PepT1 Activity Research Articles

Role of insulin on jejunal PepT1 expression and function regulation in diabetic male and female rats

The aim of this study was to determine whether the jejunal oligopeptide transporter PepT1 is regulated by insulin and whether this regulation is sex-dependent in type 1 diabetic rats. PepT1 expression, real-time polymerase chain reaction, and Western blots were performed using jejunal segments from 4 groups of male and female rats: normal (nondiabetic), insulin-treated nondiabetic, streptozotocin (STZ)-induced diabetic (type 1 diabetes), and insulin-treated diabetic models. Furthermore, the same segments from all groups underwent perfusion to assess uptake of the dipeptide glycylsarcosine through PepT1. Our results showed that insulin treatment of nondiabetic female rats decreased blood glucose level but did not affect nondiabetic male rats. In both male and female diabetic rats, insulin did not completely decrease blood glucose level. Insulin treatment decreased PepT1 mRNA level in nondiabetic male rats and increased mRNA level in nondiabetic female rats without affecting the PepT1 protein level in either sex. Inducing diabetes with STZ increased PepT1 mRNA and protein levels in female rats; however, in diabetic male rats, the increase in mRNA level was accompanied by a decrease in PepT1 protein level. Treatment of diabetic male rats with insulin partially reversed the effect of diabetes on PepT1 mRNA and protein levels, whereas the same treatment completely restored both PepT1 mRNA and protein to control levels in insulin-treated diabetic female rats. In both nondiabetic male and female rats, insulin treatment had no effect on PepT1 influx rate, and STZ treatment decreased the transporter influx rate. Treatment of diabetic male and female rats with insulin significantly increased PepT1 influx rate; however, complete recovery was found only in diabetic female rats. These results clearly show that insulin and diabetes affected blood glucose level as well as PepT1 activity, expression, and protein levels in a sex-dependent manner. These results suggest that a factor, probably estrogen, could be responsible for the sex-dependent effects of diabetes and insulin in PepT1 level and activity.

Pathogenic Bacteria Induce Colonic PepT1 Expression: An Implication in Host Defense Response

Expression of the di/tripeptide transporter PepT1 has been observed in the colon under inflammatory conditions; however, the inducing factors and underlying mechanisms remain unknown. Here, we address the effects of pathogenic bacteria on colonic PepT1 expression together with its functional consequences. Human colonic HT29-Cl.19A cells were infected with the attaching and effacing enteropathogenic Escherichia coli (EPEC). Wild-type and PepT1 transgenic mice or cultured colonic tissues derived from these mice were infected with Citrobacter rodentium, a murine attaching and effacing pathogen related to EPEC. EPEC induced PepT1 expression and activity in HT29-Cl.19A cells by intimately attaching to host cells through lipid rafts. Induction of PepT1 expression by EPEC required the transcription factor Cdx2. PepT1 expression reduced binding of EPEC to lipid rafts, as well as activation of nuclear factor-kappaB and mitogen-activated protein kinase and production of interleukin-8. Accordingly, ex vivo and in vivo experiments revealed that C rodentium induced colonic PepT1 expression and that, compared with their wild-type counterparts, PepT1 transgenic mice infected with C rodentium exhibited decreased bacterial colonization, production of proinflammatory cytokines, and neutrophil infiltration into the colon. Our findings demonstrate a molecular mechanism underlying the regulation of colonic PepT1 expression under pathologic conditions and reveal a novel role for PepT1 in host defense via its capacity to modulate bacterial-epithelial interactions and intestinal inflammation.

Responses of mRNA expression of PepT1 in small intestine to graded duodenal soybean small peptides infusion in lactating goats.

