A Pectin-Rich, Baobab Fruit Pulp Powder Exerts Prebiotic Potential on the Human Gut Microbiome In Vitro.

Increasing insight into the impact of the gut microbiota on human health has sustained the development of novel prebiotic ingredients. This exploratory study evaluated the prebiotic potential of baobab fruit pulp powder, which consists of pectic polysaccharides with unique composition as compared to other dietary sources, given that it is rich in low methoxylated homogalacturonan (HG). After applying dialysis procedures to remove simple sugars from the product (simulating their absorption along the upper gastrointestinal tract), 48 h fecal batch incubations were performed. Baobab fruit pulp powder boosted colonic acidification across three simulated human adult donors due to the significant stimulation of health-related metabolites acetate (+18.4 mM at 48 h), propionate (+5.5 mM at 48 h), and to a lesser extent butyrate (0.9 mM at 48 h). Further, there was a trend of increased lactate levels (+2.7 mM at 6h) and reduced branched chain fatty acid (bCFA) levels (-0.4 mM at 48 h). While Bacteroidetes levels increased for all donors, donor-dependent increases in Bifidobacteria, Lactobacilli, and Firmicutes were observed, stressing the potential interindividual differences in microbial composition modulation upon Baobab fruit pulp powder treatment. Overall, Baobab fruit pulp powder fermentation displayed features of selective utilization by host microorganisms and, thus, has promising prebiotic potential (also in comparison with the 'gold standard' prebiotic inulin). Further research will be required to better characterize this prebiotic potential, accounting for the interindividual differences, while aiming to unravel the potential resulting health benefits.

Rapid and sustained systemic circulation of conjugated gut microbial catabolites after single-dose black tea extract consumption.

Gut microbial catabolites of black tea polyphenols (BTPs) have been proposed to exert beneficial cardiovascular bioactivity. This hypothesis is difficult to verify because the conjugation patterns and pharmacokinetics of these catabolites are largely unknown. The objective of our study was to identify, quantify, and assess the pharmacokinetics of conjugated BTP metabolites in plasma of healthy humans by means of an a priori untargeted LC-MS-based metabolomics approach. In a randomized, open, placebo-controlled, crossover study, 12 healthy men consumed a single bolus of black tea extract (BTE) or a placebo. The relative and, in several cases, absolute concentrations of a wide range of metabolites were determined using U(H)PLC-LTQ-Orbitrap-FTMS. Following BTE consumption, a kinetic response in plasma was observed for 59 BTP metabolites, 11 of these in a quantitative manner. Conjugated and unconjugated catechins appeared in plasma without delay, at 2-4 h, followed by a range of microbial catabolites. Interindividual variation in response was greater for gut microbial catabolites than for directly absorbed BTPs. The rapid and sustained circulation of conjugated catabolites suggests that these compounds may be particularly relevant to proposed health benefits of BTE. Their presence and effects may depend on individual variation in catabolic capacity of the gut microbiota.

Intragastric infusion of pea-protein hydrolysate reduces test-meal size in rats more than pea protein.

Because protein hydrolysates are digested faster than the corresponding proteins, they may increase or hasten the acute eating-inhibitory effect of protein. Potential mediating mechanisms include accelerated or greater release of satiating gut peptides and activation of metabolic signals that inhibit eating. We tested these hypotheses in adult male rats that were surgically equipped with intragastric (IG) cannulas and adapted to 30-min test meals at dark onset after 14-h food deprivation. Equiosmotic 12 ml loads of saline-urea control (C), 13.6% pea protein (PP), or 13.6% PP hydrolysate (PPH, DSM/DFS, Delft, The Netherlands) solutions were IG infused in 1 min just before test meals. PPH reduced test-meal size compared to C more than PP (-3.8±0.3 g vs. -2.6±0.4 g; P<0.0001). Plasma glutamate increased more after PPH than PP (P<0.0001). Plasma lactate, alanine, insulin, glucagon, GLP-1 and paracetemol (an index of gastric emptying) all increased similarly, and glucose decreased similarly, after PPH or PP. Finally, PPH still reduced test-meal size more than PP (-4.6±0.3 g vs. -3.1±0.4 g; P<0.001) in rats after subdiaphragmatic vagal deafferentation, indicating that abdominal vagal afferents are not necessary for the eating-inhibitory effects of PP and PPH and, by extension, that gut peptides whose satiating effects depend on intact vagal afferents (e.g., CCK and glucagon) do not play crucial roles. Thus, PPH reduced short-term food intake more than PP under our conditions, but the mechanism(s) involved remain unclear.

