Interaction of amino acids with glycyl-glycine transport in the mammalian intestine

In order to investigate a possible interaction between free amino acids and dipeptides during their mucosal uptake in man and monkey, perfusion studies in vivo and uptake studiesin vitro using labelled and non-labelled dipeptides and amino acids have been carried out. In contrast to the observations of other workers, inhibition of glycyl-glycine uptake was observed with free leucine and methioninc but not with glycine, proline, hydroxyproline or alanine. Leucine and methionine caused inhibition of cytosol glycyl-glycine hydrolase activity, while glycine had no effect. The dipeptide uptake and dipeptide hydrolysis by cytosol enzyme was competitively inhibited by leucine. Although brush border glycyl-glycine hydrolase was also inhibited by leucine, the inhibition was noncompetitive. These data indicate that a few free amino acids can interact with dipeptides during uptake. This interaction might occur either at the transport step or at the stage of intracellular dipeptide hydrolysis

Escherichia coli, an Intestinal Microorganism, as a Biosensor for Quantification of Amino Acid Bioavailability

In animal diets optimal amino acid quantities and balance among amino acids is of great nutritional importance. Essential amino acid deficiencies have negative impacts on animal physiology, most often expressed in sub-optimal body weight gains. Over supplementation of diets with amino acids is costly and can increase the nitrogen emissions from animals. Although in vivo animal assays for quantification of amino acid bioavailability are well established, Escherichia coli-based bioassays are viable potential alternatives in terms of accuracy, cost, and time input. E. coli inhabits the gastrointestinal tract and although more abundant in colon, a relatively high titer of E. coli can also be isolated from the small intestine, where primary absorption of amino acids and peptides occur. After feed proteins are digested, liberated amino acids and small peptides are assimilated by both the small intestine and E. coli. The similar pattern of uptake is a necessary prerequisite to establish E. coli cells as accurate amino acid biosensors. In fact, amino acid transporters in both intestinal and E. coli cells are stereospecific, delivering only the respective biological l-forms. The presence of free amino- and carboxyl groups is critical for amino acid and dipeptide transport in both biological subjects. Di-, tri- and tetrapeptides can enter enterocytes; likewise only di-, tri- and tetrapeptides support E. coli growth. These similarities in addition to the well known bacterial genetics make E. coli an optimal bioassay microorganism for the assessment of nutritionally available amino acids in feeds

Structure-function relationships in the oligopeptide transporter PepT1.

The proton-dependent di- and tripeptide transporter PepT1 represents the major route of dietary amino acid intake in the intestine of many species. This transporter belongs to the solute carrier family SLC15 and because of its electrogenic properties it may be studied both through electrophysiological and radiotracer uptake experiments. In this work some functional and structural aspects of PepT1 have been investigated at the molecular level. In addition to the physiological relevance, understanding of the details of its mechanisms of operation is important since PepT1 appears to be involved in the absorption of many important, orally administered, drugs such as antibiotics, angiotensin-converting inhibitors, and antiviral agents. The electrophisyiological and biophysical properties of PepT1 expressed in Xenopus oocytes were investigated with two-electrode voltage-clamp. Most of the above functional observations were derived from uptake data, in absence of control of the membrane voltage, or from electrophysiological measurement of steady transport currents, in the presence of a dipeptide substrate. Important additional informations regarding the transport mechanism may arise from measurement of presteady-state currents, the electrophysiological signals that can be observed in absence of organic substrate, and that represent the first steps in the transport cycle. A unified kinetic model for PepT1 has been devised that can describe the different characteristics of the isoforms of different species, with respect to both presteady-state and transport-associated currents. Mutational studies have provided significant evidence for the functional role of some residues in PepT1. Particularly Arg282 and Asp341, in the transmembrane domain 7 and 8 of the transporter, have been reported to form a charge pair that may break and reform during the transport cycle. The attention has been focused on these two oppositely charged aminoacids to better understand their functional role in the absorption pathway. Finally, the functional and structural basis of reverse operation of wild-type and mutated forms of PepT1 have been studied. Mutants in the putative charge pair residues Arg282 and Asp341 of rabbit PepT1 have been shown exhibit properties useful to better understand the possibility of reverse transport. This reversed mode of operation may be either the effect or the cause of abnormal or pathological conditions

Molecular physiological studies on the peptide transporter-1, PepT1.

