Characterization of an oligopeptide transporter in renal lysosomes

Renal lysosomes play a major role in catabolism of plasma proteins. Final products of this catabolism include dipeptides and tripeptides that must be exported to the cytosol for hydrolysis. The aim of the present study was to determine whether an oligopeptide transporter is present in the renal lysosomal membrane that could mediate this export. The existence of an oligopeptide transporter was probed with the uptake of glycylglutamine (Gly-Gln) by membrane vesicles prepared from renal lysosomes. Kinetic analysis showed the presence of a single transporter with a K(m) of 8.77 mM for the uptake of Gly-Gln. The Gly-Gln uptake was energized by the imposition of an inwardly directed proton gradient (pH(out) 5.0/pH(in) 7.3) and membrane potential (outside positive/inside negative) resulting in overshoot. The Gly-Gln uptake was inhibited by the presence of dipeptides and tripeptides, but not amino acids. Western blot analysis of lysosomal membrane proteins with Pept-1 (an oligopeptide transporter) antibody as the probe showed the presence of an immunoreactive protein. This immunoreaction was abolished when the antiserum was preabsorbed with the Pept-1 epitope (0.5 microg/ml). In conclusion, the present data show the existence of a low-affinity dipeptide transporter in the renal lysosomal membrane that appears to belong to the Pept family of transporters. The function of this transporter appears to be to prevent accumulation of dipeptides in renal lysosomes.     

Hormonal regulation of oligopeptide transporter Pept-1 in a human intestinal cell line

The intestinal oligopeptide transporter (cloned as Pept-1) hasmajor roles in protein nutrition and drug therapy. A key unstudied question is whether expression of Pept-1 is hormonally regulated. Inthis experiment, we investigated whether insulin has such a role. Weused a human intestinal cell monolayer (Caco-2) as the in vitro modelof human small intestine and glycylglutamine (Gly-Gln) as the modelsubstrate for Pept-1. Results showed that addition of insulin at aphysiological concentration (5 nM) to incubation medium greatlystimulates Gly-Gln uptake by Caco-2 cells. This stimulation was blockedwhen genistein, an inhibitor of tyrosine kinase, was added toincubation medium. Studies of the mechanism of insulin stimulationshowed the following. 1) Stimulationoccurred promptly (30-60 min) after exposure to insulin.2) There was no significant changein the Michaelis-Menten constant of Gly-Gln transport, but there was anearly twofold increase in its maximal velocity.3) Insulin effect persisted evenwhen Golgi apparatus, which is involved in trafficking of newlysynthesized Pept-1, was dismantled.4) However, there was completeelimination of insulin effect by disruption of microtubules involved intrafficking of preformed Pept-1. 5)Finally, with insulin treatment, there was no change in Pept-1 geneexpression, but the amount of Pept-1 protein in the apical membrane wasincreased. In conclusion, the results show that insulin, when it bindsto its receptor, stimulates Gly-Gln uptake by Caco-2 cells byincreasing the membrane population of Pept-1. The mechanism appears tobe increased translocation of this transporter from a preformedcytoplasmic pool.     

The effect of peptide absorption on PepT1 gene expression and digestive system hormones in rainbow trout (Oncorhynchus mykiss)

The present study evaluates the effect of protein source (dipeptides, free amino acids, and intact protein) on development and growth of Salmonid fish alevin. Specifically, we follow the expression of oligopeptide transporter protein PepT1 in the intestine of rainbow trout (Oncorhynchus mykiss). Fish were fed exogenously one of four diets: three formulated (lysyl–glycine dipeptide supplemented diet — PP, free lysine and glycine supplemented diet — AA, control diet with no lysine — CON) or commercial starter (Aller Futura — AF). Fish increased mean body weight 8 fold with PP- and AA-supplemented diets resulting in significantly higher weight gain than fish fed CON. Statistical analysis revealed a significant increase in relative PepT1 expression of fish fed experimental diets. Immunohistochemical staining with PepT1 antibody showed the presence of the transporter protein in the brush border membrane of the proximal intestinal enterocytes of fish from all experimental groups. Leptin immunoreactivity occurred not only in the gastric glands but also in proximal intestine and pyloric caeca of fish fed PP, AA and AF diets. Leptin immunoreactivity was also observed in hepatocyte cytoplasm and pancreatic acinar cells. Gastrin/CCK immunoreactive cells were present in the proximal intestine and pyloric caeca.     