The steroid glycoside H.g.-12 from Hoodia gordonii activates the human bitter receptor TAS2R14 and induces CCK release from HuTu-80 cells.

Steroid glycosides extracted from the succulent plant Hoodia gordonii are suggested to have appetite-suppressant effects both in animals and humans. Yet, the mechanisms underlying the putative satiety action of Hoodia steroid glycosides are not fully understood. We found that H.g.-12, a steroid glycoside purified from H. gordonii extract, initiated cholecystokinin (CCK) secretion both ex vivo in rat intestine and in vitro in the human enteroendocrine (EC) cell line HuTu-80. CCK is known to exert central effects on appetite suppression via the vagus nerve which afferents terminate in the gut wall. Recent data show that G protein-coupled receptors signaling bitter taste (T2Rs) are expressed in both rodent and human gastrointestinal tract. It was further demonstrated that bitter sensing is functional in mouse STC-1 EC cells and leads to CCK secretion via increased intracellular Ca²(+) concentrations. Based on the bitter taste of H. gordonii purified extracts, we assessed whether H.g.-12 could activate human bitter receptors. The steroid glycoside activated selectively TAS2R7 and TAS2R14, both heterologously expressed in HEK 293 cells. Removing an essential structural feature from the steroid glycoside inhibited H.g.-12-induced Ca²(+) increase in TAS2R14-expressing HEK cells and abolished H.g.-12-induced CCK secretion from human EC cells. Similarly, a nonspecific bitter receptor antagonist abolished H.g.-12-induced CCK secretion in HuTu-80 cells. These results point to a potential route of action by which components of Hoodia might influence appetite control. Our data also provide additional evidence that bitter taste-sensing mechanisms are coupled to hormone release from EC cells in the intestine. Moreover, we identified a natural agonist of TAS2R7 and TAS2R14 for further studies on the role of bitter receptors in satiety control and food intake.

Current in vitro testing of bioactive peptides is not valuable.

Dietary peptides have been suggested to possess biological activity in vivo and could affect cardiovascular disease parameters, based on data derived from in vitro experiments. Isolated peptides are often tested in in vitro cellular assays or on heterologously expressed molecular target proteins. The stimulatory or inhibitory effect on target proteins in vitro has often been used as the justification to test these compounds directly in vivo. Unfortunately, this research approach has an inherent flaw. It neglects the poor absorption, distribution, metabolism, and excretion (ADME) properties of peptides resulting in low peptide bioavailability. Because peptides are prone to extensive hydrolysis in the gastrointestinal tract by stomach, small intestinal, and brush border peptidases, most of them do not reach the absorption stage in the duodenum and jejunum. Therefore, a valid research approach should include the demonstration of stability of the peptide toward luminal and brush border peptidases and evaluate its ADME properties. Surprisingly, only very few animal and human studies determined absolute concentrations and kinetics of bioactive peptides. These studies have shown the presence of selected peptides in plasma samples at pico- and nanomolar concentrations with fast elimination kinetics in the minute range. For the correct interpretation of results, it is advised that researchers refer to the data currently available concerning bioavailability and ADME properties in humans. Two mandatory criteria for future in vitro studies investigating potential biological activities of peptides should be using physiologically relevant concentrations and times.

Modeling of the relationship between dipeptide structure and dipeptide stability, permeability, and ACE inhibitory activity.