To study the effect of circulation small peptides concentration on mRNA expression in small intestine, graded amount of soybean small peptides (SSP) were infused into lactating goats through duodenal fistulas. Peptide-bound amino acid (PBAA) concentration in arterial plasma and the mRNA expression of PepT1 was detected in the current study. The results showed that concentrations of all peptidebound amino acids (PBAA) increased and the activity of PepT1 in duodenum tissue was enhanced by SSP infusion. The PepT1expression in duodenum tissue was significantly increased with the increment of amounts of SSP infusion (P<0.05). That in ileum tissue was increased with the increment of the amounts of SSP infusion; the increment of Pep T1 expression was significant only in the 180 g/d treatment (P<0.05). mRNA abundance of PepT1 in jejunum tissue was 1.1 to 1.8 to 2.4 times in SSP infusion group as compared with the control group. In the 120 and 180 g/d treatment, the difference of PepT1 expression in jejunum was significant (P<0.05). The data suggested Pep T1 expression in duodenum, ileum and jejunum tissue was enhanced by an increase in circulation small peptide concentration, and the duodenum might be a main position of small peptides absorption in intestine.

Reduced Intestinal Absorption of Dipeptides via PepT1 in Mice with Diet-induced Obesity Is Associated with Leptin Receptor Down-regulation

Leptin is a major determinant of energy homeostasis, acting both centrally and in the gastrointestinal tract. We previously reported that acute leptin treatment enhances the absorption of di- and tripeptides via the proton-dependent PepT1 transporter. In this study, we investigated the long term effect of leptin on PepT1 levels and activity in Caco2 cell monolayers in vitro. We then assessed the significance of the regulation of PepT1 in vivo in a model of diet-induced obesity. We demonstrated that 1) leptin regulated PepT1 at the transcriptional level, via the MAPK pathway, and at the translational level, via ribosomal protein S6 activation, in Caco2 cells and 2) this activation was systematically followed by a time- and concentration-dependent loss of leptin action reflecting desensitization. Deciphering this desensitization, we demonstrated that leptin induced a down-regulation of its own receptor protein and mRNA expression. More importantly, we showed, in mice with diet-induced obesity, that a 4-week hypercaloric diet resulted in a 46% decrease in PepT1-specific transport, because of a 30% decrease in PepT1 protein and a 50% decrease in PepT1 mRNA levels. As shown in Caco2 cells, these changes in PepT1 were supported by a parallel 2-fold decrease in leptin receptor expression in mice. Taken together, these results indicate that during induction of obesity, leptin resistance may also occur peripherally in the gastrointestinal tract, disrupting the absorption of oligopeptides and peptidomimetic drugs.

Evidence for intestinal heterogenic expression of di-tripeptides transporter PepT1 during experimental cryptosporidiosis in neonatal rats

Cryptosporidium parvum is a protozoan parasite that causes intestinal malabsorptive syndrome and malnutrition. Considering the importance of di-tripeptide absorption for nutritional status, we previously investigated the regulation of PepT1 transporter in the suckling rat model of acute cryptosporidiosis and showed that PepT1 protein expression and activity were not modified in the parasitized intestine. Here we used confocal microscopy performed on intestinal villi to determine the subcellular localization of PepT1 together with f-actin and parasites. For this purpose, confocal microscopy using vibratome thick sections was developed on the distal small intestine, the preferential site of parasite implantation. Results showed major heterogeneity of apical PepT1 expression among enterocytes, which did not correlate with actin staining or parasite implantation. These results underscore the importance of considering the effect of C. parvum at the cellular scale and not only in the entire epithelium.

Butyrate Transcriptionally Enhances Peptide Transporter PepT1 Expression and Activity