Environmental and nutritional regulation of expression and function of two peptide transporter (PepT1) isoforms in a euryhaline teleost

Expression and function of the oligopeptide transporter PepT1 in response to changes in environmental salinity have received little study despite the important role that dipeptides play in piscine nutrition. We cloned and sequenced two novel full-length cDNAs that encode Fundulus heteroclitus PepT1-type oligopeptide transporters, and examined their expression and functional properties in freshwater- and seawater-acclimated fish and in response to fasting and re-feeding. Phylogenetic analysis of vertebrate SLC15A1 sequences confirms the presence of two PepT1 isoforms, named SLC15A1a and SLC15A1b, in fish. Similar to other vertebrate SLC15A1s, these isoforms have 12 transmembrane domains, and amino acids essential for PepT1 function are conserved. Expression analysis revealed novel environment-specific expression of the SLC15A1 isoforms in F. heteroclitus, with only SLC15A1b expressed in seawater-acclimated fish, and both isoforms expressed in freshwater-acclimated fish. Fasting and re-feeding induced changes in the expression of SLC15A1a and SLC15A1b mRNA. Short-term fasting resulted in up-regulation of PepT1 mRNA levels, while prolonged fasting resulted in down-regulation. The resumption of feeding resulted in up-regulation of PepT1 above pre-fasted levels. Experiments using the in vitro gut sac technique suggest that the PepT1 isoforms differ in functional characteristics. An increased luminal pH resulted in decreased intestinal dipeptide transport in freshwater-acclimated fish but suggested an increased dipeptide transport in seawater-acclimated fish. Overall, this is the first evidence of multiple isoforms of PepT1 in fish whose expression is environmentally dependent and results in functional differences in intestinal dipeptide transport.     

Characterization of oligopeptide transporter (PepT1) in grass carp (Ctenopharyngodon idella)

The oligopeptide transporter (PepT1) is located on the brush-border membrane of the intestinal epithelium, and plays an important role in dipeptide and tripeptide absorptions from protein digestion. In this study, we cloned the PepT1 cDNA from grass carp and characterized its expression profile in response to dietary protein and feed additives (sodium butyrate) treatments. The PepT1 gene encodes a protein of 714 amino acids with high sequence similarity with other vertebrate homologues. Expression analysis revealed highest levels of PepT1 mRNA expression in the foregut of grass carp. In addition, PepT1 mRNA expression exhibited diurnal variation in all three bowel segments of intestine with lower levels of expression in daytime than nighttime. During embryonic development, PepT1 showed a dynamic pattern of expression reaching maximal levels of expression in the gastrula stage and minimal levels in the organ stage. The PepT1 expression showed constant levels from 14 to 34 day post-hatch. To determine whether fish diet of different protein contents may have any effect on PepT1 expression, we extended our research to dietary regulation of PepT1 expression. We found that dietary protein levels had a significant effect on PepT1 gene expression. In addition, PepT1 mRNA levels were higher after feeding with fish meal than with soybean meal. Moreover, in vitro and in vivo sodium butyrate treatments increased PepT1 expression in the intestine of grass carp. The results demonstrate for the first time that PepT1 mRNA expression is regulated in a temporal and spatial pattern during development, and dietary protein and feed additives had a significant effects on PepT1 gene expression in grass carp.     

Pgt1, a glutathione transporter from the fission yeast Schizosaccharomyces pombe

The Schizosaccharomyces pombe ORF, SPAC29B12.10c, a predicted member of the oligopeptide transporter (OPT) family, was identified as a gene encoding the S. pombe glutathione transporter ( Pgt1 ) by a genetic strategy that exploited the requirement of the cys1a Δ strain of S. pombe (which is defective in cysteine biosynthesis) for either cysteine or glutathione, for growth. Disruption of the ORF in the cys1a Δ strain led to an inability to grow on glutathione as a source of cysteine. Cloning and subsequent biochemical characterization of the ORF revealed that a high-affinity transporter for glutathione ( K _m=63 μM) that was found to be localized to the plasma membrane. The transporter was specific for glutathione, as significant inhibition in glutathione uptake could be observed only by either reduced or oxidized glutathione, or glutathione conjugates, but not by dipeptides or tripeptides. Furthermore, although glu–cys–gly, an analogue of glutathione (γ-glu–cys–gly), could be utilized as a sulphur source, the growth was not Pgt1 dependent. This further underlined the specificity of this transporter for glutathione. The strong repression of pgt1⁺ expression by cysteine suggested a role in scavenging glutathione from the extracellular environment for the maintenance of sulphur homeostasis in this yeast.     

