RESUMEN
This study evaluated the effect of methionine supplementation, predation risk and their interaction on gut histology, whole-body cortisol levels, and intestinal gene expression in zebrafish. A total of 360 one-year-old animals were maintained under two environmental conditions and fed diets containing different methionine sources. Fish were fed either a control diet (CTL, without methionine supplementation), a diet supplemented with dl-methionine (DLM), or a diet supplemented with methionine dipeptide (MM) in the absence (AP) of a predator or in the presence of the predator (PP) for 48 h or 20 days. Predator-induced stress for 20 days resulted in lower body weight. Zebrafish fed methionine-supplemented diets had higher weight gain than control fish. We found no effect of predation stress or methionine supplementation on cortisol level. Predation risk and methionine supplementation showed no interaction effect on dipeptide transporter gene expression. After 48 h of predation pressure, zebrafish had higher mRNA expression of SOD2, CAT and GPX1 in the gut. After 20 days of exposure to the predator, zebrafish fed methionine-supplemented diets had lower expression of GPX1, SOD2 and CAT than those diet CTL. Methionine dipeptide and free methionine supplementation improved growth, intestinal health and survivability of zebrafish both conditions.
Asunto(s)
Alimentación Animal , Fenómenos Fisiológicos Nutricionales de los Animales , Metionina , Pez Cebra , Alimentación Animal/análisis , Animales , Catalasa/metabolismo , Dieta/veterinaria , Suplementos Dietéticos , Dipéptidos , Glutatión Peroxidasa/metabolismo , Intestinos , Metionina/administración & dosificación , Transportador de Péptidos 1/metabolismo , Conducta Predatoria , Superóxido Dismutasa/metabolismo , Proteínas de Pez Cebra/metabolismo , Glutatión Peroxidasa GPX1RESUMEN
A most crucial feature of biological adaptation is the maintenance of a close temporal relationship of behaviour and physiology with prevailing 24-h light-dark environment, which is rapidly changing with increasing nighttime illumination. This study investigated developmental effects of the loss of night on circadian behaviour, metabolism and gene expressions in diurnal zebra finches born and raised under LL, with controls on 12L:12D. Birds under LD were entrained, and showed normal body mass and a significant 24-h rhythm in both activity-rest pattern and mRNA expression of candidate genes that we measured. But, under LL, birds gained weight and accumulated lipid in the liver. Intriguingly, at the end of the experiment, the majority (4/5th) of birds under LL were rhythmic in activity despite arrhythmic expression in the hypothalamus of c-Fos (neuronal activity), Rhodopsin and Mel1-a genes (light perception), and clock genes (Bmal1, Per2 and Rev-erb ß). In peripheral tissues, LL induced variable clock gene expressions. Whereas 24-h mRNA rhythm was abolished for Bmal1 in both liver and gut, it persisted for Per2 and Rev-erb ß in liver, and for Per2 in gut. Further, we found under LL, the loss of 24-h rhythm in hepatic expression of Fasn and Cd36/Fat (biosynthesis and its uptake), and gut expression of Sglt1, Glut5, Cd36 and Pept1 (nutrient absorption) genes. As compared to LD, baseline mRNA levels of Fasn and Cd36 genes were attenuated under LL. Among major transporter genes, Sglt1 (glucose) and Cd36 (fat) genes were arrhythmic, while Glut5 (glucose) and Pept1 (protein) genes were rhythmic but with phase differences under LL, compared to LD. These results demonstrate dissociation of circadian behaviour from clock gene rhythms, and provide molecular insights into possible mechanisms at different levels (behaviour and physiology) that diurnal animals might employ in order to adapt to an emerging overly illuminated-night urban environment.
