Intestinal Absorption Of Peptides Research Articles

The effect of granulated collagen tripeptide on sarcopenia in aged mice compared with collagen tripeptide

This study investigates the efficacy of granulated collagen tripeptide (gCTP) in mitigating sarcopenia in aged mice compared to CTP. 58 weeks (male, n = 38) C57BL/6J mice were divided into 4 groups: an aging control, 0.03 % Gly-Pro-Hyp, 0.9 % CTP, and 0.9 % gCTP-supplemented group. gCTP administration significantly increased lean mass without altering body weight, indicating potential benefits in improving body composition in aged mice. Moreover, gCTP enhanced muscle synthesis pathways, including IGF1/PI3K/mTOR axis and myogenin expression, increasing muscle mass and strength. Additionally, gCTP reduced muscle and systemic inflammation, as evidenced by decreased levels of pro-inflammatory cytokines and myokines. Notably, gCTP enhanced intestinal absorption of peptides, potentially contributing to improved protein synthesis. gCTP demonstrated superior efficacy in alleviating sarcopenia and related symptoms in aged mice compared to CTP, attributed to differences in absorption. These findings provide insights into the potential of gCTP supplementation as a therapeutic strategy for sarcopenia.

In Vivo Mechanism of Action of Sodium Caprate for Improving the Intestinal Absorption of a GLP1/GIP Coagonist Peptide

Sodium caprate (C10) has been widely evaluated as an intestinal permeation enhancer for the oral delivery of macromolecules. However, the effect of C10 on the intestinal absorption of peptides with different physicochemical properties and its permeation-enhancing effect in vivo remains to be understood. Here, we evaluated the effects of C10 on intestinal absorption in rats with a glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GIP-GLP1) dual agonist peptide (LY) and semaglutide with different enzymatic stabilities and self-association behaviors as well as the oral exposure of the LY peptide in minipigs. Furthermore, we investigated the mechanism of action (MoA) of C10 for improving the intestinal absorption of the LY peptide in vivo via live imaging of the rat intestinal epithelium and tissue distribution of the LY peptide in minipigs. The LY peptide showed higher proteolytic stability in pancreatin and was a monomer in solution compared to that in semaglutide. C10 increased in vitro permeability in the minipig intestinal organoid monolayer to a greater extent for the LY peptide than for semaglutide. In the rat jejunal closed-loop model, C10 increased the absorption of LY peptide better than that of semaglutide, which might be attributed to higher in vitro proteolytic stability and permeability of the LY peptide. Using confocal live imaging, we observed that C10 enabled the rapid oral absorption of a model macromolecule (FD4) in the rat intestine. In the duodenum tissues of minipigs, C10 was found to qualitatively reduce the tight junction protein level and allow peptide uptake to the intestinal cells. C10 decreased the transition temperature of the artificial lipid membrane, indicating an increase in membrane fluidity, which is consistent with the above in vivo imaging results. These data indicated that the LY's favorable physicochemical properties combined with the effects of C10 on the intestinal mucosa resulted in an ∼2% relative bioavailability in minipigs.

Intestinal permeation enhancers enable oral delivery of macromolecules up to 70 kDa in size

The decades-long effort to deliver peptide drugs orally has resulted in several clinically successful formulations. These formulations are enabled by the inclusion of permeation enhancers that facilitate the intestinal absorption of peptides. Thus far, these oral peptide drugs have been limited to peptides less than 5 kDa, and it is unclear whether there is an upper bound of protein size that can be delivered with permeation enhancers. In this work, we examined two permeation enhancers, 1-phenylpiperazine (PPZ) and sodium deoxycholate (SDC), for their ability to increase intestinal transport of a model macromolecule (FITC-Dextran) as a function of its size. Specifically, the permeability of dextrans with molecular weights of 4, 10, 40, and 70 kDa was assessed in an in vitro and in vivo model of the intestine. In Caco-2 monolayers, both PPZ and SDC significantly increased the permeability of only FD4 and FD10. However, in mice, PPZ and SDC behaved differently. While SDC improved the absorption of all tested sizes of dextrans, PPZ was effective only for FD4 and FD10. This work is the first report of PPZ as a permeation enhancer in vivo, and it highlights the ability of permeation enhancers to improve the absorption of macromolecules across a broad range of sizes relevant for protein drugs.