BackgroundPepT1, an intestinal epithelial apical di/tripeptide transporter, is normally expressed in the small intestine and induced in colon during chronic inflammation. This study aimed at investigating PepT1 regulation by butyrate, a short-chain fatty acid produced by commensal bacteria and accumulated inside inflamed colonocyte.ResultsWe found that butyrate treatment of human intestinal epithelial Caco2-BBE cells increased human PepT1 (hPepT1) promoter activity in a dose- and time-dependent manner, with maximal activity observed in cells treated with 5 mM butyrate for 24 h. Under this condition, hPepT1 promoter activity, mRNA and protein expression levels were increased as assessed by luciferase assay, real-time RT-PCR and Western blot, respectively. hPepT1 transport activity was accordingly increased by ∼2.5-fold. Butyrate did not alter hPepT1 mRNA half-life indicating that butyrate acts at the transcriptional level. Molecular analyses revealed that Cdx2 is the most important transcription factor for butyrate-induced increase of hPepT1 expression and activity in Caco2-BBE cells. Butyrate-activated Cdx2 binding to hPepT1 promoter was confirmed by gel shift and chromatin immunoprecipitation. Moreover, Caco2-BBE cells overexpressing Cdx2 exhibited greater hPepT1 expression level than wild-type cells. Finally, treatment of mice with 5 mM butyrate added to drinking water for 24 h increased colonic PepT1 mRNA and protein expression levels, as well as enhanced PepT1 transport activity in colonic apical membranes vesicles.ConclusionsCollectively, our results demonstrate that butyrate increases PepT1 expression and activity in colonic epithelial cells, which provides a new understanding of PepT1 regulation during chronic inflammation.

PDZK1 Regulates Two Intestinal Solute Carriers (Slc15a1 and Slc22a5) in Mice

Gastrointestinal (GI) absorption of certain therapeutic agents is thought to be mediated by solute carrier (SLC) transporters, although minimal in vivo evidence has been reported. Here, we show key roles of postsynaptic density 95/disk-large/ZO-1 (PDZ) domain-containing protein, PDZK1, as a regulatory mechanism of two solute carriers, Slc15a1 (oligopeptide transporter PEPT1) and Slc22a5 (carnitine/organic cation transporter OCTN2) in mouse small intestine by using pdzk1 gene knockout (pdzk1(-/-)) mice. GI absorption of cephalexin, a substrate of PEPT1, after p.o. administration was delayed in pdzk1(-/-) mice compared with wild-type mice. Absorption of carnitine, a substrate of OCTN2, was also decreased in pdzk1(-/-) mice. Immunohistochemical analysis revealed the localization of both PEPT1 and OCTN2 at apical membrane of small intestinal epithelial cells in wild-type mice, whereas such apical localization was reduced in pdzk1(-/-) mice, with a concomitant decrease in their protein levels assessed by Western blotting in intestinal brush-border membranes. Electron microscopy revealed localization of PEPT1 in intracellular vesicular structures in pdzk1(-/-) mice. In addition, we first identified interaction between PEPT1 and PDZK1 in mouse small intestine and found that PDZK1 stimulates transport activity of PEPT1 by increasing its expression level in human embryonic kidney 293 cells. Taken together, the present findings provide direct evidence that PDZK1 regulates two intestinal SLC transporters in vivo as an adaptor protein for these transporters and affects oral absorption of their substrates. These findings also raise the possibility that intestinal absorption of the substrate drugs for PEPT1 and OCTN2 is governed by the protein network of these transporters and their adaptor PDZK1.

Sodium butyrate increases di/tripeptide transporter PepT1 expression and function

PepT1 is a di/tripeptide transporter normally expressed in the small intestine but not in the large intestine. This study aimed at investigating PepT1 regulation by sodium butyrate (NaB). We found in vitro, that NaB treatment increased PepT1 promoter activity in a dose‐ and time‐dependent manner, with maximal activity observed in cells treated with 5 mM NaB for 24 hours. Under these conditions, we observed a ~21‐fold increase in hPepT1 promoter activity and a ~2.5‐fold increase in hPepT1 transport activity. These results were confirmed by real‐time RT‐PCR and Western‐blot. Molecular analyses revealed that Cdx2 and CREB transcription factors are essential for NaB‐induced increase of hPepT1 expression and activity. Butyrate‐induced CREB and Cdx2‐promoter binding was confirmed by gel shift and chromatin immunoprecipitation. Moreover, in Cdx2‐overexpressing cells, hPepT1 protein level is higher compared to control cells. Finally, 5 mM butyrate added to drinking water during 24 hours increases the levels of colonic PepT1 expression and activity in mice. Collectively, these data indicate that intracellular accumulation of NaB increases PepT1 expression and activity in colonic epithelial cells.