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Relojes Circadianos/fisiología , Ritmo Circadiano/fisiología , Regulación de la Expresión Génica/fisiología , Hipotálamo/fisiología , Metabolismo/fisiología , Animales , Antígenos CD36/genética , Antígenos CD36/metabolismo , Proteínas CLOCK/genética , Proteínas CLOCK/metabolismo , Pinzones , Transportador de Glucosa de Tipo 5/genética , Transportador de Glucosa de Tipo 5/metabolismo , Luz , Hígado , Transportador de Péptidos 1/genética , Transportador de Péptidos 1/metabolismo , Fotoperiodo , ARN Mensajero/metabolismo , Rodopsina/genética , Rodopsina/metabolismo , Transportador 1 de Sodio-Glucosa/genética , Transportador 1 de Sodio-Glucosa/metabolismo , EstómagoRESUMEN
Naturally occurring dietary peptides derived from gastrointestinal digestates of common bean milk and yogurt were studied for their bioaccessibility, bioavailability, and anti-inflammatory activity in both Caco-2 mono- and Caco-2/EA.hy926 co-culture cell models. Anti-inflammatory activities of these peptide extracts were found to be strongly associated with cellular uptake by the intestinal epithelial cells. Mechanisms underlying the cellular uptake were studied by examining the role of peptide transporter 1 and calcium sensing reporter. Three peptides, including γ-glutamyl-S-methylcysteine, γ-glutamyl-leucine, and leucine-leucine-valine, were found to be transported across the Caco-2 cell monolayer and detected by liquid chromatography-tandem mass spectrometry. A strong anti-inflammatory effect was observed in the basolateral EA.hy926 cells (co-culture model), as shown in their inhibition of tumor necrosis factor α-induced pro-inflammatory mediators of the nuclear factor κB and mitogen-activated protein kinase signal cascades. The results suggest that these peptides can be absorbed and possibly have systemic inhibition on inflammatory responses in vascular endothelial cells, indicating potential preventive effects on vascular diseases.
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Antiinflamatorios/metabolismo , Células Endoteliales/metabolismo , Péptidos/metabolismo , Phaseolus/química , Extractos Vegetales/metabolismo , Yogur/análisis , Transporte Biológico , Células CACO-2 , Técnicas de Cocultivo , Células Epiteliales/metabolismo , Humanos , Mucosa Intestinal/metabolismo , Intestinos/citología , FN-kappa B/genética , FN-kappa B/metabolismo , Transportador de Péptidos 1/genética , Transportador de Péptidos 1/metabolismo , Péptidos/química , Phaseolus/metabolismo , Extractos Vegetales/químicaRESUMEN
It is difficult to predict the pharmacokinetics and plasma concentration-time profiles of new chemical entities in humans based on animal data. Some pharmacokinetic parameters, such as clearance and volume of distribution, can be scaled allometrically from rodents, mammals, and nonhuman primates with good success. However, it is far more challenging to predict the oral pharmacokinetics of experimental drug candidates. In the present study, we used in situ estimates of intestinal permeability, obtained in silico and from rat, wild-type (WT), and humanized PepT1 (huPepT1) mice, to predict the systemic exposure of cefadroxil, an orally administered model compound, under a variety of conditions. Using the GastroPlus simulation software program (Simulations Plus, Lancaster, CA), we found that the C max and area under the plasma concentration-time curve from time zero to the last measurable concentration of cefadroxil were better predicted using intestinal permeability estimates (both segmental and jejunal) from huPepT1 than from WT mice, and that intestinal permeabilities based on in silico and rat estimates gave worse predictions. We also observed that accurate predictions were possible for cefadroxil during oral dose escalation (i.e., 5, 15, and 30 mg/kg cefadroxil), a drug-drug interaction study (i.e., 5 mg/kg oral cefadroxil plus 45 mg/kg oral cephalexin), and an oral multiple dose study [i.e., 500 mg (6.7 mg/kg) cefadroxil every 6 hours]. Finally, the greatest amount of cefadroxil was absorbed in duodenal and jejunal segments of the small intestine after a 5 mg/kg oral dose. Thus, by combining a humanized mouse model and in silico software, the present study offers a novel strategy for better translating preclinical pharmacokinetic data to oral drug exposure during first-in-human studies.