The peptide transporter-1, PepT1, is responsible for the uptake of peptides in the mammalian intestine after protein digestion. It has also been shown to transport a wide range of pharmacological agents; thus making it important in drug design and delivery. Many studies have addressed structure-function relationships in the transporter, using biochemical and uptake assays; however PepT1 is an electrogenic transporter whose activities could be more clearly studied using electrophysiological methods. In this work therefore, the transporter has been studied using the two electrode voltage clamp technique on the transporter expressed in Xenopus laevis oocytes. Temperature effects on the kinetics of the three isoforms studied (rabbit, zebrafish, seabass) is reported here with a suggestion of a possible structural adaptation in the transporter. Since the transporter is also of interest in animal feed formulation, transport characteristics of a range of dipeptide combinations are also presented; with a recommendation on the possible optimal lysine supplementation in feeds that require essential amino acids supplementation. Reverse operation in transporters may be the result of or lead to pathophysiological states. Experimental data here show that mutants in Arginine 282 and aspartate 341 exhibit properties that make them suitable as models to study possible reverse operation in PepT1. To understand the physiological significance of reverse operation, the structural and functional basis of this phenomenon was also explored

Dipeptide transport in Yucatan miniature piglet intestine

The PepT1 transporter is getting much attention in modem nutrition due to its universal substrate affinity for di/tripeptides which facilitates efficient transport of amino acids. As such, di/tripeptides may be an important component of amino acid nutrition because they are transported into enterocytes more efficiently than a mixture of free amino acids. However, limited data are available on the contribution of PepT1 to total peptide uptake. We studied the contribution of PepT1-mediated transport versus paracellular movement to total dipeptide uptake in intestinal samples excised from suckling piglets. Using an in vitro Ussing chamber model, we determined that PepT1 was responsible for 46% of the glycyl-sarcosine uptake by piglet jejunum and 73% of the glycyl-sarcosine uptake by piglet ileum; these values are lower than previously reported in cell culture models, suggesting that paracellular peptide uptake is quantitatively important in young piglets. -- PepT1 and amino acid transport systems both contribute to amino acid uptake by enterocytes, but the contribution of the different routes to overall amino acid absorption has not yet been defined. Furthermore, very little is known about the interaction between free amino acid and peptide uptake at the cellular level. Using an in vivo gut loop perfusion model in piglets, we demonstrated that arginine uptake was enhanced by 81% when perfused simultaneously with 20 mM lysyl-lysine, compared to control. In contrast, perfusing loops with equimolar lysyl-glycine did not alter arginine uptake. We speculated that enhanced uptake of arginine was likely due to trans-stimulation of rBAT/b⁰⁺ transporter. Dipeptides are taken up by enterocytes via PepT1 and are then hydrolyzed to release free lysine. High intracellular free lysine trans-stimulates the rBAT/b⁰⁺ anti-transporter to enhance arginine uptake. When lysyl-lysine was perfused with an amino peptidase inhibitor (amastatin), the potentiating effect was abolished, suggesting that this trans-stimulation activity was impeded by reducing intracellular hydrolysis of dipeptides. To the best of my knowledge we are the first to demonstrate the interaction between arginine absorption and lysine-containing dipeptides at the cellular level in an in situ model

Transport and utilization of arginine and arginine-containing peptides by rat alveolar macrophages

Purpose. To demonstrate that alveolar macrophages (AM) from rats exhibit pepT1-like transporter for the uptake of small arginine-containing peptides (ACPs) and utilized these peptides as direct substrates for nitric oxide (NO) production.;Method. A HPLC assay was developed for quantitative measurement of Arg and ACPs in rat plasma and bronchoalveolar lavage (BAL) fluid. The uptake of small peptides by rat AM was evaluated using fluorescein isothiocynate (FITC)-labeled (*) peptides (Arg-Lys*, beta-Ala-Lys*, and Gly-Sar-Lys*), HPLC analysis of potential peptide degradation, and known inhibitors on arginine (Arg) and PepT1 transport. NO production by AM through Arg and ACPs was studied with and without inhibition by transport inhibitors. The presence of PepT1-like transporter on AM was evaluated using antipepT1 antisera and Western blot analysis. The substrate specificity of Arg-Gly and Arg-Gly-Asp was determined using purified inducible nitric oxide synthase (iNOS). The availability of ACPs in the lung was determined by the HPLC analysis of plasma and (BAL) fluid.;Results. The FITC-labeled peptides were internalized by AM without degradation. Uptake of Arg-Lys*, beta-Ala-Lys*, and Gly-Sar* was blocked (∼50%) by cephradine, but not by Lys (an inhibitor on CAT-2B for arginine transport). The NO production by AM through ACPs was significantly blocked by PepT1 inhibitors and by an antiPepT1 antibody in a dose-dependent manner. These inhibitors had no effect on AM production of NO using Arg as a substrate. Arg-Gly and Arg-Gly-Asp were found to be direct substrates for iNOS with similar Km and Vmax values to those of Arg. But the production of NO by AM using ACPs as substrate was 2-fold higher than using Arg as a substrate. Both Arg-Gly and Arg-Gly-Asp were found in rat plasma and BAL fluid. The presence of a PepT1-like transporter on AM was confirmed by Western blot.;Conclusion. This study shows that AM exhibits PepT1-like transporter for small peptide uptake. ACPs, through PepT1-like transporter, can serve as direct substrates for AM production of NO, an important mediator on both protection the lung from bacteria infection and augments inflammation lung injury