Developmental changes in peptide transport in the small intestine of the Yucatan miniature pig

Di/tripeptides in the small intestine (SI) are transported via the oligopeptide transporter PepT1. Our objectives were to characterize the activity of PepT1 across the length of the SI and to describe ontological changes in PepT1 activity in early life. A laparotomy was conducted in piglets at 1,2,3 and 6 wk of age (n=4 per group). Four 10 cm ligated SI segments were created with the first at 15 cm distal to the ligament of Trietz and at 25, 50, and 75% of SI length. 5 mM glycylsarcosine (gly‐sar) with 3H‐gly‐sar was perfused through each segment for 2 h. The perfusate was sampled every 10 min. Gly‐sar uptake was determined from 3H‐gly‐sar disappearance. Uptake in specific segments and between age groups were compared by ANOVA. In 1 wk piglets, greater dipeptide uptake occurred in the mid gut compared to the initial (p &lt; 0.05) and distal segments (75%) (p &lt; 0.05). In the mid gut, uptake was greater at 1 wk than at 2 and 3 wk (p &lt; 0.05). At 2 and 3 wk, uptake was greatest in the initial segment compared to the other three sites (p &lt;0.001). In 6 wk pigs, gly‐sar uptake was again greater in the mid gut compared to younger pigs (wk 2, p &lt; 0.001). These observations suggest that PepT1‐mediated amino acid uptake in the mid gut may be important during gut stress such as at birth and weaning. Identifying factors that can induce PepT1, and providing amino acids as dipeptides might provide a nutritional advantage when the SI is compromised. (NSERC)

Butyrate transcriptionally induces peptide transporter PepT1 expression and activity via Cdx2 and CREB transcription factors

Butyrate transcriptionally induces peptide transporter PepT1 expression and activity via Cdx2 and CREB transcription factors

Butyrate transcriptionally induces peptide transporter PepT1 expression and activity via Cdx2 and CREB transcription factors

Butyrate transcriptionally induces peptide transporter PepT1 expression and activity via Cdx2 and CREB transcription factors

Inhibitory Effect of Zinc on the Absorption of β-Lactam Antibiotic Ceftibuten via the Peptide Transporters in Rats

Zinc is an essential metal ion for the body, and is widely used for nutritional and clinical purposes. Previously, we showed that zinc inhibits the transport of glycylsarcosine via the intestinal peptide transporter PEPT1 in the human intestinal cell line Caco-2. In this study, we examined the effect of zinc on the activity of peptide transporters in rats using the oral beta-lactam antibiotic ceftibuten as a model drug. The plasma ceftibuten concentration after intraintestinal administration was decreased in the presence of zinc. The maximum plasma concentration (C(max)) was significantly decreased and the time required to reach C(max) (T(max)) was prolonged by zinc coadministration. The plasma ceftibuten concentration after iron coadministration or two hours after zinc administration was not affected. The in situ loop technique revealed 50% inhibition of ceftibuten absorption by zinc. In conclusion, zinc inhibits the transport activity of PEPT1 in vivo as well in vitro.

