extracellular space.

Effect of catechin on dietary AGEs absorption and cytotoxicity in Caco-2 cells.

Maillard reaction produces advanced glycation end products (AGEs) that endanger human health. This study investigated the protective effect of (+)-catechin (CC) on different types of dietary AGEs absorption and cytotoxicity in Caco-2 cells. Our results showed that CC had higher inhibitory rate on peptide bound-AGEs absorption than free Nɛ-carboxymethyl lysine (CML), which dropped to 36.24% and 32.21% when treated with 20 and 50 µM CC. The reasons might be that CC could repair the loose tight junction (ZO-1) and down-regulation of protein-coupling peptide carrier 1 (PEPT-1) expression in Caco-2 cells which were in accordance with molecular docking results. Additionally, CC could remarkably decreased the protein levels of receptor of AGEs (RAGE), mitogen-activated protein kinases (MAPK) and nuclear factor-kappa B (NF-κB) that detected by western blotting and immunohistochemical staining method. Taken together, these findings demonstrated that CC may inhibit AGEs absorption and protected Caco-2 cells against RAGE-MAPK-NF-κB signaling suppression.

PepT1-knockout mice harbor a protective metabolome beneficial for intestinal wound healing.

Genetic knockout (KO) of peptide transporter-1 (PepT1) protein is known to provide resistance to acute colitis and colitis-associated cancer (CAC) in mouse models. However, it was unclear which molecule(s) or pathway(s) formed the basis for these protective effects. Recently, we demonstrated that the PepT1-/- microbiota is sufficient to protect against colitis and CAC. Given that PepT1 KO alters the gut microbiome and thereby changes the intestinal metabolites that are ultimately reflected in the feces, we investigated the fecal metabolites of our PepT1 KO mice. Using a liquid chromatography-mass spectrometry (LC-MS)-based untargeted-metabolomics technique, we found that the fecal metabolites were significantly different between the KO and normal wild-type (WT) mice. Among the altered fecal metabolites, tuberonic acid (TA) was sevenfold higher in KO mouse feces than in WT mouse feces. Accordingly, we studied whether the increased TA could direct an anti-inflammatory effect. Using in vitro models, we discovered that TA not only prevented lipopolysaccharide (LPS)-induced inflammation in macrophages but also improved the epithelial cell healing processes. Our results suggest that TA, and possibly other fecal metabolites, play a crucial role in the pathway(s) associated with the anticolitis effects of PepT1 KO.NEW & NOTEWORTHY Fecal metabolites were significantly different between the KO and normal wild-type (WT) mice. One fecal metabolite, tuberonic acid (TA), was sevenfold higher in KO mouse feces than in WT mouse feces. TA prevented lipopolysaccharide (LPS)-induced inflammation in macrophages and improved the epithelial cell healing process.

Developmental effects of constant light on circadian behaviour and gene expressions in zebra finches: Insights into mechanisms of metabolic adaptation to aperiodic environment in diurnal animals.

A most crucial feature of biological adaptation is the maintenance of a close temporal relationship of behaviour and physiology with prevailing 24-h light-dark environment, which is rapidly changing with increasing nighttime illumination. This study investigated developmental effects of the loss of night on circadian behaviour, metabolism and gene expressions in diurnal zebra finches born and raised under LL, with controls on 12L:12D. Birds under LD were entrained, and showed normal body mass and a significant 24-h rhythm in both activity-rest pattern and mRNA expression of candidate genes that we measured. But, under LL, birds gained weight and accumulated lipid in the liver. Intriguingly, at the end of the experiment, the majority (4/5th) of birds under LL were rhythmic in activity despite arrhythmic expression in the hypothalamus of c-Fos (neuronal activity), Rhodopsin and Mel1-a genes (light perception), and clock genes (Bmal1, Per2 and Rev-erb ß). In peripheral tissues, LL induced variable clock gene expressions. Whereas 24-h mRNA rhythm was abolished for Bmal1 in both liver and gut, it persisted for Per2 and Rev-erb ß in liver, and for Per2 in gut. Further, we found under LL, the loss of 24-h rhythm in hepatic expression of Fasn and Cd36/Fat (biosynthesis and its uptake), and gut expression of Sglt1, Glut5, Cd36 and Pept1 (nutrient absorption) genes. As compared to LD, baseline mRNA levels of Fasn and Cd36 genes were attenuated under LL. Among major transporter genes, Sglt1 (glucose) and Cd36 (fat) genes were arrhythmic, while Glut5 (glucose) and Pept1 (protein) genes were rhythmic but with phase differences under LL, compared to LD. These results demonstrate dissociation of circadian behaviour from clock gene rhythms, and provide molecular insights into possible mechanisms at different levels (behaviour and physiology) that diurnal animals might employ in order to adapt to an emerging overly illuminated-night urban environment.