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Antibacterianos/farmacocinética , Cefadroxilo/farmacocinética , Mucosa Intestinal/metabolismo , Modelos Biológicos , Transportador de Péptidos 1/genética , Administración Oral , Animales , Antibacterianos/administración & dosificación , Área Bajo la Curva , Cefadroxilo/administración & dosificación , Cefalexina/administración & dosificación , Cefalexina/farmacología , Simulación por Computador , Evaluación Preclínica de Medicamentos/métodos , Interacciones Farmacológicas , Duodeno/metabolismo , Humanos , Yeyuno/metabolismo , Ratones , Ratones Transgénicos , Transportador de Péptidos 1/metabolismo , Permeabilidad , Ratas , Programas InformáticosRESUMEN
Immunomodulatory protein hydrolysate consumption may delay or prevent western immune-related diseases. In order to purposively develop protein hydrolysates with an optimal and reproducible immunomodulatory effect, knowledge is needed on which components in protein hydrolysates are responsible for the immune effects. Important advances have been made on this aspect. Also, knowledge on mechanisms underlying the immune modulating effects is indispensable. In this review, we discuss the most promising application possibilities for immunomodulatory protein hydrolysates. In order to do so, an overview is provided on reported in vivo immune effects of protein hydrolysates in both local intestinal and systemic organs, and the current insights in the underlying mechanisms of these effects. Furthermore, we discuss current knowledge and physicochemical approaches to identify the immune active protein sequence(s). We conclude that multiple hydrolysate compositions show specific immune effects. This knowledge can improve the efficacy of existing hydrolysate-containing products such as sports nutrition, clinical nutrition, and infant formula. We also provide arguments for why immunomodulatory protein hydrolysates could be applied to manage the immune response in the increasing number of individuals with a higher risk of immune dysfunction due to, for example, increasing age or stress.
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Suplementos Dietéticos , Sistema Inmunológico/efectos de los fármacos , Factores Inmunológicos/administración & dosificación , Hidrolisados de Proteína/administración & dosificación , Animales , Suplementos Dietéticos/efectos adversos , Endocitosis , Humanos , Sistema Inmunológico/inmunología , Sistema Inmunológico/metabolismo , Inmunidad Mucosa/efectos de los fármacos , Factores Inmunológicos/efectos adversos , Factores Inmunológicos/inmunología , Factores Inmunológicos/metabolismo , Mucosa Intestinal/metabolismo , Intestinos/efectos de los fármacos , Intestinos/inmunología , Ganglios Linfáticos/efectos de los fármacos , Ganglios Linfáticos/inmunología , Ganglios Linfáticos/metabolismo , Transportador de Péptidos 1/metabolismo , Hidrolisados de Proteína/efectos adversos , Hidrolisados de Proteína/inmunología , Hidrolisados de Proteína/metabolismo , Receptores Inmunológicos/efectos de los fármacos , Receptores Inmunológicos/inmunología , Receptores Inmunológicos/metabolismoRESUMEN
The purpose of this work was to evaluate the intestinal permeability, oral absorption and disposition of the ester prodrug valacyclovir in wildtype mice and a huPepT1 transgenic mouse model. PepT1 (SLC15A1) is a transporter apically expressed along the lumen of the gastrointestinal tract and is responsible for the absorption of di-/tripeptides, ACE inhibitors, ß-lactam antibiotics and numerous prodrugs. Unfortunately, PepT1-mediated substrates that have been extensively studied were shown to exhibit species-dependent absorption and pharmacokinetics. Accordingly, in situ intestinal perfusion studies were conducted and valacyclovir uptake was shown to have a 30-fold lower Km and 100-fold lower Vmax in huPepT1 compared to wildtype mice. Moreover, inhibition studies demonstrated that the huPepT1 transporter alone was responsible for valacyclovir uptake, and segment-dependent studies reported significant reductions in permeability along the length of small intestine in huPepT1 mice. Subsequent oral administration studies revealed that the in vivo rate and extent of valacyclovir absorption were lower in huPepT1 mice, as indicated by 3-fold lower Cmax and 3-fold higher Tmax values, and an AUC0-180 that was 80% of that observed in wildtype mice. However, no significant changes in drug disposition were observed between genotypes after intravenous bolus administration of acyclovir. Lastly, mass balance studies established that the bioavailability of acyclovir, after oral dosing of valacyclovir, was 77.5% in wildtype mice and 52.8% in huPepT1 mice, which corroborated values of 51.3% in clinical studies. Thus, it appears the huPepT1 transgenic mice may be a better model to study prodrug absorption and disposition in humans than wildtype mice.