Selected di- and tripeptides exhibit angiotensin-I converting enzyme (ACE) inhibitory activity in vitro. However, the efficacy in vivo is most likely limited for most peptides due to low bioavailability. The purpose of this study was to identify descriptors of intestinal stability, permeability, and ACE inhibitory activity of dipeptides. A total of 228 dipeptides were synthesized; intestinal stability was obtained by in vitro digestion, intestinal permeability using Caco-2 cells and ACE inhibitory activity by an in vitro assay. Databases were constructed to study the relationship between structure and activity, permeability, and stability. Quantitative structure-activity relationship (QSAR) modeling was performed based on computed models using partial least squares regression based on 400 molecular descriptors. QSAR modeling of dipeptide stability revealed high correlation coefficients (R > 0.65) for models based on Z and X scales. However, amino acid (AA) clustering showed the best results in describing stability of dipeptides. The N-terminal AA residues Asp, Gly, and Pro as well as the C-terminal residues Pro, Ser, Thr, and Asp stabilize dipeptides toward luminal enzymatic peptide hydrolysis. QSAR modeling did not reveal significant correlation models for intestinal permeability. 2D-fingerprint models were identified describing ACE inhibitory activity of dipeptides. The intestinal stability of 12 peptides was predicted. Peptides were synthesized and stability was confirmed in simulated digestion experiments. Based on the results, specific dipeptides can be designed to meet both stability and activity criteria. However, postabsorptive ACE inhibitory activities of dipeptides in vivo are most likely limited due to the very low intestinal permeability of dipeptides.

Intragastric layering of lipids delays lipid absorption and increases plasma CCK but has minor effects on gastric emptying and appetite.

Intestinal intubation studies have demonstrated that lipids induce satiety, but the contribution of lipid processing by the stomach on satiety remains poorly understood. In this explorative, randomized, placebo-controlled, crossover study we tested whether delayed lipid absorption, increased cholecystokinin (CCK), decelerated gastric emptying (GE), and increased satiety can be achieved by controlling lipid distribution in the stomach. Six healthy men were intubated nasogastrically. Two treatments were performed and repeated in duplicate. In the oil-on-top treatment (OT), subjects received a fat-free liquid meal (LM, 325 ml, 145 kcal) followed by intragastric infusion of 4 g of high-oleic-acid rapeseed oil (4.6 ml, 36 kcal) labeled with 77 mg glyceryl-[(13)C]trioleate. In the emulsion treatment (EM, control), 4 g of labeled rapeseed oil was incorporated into the LM (325 ml, 181 kcal); 4.6 ml of saline was infused as a control. In OT and EM a second LM was consumed at time t = 270 min. Plasma (13)C-C18:1, CCK and satiety were measured over 480 min. GE was determined by the paracetamol absorption test. OT delayed oleic acid absorption shown by an increased lag time of absorption (EM: 37 +/- 7 min; OT: 75 +/- 10 min; P < 0.01) and time at maximum concentration (EM: 162 +/- 18 min; OT: 280 +/- 33 min; P = 0.01). OT released more CCK than EM (P = 0.03), including increased CCK after the second meal. OT accelerated initial GE until 30 min postprandial. OT showed a tendency (P = 0.06) to suppress hunger and increase satiety and fullness 120-270 min postprandially. The results demonstrate that low amounts of lipids, when separated from the aqueous phase of a meal, delay lipid absorption and increase CCK. An escalating-dose study should determine whether this could have implications for the development of weight-control foods.

The angiotensin converting enzyme inhibitory tripeptides Ile-Pro-Pro and Val-Pro-Pro show increasing permeabilities with increasing physiological relevance of absorption models.

