Effect of N-glycosylation on the transport activity of the peptide transporter PEPT1.

The intestinal peptide transporter PEPT1 provides bulk quantities of amino acids to epithelial cells. PEPT1 is a high-capacity and low-affinity solute carrier of the SLC15 family found in apical membranes of enterocytes in small intestine and distal colon. Surprisingly, murine PEPT1 (mPEPT1) has an apparent molecular mass of ∼95 kDa in the small intestine but ∼105 kDa in the large intestine. Here we describe studies on mPEPT1 protein glycosylation and how glycans affect transport function. Putative N-glycosylation sites of mPEPT1 were altered by site-directed mutagenesis followed by expression in Xenopus laevis oocytes. Replacement of six asparagine residues (N) at positions N50, N406, N439, N510, N515, and N532 by glutamine (Q) resulted in a decrease of the mPEPT1 mass by around 35 kDa. Electrophysiology revealed all glycosylation-deficient transporters to be functional with comparable expression levels in oocyte membranes. Strikingly, the mutant protein with N50Q exhibited a twofold decreased affinity for Gly-Sar but a 2.5-fold rise in the maximal inward currents compared with the wild-type protein. Elevated maximal transport currents were also recorded for cefadroxil and tri-l-alanine. Tracer flux studies performed with [(14)C]-Gly-Sar confirmed the reduction in substrate affinity and showed twofold increased maximal transport rates for the N50Q transporter. Elimination of individual N-glycosylation sites did not alter membrane expression in oocytes or overall transport characteristics except for the mutant protein N50Q. Because transporter surface density was not altered in N50Q, removal of the glycan at this location appears to accelerate the substrate turnover rate.

Inhibition of the intestinal sodium-coupled glucose transporter 1 (SGLT1) by extracts and polyphenols from apple reduces postprandial blood glucose levels in mice and humans.

SCOPE: There is a growing interest in food constituents that could reduce intestinal glucose absorption to prevent overshooting plasma glucose and insulin levels in patients with prediabetes and diabetes mellitus type 2. METHODS AND RESULTS: We here demonstrate that an extract and individual polyphenols from apple diminish sodium-coupled glucose transporter 1 (SGLT1) mediated glucose uptake in vitro and in vivo. Inhibition of transport of sugars by SGLT1 was shown in Xenopus oocytes and in mice jejunal segments. Strongest inhibition was observed for phlorizin with IC50 values for transport inhibition of 0.46 ± 0.19 and 4.1 ± 0.6 µM in oocytes and intestinal segments, respectively. An oral glucose tolerance test performed in volunteers with prior administration of the apple extract reduced venous blood glucose and plasma insulin levels, similar to findings obtained in C57BL/6N mice. Analysis of human urine samples revealed that the extract increased modestly renal glucose loss that is most likely a result of inhibition of renal glucose reabsorption by phloretin derivatives found in plasma of the volunteers. CONCLUSION: Although the apple extract substantially decreased intestinal glucose absorption in all test systems, the finding that there are systemic effects that relate to inhibition of glucose transport processes beyond the intestine addresses safety issues that need further exploitation.

Gymnemic acids inhibit sodium-dependent glucose transporter 1.

To evaluate the activity of botanicals used in Chinese Traditional Medicine as hypoglycemic agents for diabetes type II prevention and/or treatment, extracts prepared from 26 medicinal herbs were screened for their inhibitory activity on sodium-dependent glucose transporter 1 (SGLT1) by using two-electrode voltage-clamp recording of glucose uptake in Xenopus laevis oocytes microinjected with cRNA for SGLT1. Showing by far the strongest SGLT1 inhibitory effect, the phytochemicals extracted from Gymnema sylvestre (Retz.) Schult were located by means of activity-guided fractionation and identified as 3-O-ß-D-glucuronopyranosyl-21-O-2-tigloyl-22-O-2-tigloyl gymnemagenin (1) and 3-O-ß-D-glucuronopyranosyl-21-O-2-methylbutyryl-22-O-2-tigloyl gymnemagenin (2) by means of LC-MS/MS, UPLC-TOF/MS, and 1D/2D-NMR experiments. Both saponins exhibited low IC50 values of 5.97 (1) and 0.17 µM (2), the latter of which was in the same range as found for the high-affinity inhibitor phlorizin (0.21 µM). As SGLT1 is found in high levels in brush-border membranes of intestinal epithelial cells, these findings demonstrate for the first time the potential of these saponins for inhibiting electrogenic glucose uptake in the gastrointestinal tract.