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Antivirales/metabolismo , Absorción Intestinal/fisiología , Mucosa Bucal/metabolismo , Transportador de Péptidos 1/biosíntesis , Profármacos/metabolismo , Valaciclovir/metabolismo , Administración Oral , Animales , Antivirales/administración & dosificación , Relación Dosis-Respuesta a Droga , Evaluación Preclínica de Medicamentos , Humanos , Absorción Intestinal/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Mucosa Bucal/efectos de los fármacos , Transportador de Péptidos 1/genética , Profármacos/administración & dosificación , Valaciclovir/administración & dosificaciónRESUMEN
As the organ with one of the largest surface areas facing the environment and responsible for nutrient uptake, the small intestine expresses numerous transport proteins in its brush-border membrane for efficient absorption and supply of dietary macro- and micronutrients. The understanding of regulation and functional interplay of these nutrient transporters is of emerging interest in nutrition and medical physiology research in respect to development of diabetes, obesity, and inflammatory bowel disease worldwide. The peptide transporter 1 (PepT1, SLC15A1) is abundantly expressed particularly in the intestinal tract and provides highly effective transport of amino acids in the form of di- and tripeptides and features a substantial acceptance for structurally related compounds and drugs. These characteristics bring PepT1 into focus for nutritional and medical/pharmaceutical approaches, as it is the essential hub responsible for oral bioavailability of dietary protein/peptide supplements and peptide-like drugs in eukaryotic organisms. Detailed analysis of molecular processes regulating PepT1 expression and function achieved in the last two decades has helped to define and use adjusting tools and to better integrate the transporter's role in cell and organ physiology. In this article, we provide an overview of the current knowledge on PepT1 function in health and disease, and on regulatory factors modulating its gene and protein expression as well as transport activity. © 2018 American Physiological Society. Compr Physiol 8:843-869, 2018.
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Absorción Intestinal/fisiología , Transportador de Péptidos 1/fisiología , Animales , Transporte Biológico/fisiología , Diabetes Mellitus/metabolismo , Regulación de la Expresión Génica/fisiología , Homeostasis/fisiología , Humanos , Enfermedades Inflamatorias del Intestino/metabolismo , MicroARNs/genética , Fenómenos Fisiológicos de la Nutrición/fisiología , Obesidad/metabolismo , Transportador de Péptidos 1/genéticaRESUMEN
In humans, peptides derived from dietary proteins and peptide-like drugs are transported via the proton-dependent oligopeptide transporter hPepT1 (SLC15A1). hPepT1 is located across the apical membranes of the small intestine and kidney, where it serves as a high-capacity low-affinity transporter of a broad range of di- and tripeptides. hPepT1 is also overexpressed in the colon of inflammatory bowel disease (IBD) patients, where it mediates the transport of harmful peptides of bacterial origin. Therefore, hPepT1 is a drug target for prodrug substrates interacting with intracellular proteins or inhibitors blocking the transport of toxic bacterial products. In this study, we construct multiple structural models of hPepT1 representing different conformational states that occur during transport and inhibition. We then identify and characterize five ligands of hPepT1 using computational methods, such as virtual screening and QM-polarized ligand docking (QPLD), and experimental testing with uptake kinetic measurements and electrophysiological assays. Our results improve our understanding of the substrate and inhibitor specificity of hPepT1. Furthermore, the newly discovered ligands exhibit unique chemotypes, providing a framework for developing tool compounds with optimal intestinal absorption as well as future IBD therapeutics against this emerging drug target.