Effects of Ghrelin on intestinal cell proliferation, the expression of protein absorption and metabolism factors in juvenile grass carp (Ctenopharyngodon idella)

Ghrelin can stimulate pituitary growth hormone (GH) release, and has a key role in the regulation of food intake and adiposity in vertebrates. At present, no relevant studies have reported the effects of Ghrelin on intestinal cell proliferation, protein absorption and metabolism. In this paper, firstly we established the intestinal epithelial cell lines of juvenile grass carp (Ctenopharyngodon idella) that can be passed stably and identified by the intestinal specific expression proteins E-cadherin and ZO-1. And we used this cell line as a model to investigate the effects of Ghrelin, Ala-Gln, Ala-Gln+Ghrelin on the proliferation of juvenile grass carp intestinal cells. We found that the use of Ghrelin, Ala-Gln and Ala-Gln+Ghrelin could promote the proliferation of intestinal cells of juvenile grass carp, but Ala-Gln+Ghrelin had the strongest promoting effect. Further, we investigated the effects of Ghrelin on the expression of cdx2, PepT1 and mTOR which are related to intestinal cell proliferation, protein absorption and metabolism of grass carp in vitro and in vivo experiments. And we studied the effects of different protein sources on the expression of Ghrelin, PepT1 and mTOR in grass carp at individual level. The results suggested that Ghrelin can promote grass carp intestinal cell proliferation, and affect the expression of genes which are related to intestinal peptide absorption and metabolism in juvenile grass carp.

New Nanocarrier System for Liposomes Coated with Lactobacillus acidophilus S-Layer Protein to Improve Leu-Gln-Pro-Glu Absorption through the Intestinal Epithelium.

The present study describes the development of a novel liposome nanocarrier system. The liposome was coated with Lactobacillus acidophilus CICC 6074 S-layer protein (SLP) to improve the intestinal absorption of the cholesterol-lowering peptide Leu-Gln-Pro-Glu (LQPE). The SLP-coated liposomes were prepared and characterized with morphology, particle size, zeta potential, membrane stability, Fourier transform infrared spectroscopy, and dual-channel surface plasma resonance. The results showed that SLP could successfully self-assemble on liposomes. Then, LQPE liposomes and SLP-coated LQPE liposomes (SLP-L-LQPE) were prepared. SLP-L-LQPE not only showed better sustained release properties and gastrointestinal tolerance in vitro but also increased the retention time in mice intestine. Transepithelial transport experiment indicates that the transshipment of LQPE increased significantly after being embedded by liposomes and coated with SLP. The research provides a theoretical basis for the study of SLP-coated liposomes and a potential drug delivery system for improving the intestinal absorption of peptides.

Properties and regulation of Gly-Sar uptake and transport in bovine intestinal epithelial cells.

Intestinal absorption of peptides is vital for the overall health and productivity of dairy cows. This study investigated the regulation, uptake and transport of dipeptides in bovine intestinal epithelial cells (BIECs). We also evaluated the effects of time, pH, concentration of the dipeptides, temperature, presence of diethylpyrocarbonate (DEPC)-an inhibitor of PepT1, and other dipeptides (Met-Met, Lys-Lys or Met-Lys), on the uptake and transport of Gly-Sar-FITC, which was a common fluorophore-labelled dipeptide. Under controlled experiments, BIECs were treated with 25μM LY294002 (a phosphatidylinositol 3-kinase (PI3K) inhibitor) and 25μM Perifosine (a protein kinase B (AKT) inhibitor). The subsequent expression of PepT1 in the BIECs was assessed by reverse transcriptase polymerase chain reaction (RT-PCR) and Western blotting. It was found that the uptake and transport of Gly-Sar-FITC were significant high at 37℃ than that at 4℃. The optimal pH for transport and uptake of Gly-Sar-FITC was 6.0-6.5, whereas the two properties decreased significantly in the presence of DEPC, Met-Met, Lys-Lys and Met-Lys (p<0.05). The apical-to-basolateral transport was also found to be significantly higher than the reverse transport (p<0.05). PI3K and AKT inhibitors were found to significantly suppress the expression of PepT1, thus impairing uptake and transport of Gly-Sar-FITC. Findings of this study thus suggest that the uptake and transport of Gly-Sar-FITC in BIECs are mediated by PepT1, and the PI3K/AKT signalling pathway regulates the absorption of small peptides.

Chitosan/sodium tripolyphosphate nanoparticles as efficient vehicles for enhancing the cellular uptake of fish-derived peptide.