Protein cold adaptation strategy via a unique seven-amino acid domain in the icefish (Chionodraco hamatus) PEPT1 transporter.

Adaptation of organisms to extreme environments requires proteins to work at thermodynamically unfavorable conditions. To adapt to subzero temperatures, proteins increase the flexibility of parts of, or even the whole, 3D structure to compensate for the lower thermal kinetic energy available at low temperatures. This may be achieved through single-site amino acid substitutions in regions of the protein that undergo large movements during the catalytic cycle, such as in enzymes or transporter proteins. Other strategies of cold adaptation involving changes in the primary amino acid sequence have not been documented yet. In Antarctic icefish (Chionodraco hamatus) peptide transporter 1 (PEPT1), the first transporter cloned from a vertebrate living at subzero temperatures, we came upon a unique principle of cold adaptation. A de novo domain composed of one to six repeats of seven amino acids (VDMSRKS), placed as an extra stretch in the cytosolic COOH-terminal region, contributed per se to cold adaptation. VDMSRKS was in a protein region uninvolved in transport activity and, notably, when transferred to the COOH terminus of a warm-adapted (rabbit) PEPT1, it conferred cold adaptation to the receiving protein. Overall, we provide a paradigm for protein cold adaptation that relies on insertion of a unique domain that confers greater affinity and maximal transport rates at low temperatures. Due to its ability to transfer a thermal trait, the VDMSRKS domain represents a useful tool for future cell biology or biotechnological applications.

Peptide transporter isoforms are discriminated by the fluorophore-conjugated dipeptides ß-Ala- and d-Ala-Lys-N-7-amino-4-methylcoumarin-3-acetic acid.

Peptide transporters of the SLC15 family are classified by structure and function into PEPT1 (low-affinity/high-capacity) and PEPT2 (high-affinity/low-capacity) isoforms. Despite the differences in kinetics, both transporter isoforms are reckoned to transport essentially all possible di- and tripeptides. We here report that the fluorophore-conjugated dipeptide derivatives ß-Ala-Lys-N-7-amino-4-methylcoumarin-3-acetic acid (ß-AK-AMCA) and d-Ala-Lys-N-7-amino-4-methylcoumarin-3-acetic acid (d-AK-AMCA) are transported by distinct PEPT isoforms in a species-specific manner. Transport of the fluorophore peptides was studied (1) in vitro after heterologous expression in Xenopus oocytes of PEPT1 and PEPT2 isoforms from different vertebrate species and of PEPT1 and PEPT2 transporters from Caenorhabditis elegans by using electrophysiological and fluorescence methods and (2) in vivo in C. elegans by using fluorescence methods. Our results indicate that both substrates are transported by the vertebrate "renal-type" and the C. elegans "intestinal-type" peptide transporter only. A systematic analysis among species finds four predicted amino acid residues along the sequence that may account for the substrate uptake differences observed between the vertebrate PEPT1/nematode PEPT2 and the vertebrate PEPT2/nematode PEPT1 subtype. This selectivity on basis of isoforms and species may be helpful in better defining the structure-function determinants of the proteins of the SLC15 family.

In vitro characterization of human peptide transporter hPEPT1 interactions and passive permeation studies of short cationic antimicrobial peptides.

The present study assesses the permeation of cationic antimicrobial di- and tripeptides derived from lactoferricin via interaction with the human intestinal peptide transporter hPEPT1 and via passive routes. While some tested peptides displayed moderate affinity (0.6 and 2.7 mM) for interaction with hPEPT1, none served as substrate for hPEPT1 expressed by Xenopus laevis oocytes. It is shown that structural strategies employed to generate sufficient biological activity and metabolic stability such as introduction of large hydrophobic unnatural amino acids and different C-terminal modifications counteracted hPEPT1 mediated uptake. Most of the included peptides were nevertheless shown to permeate at rates suggesting moderate to excellent human oral absorption in the applied phospholipid vesicle-based passive permeation assay. Although the main factor governing passive permeation appears to be the hydrophobicity, peptide structure was also important and the overall permeation behavior was difficult to predict. Comparisons with a theoretical prediction model were also performed.

Amino acid absorption and homeostasis in mice lacking the intestinal peptide transporter PEPT1.