Transepithelial transport of the ACE inhibitory peptides Ile-Pro-Pro and Val-Pro-Pro was studied in different models of absorption. Apparent permeability (P(app)) values for absorptive transport across Caco-2 monolayers were 1.0+/-0.9 x 10(-8) (Ile-Pro-Pro) and 0.5+/-0.1 x 10(-8)cms(-1) (Val-Pro-Pro). Ex vivo transport across jejunal segments in the Ussing chamber was 5-times (Ile-Pro-Pro) to 10-times (Val-Pro-Pro) higher with no significant differences (p>0.05) observed between both peptides. The peptidase inhibitor bestatin increased permeability for the absorptive direction for Ile-Pro-Pro by twofold. Neither a transepithelial pH gradient nor increased apical tripeptide concentration nor longitudinal localization of the intestinal segment influenced P(app) in the ex vivo experiments. Val-Pro-Pro transport across Peyer's patches, however, was 4-times higher (P(app)=21.0+/-9.3 x10(-8)cms(-1)) as compared to duodenum (P(app)=4.8+/-1.4 x 10(-8)cms(-1)). In the in situ perfusion experiments P(app) values varied greatly among different animals ranging from 0.5 to 24.0 x10(-8)cms(-1) (Ile-Pro-Pro) and from 1.0 to 15.6 x 10(-8)cms(-1) (Val-Pro-Pro). In summary, Caco-2 and ex vivo absorption models differ considerably regarding their peptide permeability. The in situ model seems to be less appropriate because of the observed large variability in peptide permeability. The results of this study demonstrate that the ACE inhibitory peptides Ile-Pro-Pro and Val-Pro-Pro are absorbed partially undegraded.

Protein hydrolysates induce CCK release from enteroendocrine cells and act as partial agonists of the CCK1 receptor.

Protein has been reported to be the most satiating of all macronutrients. Upon gastrointestinal digestion, peptides are generated that stimulate the release of satiety hormones such as cholecystokinin (CCK) from enteroendocrine cells. As such, bioactive peptides could be the target of Functional Food ingredients with satiating effects. We set up an in vitro assay system to investigate if different protein hydrolysates exhibit varying CCK-releasing properties. Soy, pea, potato, casein, and whey protein hydrolysates were incubated with the enteric endocrine cell line STC-1 that endogenously expresses and secretes CCK. Release of CCK was measured by ELISA. All hydrolysates induced CCK release at low concentrations (>0.1 mg.L -1)); however, no significant differences in CCK-releasing properties between the different protein hydrolysates were found, suggesting a generic, nonspecific peptide-sensing mechanism in the STC-1 cells on hydrolyzed protein. As the ELISA exhibits sensitivity to all CCK isoforms possessing the C-terminal CCK octapeptide but varying in biological activity at the CCK 1 receptor (CCK 1R), a secondary module was added to the STC-1 cell assay. Intracellular calcium measurements were performed in CHO-CCK 1R cells. Following exposure of the STC-1 cells to the protein hydrolysates, the medium was tested on the CCK 1R assay. Released CCK was measured with higher sensitivity and lower variability than in the ELISA. Surprisingly, we found that some protein hydrolysates (soy > potato >> casein) also directly stimulated CCK 1R-expressing cells, while whey and pea protein hydrolysates were inactive. As CCK 1R is expressed in the GI tract, direct interaction of CCK 1R with dietary peptides may contribute to their satiety effects. Future experiments developing bioactive ingredients for Functional Foods for weight management could involve isolation of the active, CCK 1R-activating peptides in, for example, soy protein hydrolysates.

Angiotensin converting enzyme inhibitory peptides from a lactotripeptide-enriched milk beverage are absorbed intact into the circulation.