Methodology to enhance the intestinal absorption of peptides is an important challenge due to their easily degradation and poor permeability across the intestinal epithelium. In this study, the fish-derived peptide (DGDDGEAGKIG)-loaded chitosan (CS) nanoparticles (CS/PEP-NPs) were prepared and investigated in Caco-2 monolayer model. The results indicated zeta potential of CS/PEP-NPs increased with the increase in molecular weight of CS (10-50kDa). Transmission electron microscopy images revealed the CS/PEP-NPs were uniform spherical-shaped nanoparticles with a diameter of 50-200nm (150kDa). Compared to other CS/PEP-NPs, 150-kDa CS/PEP-NPs performed an outstanding apparent permeability coefficient (Papp, 2.29×10-5 cms-1 ) and cumulative amount of peptide (120min, 2,987ng) in Caco-2 cells. CS/PEP-NPs could reduce the tight junction integrity of Caco-2 cells and enhance the intracellular fluorescence intensities of fluorescein isothiocyanate-labeled peptide. These findings suggest that chitosan nanoparticles are promising carriers to promote intestinal absorption of fish-derived peptide via paracellular pathway mediated by tight junctions. PRACTICAL APPLICATIONS: Chitosans are promising carriers to promote intestinal absorption of fish-derived peptide. The 150-kDa CS/PEP-NPs performed an outstanding apparent permeability coefficient (Papp, 2.29×10-5 cms-1 ) and cumulative amount of peptide (120min, 2,987ng) in Caco-2 cells. CS/PEP-NPs could reduce the tight junction integrity of Caco-2 cells and enhance the peptide uptake by paracellular pathway. Chitosan nanoparticles can be developed as vehicles for enhancing the cellular uptake of peptide in food industry.

Luminal Na+ homeostasis has an important role in intestinal peptide absorption in vivo.

Intestinal cell line studies indicated luminal Na+ homeostasis is essential for proton-coupled peptide absorption, because the driving force of PepT1 activity is supported by the apical Na+/H+ exchanger NHE3. However, there is no direct evidence demonstrating the importance of in vivo luminal Na+ for peptide absorption in animal experiments. To investigate the relationship between luminal Na+ homeostasis and peptide absorption, we took advantage of claudin 15-deficient (cldn15-/-) mice, whereby Na+ homeostasis is disrupted. We quantitatively assessed the intestinal segment responsible for peptide absorption using radiolabeled nonhydrolyzable dipeptide (glycylsarcosine, Gly-Sar) and nonabsorbable fluid phase marker polyethylene glycol (PEG) 4000 in vivo. In wild-type (WT) mice, the concentration ratio of Gly-Sar to PEG 4000 decreased in the upper jejunum, suggesting the upper jejunum is responsible for peptide absorption. Gly-Sar absorption was decreased in the jejunum of cldn15-/- mice. To elucidate the mechanism underlining these impairments, a Gly-Sar-induced short-circuit ( Isc) current was measured. In WT mice, increments of Gly-Sar-induced Isc were inhibited by the luminal application of a NHE3-specific inhibitor S3226 in a dose-dependent fashion. In contrast to in vivo experiments, robust Gly-Sar-induced Isc increments were observed in the jejunal mucosa of cldn15-/- mice. Gly-Sar-induced Isc was inhibited by S3226 or a reduction of luminal Na+ concentration, which mimics low luminal Na+ concentrations in vivo . Our study demonstrates that luminal Na+ homeostasis is important for peptide absorption in native epithelia and that there is a cooperative functional relationship between PepT1 and NHE3. NEW & NOTEWORTHY Our study is the first to demonstrate that luminal Na+ homeostasis is important for proton-coupled peptide absorption in in vivo animal experiments.

Enhanced Visualization of Small Peptides Absorbed in Rat Small Intestine by Phytic-Acid-Aided Matrix-Assisted Laser Desorption/Ionization-Imaging Mass Spectrometry