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Modelos Químicos , Oligopéptidos/química , Transportador de Péptidos 1/química , Profármacos/química , Transporte Biológico Activo/efectos de los fármacos , Evaluación Preclínica de Medicamentos/métodos , Humanos , Concentración 50 Inhibidora , Absorción Intestinal/efectos de los fármacos , Absorción Intestinal/fisiología , Cinética , Ligandos , Modelos Moleculares , Simulación del Acoplamiento Molecular , Oligopéptidos/metabolismo , Transportador de Péptidos 1/antagonistas & inhibidores , Transportador de Péptidos 1/fisiología , Profármacos/farmacologíaRESUMEN
High ambient temperature adversely influences poultry production. In the present study, gamma amino butyric acid (GABA) supplementation was used to alleviate the adverse changes due to heat stress (HS) in a broiler chicken strain (Ross 308). At 21 days of age, the birds were divided into four groups of 13. Two groups were housed under normal room temperature, one group was given orally 0.2 ml 0.9% physiological saline (CN) daily, the other group received 0.2 ml of 0.5% GABA solution orally (GN). A third group was exposed to environmental HS (33 ± 1 °C lasting for 2 weeks) + physiological saline (CH) and a fourth group was exposed to HS + GABA supplementation (GH). GABA supplementation during HS significantly reduced the birds' increased body temperature (p <.0001) and increased their body weight gain (p <.0001). This effect was associated with increases in the heat stress-induced reductions in jejunal villus length, crypt depth and mucous membrane thickness, and decreases in the vascular changes occurred due to HS. Additionally, GABA supplementation significantly modulated HS-induced changes in glucose facilitated transporter 2 (GLUT2), peptide transporter 1 (PEPT1) and heat shock protein 70 (HSP70) mRNA expression in the jejunal mucosa (p < .0001). GABA supplementation also significantly elevated the triiodothyronine (T3) hormone level and hemoglobin levels and decreased the heterophil-lymphocyte ratio (H/L ratio) (p <.0001). Furthermore, it induced higher hepatic glutathione peroxidase enzyme (GSH-Px) activities and decreased the malondialdehyde dehydrogenase (MDA) content. These results indicate that GABA supplementation during HS may be used to alleviate HS-related changes in broiler chickens.
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GABAérgicos/farmacología , Transportador de Glucosa de Tipo 2/efectos de los fármacos , Proteínas HSP70 de Choque Térmico/efectos de los fármacos , Respuesta al Choque Térmico/efectos de los fármacos , Calor/efectos adversos , Mucosa Intestinal/efectos de los fármacos , Yeyuno/efectos de los fármacos , Transportador de Péptidos 1/efectos de los fármacos , ARN Mensajero/efectos de los fármacos , Ácido gamma-Aminobutírico/farmacología , Animales , Pollos , Transportador de Glucosa de Tipo 2/genética , Transportador de Glucosa de Tipo 2/metabolismo , Glutatión Peroxidasa/efectos de los fármacos , Glutatión Peroxidasa/metabolismo , Proteínas HSP70 de Choque Térmico/genética , Proteínas HSP70 de Choque Térmico/metabolismo , Trastornos de Estrés por Calor , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patología , Yeyuno/metabolismo , Yeyuno/patología , Hígado/efectos de los fármacos , Masculino , Malondialdehído/metabolismo , Oxidorreductasas/efectos de los fármacos , Oxidorreductasas/metabolismo , Transportador de Péptidos 1/genética , Transportador de Péptidos 1/metabolismo , ARN Mensajero/metabolismo , Estrés Fisiológico/efectos de los fármacosRESUMEN
Synergistic effect is main pharmacological mechanism of traditional Chinese medicine(TCM). The research method based on the key targets combination is an important method to explore the synergistic effect of TCM. Peptide transporter 1 (PepT1) is an essential target for drug uptake into the bloodstream, accounting for about 50% of the total transporter protein content from the small intestine. Peroxisome proliferator-activated receptor α(PPARα) is the lipid-lowering target of fibrates, which have a good hypolipidemic effect by activating PPARα. It has been reported that PPARα could activate the gene expression of PepT1s, and PPARα agonists can promote the uptake of PepT1 substrates, indicating their synergistic effect. In this paper, PepT1 substrates and PPARα agonists from TCM were discovered, and their synergistic mechanism was also been discussed based on the target combination of PepT1 and PPARα. The support vector machine(SVM) model of PepT1 substrates was first constructed and utilized to predict potential TCM components. Meanwhile, merged pharmacophore and docking model of PPARα agonists was used to screen the potential active ingredients from TCM. According to the analysis results of two groups, the TCM combination of Panax notoginseng and Ganoderma lucidum, as well as TCM combination of P. notoginseng and Salvia miltiorrhiza were identified to have the synergistic mechanism based on target combination of PepT1 and PPARα. In this study, synergistic mechanism of TCM was analyzed for absorption and hypolipidemic effect based on target combination, which provides a new way to explore the synergetic mechanism of TCM related to pharmacokinetics.