Long-Term Effect of Leptin on H+-Coupled Peptide Cotransporter 1 Activity and Expression in Vivo: Evidence in Leptin-Deficient Mice

The H+-coupled peptide cotransporter 1 (PepT1) mediates absorption of peptides and peptidomimetic drugs. Acute luminal leptin was reported to induce translocation of PepT1 to the enterocyte membrane in vitro and in vivo in the rat, resulting in enhanced peptide and peptidomimetic drug absorption. In this study, we analyzed chronic effects of leptin and leptin deficiency on PepT1 activity and expression in the small intestine. Wistar rats and ob/ob mice were used. Activity of PepT1 was determined by monitoring [3H]glycyl-sarcosine (Gly-Sar) transport using the jejunal loop method. The levels of PepT1 mRNA and protein were quantified by real-time quantitative reverse transcription-polymerase chain reaction and Western blot analysis, respectively. Induction of chronic hyperleptinemia in rats (1 microg/g/day for 7 days; subcutaneous continuous infusion), caused a significant 25% increase (P < 0.05 versus control) in Gly-Sar transport and uptake. This effect was associated with a significant 2-fold increase in the abundance of PepT1 protein and a 6-fold increase in the levels of PepT1 mRNA. In the leptin-deficient ob/ob mice, PepT1 activity and expression were significantly reduced, and replacement of leptin (10 microg/day for 7 days; subcutaneous continuous infusion) completely restored full PepT1 expression and activity. Moreover, we showed that a 7-day challenge of the Caco-2 cells with 0.2 nM leptin induced a significant increase in PepT1 activity and protein expression, arguing for a direct action. These data demonstrate, for the first time, an impaired activity/expression of PepT1 in leptin-deficient ob/ob mice that could be restored by leptin replacement. These findings may have relevance in modulation of dietary nitrogen supply and PepT1 substrate bioavailability in obesity.

Leptin Transcriptionally Enhances Peptide Transporter (hPepT1) Expression and Activity via the cAMP-response Element-binding Protein and Cdx2 Transcription Factors

PepT1 is an intestinal epithelial apical membrane transporter that is expressed in the small intestine, with little or no expression in the normal colon. However, we previously demonstrated that colonic PepT1 may be expressed during chronic inflammation. To begin elucidating inflammatory hPepT1 signaling, we herein investigated the long term leptin treatments, on PepT1 expression and activity in Caco2-BBE cells, and began to reveal the involved signaling pathways. We successfully cloned the 723-bp hPepT1 promoter region and identified the human transcription initiation site 86 bp upstream from the translation start site. Leptin treatment dose- and time-dependently increased hPepT1 promoter and transport activities in Caco2-BBE cells, with maximal activity observed in cells treated with 100 nM leptin for 8 h. Under these conditions, we observed 2-fold increases in hPepT1 mRNA and protein expression, as well as increased transport activity. Our molecular analyses of possible signal-transduction pathways revealed that leptin treatment enhanced the intracellular levels of cAMP and phosphorylated cAMP-response element-binding protein (CREB) protein in Caco2-BBE cells, whereas our deletion, mutation, and CDX2 overexpression analyses demonstrated that interaction of the Cdx2 and phosphorylated CREB transcription factors was essential for leptin-induced hPepT1 transcription in Caco2-BBE cells. Our results indicate that leptin, which is increased in inflamed colonic mucosa, triggers colonic expression of hPepT1 via the CREB and Cdx2 transcription factors. These findings provide important new insights into the mechanisms of intestinal inflammation and may suggest new therapeutic modalities in the future.

Tumor necrosis factor-α and interferon-γ increase PepT1 expression and activity in the human colon carcinoma cell line Caco-2/bbe and in mouse intestine