Enhanced visualization of small peptides absorbed through a rat intestinal membrane was achieved by matrix-assisted laser desorption/ionization time-of-flight imaging mass spectrometry (MALDI-IMS) with the aid of phytic acid as a matrix additive. Penetrants through intestinal peptide transporter 1, i.e., glycyl-sarcosine (Gly-Sar, 147.1 m/z) and antihypertensive dipeptide, Val-Tyr (281.2 m/z), were chosen for MALDI-IMS. The signal-to-noise (S/N) ratios of dipeptides Gly-Sar and Val-Tyr were seen to increase by 2.4- and 8.0-fold, respectively, when using a 2',4',6'-trihydroxyacetophenone (THAP) matrix containing 5.0 mM phytic acid, instead of the THAP matrix alone. Owing to the phytic-acid-aided MALDI-IMS method, Gly-Sar and Val-Tyr absorbed in the rat intestinal membrane were successfully visualized. The proposed imaging method also provided useful information on intestinal peptide absorption; to some extent, Val-Tyr was rapidly hydrolyzed to Tyr by peptidases located at the intestinal microvillus during the absorption process. In conclusion, the strongly acidic additive, phytic acid, is beneficial for enhancing the visualization of small peptides using MALDI-IMS, owing to the suppression of ionization-interfering salts in the tissue.

Complex Dietary Protein Improves Growth Through a Complex Mechanism of Intestinal Peptide Absorption and Protein Digestion

The small intestinal epithelium has an impressive ability to adapt to changes resulting from loss of length or alteration in dietary load. We sought to determine the potential influence of dietary protein absorption on growth and development. We hypothesized that a complex protein diet would improve growth over that of an elemental diet. Using the nematode Caenorhabditis elegans, a transparent worm with a rudimentary intestinal tube lined by enterocytes that function remarkably similar to humans, we measured daily growth, intestinal peptide, and amino acid transporter expression and intestinal proteolysis to compare the effects of a complex protein diet to a diet containing only amino acids. Nematodes raised on a complex protein diet grew to adulthood faster than those raised solely on amino acids, which did not require an overall change in intestinal peptide or amino acid transporter expression. Despite no overall change in transporter expression, a shift in location of peptide transporter expression was noted between diets that corresponded to decreased rate of intestinal proteolysis seen in the complex protein-fed group. A complex protein diet stimulates in an altered pattern of intestinal peptide transporter expression and intestinal proteolytic activity that results in improved growth compared with a diet of elemental amino acids. This improved growth appears to be the result of increased efficiency of a smaller number of enterocytes. These data might be useful in targeting intestinal rehabilitation therapies for short bowel syndrome and other risk factors for intestinal failure.

Dietary inorganic phosphorus and intestinal peptide absorption

Both organic and inorganic phosphorus (Pi) are present in regularly consumed foods such as meats, eggs, and dairy products. Pi is often included in foods as an additive, as hidden phosphorus. The intestinal peptide transporter PepT1 mediates protein absorption, which is disturbed in renal insufficiency. In the present study, we examined the effects of dietary Pi content on the intestinal peptide transport activity and expression of PepT1. The following animal models were used: 1) 7-week-old, male Wistar rats; and 2) rats that underwent 3/4 nephrectomy (3/4NR) to induce chronic kidney disease (CKD). Rats were fed a normal-protein (20%) diet containing low (0.02%), normal (0.6%) or high (1.2%) Pi levels. Rats were also fed diets containing varying amounts of protein and either low or normal Pi levels, as follows: 1) low Pi/normal protein, 2) low Pi/high (50%) protein, 3) normal Pi/normal protein, and 4) normal Pi/high protein. Intestinal peptide transport activity and PepT1 expression levels were significantly higher in CKD rats than in sham-operated controls. Compared with the normal diet, the high-protein diet increased PepT1 expression in CKD rats. Intestinal di-peptide transport activity and PepT1 protein levels did not increase in rats fed the low-Pi/high-protein diet. In contrast, intestinal di-peptide transport activity and PepT1 protein expression were markedly increased in rats fed the normal-Pi/high-protein diet. In conclusion, dietary Pi levels regulate intestinal peptide transport activity via PepT1.

Importance of intermolecular interaction on the improvement of intestinal therapeutic peptide/protein absorption using cell-penetrating peptides

Our previous reports showed that the absorption of therapeutic peptides and proteins was significantly improved by coadministration of cell-penetrating peptides (CPPs) as the physical mixture. However, the mechanisms for this improvement are not clear. In the present study, we verified the hypothesis that the electrostatic interaction between drug and CPP is related to the enhancing effect of the CPP on the intestinal absorption of therapeutic peptides and proteins. In this study, the intermolecular binding was analyzed by surface plasmon resonance (SPR)-based binding assay, and the effect of CPPs on the intestinal absorption of peptide drugs was examined by in situ absorption study using a rat intestinal loop. Among the 16 peptide drugs possessing different isoelectric points, it was observed that only gastrin, insulin and glucagon-like peptide-1 (GLP-1) bound to D-R8 (D-form arginine octamer, a typical CPP), and subsequently their intestinal absorption increased by coadministration of D-R8. In contrast, the intestinal absorption of other peptide drugs that did not bind to D-R8 was not affected in the presence of D-R8. Thus, this study suggests that intermolecular binding between drug and CPP is an important factor governing the enhancing effect of the CPP on the intestinal absorption of therapeutic peptides and proteins.