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Medicamentos Herbarios Chinos/farmacología , PPAR alfa/metabolismo , Transportador de Péptidos 1/metabolismo , Sinergismo Farmacológico , Ganoderma , Humanos , Medicina Tradicional China , Simulación del Acoplamiento Molecular , PPAR alfa/agonistas , Panax notoginseng , Máquina de Vectores de SoporteRESUMEN
Curcuma comosa has been widely used as a herbal medicine in Thailand; however, it remains unclear whether C. comosa influences the absorption of drugs that are substrates for the transporters in the small intestine. In this study, we investigated the effect of C. comosa extracts on the functioning of peptide transporter 1 (PEPT1), an influx transporter, and P-glycoprotein (P-gp), an efflux transporter, in Caco-2 cells and rat intestine. In Caco-2 cells, the ethanolic extract of C. comosa (CCE) lowered the uptake of glycylsarcosine (Gly-Sar), a PEPT1 substrate, while it enhanced the uptake of rhodamine 123 (Rho123), a P-gp substrate, in a concentrationdependent manner. In addition, CCE inhibited apical-to-basal transport of Gly-Sar and basal-to-apical transport of Rho123. Furthermore, the absorption of cephalexin, another PEPT1 substrate, and the exsorption of Rho123 across the rat intestine were inhibited by CCE. Conversely, CCW, the hot water extract of C. comosa, suppresses the function of PEPT1 but not of P-gp in Caco-2 cells. These results suggest that C. comosa used as a herbal medicine in Thailand may affect the intestinal absorption of certain drugs.
Asunto(s)
Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/efectos de los fármacos , Curcuma/química , Extractos Vegetales/farmacología , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Animales , Células CACO-2 , Relación Dosis-Respuesta a Droga , Interacciones Farmacológicas , Humanos , Absorción Intestinal/efectos de los fármacos , Mucosa Intestinal/citología , Mucosa Intestinal/efectos de los fármacos , Medicina Tradicional de Asia Oriental , Transportador de Péptidos 1/efectos de los fármacos , Transportador de Péptidos 1/metabolismo , Extractos Vegetales/administración & dosificación , Ratas , Ratas Sprague-Dawley , Rodamina 123/farmacocinética , TailandiaRESUMEN
The impact of increased incorporation of plant ingredients on diets for rainbow trout was evaluated in terms of gene expression of gastric (gastric lipase, pepsinogen) and intestinal (prolidase, maltase, phospholipase A2) digestive enzymes and nutrient transporters (peptide and glucose transporters), as well as of postprandial levels of plasma glucose, triglycerides and total free amino acids. For that purpose, trout alevins were fed from the start of exogenous feeding one of three different experimental diets: a diet rich in fish meal and fish oil (FM-FO), a plant-based diet (noFM-noFO) totally free from fish meal and fish oil, but containing plant ingredients and a Mixed diet (Mixed) intermediate between the FM-FO and noFM-noFO diets. After 16 months of rearing, all fish were left unfed for 72 h and then given a single meal to satiation. Blood, stomach and anterior intestine were sampled before the meal and at 2, 6 and 12 h after this meal. The postprandial kinetics of gene expression of gastric and intestinal digestive enzymes and nutrient transporters were then followed in trout fed the FM-FO diet. The postprandial profiles showed that the expression of almost all genes studied was stimulated by the presence of nutrients in the digestive tract of trout, but the timing (appearance of peaks) varied between genes. Based on these data, we have focused on the molecular response to dietary factors in the stomach and the intestine at 6 and 12 h after feeding, respectively. The reduction in FM and FO levels of dietary incorporation induced a significant decrease in the gene expression of gastric lipase, GLUT2 and PEPT1. The plasma glucose and triglycerides levels were also reduced in trout fed the noFM-noFO diet. Consequently, the present study suggests a decrease in digestive capacities in trout fed a diet rich in plant ingredients.