The intestinal peptide transporter PEPT1 mediates the uptake of di- and tripeptides derived from dietary protein breakdown into epithelial cells. Whereas the transporter appears to be essential to compensate for the reduced amino acid delivery in patients with mutations in amino acid transporter genes, such as in cystinuria or Hartnup disease, its physiological role in overall amino acid absorption is still not known. To assess the quantitative importance of PEPT1 in overall amino acid absorption and metabolism, PEPT1-deficient mice were studied by using brush border membrane vesicles, everted gut sacs, and Ussing chambers, as well as by transcriptome and proteome analysis of intestinal tissue samples. Neither gene expression nor proteome profiling nor functional analysis revealed evidence for any compensatory changes in the levels and/or function of transporters for free amino acids in the intestine. However, most plasma amino acid levels were increased in Pept1(-/-) compared with Pept1(+/+) animals, suggesting that amino acid handling is altered. Plasma appearance rates of (15)N-labeled amino acids determined after intragastric administration of a low dose of protein remained unchanged, whereas administration of a large protein load via gavage revealed marked differences in plasma appearance of selected amino acids. PEPT1 seems, therefore, important for overall amino acid absorption only after high dietary protein intake when amino acid transport processes are saturated and PEPT1 can provide additional absorption capacity. Since renal amino acid excretion remained unchanged, elevated basal concentrations of plasma amino acids in PEPT1-deficient animals seem to arise mainly from alterations in hepatic amino acid metabolism.

Unified modeling of the mammalian and fish proton-dependent oligopeptide transporter PepT1.

Electrophysiological and biophysical analyses were used to compare the partial and complete transport cycles of the intestinal oligopeptide transporter PepT1 among three species (seabass, zebrafish and rabbit). On the whole, the presteady-state currents of the fish transporters were similar to each other. Rabbit PepT1 differed from the fish transporters by having slower-decaying currents, and the charge vs. potential (Q/V) and time constant vs. potential (τ/V) curves shifted to more positive potentials. All of the isoforms were similarly affected by external pH, showing acidity-induced slowing of the transients and positive shifts in the Q/V and τ/V curves. Analysis of the pH-dependence of the unidirectional rates of the intramembrane charge movement suggested that external protonation of the protein limits the speed of this process in both directions. The complete cycle of the transporter was studied using the neutral dipeptide Gly-Gln. Michaelis-Menten analysis confirmed that, in all species, acidity significantly increases the apparent affinity for the substrate but does not strongly impact maximal transport current. Simulations using a kinetic model incorporating the new findings showed good agreement with experimental data for all three species, both with respect to the presteady-state and the transport currents.

Functional and structural determinants of reverse operation in the pH-dependent oligopeptide transporter PepT1.

The functional and structural basis of reverse operation of PepT1 has been studied in Xenopus oocytes expressing the wild-type and mutated forms of this protein. Using brief pulses from a negative holding potential, wild-type and Arg282 mutants exhibit outward currents in the presence of Gly-Gln. The reversal potential of these currents is affected by both pH and substrate concentration, confirming coupled transport in the wild type and in the mutants as well. Long-lasting voltage and current-clamp experiments show that the outward currents are only temporary, and reflect accumulation and/or depletion effects near the membrane. The ability to operate in reverse mode was confirmed in all isoforms by intracellular injection of substrate. The role of Arg282 and Asp341 in the reverse transport was also investigated using charged substrates. Positive Lys-Gly (but not Gly-Lys) showed enhanced transport currents in the Arg282 mutants. In contrast, negative Gly-Asp and Asp-Gly elicited modest currents in all isoforms.

Residues R282 and D341 act as electrostatic gates in the proton-dependent oligopeptide transporter PepT1.

The oligopeptide transporter PepT1 is a protein found in the membrane of the cells of the intestinal walls, and represents the main route through which proteic nutrients are absorbed by the organism. Along the polypeptidic chain of this protein, two oppositely charged amino acids, an arginine in position 282 and an aspartate in position 341 of the sequence, have been hypothesised to form a barrier in the absorption pathway. In this paper we show that appropriate mutations of these amino acids change the properties of PepT1 in a way that confirms that these parts of the protein indeed act as an electrostatic gate in the transport process. The identification of the structural basis of the functional mechanism of this transporter is important because, in addition to its role in nutrient uptake, PepT1 represents a major pathway for the absorption of several therapeutic drugs.