Interferon-γ increases expression of the di/tri-peptide transporter, h-PEPT1, and dipeptide transport in cultured human intestinal monolayers

The di/tri-peptide transporter h-PEPT1 plays an important role in the oral absorption of di/tri-peptides and numerous drugs. Inflammatory conditions may influence intestinal xenobiotic transporter function; however, the effects of inflammation on h-PEPT1 have not been well described. This study was conducted to determine the effects of the inflammatory cytokine interferon-γ (IFN-γ) on h-PEPT1 mediated dipeptide absorption. Caco-2 monolayers were grown on permeable supports. The effective apical-to-basolateral permeability ( P eff) of glycylsarcosine (Gly-Sar) was measured following incubation with IFN-γ or control media. Additional experiments were conducted at 4 °C, and with escalating concentrations of Gly-Sar. h-PEPT1 expression was determined using semiquantitative RT-PCR. IFN-γ 50 ng/ml increased Gly-Sar P eff 28.6% compared to controls ( p = 0.03). In experiments conducted at 4 °C, Gly-Sar P eff decreased 39.6% in IFN-γ treated cells ( p = 0.003) and 28.4% in controls ( p = 0.006). In controls and IFN-γ treated cells, concentration dependent transport was seen with escalating concentrations of Gly-Sar. Compared to controls, IFN-γ 50 and 100 ng/ml increased h-PEPT1 mRNA expression by 14.2% and 11.5%, respectively ( p = 0.019). In summary, IFN-γ increases h-PEPT1 expression and permeation of the dipeptide Gly-Sar in Caco-2 monolayers. These findings imply that intestinal absorption of peptides and peptidomimetic drugs may be increased in certain inflammatory conditions.

Nanoparticles based on N-trimethylchitosan: Evaluation of absorption properties using in vitro (Caco-2 cells) and ex vivo (excised rat jejunum) models

Among the chitosan derivatives, trimethylchitosan (TMC) has been shown to have penetration enhancement properties also in intestinal environment. In addition, the use of nanoparticulate systems has the advantage of protecting peptidic drugs from intestinal degradations, due to internalisation behaviour. Therefore, the aim of this paper was to evaluate nanoparticulate systems based on TMC. In particular the mucoadhesive and absorption enhancement properties of nanoparticles based on TMC with different quaternization degree (QD) intended for the intestinal administration of macromolecules (peptides) have been evaluated. Comparison with chitosan (CS·HCl) nanoparticles was made. The nanoparticles were loaded with fluorescein isothiocyanate dextran (FD4, MW 4400 Da), used as the model macromolecule. The intestinal penetration enhancement properties of nanoparticles were investigated in an in vitro Caco-2 cell model and an ex vivo rat jejunum model. The mucoadhesion of the nanosystems was evaluated using excised rat jejunum. All of the nanoparticulate systems interacted with the Caco-2 cells decreasing the transepithelial electric resistance (TEER) and increasing Lucifer Yellow (LY) Papp (paracellular pathway marker). All the nanosystems improved FD4 Papp, with the exception of the nanoparticles based on TMC with the highest QD. In this case an entrapment of nanoparticles into Caco-2 cells was supposed. Analogous results were obtained using the excised rat jejunum model. The increase in QD of TMC was seen to favour the mucoadhesion, resulting in a prolonged residence time on intestinal mucosa. The nanoparticle penetration into excised rat jejunum tissue, observed by means of CLSM, suggested that the mucoadhesive properties delayed the absorption of nanoparticles, however they produced an increase in the contact time with intestinal epithelium, offering a better chance for internalisation. The improvement of mucoadhesion and of nanoparticle internalisation with respect to chitosan nanosystems makes the TMCs nanosystems suitable carriers for the intestinal absorption of peptides.