A major mechanism for apical peptide absorption by small intestine is via the proton-coupled transporter PepT1. PepT1 is expressed at a high level in proximal small intestine, but it is not expressed in the healthy colon. However, in chronic states of intestinal inflammation, such as in Crohn's disease and ulcerative colitis, PepT1 expression in colonic epithelia is increased, serving as a pathway for entry of bacteria-derived molecules such as muramyl dipeptide (MDP) and fMet-Leu-Phe (fMLP). As little is known of how inflammation induces PepT1, we investigated whether or not inflammatory cytokines and mediators such as interleukins (IL)-1beta, IL-2, IL-8, IL-10, tumor necrosis factor-alpha, (TNF-alpha) and interferon-gamma (IFN-gamma ) up-regulate PepT1 activity and expression. Uptake of the PepT1 substrate glycylsarcosine [(3)H]-Gly-Sar was studied in vitro in the human colon carcinoma cell line Caco2/bbe monolayers as well as in vivo in mice injected with cytokines. TNF-alpha and IFN-gamma increased the activity, and total and apical membrane protein expression of PepT1 protein in a concentration- and time-dependent fashion. No changes in PepT1 mRNA were observed, suggesting post-transcriptional regulation. All three cytokines increased PepT1 protein expression in mouse proximal and distal colon but not in jejunum or ileum. TNF-alpha and IFN-gamma, but not IL-1beta, increased Gly-Sar uptake in mouse proximal and distal colon; however, no changes were observed in the small intestine with any cytokine treatment. Whereas neither TNF-alpha nor IFN-gamma increased PepT1 mRNA expression in any segment of the intestine, treatment with IL-1beta increased PepT1 mRNA expression in mouse proximal and distal colon and decreased PepT1 mRNA expression in jejunum and ileum. Since PepT1 transports bacteria-derived peptides, the up-regulation of protein expression and activity observed after treatment with TNF-alpha or IFN-gamma may play a role in activating host responses in involved colon.

Extracellular Glucose Concentration Alters Functional Activity of the Intestinal Oligopeptide Transporter (PepT-1) in Caco-2 Cells

The objective of this study was to determine the effect of different cell culture media glucose concentrations on the functional activity of PepT-1 in Caco-2 cells. Uptake kinetics of Gly-Sar into Caco-2 cells that were maintained in iso-osmotic media containing 25 or 5.5 mM glucose were determined in the presence and absence of amino acid-selective chemical modifiers and dithiothreitol. Inhibition of Gly-Sar uptake into Caco-2 cells was measured in the presence of dipeptides and xenobiotics exhibiting various binding affinities for the PepT-1. The effect of extracellular glucose on PepT-1 gene expression was assessed using comparative RT-PCR. Long-term exposure of Caco-2 cells to 25 mM glucose reduced maximum transport capacity for Gly-Sar uptake without altering PepT-1 gene expression. In contrast, binding affinity of Gly-Sar and other dipeptides or xenobiotics was not significantly changed. Chemical modification of Lys and Tyr residues decreased V(max), while Cys modification increased the maximum transport capacity of the carrier. Preincubation of Caco-2 cells with dithiothreitol restored PepT-1 activity in cells maintained at 25 mM glucose. In conclusion, cell culture media containing 25 mM glucose decreases maximum transport capacity of PepT-1 in Caco-2 cells without affecting substrate recognition, at least in part, mediated via an oxidative pathway.

Regulation of oligopeptide transporter (Pept-1) in experimental diabetes.

The knowledge of expression and biology of the intestinal oligopeptide transporter (Pept-1) in a metabolic disorder such as diabetes may have nutritional and pharmacological implications. To study this problem, rats were made diabetic by streptozotocin injection, and Western and Northern blot analyses and nuclear run-on assay were used to determine the protein and gene expressions of Pept-1 and its rate of transcription, respectively. Uncontrolled diabetes for 96 h increased the activity of Pept-1 in the brush-border membrane of intestinal mucosa. Studies of Michaelis-Menten constant, maximal velocity, and protein expression of Pept-1 indicated that an increase in the abundance of this transporter was responsible for the increased activity. Studies of the gene expression showed that uncontrolled diabetes increased the abundance of mRNA encoding Pept-1 without altering its rate of transcription. Lastly, studies of the specificity of the above effect showed that uncontrolled diabetes similarly affected the protein and gene expressions of Pept-1 located in the kidney. In conclusion, the data show that 1) uncontrolled diabetes has a tropic effect on Pept-1 and 2) the effect is systemic, and its molecular mechanism appears to be an increase in the stabilization of mRNA encoding Pept-1.