Food products containing angiotensin converting enzyme (ACE) inhibitory peptides reportedly play a role in treatment of mild hypertension. The aim of this placebo-controlled crossover study was to assess the bioavailability of Ile-Pro-Pro and 7 other ACE-inhibiting peptides present in a lactotripeptide (LTP)-enriched yogurt beverage and whether meal intake affects Ile-Pro-Pro bioavailability. Six male and female subjects randomly consumed an LTP-enriched yogurt beverage or a placebo in the fasted state and an LTP-enriched yogurt beverage in the fed or fasted state. The area under the curve (AUC) of Ile-Pro-Pro after the LTP treatment in the fasted state was 2.1-fold of that after the placebo treatment (P < 0.001). The maximum peptide plasma concentration (C(max)) value was greater after consumption of the LTP-enriched beverage (897 +/- 157 pmol/L) than after the placebo treatment (555 +/- 0.09 pmol/L; P < 0.001) with a greater time after ingestion when reaching C(max) (T(max)) in the placebo treatment. Plasma concentrations of the peptides Leu-Trp, Phe-Tyr, Ile-Tyr, and Leu-Pro-Pro increased compared with baseline (P < 0.05) in the LTP-enriched and placebo treatment when consumed in the fasted state. However, DeltaC(max) values differed significantly between the placebo and LTP-enriched treatment only for Leu-Pro-Pro. Meal intake affected Ile-Pro-Pro concentrations. When the beverage was consumed after a meal, the AUC of Ile-Pro-Pro was 1.3-fold (P < 0.05) of the AUC derived from premeal intake. This was due to an increase in the plasma elimination half-life (P < 0.05); C(max) and T(max) were not affected by meal intake. In summary, this is the first demonstration, to our knowledge, that the tripeptide Ile-Pro-Pro selectively escapes from intestinal degradation and reaches the circulation undegraded.

Kinetics of bidirectional H+ and substrate transport by the proton-dependent amino acid symporter PAT1.

PAT1 is a recently identified member of the PAT family of proton/amino acid co-transporters with predominant expression in the plasma membrane of enterocytes and in lysosomal membranes of neurons. Previous studies in Xenopus oocytes expressing PAT1 established proton/substrate co-transport associated with positive inward currents for a variety of small neutral amino acids. Here we provide a detailed analysis of the transport mode of the murine PAT1 in oocytes using the two-electrode voltage-clamp technique to measure steady-state and pre-steady-state currents. The GPC (giant patch clamp) technique and efflux studies were employed to characterize the reversed transport mode. Kinetic parameters [K(m) (Michaelis constant) and I(max) (maximum current)] for transport of various substrates revealed a dependence on membrane potential: hyperpolarization increases the substrate affinity and maximal transport velocity. Proton affinity for interaction with PAT1 is almost 100 nM, corresponding to a pH of 7.0 and is independent of substrate. Kinetic analysis revealed that binding of proton most likely occurs before substrate binding and that the proton and substrate are translocated in a simultaneous step. No evidence for a substrate-uncoupled proton shunt was observed. As shown by efflux studies and current measurements by the GPC technique, PAT1 allows bidirectional amino acid transport. Surprisingly, PAT1 exhibits no pre-steady-state currents in the absence of substrate, even at low temperatures, and therefore PAT1 takes an exceptional position among the ion-coupled co-transporters.

H+/amino acid transporter 1 (PAT1) is the imino acid carrier: An intestinal nutrient/drug transporter in human and rat.

BACKGROUND AND AIMS: Amino acid (and related drug) absorption across the human small intestinal wall is an essential intestinal function. Despite the revelation of a number of mammalian genomes, the molecular identity of the classic Na(+)-dependent imino acid transporter (identified functionally in the 1960s) remains elusive. The aims of this study were to determine whether the recently isolated complementary DNA hPAT1 (human proton-coupled amino acid transporter 1), or solute carrier SLC36A1, represents the imino acid carrier; the Na(+) -dependent imino acid transport function measured at the brush-border membrane of intact intestinal epithelia results from a close functional relationship between human proton-coupled amino acid transporter-1 and N(+) /H(+) exchanger 3 (NHE3). METHODS: PAT1 function was measured in isolation ( Xenopus laevis oocytes) and in intact epithelia (Caco-2 cell monolayers and rat small intestine) by measurement of amino acid and/or H(+) influx. Tissue and membrane expression of PAT1 were determined by reverse-transcription polymerase chain reaction and immunohistochemistry. RESULTS: PAT1-specific immunofluorescence was localized exclusively to the luminal membrane of Caco-2 cells and human and rat small intestine. The substrate specificity of hPAT1 is identical to that of the imino acid carrier. In intact epithelia, PAT1-mediated amino acid influx is reduced under conditions in which NHE3 is inactive. CONCLUSIONS: The identification in intact epithelia of a cooperative functional relationship between PAT1 (H(+) /amino acid symport) and NHE3 (N(+) /H(+) exchange) explains the apparent Na + dependence of the imino acid carrier in studies with mammalian intestine. hPAT1 is the high-capacity imino acid carrier localized at the small intestinal luminal membrane that transports nutrients (imino/amino acids) and orally active neuromodulatory agents (used to treat affective disorders).