Gene ablation for PEPT1 in mice abolishes the effects of dipeptides on small intestinal fluid absorption, short-circuit current, and intracellular pH.

PEPT1 function in mouse intestine has not been assessed by means of electrophysiology and methods to assess its role in intracellular pH and fluid homeostasis. Therefore, the effects of the dipeptide glycilsarcosin (Gly-Sar) on jejunal fluid absorption and villous enterocyte intracellular pH (pH(i)) in vivo, as well as on enterocyte[(14)C]Gly-Sar uptake, short-circuit current (I(sc)) response, and enterocyte pH(i) in vitro were determined in wild-type and PEPT1-deficient mice and in mice lacking PEPT1. Immunohistochemistry for PEPT1 failed to detect any protein in enterocyte apical membranes in Slc15a1(-/-) animals. Saturable Gly-Sar uptake in Slc15a1(-/-) everted sac preparations was no longer detectable. Similarly, Gly-Sar-induced jejunal I(sc) response in vitro was abolished. The dipeptide-induced increase in fluid absorption in vivo was also abolished in animals lacking PEPT1. Since PEPT1 acts as an acid loader in enterocytes, enterocyte pH(i) was measured in vivo by two-photon microscopy in SNARF-4-loaded villous enterocytes of exteriorized jejuni in anesthetized mice, as well as in BCECF-loaded enterocytes of microdissected jejunal villi. Gly-Sar-induced pH(i) decrease was no longer observed in the absence of PEPT1. A reversal of the proton gradient across the luminal membrane did not significantly diminish Gly-Sar-induced I(sc) response, whereas a depolarization of the apical membrane potential by high K(+) or via Na(+)-K(+)-ATPase inhibition strongly diminished Gly-Sar-induced I(sc) responses. This study demonstrates for the first time that proton-coupled electrogenic dipeptide uptake in the native small intestine, mediated by PEPT1, relies on the negative apical membrane potential as the major driving force and contributes significantly to intestinal fluid transport.

Molecular cloning and functional expression of atlantic salmon peptide transporter 1 in Xenopus oocytes reveals efficient intestinal uptake of lysine-containing and other bioactive di- and tripeptides in teleost fish.

Atlantic salmon (Salmo salar L.) is one of the most economically important cultured fish and also a key model species in fish nutrition. During digestion, dietary proteins are enzymatically cleaved and a fraction of degradation products in the form of di- and tripeptides translocates from the intestinal lumen into the enterocyte via the Peptide Transporter 1 (PepT1). With this in mind, a full-length cDNA encoding the Atlantic salmon PepT1 (asPepT1) was cloned and functionally characterized. When overexpressed in Xenopus laevis oocytes, asPepT1 operated as a low-affinity/high-capacity transport system, and its maximal transport activity slightly increased as external proton concentration decreased (varying extracellular pH from 6.5 to 8.5). A total of 19 tested di- and tripeptides, some with acknowledged bioactive properties, some containing lysine, which is conditionally growth limiting in fish, were identified as well transported substrates, with affinities ranging between approximately 0.5 and approximately 1.5 mmol/L. Analysis of body tissue distribution showed the highest levels of asPepT1 mRNA in the digestive tract. In particular, asPepT1 mRNA was present in all segments after the stomach, with higher levels in the pyloric caeca and midgut region and lower levels in the hindgut. Depriving salmon of food for 6 d resulted in a approximately 70% reduction of intestinal PepT1 mRNA levels. asPepT1 will allow systematic in vitro analysis of transport of selected di- and tripeptides that may be generated in Atlantic salmon intestine during gastrointestinal transit. Also, asPepT1 will be useful as a marker to estimate protein absorption function along the intestine under various physiological and pathological conditions.

Sodium translocation by the iminoglycinuria associated imino transporter (SLC6A20).