Thyroid hormone regulates the activity and expression of the peptide transporter PEPT1 in Caco-2 cells.

An oligopeptide transporter (PEPT1) in the small intestine plays an important role in the absorption of small peptides and peptide-like drugs. We examined the effect of thyroid hormone 3,5,3'-L-triiodothyronine (T(3)) on the activity and expression of PEPT1 in human intestinal Caco-2 cells. Treatment of Caco-2 cells with T(3) inhibited [(14)C]glycylsarcosine uptake in a time- and dose-dependent manner. [(14)C]glycylsarcosine uptake was reduced by pretreatment of the cells with 100 nM T(3) for 4 days (67% of control value), whereas methyl-alpha-D-[U-(14)C]glucopyranoside and [(3)H]threonine uptake were not decreased. Kinetic analysis showed that T(3) treatment significantly decreased the maximum uptake (V(max)) value for [(14)C]glycylsarcosine uptake but had no effect on the K(m) value. Moreover, T(3) treatment caused a significant decrease in the amount of PEPT1 mRNA (25% of the control). Western blotting indicated that the amount of PEPT1 protein in the apical membrane was decreased (70% of the control). These findings indicate that T(3) treatment inhibits the uptake of [(14)C]glycylsarcosine by decreasing the transcription and/or stability of PEPT1 mRNA.

Effect of Lipopolysaccharide on Peptide Transporter 1 Expression in Rat Small Intestine and Its Attenuation by Dexamethasone

Background/Aim: The peptide transporter (PepT1) activity is upregulated or preserved under certain pathological conditions. The aim of this study was to investigate the PepT1 expression during lipopolysaccharide (LPS) treatment and the effect of dexamethasone on PepT1 expression. Methods: Rats were injected daily for 3 days with (1) LPS; (2) LPS plus dexamethasone; (3) dexamethasone, and (4) saline only. Mucosal specimens were used for Northern blot and Western blot analyses. Tissues of small intestine were taken for histological studies. In addition, tumor necrosis factor alpha and interleukin 1β levels were determined 3 h after single injections of the above mediators. Results: Northern blot analysis revealed that LPS treatment significantly decreased the expression of mRNA for PepT1 (32–62% of controls) at different time points. In the LPS + dexamethasone group, it was 66–95% of controls. The protein level of PepT1 in the jejunum was consistent with the mRNA expression level. Imunohistochemistry also showed a reduction of PepT1 immunoreactivity in the LPS group. LPS treatment resulted in increased tumor necrosis factor alpha and interleukin 1β levels, but dexamethasone treatment profoundly counteracted the cytokine production. Conclusions: This is the first report that LPS treatment reduces PepT1 expression in the rat small intestine. Short-term treatment with dexamethasone attenuated the effects of LPS by decreasing the cytokine levels.

Functional Roles of Histidine and Tyrosine Residues in the H+-Peptide Transporter PepT1

Histidyl residues in peptide transporters PepT1 and PepT2 are believed to participate in proton and substrate binding and to be crucial to the transporters' functional activities. In the present study, we performed mutagenesis of rabbit PepT1. We mutated three histidine residues (H57, H111, and H121) predicted to reside in transmembrane segments, as well as tyrosine residues adjacent to H57. Functional analysis of wild-type and mutant PepT1 expressed in Xenopus oocytes, using both the radiotracer methods and two-microelectrode voltage-clamping, revealed that not only the H57 but also the aromatic residues near H57 were essential for the normal function of PepT1, in agreement with the concept that aromatic residues stabilize the charge on H+ when interacting with H57. While mutagenesis at H111 did not significantly affect the activity of PepT1, mutagenesis at H121 had profound implications. The substrate affinities for H121 mutants were decreased depending both on the charge of the substrate and the charge on the substituted residues at position 121. We propose that H57 and H121 are intimately involved in the binding of the coupling ion H+ and the recognition of transportable peptide substrates, respectively.