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Digestión/genética , Proteínas de Peces/genética , Oncorhynchus mykiss/genética , Periodo Posprandial/genética , Aminoácidos/sangre , Animales , Glucemia/análisis , Dieta , Aceites de Pescado , Productos Pesqueros , Mucosa Gástrica/metabolismo , Expresión Génica , Transportador de Glucosa de Tipo 2/genética , Hidrolasas/genética , Mucosa Intestinal/metabolismo , Oncorhynchus mykiss/sangre , Oncorhynchus mykiss/fisiología , Transportador de Péptidos 1 , Aceites de Plantas , Proteínas de Plantas , Transportador 1 de Sodio-Glucosa/genética , Simportadores/genética , Triglicéridos/sangreRESUMEN
USP18 (Ubiquitin-like specific protease 18) is an enzyme cleaving ubiquitin from target proteins. USP18 plays a pivotal role in antiviral and antibacterial immune responses. On the other hand, ubiquitination participates in the regulation of several ion channels and transporters. USP18 sensitivity of transporters has, however, never been reported. The present study thus explored, whether USP18 modifies the activity of the peptide transporters PEPT1 and PEPT2, and whether the peptide transporters are sensitive to the ubiquitin ligase Nedd4-2. To this end, cRNA encoding PEPT1 or PEPT2 was injected into Xenopus laevis oocytes without or with additional injection of cRNA encoding USP18. Electrogenic peptide (glycine-glycine) transport was determined by dual electrode voltage clamp. As a result, in Xenopus laevis oocytes injected with cRNA encoding PEPT1 or PEPT2, but not in oocytes injected with water or with USP18 alone, application of the dipeptide gly-gly (2 mM) was followed by the appearance of an inward current (Igly-gly). Coexpression of USP18 significantly increased Igly-gly in both PEPT1 and PEPT2 expressing oocytes. Kinetic analysis revealed that coexpression of USP18 increased maximal Igly-gly. Conversely, overexpression of the ubiquitin ligase Nedd4-2 decreased Igly-gly. Coexpression of USP30 similarly increased Igly-gly in PEPT1 expressing oocytes. In conclusion, USP18 sensitive cellular functions include activity of the peptide transporters PEPT1 and PEPT2.