The intestinal peptide absorption is critical for the modulation of growth, development and lifespan in Caenorhabditis elegans

The intestinal peptide absorption is critical for the modulation of growth, development and lifespan in Caenorhabditis elegans

Current Perspectives on Established and Putative Mammalian Oligopeptide Transporters

Peptides and peptide‐based drugs are increasingly being utilized as therapeutic agents for the treatment of numerous disorders. The increasing development of peptide‐based therapeutic agents is largely due to technological advances including the advent of combinatorial peptide libraries, peptide synthesis strategies, and peptidomimetic design. Peptides and peptide‐based agents have a broad range of potential clinical applications in the treatment of many disorders including AIDS, hypertension, and cancer. Peptides are generally hydrophilic and often exhibit poor passive transcellular diffusion across biological barriers. Insights into strategies for increasing their intestinal absorption have been derived from the numerous studies demonstrating that the absorption of protein digestion products occurs primarily in the form of small di‐ and tripeptides. The characterization of the pathways of intestinal, transepithelial transport of peptides and peptide‐based drugs have demonstrated that a significant degree of absorption occurs through the role of proteins within the proton‐coupled, oligopeptide transporter (POT) family. Considerable focus has been traditionally placed on Peptide Transporter 1 (PepT1) as the main mammalian POT member regulating intestinal peptide absorption. Recently, several new POT members, including Peptide/Histidine Transporter 1 (PHT1) and Peptide/Histidine Transporter 2 (PHT2) and their splice variants have been identified. This has led to an increased need for new experimental methods enabling better characterization of the biophysical and biochemical barriers and the role of these POT isoforms in mediating peptide‐based drug transport. © 2003 Wiley‐Liss, Inc. and the American Pharmaceutical Association J Pharm Sci 92:691–714, 2003

Possible degradative process of cholecystokinin analogs in rabbit jejunum brush-border membrane vesicles

Our recent work on the intestinal metabolism and absorption of cholecystokinin analogs, sulfated C-terminal octapeptide (CCK8; Asp-Tyr(SO 3H)-Met-Gly-Trp-Met-Asp-Phe(NH 2) = DY SO 3H MGWMDF NH 2 ) and tetrapeptide (CCK4; Trp-Met-Asp-Phe(NH 2) = WMDF NH 2 ), was extended to investigate the degradative process of these analogs using rabbit jejunum brush-border membrane vesicles and to find a better enzyme-inhibitor system for intestinal absorption of peptide drugs. Various enzyme inhibitors and a lower pH buffer were applied to discover the major enzyme(s) involved in each process. Metabolic pathways showing degradative processes were proposed for both analogs. The major cleavage site occurs at the W 1-M 2 for CCK4. At least three metabolic pathways occur independently for CCK8 and appear at peptides bonds between G 4-W 5, M 6-D 7, and D 7-F NH 2 8. Many different enzymes of aminopeptidase, endopeptidase, angiotensin-converting enzyme, metalloenzyme, and others were involved in each process. Identification of more specific yet safe enzyme inhibitors and co-administration of various these inhibitors may lead to further enhancement in intestinal peptide absorption when administered orally.

Improvement of intestinal absorption of peptides: adsorption of B1-Phe monoglucosylated insulin to rat intestinal brush-border membrane vesicles