The system IMINO transporter plays an essential role in the transport of proline and hydroxyproline in the intestine and kidney. Its molecular correlate has been identified and named SIT1 or IMINO (SLC6A20). Initial characterization of the transporter showed it to be Na(+) and Cl(-)-dependent, but the stoichiometry remained unresolved. Using homology modeling along the structure of the bacterial leucine transporter LeuT, we identified two highly conserved Na(+)-binding sites and a putative Cl(-)-binding site. Mutation of all residues in the two proposed Na(+)-binding sites revealed that most of them were essential for uptake and completely inactivated the transporter. However, mutants A22V (Na(+)-binding site 1) and mutants S20A, S20G, S20G/G405S (Na(+)-binding site 2) were partially active and characterized further. Flux studies suggested that mutations of Na(+)-binding site 1 caused a decrease of the Na(+)-K(0.5), whereas mutations of site 2 increased the K(0.5). Mutation of Na(+)-binding site 1 also changed the ion selectivity of the IMINO transporter. IMINO actively translocates (36)Cl(-) demonstrating that the proposed chloride binding site is used in the transporter. Accumulation experiments and flux measurements at different holding potentials showed that the transporter can work as a 2Na(+)/1Cl(-)-proline cotransporter. The proposed homology model allows to study mutations in IMINO associated with iminoglycinuria.

High-affinity interaction of sartans with H+/peptide transporters.

Sartans are very effective drugs for treatment of hypertension, heart failure, and other cardiovascular disorders. They antagonize the effects of angiotensin II at the AT(1) receptor and display p.o. bioavailability rates of 13 to 80%. Because some sartans sterically resemble dipeptide derivatives, we investigated whether they are transported by peptide transporters. We first assessed the effects of sartans on [(14)C]glycylsarcosine uptake into Caco-2 cells expressing H(+)/peptide transporter (PEPT) 1 and into SKPT cells expressing PEPT2. Losartan, irbesartan, valsartan, and eprosartan inhibited [glycine-1-(14)C]glycylsarcosine ([(14)C]Gly-Sar) uptake into Caco-2 cells in a competitive manner with K(i) values of 24, 230, 390, and >1000 microM. Losartan and valsartan also strongly inhibited the total transepithelial flux of [(14)C]Gly-Sar across Caco-2 cell monolayers. In SKPT cells, [(14)C]Gly-Sar uptake was inhibited with K(i) values of 2.2 microM (losartan), 65 microM (irbesartan), 260 microM (valsartan), and 490 microM (eprosartan). We determined by the two-electrode voltage-clamp technique whether the compounds elicited transport currents by PEPT1 or PEPT2 when expressed in Xenopus laevis oocytes. No currents were observed for any of the sartans, but the compounds strongly and reversibly inhibited peptide-induced currents. Uptake of valsartan, losartan, and cefadroxil was quantified in HeLa cells after heterologous expression of human PEPT1 (hPEPT1). In contrast to cefadroxil, no PEPT1-specific uptake of valsartan and losartan was found. We conclude that the sartans tested in this study display high-affinity interaction with PEPTs but are not transported themselves. However, they strongly inhibit hPEPT1-mediated uptake of dipeptides and cefadroxil.

Inhibition of intracellular dipeptide hydrolysis uncovers large outward transport currents of the peptide transporter PEPT1 in Xenopus oocytes.

The "reversed transport mode" of electrogenic carriers is usually difficult to assess, as substrates are metabolized after reaching the cell, and the cytosolic surface is only accessible in special experimental settings such as giant-patch techniques. In the present experiments with the two-electrode voltage clamp, we demonstrate a unique feature of the peptide transporter PEPT1 that produces huge outward transport currents when oocytes are preloaded with hydrolysis-resistant dipeptides or when intracellular hydrolysis is prevented by aminopeptidase inhibition. A rapid intracellular degradation of dipeptides in oocytes and a parallel decline of outward currents were observed by analysis of amino acids in the cells. Dipeptide hydrolysis could almost completely be blocked by preincubation of oocytes with the aminopeptidase-inhibitor bestatin, itself a substrate of PEPT1. Dipeptide-driven outward currents of bestatin-treated oocytes remained stable over at least 10 min. Unexpectedly, the outward currents at a membrane potential of +60 mV were about five times higher than the corresponding inward currents measured before preloading at -60 mV under symmetrical conditions. The huge outward current was carried by PEPT1 and did not result from opening of potassium or chloride conductances in the membrane. Dipeptide-preloading of oocytes also increased inward currents evoked by substrates provided on the outside and equal substrate concentrations on both membrane surfaces in the absence of a pH gradient resulted in a linear current-voltage relation crossing the current axis at the origin. Our data and preliminary model calculations suggest a faster turnover rate of rPEPT1 in the presence of high substrate concentrations on the cytosolic surface.