A novel pineal-specific product of the oligopeptide transporter PepT1 gene: circadian expression mediated by cAMP activation of an intronic promoter

The oligopeptide transporter 1, PepT1, is a member of the Slc15 family of 12 membrane-spanning domain transporters; PepT1 has proton/peptide cotransport activity and is selectively expressed in intestinal epithelial cells, where it is responsible for the nutritional absorption of di- and tri-peptides. Here, a novel PepT1 gene product has been identified in the rat pineal gland, termed pgPepT1. It encodes a 150-amino acid protein encompassing the C-terminal 3 membrane-spanning domains of intestinal PepT1 protein, with 3 additional N-terminal residues. Expression of pgPepT1 appears to be restricted to the pineal gland and follows a marked circadian pattern with >100-fold higher levels of mRNA occurring at night; this is accompanied by an accumulation of membrane-associated pgPepT1 protein ( approximately 16 kDa). The daily rhythm in pgPepT1 mRNA is regulated by the well described neural pathway that controls pineal melatonin production. This includes the retina, the circadian clock in the suprachiasmatic nucleus, central structures, and projections from the superior cervical ganglia; activation of this pathway results in the release of norepinephrine. Here it was found that pgPepT1 expression is mediated by a norepinephrine-->cyclic AMP mechanism that activates an alternative promoter located in intron 20 of the gene. pgPepT1 protein was found to have transporter-modulator activity; it could contribute to circadian changes in pineal function through this mechanism.     

Luminal leptin enhances CD147/MCT-1-mediated uptake of butyrate in the human intestinal cell line Caco2-BBE

In the intestine, butyrate constitutes the major energy fuel for colonocytes. However, little is known about the transport of butyrate and its regulation in the intestine. In this study we demonstrate that the monocarboxylate transporter (MCT-1) is apically polarized in model human intestinal epithelia and is involved in butyrate uptake by Caco2-BBE cell monolayers. The butyrate uptake by Caco2-BBE cell monolayers displayed conventional Michaelis-Menten kinetics and was found to be pH-dependent, Na(+)-independent, 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid-insensitive, and inhibited by the monocarboxylate transporter inhibitor alpha-cyano-4-hydroxycinnamate and by an excess of unlabeled butyrate. We show that MCT-1 associates with CD147 at the apical plasma membrane in Caco2-BBE cell monolayers. Using antisense CD147, we demonstrate that the association of CD147 with MCT-1 is critical for the butyrate transport activity. Interestingly, we show for the first time hormonal regulation of CD147/MCT-1 mediated butyrate uptake. Specifically, luminal leptin significantly up-regulates MCT-1-mediated butyrate uptake by increasing its maximal velocity (V(max)) without any modification in the apparent Michaelis-Menten constant (K(m)). Finally, we show that luminal leptin up-regulates butyrate uptake in Caco2-BBE monolayers by two distinct actions: (i) increase of the intracellular pool of MCT-1 protein without affecting CD147 expression and (ii) translocation of CD147/MCT-1 to the apical plasma membrane of Caco2-BBE cell monolayers.     

Functional expression of the intestinal peptide-proton co-transporter in Xenopus laevis oocytes.

The expression of the intestinal peptide-proton cotransporter was examined in Xenopus laevis oocytes by microinjection of poly(A)+ mRNA prepared from rabbit intestinal mucosal cells. The concomitant expression of the glucose-sodium co-transporter was used as the control for the effectiveness of the expression technique. There was significant endogenous activity of Gly-Sar uptake in water-injected oocytes, but the uptake activity increased nearly 3-fold in poly(A)+ mRNA-injected oocytes. The expression of the peptide transporter was time-dependent. There was no detectable expression on day 1 after injection. The expression became noticeable on day 2 and increased with time, reaching a maximum on day 4. There was no further change on days 5 and 6. The endogenous uptake rate measured in water-injected oocytes, on the contrary, showed a slight decrease during this time. The expressed peptide transporter retained its substrate specificity, having affinity for the dipeptides, Gly-Sar and Gly-Pro, and no or little affinity for the free amino acids, Gly and Sar. The expressed peptide transporter also showed a dependence on a transmembrane H+ gradient for maximal activity. These data demonstrate that the mammalian intestinal peptide-proton co-transporter can be successfully expressed in Xenopus laevis oocytes. This expression system can provide an effective assay procedure to clone the gene encoding the transporter.     

Evidence for two different broad-specificity oligopeptide transporters in intestinal cell line Caco-2 and colonic cell line CCD841

Recently the existence of two different Na(+)-coupled oligopeptide transport systems has been described in mammalian cells. These transport systems are distinct from the previously known H(+)/peptide cotransporters PEPT1 and PEPT2, which transport only dipeptides and tripeptides. To date, the only peptide transport system known to exist in the intestine is PEPT1. Here we investigated the expression of the Na(+)-coupled oligopeptide transporters in intestinal cell lines, using the hydrolysis-resistant synthetic oligopeptides deltorphin II and [d-Ala(2),d-Leu(5)]enkephalin (DADLE) as model substrates. Caco-2 cells and CCD841 cells, both representing epithelial cells from human intestinal tract, were able to take up these oligopeptides. Uptake of deltorphin II was mostly Na(+) dependent, with more than 2 Na(+) involved in the uptake process. In contrast, DADLE uptake was only partially Na(+) dependent. The uptake of both peptides was also influenced by H(+) and Cl(-), although to a varying degree. The processes responsible for the uptake of deltorphin II and DADLE could be differentiated not only by their Na(+) dependence but also by their modulation by small peptides. Several dipeptides and tripeptides stimulated deltorphin II uptake but inhibited DADLE uptake. These modulating small peptides were, however, not transportable substrates for the transport systems that mediate deltorphin II or DADLE uptake. These two oligopeptide transport systems were also able to take up several nonopioid oligopeptides, consisting of 9-17 amino acids. This represents the first report on the existence of transport systems in intestinal cells that are distinct from PEPT1 and capable of transporting oligopeptides consisting of five or more amino acids.     