In a previous study we glycosylated insulin to improve its intestinal absorption. When the glycosylated product, p-(succinylamido)-phenyl-α- d-glucopyranoside (SAPG)-substituted insulin (SAPG-INS), was administered intra-intestinally to rats, it showed a greater hypoglycemic effect than native bovine insulin. The enhanced hypoglycemic effect of SAPG-INS was considered to be due to an increase in membrane permeability as well as an increase in resistance to enzymatic degradation. In particular, membrane permeability may be related to an interaction with the Na +-dependent d-glucose transporter (SGLT-1) which is located in the brush-border membrane of epithelial cells. The insulin product used in the previous study, however, comprised a mixture of mono-, di- and tri-SAPG-substituted insulin. In this study SAPG-INS with a defined substitution number and position was synthesized to examine the interaction between the transporter and glycosylated insulin in more detail. The new product was mono-SAPG-substituted insulin substituted at the B1-phenylalanine position (B1-SAPG-INS) and was selectively synthesized after protection of the A1-glycine and εB29-lysine amino acids. The hypoglycemic effect of B1-SAPG-INS in rats after an intravenous dose of 71 μg/kg was almost the same as that of native bovine insulin at a dose of 1 U/kg and B1-SAPG-INS retained about 60% of the immunoreactivity of native bovine insulin. The interaction of B1-SAPG-INS with the intestinal transporter was examined by a rapid filtration technique using 125I-labeled B1-SAPG-INS and brush-border membrane vesicles (BBMVs) which were prepared from rat small intestine by the Mg-precipitation method. The amount of B1-SAPG-INS adsorbed or absorbed by BBMVs in the presence of an inward Na +-gradient into BBMVs was greater than that of native bovine insulin. This adsorption/absorption was significantly inhibited by the presence of 1 mM phloridzin. A similar inhibition was observed when Na + was replaced with K + and when B1-SAPG-INS was incubated with BBMVs at 4°C. From the effect of osmolarity on the extent of adsorption/absorption, it was considered that B1-SAPG-INS was not taken up into the intravesicular space but adsorbed onto the external membrane surface of BBMVs. These findings suggested that B1-SAPG-INS was adsorbed specifically onto the transporter. The hypoglycemic effect of insulin was enhanced by glycosylation at the B1 position in in situ experiments using normal and diabetic rats. Consequently, it is suggested that B1-SAPG-INS was adsorbed specifically onto the glucose transporter of intestinal BBM. This specific adsorption may be involved in the mechanism of the enhanced hypoglycemic effect of B1-SAPG-INS both in normal and diabetic rats.

Intestinal absorption of peptides through the enterocytes.

Transport of intact peptides and proteins from the intestinal lumen into the blood is a unique phenomenon, which differs from the regular process of food digestion and absorption. Intestinal absorption of minute amounts of proteins is, however, being considered as a normal physiological process. It is thus important to define and understand the routes for protein transfer from the intestinal lumen to the blood and the mechanisms by which the macromolecules overcome the sieving barrier of the intestinal wall. The study on insulin has demonstrated that, upon proper introduction into the intestinal lumen, insulin is absorbed by the epithelial cells and transferred to the circulation. The peptides absorbed and transferred to the blood retained their biological activity and induced significant lowering of blood glucose levels. The efficiency of the absorption does not differ among the ileum, duodenum, and colon. Morphological examination demonstrated no alteration of the structural integrity of the epithelia, the enterocytes stay intact with well-developed microvilli, and the cells remain joined by tightly closed junctions. Application of immunocytochemistry on thin tissue sections revealed insulin antigenic sites at different locations depending on the time point. Insulin detected in the lumen of the intestinal tract is absorbed through the endosomal compartment of the epithelial cells rather than passing between cells. Internalization occurs through invaginations of the luminal plasma membrane and vesicular structures of the endosomal compartment. In 5-10 minutes, insulin is transferred to the basolateral membrane and released into the interstitial space to reach the circulation. Definition of the transcytotic pathway will contribute to a better understanding of drug delivery for potential therapeutic applications.

Intestinal absorption of peptides through the enterocytes

Transport of intact peptides and proteins from the intestinal lumen into the blood is a unique phenomenon, which differs from the regular process of food digestion and absorption. Intestinal absorption of minute amounts of proteins is, however, being considered as a normal physiological process. It is thus important to define and understand the routes for protein transfer from the intestinal lumen to the blood and the mechanisms by which the macromolecules overcome the sieving barrier of the intestinal wall. The study on insulin has demonstrated that, upon proper introduction into the intestinal lumen, insulin is absorbed by the epithelial cells and transferred to the circulation. The peptides absorbed and transferred to the blood retained their biological activity and induced significant lowering of blood glucose levels. The efficiency of the absorption does not differ among the ileum, duodenum, and colon. Morphological examination demonstrated no alteration of the structural integrity of the epithelia, the enterocytes stay intact with well-developed microvilli, and the cells remain joined by tightly closed junctions. Application of immunocytochemistry on thin tissue sections revealed insulin antigenic sites at different locations depending on the time point. Insulin detected in the lumen of the intestinal tract is absorbed through the endosomal compartment of the epithelial cells rather than passing between cells. Internalization occurs through invaginations of the luminal plasma membrane and vesicular structures of the endosomal compartment. In 5–10 minutes, insulin is transferred to the basolateral membrane and released into the interstitial space to reach the circulation. Definition of the transcytotic pathway will contribute to a better understanding of drug delivery for potential therapeutic applications. Microsc. Res. Tech. 49:346–352, 2000. © 2000 Wiley-Liss, Inc.