A novel bifunctionality: PAT1 and PAT2 mediate electrogenic proton/amino acid and electroneutral proton/fatty acid symport.

Recently, the PAT family of proton-dependent amino acid transporters has been identified as a novel class of mammalian amino acid symporters. PAT1 and PAT2 members mediate electrogenic uptake of small, neutral amino acids and derivatives by cotransport of protons. Analysis of the structural requirements for substrate recognition by PAT1 identified that a free amino group in a substrate is not essential for recognition. We therefore hypothesized that PAT1 and its ortholog PAT2 may also be able to recognize and transport the homologous short-chain fatty acids (SCFAs) such as acetate, propionate, and butyrate. We examined in Xenopus laevis oocytes whether the SCFAs interact with the transporter by employing flux studies, electrophysiology and intracellular pH recordings. SCFAs did not induce positive inward currents but inhibited glycine-induced transport currents. PAT-mediated uptake of radiolabeled proline was also dose-dependently reduced by SCFA and could be described by first order competition kinetics with apparent Ki-values for butyrate of 6.0 +/- 0.7 and 7.6 +/- 1.3 mM for PAT1 and PAT2, respectively. Acetate as well as propionate uptake was significantly enhanced in oocytes expressing PAT1 or PAT2. An electroneutral H+/SCFA symport mode was demonstrated by recording intracellular pH changes under voltage clamp conditions with rate constants for the initial intracellular acidification in the presence of SCFAs significantly increased in PAT-expressing oocytes. In conclusion, our data demonstrate that the PAT1 and PAT2 proteins are capable to transport selected SCFAs in an electroneutral and the homologous amino acids in an electrogenic mode and are therefore a paradigm for bifunctional solute carriers.

Substrate specificity and transport mode of the proton-dependent amino acid transporter mPAT2.

The PAT2 transporter has been shown to act as an electrogenic proton/amino acid symporter. The PAT2 cDNA has been cloned from various human, mouse and rat tissues and belongs to a group of four genes (pat1 to pat4) with PAT3 and PAT4 still resembling orphan transporters. The first immunolocalization studies demonstrated that the PAT2 protein is found in the murine central nervous system in neuronal cells with a proposed role in the intra and/or intercellular amino acid transport. Here we provide a detailed analysis of the transport mode and substrate specificity of the murine PAT2 transporter after expression in Xenopus laevis oocytes, by electrophysiological techniques and flux studies. The structural requirements to the PAT2 substrates - when considering both low and high affinity type substrates - are similar to those reported for the PAT1 protein with the essential features of a free carboxy group and a small side chain. For high affinity binding, however, PAT2 requires the amino group to be located in an alpha-position, tolerates only one methyl function attached to the amino group and is highly selective for the L-enantiomers. Electrophysiological analysis revealed pronounced effects of membrane potential on proton binding affinity, but substrate affinities and maximal transport currents only modestly respond to changes in membrane voltage. Whereas substrate affinity is dependent on extracellular pH, proton binding affinity to PAT2 is substrate-independent, favouring a sequential binding of proton followed by substrate. Maximal transport currents are substrate-dependent which suggests that the translocation of the loaded carrier to the internal side is the rate-limiting step.

A rapid in vitro screening for delivery of peptide-derived peptidase inhibitors as potential drug candidates via epithelial peptide transporters.