Substrate-induced changes in the density of peptide transporter PEPT1 expressed in Xenopus oocytes.

The adaptation of the capacity of the intestinal peptide transporter PEPT1 to varying substrate concentrations may be important with respect to its role in providing bulk quantities of amino acids for growth, development, and other nutritional needs. In the present study, we describe a novel phenomenon of the regulation of PEPT1 in the Xenopus oocyte system. Using electrophysiological and immunofluorescence methods, we demonstrate that a prolonged substrate exposure of rabbit PEPT1 (rPEPT1) caused a retrieval of transporters from the membrane. Capacitance as a measure of membrane surface area was increased in parallel with the increase in rPEPT1-mediated transport currents with a slope of approximately 5% of basal surface per 100 nA. Exposure of oocytes to the model peptide Gly-l-Gln for 2 h resulted in a decrease in maximal transport currents with no change of membrane capacitance. However, exposure to substrate for 5 h decreased transport currents but also, in parallel, surface area by endocytotic removal of transporter proteins from the surface. The reduction of the surface expression of rPEPT1 was confirmed by presteady-state current measurements and immunofluorescent labeling of rPEPT1. A similar simultaneous decrease of current and surface area was also observed when endocytosis was stimulated by the activation of PKC. Cytochalasin D inhibited all changes evoked by either dipeptide or PKC stimulation, whereas the PKC-selective inhibitor bisindolylmaleimide only affected PKC-stimulated endocytotic processes but not substrate-dependent retrieval of rPEPT1. Coexpression experiments with human Na(+)-glucose transporter 1 (hSGLT1) revealed that substrate exposure selectively affected PEPT1 but not the activity of hSGLT1.

Transport of angiotensin-converting enzyme inhibitors by H+/peptide transporters revisited.

Angiotensin-converting enzyme (ACE) inhibitors are often regarded as substrates for the H+/peptide transporters (PEPT)1 and PEPT2. Even though the conclusions drawn from published data are quite inconsistent, in most review articles PEPT1 is claimed to mediate the intestinal absorption of ACE inhibitors and thus to determine their oral availability. We systematically investigated the interaction of a series of ACE inhibitors with PEPT1 and PEPT2. First, we studied the effect of 14 ACE inhibitors including new drugs on the uptake of the dipeptide [14C]glycylsarcosine into human intestinal Caco-2 cells constitutively expressing PEPT1 and rat renal SKPT cells expressing PEPT2. In a second approach, the interaction of ACE inhibitors with heterologously expressed human PEPT1 and PEPT2 was determined. In both assay systems, zofenopril and fosinopril were found to have very high affinity for binding to peptide transporters. Medium to low affinity for transporter interaction was found for benazepril, quinapril, trandolapril, spirapril, cilazapril, ramipril, moexipril, quinaprilat, and perindopril. For enalapril, lisinopril, and captopril, very weak affinity or lack of interaction was found. Transport currents of PEPT1 and PEPT2 expressed in Xenopus laevis oocytes were recorded by the two-electrode voltage-clamp technique. Statistically significant, but very low currents were only observed for lisinopril, enalapril, quinapril, and benazepril at PEPT1 and for spirapril at PEPT2. For the other ACE inhibitors, electrogenic transport activity was extremely low or not measurable at all. The present results suggest that peptide transporters do not control intestinal absorption and renal reabsorption of ACE inhibitors.

Synthesis and characterization of a new and radiolabeled high-affinity substrate for H+/peptide cotransporters.