Molecular and cellular regulation of glucose transporter (GLUT) proteins in cancer

Malignant cells are known to have accelerated metabolism, high glucose requirements, and increased glucose uptake. Transport of glucose across the plasma membrane of mammalian cells is the first rate-limiting step for glucose metabolism and is mediated by facilitative glucose transporter (GLUT) proteins. Increased glucose transport in malignant cells has been associated with increased and deregulated expression of glucose transporter proteins, with overexpression of GLUT1 and/or GLUT3 a characteristic feature. Oncogenic transformation of cultured mammalian cells causes a rapid increase of glucose transport and GLUT1 expression via interaction with GLUT1 promoter enhancer elements. In human studies, high levels of GLUT1 expression in tumors have been associated with poor survival. Studies indicate that glucose transport in breast cancer is not fully explained by GLUT1 or GLUT3 expression, suggesting involvement of another glucose transporter. Recently, a novel glucose transporter protein, GLUT12, has been found in breast and prostate cancers. In human breast and prostate tumors and cultured cells, GLUT12 is located intracellularly and at the cell surface. Trafficking of GLUT12 to the plasma membrane could therefore contribute to glucose uptake. Several factors have been implicated in the regulation of glucose transporter expression in breast cancer. Hypoxia can increase GLUT1 levels and glucose uptake. Estradiol and epidermal growth factor, both of which can play a role in breast cancer cell growth, increase glucose consumption. Estradiol and epidermal growth factor also increase GLUT12 protein levels in cultured breast cancer cells. Targeting GLUT12 could provide novel methods for detection and treatment of breast and prostate cancer.     

Functional characterization of the chicken peptide transporter 1 (pept1, slc15a1) gene

Dietary amino acids can be transported into intestinal epithelial cells as di- and tripeptides by the action of the peptide transporter, PepT1 (SLC15A1). Expression of the chicken PepT1 (cPepT1) gene changes in response to dietary crude protein level; however, the molecular mechanism governing this regulation is unknown. This study analyzed the promoter region of the cPepT1 gene. Using deletion analysis, positive-acting (-314 to -261, -169 to -155, and -120 to -60) and negative-acting (-419 to -386 and -214 to -169) regions were mapped in transfected chick embryo fibroblasts (CEF). The addition of neither amino acids Phe, Arg, or Val, nor the dipeptides Gly-Sar (glycyl-sarcosine), Gly-Pro, Gly-Phe, Met-Pro, Met-Lys or Lys-Lys, had an effect on cPepT1 promoter activity in transfected CEF. The cPepT1 promoter was more active in CEF and primary chicken intestinal cells than in chicken liver cells. This study represents a functional characterization of the molecular regulation of the chicken PepT1 gene.     

Curcumin prevents leptin-induced tight junction dysfunction in intestinal Caco-2 BBe cells

Maintaining tight junction (TJ) integrity in the intestine is critical for nutrient absorption, host defense, and host immunity. While leptin secreted from adipose tissue is associated with obesity and obesity-related intestinal inflammation, the role of luminal leptin in intestinal TJ function is elusive. Here, we examined the role of leptin in intestinal TJ function in Caco-2 BBe cells and further explored the function of curcumin (CCM) in leptin-induced TJ dysfunction. Apical leptin, but not basolateral leptin, treatment at a concentration of 100 ng/ml deteriorated TJ function in Caco-2 BBe cells. Leptin-impaired TJ alteration was resulted from induction of leptin receptor-dependent JAK2/STAT3 signaling pathway and its-related PI3K/Akt/ERK1/2 signaling pathways. Apical leptin also lowered the expression levels of genes encoding TJ-associated proteins such as zonula occludens-3, claudin-5, and occludin, and elevated expression of pro-inflammatory genes such as IL-6 and TNF-α. Leptin-impaired TJ junction in Caco-2 BBe cells was blunted by a 30-min CCM pretreatment through inhibition of leptin receptor-dependent signaling pathway, and its-associated induction of expression of genes encoding TJ-associated proteins and pro-inflammatory cytokines. Our results elucidate a novel function of luminal leptin in intestinal TJ dysfunction, and further identify CCM as an effective dietary compound that prevents leptin-impaired TJ function in intestinal cells.