Targeting drugs or prodrugs to a specific enzyme by simultaneously targeting cell membrane carriers for efficient transport should provide the highest bioavailability along with specificity at the site of action. The peptide transporters PEPT1 and PEPT2 are expressed in a variety of tissues, including the brush-border membranes of epithelial cells of the small intestine and kidney. The transporters accept a wide range of substrates and are therefore good targets for a transporter-mediated drug delivery. Here, we report a screening procedure for peptidomimetic drug candidates combining two independent expression systems: 1) a competition assay in transgenic Pichia pastoris yeast cells expressing either mammalian PEPT1 or PEPT2 for identifying substrate interaction with the transporter binding site; and 2) a Xenopus laevis-based oocyte expression of the peptide transporter for assessing electrogenic transport of drug candidates. Based on the known oral availability and in vivo efficacy of the dipeptidyl peptidase IV (DPIV) inhibitor isoleucine-thiazolidide and its peptide-like structure, we first tested whether this compound is a substrate of epithelial peptide transporters. Additionally, a series of structurally related inhibitors were analyzed for transport. We identified various compounds that serve as substrates of the intestinal peptide transporter PEPT1. In contrast, none of these DPIV inhibitors showed electrogenic transport by PEPT2, although a variety of the compounds displayed good affinities for competition in peptide uptake in PEPT2-expressing cells, suggesting that they may serve as efficient inhibitors. In conclusion, we have applied an in vitro screening system that predicts efficient intestinal absorption of peptide-derived peptidase inhibitors via PEPT1 in vivo.

Analysis of the transport properties of side chain modified dipeptides at the mammalian peptide transporter PEPT1.

This study was initiated to examine systematically the effect of side chain modifications at dipeptides on their transport via PEPT1. We synthesized a series of Xaa(R)-Ala and Ala-Xaa(R) dipeptides with the functional groups of the side chains modified by structurally different blocking groups R. Recognition and transport of these derivatives by PEPT1 was measured in Caco-2 cells, in transgenic Pichia pastoris cells and in Xenopus laevis oocytes expressing PEPT1. The dipeptide derivatives displayed K(i) values between 0.002 and 4 mM. Electrophysiological analyses showed that the Ala-Xaa(R) derivatives were transported by PEPT1. In contrast, most Xaa(R)-Ala derivatives--although recognized--did not show significant transport rates. Substitution of a terminal phenyl residue in the side chain blocking group by a p-nitrophenyl residue enhanced the affinity of several dipeptide derivatives for interaction with PEPT1. However, none of these compounds showed electrogenic transport in oocytes. With a K(i) value of 0.002 mM, Lys[Z(NO(2))]-Val displayed the highest affinity to PEPT1 ever reported. We conclude that the transport of side chain modified dipeptides into enterocytes depends (a) on the position of the modified trifunctional amino acid in the dipeptide, (b) the distance between its alpha-carbon and the side chain blocking group and (c) the hydrophobic character of the side chain modification.

The proton/amino acid cotransporter PAT2 is expressed in neurons with a different subcellular localization than its paralog PAT1.

The new member of the mammalian amino acid/auxin permease family, PAT2, has been cloned recently and represents an electrogenic proton/amino acid symporter. PAT2 and its paralog, PAT1/LYAAT-1, are transporters for small amino acids such as glycine, alanine, and proline. Our immunodetection studies revealed that the PAT2 protein is expressed in spinal cord and brain. It is found in neuronal cell bodies in the anterior horn in spinal cord and in brain stem, cerebellum, hippocampus, hypothalamus, rhinencephalon, cerebral cortex, and olfactory bulb in the brain. PAT2 is expressed in neurons positive for the N-methyl-d-aspartate subtype glutamate receptor subunit NR1. PAT2 is not found in lysosomes, unlike its paralog PAT1, but is present in the endoplasmic reticulum and recycling endosomes in neurons. PAT2 has a high external proton affinity causing half-maximal transport activation already at a pH of 8.3, suggesting that its activity is most likely not altered by physiological pH changes. Transport of amino acids by PAT2 activity is dependent on membrane potential and can occur bidirectionally; membrane depolarization causes net glycine outward currents. Our data suggest that PAT2 contributes to neuronal transport and sequestration of amino acids such as glycine, alanine, and/or proline, whereby the transport direction is dependent on the sum of the driving forces such as substrate concentration, pH gradient, and membrane potential.