Amino acids in the development of Prodrugs

Although drugs currently used for the various types of diseases (e.g., antiparasitic, antiviral, antibacterial, etc.) are effective, they present several undesirable pharmacological and pharmaceutical properties. Most of the drugs have low bioavailability, lack of sensitivity, and do not target only the damaged cells, thus also affecting normal cells. Moreover, there is the risk of developing resistance against drugs upon chronic treatment. Consequently, their potential clinical applications might be limited and therefore, it is mandatory to find strategies that improve those properties of therapeutic agents. The development of prodrugs using amino acids as moieties has resulted in improvements in several properties, namely increased bioavailability, decreased toxicity of the parent drug, accurate delivery to target tissues or organs, and prevention of fast metabolism. Herein, we provide an overview of models currently in use of prodrug design with amino acids. Furthermore, we review the challenges related to the permeability of poorly absorbed drugs and transport and deliver on target organs.NV acknowledges support from Fundação para a Ciência e Tecnologia (FCT, Lisbon, Portugal) and FEDER (European Union), award number IF/00092/2014/CP1255/CT0004. NV also thanks FCT for the IF position and Fundação Manuel António da Mota (FMAM, Porto, Portugal) and Pfizer (Portugal) for support for the Nuno Vale Research Group. The contents of this report are solely the responsibility of the authors and do not necessarily represent the official views of the FCT, FMAM and Pfizer

Studies of the Di/tripeptide Transporter in \u3cem\u3eSaccharomyces cerevisiae\u3c/em\u3e: The N-terminal Cytoplasmic Domain of Ptr2p is Involved in Post-Translational Regulation

Throughout nature cells use peptides as a source of nutrition. For microbes, an ability to utilize peptides is especially important in nitrogen-poor environments, as peptides can be catabolized for their use as a nitrogen source. The yeast Saccharomyces cerevisiae imports di/tripeptides from the environment using the peptide transporter Ptr2p. Cellular levels of Ptr2p are highest under poor-nitrogen conditions. Here we report that the addition of a rich nitrogen source to the growth medium results in a down-regulation of Ptr2p, wherein plasma membrane Ptr2p is ubiquitinated, endocytosed, and delivered to the vacuole for destruction. We report evidence that the N-terminal portion of Ptr2p that is cytoplasmic is involved in mediating this effect. Mutations to known phosphorylation sites (Y37, S39, and S45) and suspected ubiquitination sites (K27 and K34) on Ptr2p’s Nterminal region greatly impair both the normal turnover of Ptr2p and its down-regulation in response to a rich nitrogen source. The data presented support the notion that Ptr2p turnover from the cytoplasmic membrane requires phosphorylation and ubiquitination of a discrete N-terminal cytoplasmic domain

The role of peptide transporter 1 (PepT1) in maintaining health in neonates

Peptide transporter 1 (PepT1) carries dietary tri-/dipeptides, bacterial peptides and peptidomimetic drugs in the small intestine (SI). The first study in this thesis describes the interaction between the dipeptide lysyl-lysine and arginine uptake from the SI in Yucatan miniature piglets. This study confirmed that arginine uptake was significantly higher when delivered with lysyl-lysine compared to equimolar amount of free lysine. We speculated that more efficient uptake of lysine as a dipeptide led to enhanced arginine uptake through the trans-stimulation of the b⁰, ⁺ transporter. The activity of PepT1 was preserved during the use of parenteral nutrition (PN). As such, the objective of the second study was to determine if there were advantages to providing lysine as dipeptide in neonatal piglets with PN-induced atrophied gut. PN was provided for four days to induce intestinal atrophy and then piglets were switched to enteral diets. When lysine was provided at 75% of requirement, it was found that providing lysyl-lysine during the transition period from PN to enteral feeding improved villus height, mucosal weight, whole-body protein synthesis and reduced the MPO activity compared to free lysine. The addition of glycyl-sarcosine to competitively inhibit lysyl-lysine uptake completely abolished the beneficial effects of lysyl-lysine. Thus, delivering lysine as dipeptide during the high-risk transition period from PN to enteral feeding may serve to enhance gut recovery and avoid complications related to refeeding. Recently, researchers identified that PepT1 influences the secretion of peptide hormones from the enteroendocrine cells, which may affect appetite regulation and glucose homeostasis in adult rodents. The final part of the thesis characterized the role of PepT1 activation on the protein-mediated gut hormone release and glucose kinetics in neonatal piglets using an in-situ perfusion model. Duodenal casein hydrolysate infusion stimulated the release of incretin hormones GLP-1 and GIP as measured in the portal blood, and the PepT1 inhibitor 4-AMBA hindered this protein-mediated effect on gut hormone release, confirming the involvement of PepT1. Higher baseline plasma insulin and higher insulin response to a glucose challenge strongly suggest that PepT1 activity induces insulin secretion and faster glucose disposal. This mechanism may be advantageous for adults with type 2 diabetes, but in neonates taking in high protein from infant formulas, it contributes to the understanding of the “early protein hypothesis” of childhood obesity. Overall, these studies shed light on the understanding of the role of PepT1 in the neonatal small intestine and characterize novel influences on neonatal metabolism that may be important in conditions of health and disease