In this study we described the design, rational synthesis and functional characterization of a novel radiolabeled hydrolysis-resistant high-affinity substrate for H(+)/peptide cotransporters. L-4,4'-Biphenylalanyl-L-Proline (Bip-Pro) was synthesized according to standard procedures in peptide chemistry. The interaction of Bip-Pro with H(+)/peptide cotransporters was determined in intestinal Caco-2 cells constitutively expressing human H(+)/peptide cotransporter 1 (PEPT1) and in renal SKPT cells constitutively expressing rat H(+)/peptide cotransporter 2 (PEPT2). Bip-Pro inhibited the [(14)C]Gly-Sar uptake via PEPT1 and PEPT2 with exceptional high affinity (K(i) = 24 microm and 3.4 microm, respectively) in a competitive manner. By employing the two-electrode voltage clamp technique in Xenopus laevis oocytes expressing PEPT1 or PEPT2 it was found that Bip-Pro was transported by both peptide transporters although to a much lower extent than the reference substrate, Gly-Gln. Bip-Pro remained intact to > 98% for at least 8 h when incubated with intact cell monolayers. Bip-[(3)H]Pro uptake into SKPT cells was linear for up to 30 min and pH dependent with a maximum at extracellular pH 6.0. Uptake was strongly inhibited, not only by unlabeled Bip-Pro but also by known peptide transporter substrates such as dipeptides, cefadroxil, Ala-4-nitroanilide and delta-aminolevulinic acid, but not by glycine. Bip-Pro uptake in SKPT cells was saturable with a Michaelis-Menten constant (K(t)) of 7.6 microm and a maximal velocity (V(max)) of 1.1 nmol x 30 min(-1) x mg of protein(-1). Hence, the uptake of Bip-Pro by PEPT2 is a high-affinity, low-capacity process in comparison to the uptake of Gly-Sar. We conclude that Bip-[(3)H]Pro is a valuable substrate for both mechanistic and structural studies of H(+)/peptide transporter proteins.

Tripeptides of RS1 (RSC1A1) inhibit a monosaccharide-dependent exocytotic pathway of Na+-D-glucose cotransporter SGLT1 with high affinity.

The human gene RSC1A1 codes for a 67-kDa protein named RS1 that mediates transcriptional and post-transcriptional regulation of Na(+)-D-glucose cotransporter SGLT1. The post-transcriptional regulation occurs at the trans-Golgi network (TGN). We identified two tripeptides in human RS1 (Gln-Cys-Pro (QCP) and Gln-Ser-Pro (QSP)) that induce posttranscriptional down-regulation of SGLT1 at the TGN leading to 40-50% reduction of SGLT1 in plasma membrane. For effective intracellular concentrations IC(50) values of 2.0 nM (QCP) and 0.16 nm (QSP) were estimated. Down-regulation of SGLT1 by tripeptides was attenuated by intracellular monosaccharides including non-metabolized methyl-alpha-D-glucopyranoside and 2-deoxyglucose. In small intestine post-transcriptional regulation of SGLT1 may contribute to glucose-dependent regulation of liver metabolism and intestinal mobility. QCP and QSP are transported by the H(+)-peptide cotransporter PepT1 that is colocated with SGLT1 in small intestinal enterocytes. Using coexpression of SGLT1 and PepT1 in Xenopus oocytes or polarized Caco-2 cells that contain both transporters we demonstrated that the tripeptides were effective when applied to the extracellular compartment. After a 1-h perfusion of intact rat small intestine with QSP, glucose absorption was reduced by 30%. The data indicate that orally applied tripeptides can be used to down-regulate small intestinal glucose absorption, e.g. in diabetes mellitus.

Flavonoid glycosides are not transported by the human Na+/glucose transporter when expressed in Xenopus laevis oocytes, but effectively inhibit electrogenic glucose uptake.

There is controversy as to whether intestinal absorption of glycosylated flavonoids, and particularly quercetin glycosides, involves their uptake in intact form via the human sodium-coupled glucose transporter hSGLT1. We here describe studies using Xenopus oocytes that express hSGLT1 and the two-electrode voltage clamp technique to determine the transport characteristics of a variety of flavonoids carrying glucose residues at different positions as well as of their aglycones (altogether 27 compounds). Neither quercetin, luteolin, apigenin, naringenin, pelarginidin, daidzein, or genistein, nor any of their glycosylated derivatives generated significant transport currents. However, the inward current evoked by 1 mM of the hSGLT1 substrate alpha-methyl-D-glucopyranoside was potently reduced by the simultaneous application of not only various flavonoid glycosides but also by some aglycones. The inhibitory potency remained unchanged when the attached glucose was replaced by galactose, suggesting that these residues may bind to SGLT1. Kinetic analysis by Dixon plots revealed inhibition of competitive type with high affinities, particularly when the glucose was attached to the position 4' of the aromatic ring of the flavonoids. The affinities became lower when the glucose was attached to a different position. Unexpectedly, the aglycone form of luteolin also inhibited the transport competitively with high affinity. These data show that hSGLT1 does not transport any of the flavonoids and seems therefore not involved in their intestinal absorption. However, not only glycosylated but also a few nonglycosylated flavonoids show a structure-dependent capability for effective inhibition of SGLT1.