Substrate recognition by the mammalian proton-dependent amino acid transporter PAT1.

The PAT family of proton-dependent amino acid transporters has recently been identified at the molecular level. This paper describes the structural requirements in substrates for their interaction with the cloned murine intestinal proton/amino acid cotransporter (PAT1). By using the Xenopus laevis oocytes as an expression system and by combining the two-electron voltage clamp technique with radiotracer flux studies, it was demonstrated that the aliphatic side chain of L-alpha-amino acids substrates can consist maximally of only one CH2-unit for high affinity interaction with PAT1. With respect to the maximal separation between the amino and carboxyl groups, only two CH2-units, as in gamma-aminobutyric acid (GABA), are tolerated. PAT1 displays no or even a reversed stereoselectivity, tolerating serine and cystein only in the form of D-enantiomers. A methyl-substitution of the carboxyl group (e.g. O-methyl-glycine) markedly diminishes substrate affinity and transport rates, whereas methyl-substitutions at the amino group (e.g. sarcosine or betaine) have only minor effects on substrate interaction with the transporter binding site. Furthermore, it has been shown (by kinetic analyses of radiolabelled betaine influx and inhibition studies) that the endogenous PAT system of human Caco-2 cells has very similar transport characteristics to mouse PAT1. In summary, one has defined the structural requirements and limitations thet determine the substrate specificity of PAT1. A critical recognition criterion of PAT1 is the backbone charge separation distance and the side chain size, whereas substitutions on the amino group are well tolerated.

A cluster of proton/amino acid transporter genes in the human and mouse genomes.

We recently cloned and functionally characterized two novel proton/amino acid transporters (PAT1 and PAT2) from mouse. Here we report the isolation of the corresponding cDNAs of the human orthologues and one additional mouse and human PAT-like transporter cDNA, designated PAT3. The PAT proteins comprise 470 to 483 amino acids. The mouse PAT3 mRNA is expressed in testis of adult mice. In the human and mouse genomes the genes of the PAT transporters (designated SLC36A1-3 and Slc36a1-3, respectively) are clustered on human chromosome 5q33.1 and in the syntenic region of mouse chromosome 11B1.3. PAT-like transporter genes are present as well in the genomes of other eukaryotic organisms such as Drosophila melanogaster and Caenorhabditis elegans. For the PAT3 subtype transporter, we could not yet identify its function. The human PAT1 and PAT2 transporters when functionally expressed in Xenopus laevis oocytes show characteristics similar to those of their mouse counterparts.

Functional characterization of two novel mammalian electrogenic proton-dependent amino acid cotransporters.

We cloned two cDNAs encoding proton/amino acid cotransporters, designated as mPAT1 and mPAT2, from murine tissues. They were identified by sequence similarity to the amino acid/auxin permease family member of lower eukaryotes. We functionally characterized both transporters by flux studies and electrophysiology after expression in Xenopus laevis oocytes. Both mPAT1 and mPAT2 induced a pH-dependent electrogenic transport activity for small amino acids (glycine, alanine, and proline) that is altered by membrane potential. Direct evidence for amino acid/H(+)-symport was shown by intracellular acidification, and a flux coupling stoichiometry for proline/H(+)-symport of 1:1 was determined for both transporters. Besides small apolar L-amino acids, the transporters also recognize their D-enantiomers and selected amino acid derivatives such as gamma-aminobutyric acid. The mPAT1 transporter, the murine orthologue of the recently cloned rat LYAAT-1 transporter, can be considered as a low affinity system when compared with mPAT2. The mRNA of mPAT1 is highly expressed in small intestine, colon, kidney, and brain; the mPAT2-mRNA is mainly found in heart and lung. Phenotypically, the PAT1 transporter possesses the same functional characteristics as the previously described proton-dependent amino acid transport process in apical membranes of intestinal and renal epithelial cells.