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Dipéptidos/metabolismo , Endopeptidasas/metabolismo , Simportadores/metabolismo , Animales , Transporte Biológico , Dipéptidos/farmacología , Endopeptidasas/genética , Complejos de Clasificación Endosomal Requeridos para el Transporte/genética , Complejos de Clasificación Endosomal Requeridos para el Transporte/metabolismo , Femenino , Humanos , Inyecciones , Canal de Potasio KCNQ1/genética , Canal de Potasio KCNQ1/metabolismo , Mediciones Luminiscentes/métodos , Potenciales de la Membrana/efectos de los fármacos , Ubiquitina-Proteína Ligasas Nedd4 , Oocitos/efectos de los fármacos , Oocitos/metabolismo , Oocitos/fisiología , Técnicas de Placa-Clamp , Transportador de Péptidos 1 , Canales de Potasio con Entrada de Voltaje/genética , Canales de Potasio con Entrada de Voltaje/metabolismo , ARN Complementario/administración & dosificación , ARN Complementario/genética , Conejos , Simportadores/genética , Ubiquitina Tiolesterasa , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismo , Proteínas de Xenopus , Xenopus laevisRESUMEN
SCOPE: The basic dipeptide, Trp-His, was found to show an in vivo anti-atherosclerotic effect when orally administered to apo E-deficient mice. In addition, this dipeptide causes vasorelaxation in contracted rat aorta via suppression of intracellular Ca(2+) signaling cascades. In this study, we attempted to determine whether Trp-His can be absorbed after single oral administration in Sprague-Dawley (SD) rats. METHODS AND RESULTS: Trp-His and His-Trp (10 or 50 mg/kg) was orally administered to 8-week-old male SD rats. Both peptides in plasma were assayed by LC-MS/MS in combination with 2,4,6-trinitrobenzene sulfonate derivatization technique. In vitro transport experiments using Caco-2 cell monolayers were performed to evaluate the apparent permeability (Papp ). A phytic acid-aided MALDI-MS imaging (MSI) was conducted to visualize the distribution of dipeptides in the rat intestinal membrane. Trp-His was absorbed intact into SD rat blood, showing a maximal level at 1 h after administration at 10 mg/kg dose (Cmax , 28.7 ± 8.9 pmol/mL-plasma; area under the curve, 71.3 ± 18.7 pmol·h/mL-plasma). In contrast, His-Trp was surprisingly not detected, although the Papp was compatible to that of Trp-His. MSI analysis provided crucial evidence that Trp-His was visualized in the overall intestinal membrane. The Trp-His peptide was not visualized in the presence of Gly-Sar, which is a model peptide that is transported via the intestinal proton-coupled peptide transporter 1 (PepT1) transporter. The His-Trp molecular ion was not observed at the intestinal membrane. The MSI analysis illustrated that there is no absorption of His-Trp due to its unexpected hydrolysis by brush border proteases. CONCLUSION: To the best of our knowledge, this is the first study demonstrating that the vasoactive Trp-His is preferably transported across the rat intestinal membrane by PepT1 and is absorbed intact into the circulation. However, no absorption of His-Trp, a reverse sequence of absorbable Trp-His, is observed owing to hydrolysis by intestinal proteases. This suggests that the bioavailability of peptides may be determined in part by their protease resistance in the intestinal membrane.
Asunto(s)
Suplementos Dietéticos , Dipéptidos/metabolismo , Absorción Intestinal , Mucosa Intestinal/metabolismo , Simportadores/metabolismo , Vasodilatadores/metabolismo , Métodos Analíticos de la Preparación de la Muestra , Animales , Aterosclerosis/prevención & control , Células CACO-2 , Permeabilidad de la Membrana Celular , Cromatografía Líquida de Alta Presión , Dipéptidos/administración & dosificación , Dipéptidos/sangre , Dipéptidos/química , Humanos , Indicadores y Reactivos/química , Mucosa Intestinal/citología , Masculino , Transportador de Péptidos 1 , Ácido Fítico/química , Ratas Sprague-Dawley , Espectrometría de Masa por Ionización de Electrospray , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Espectrometría de Masas en Tándem , Ácido Trinitrobencenosulfónico/química , Vasodilatadores/administración & dosificación , Vasodilatadores/sangre , Vasodilatadores/químicaRESUMEN
SCOPE: Dietary peptides are potent stimulators of cholecystokinin (CCK) secretion, but the sensing mechanism in CCK-producing cells is poorly understood. Recently, it has been demonstrated that the calcium-sensing receptor (CaSR) mediates CCK secretion induced by amino acids. We investigated the role of CaSR in CCK secretions induced by various protein hydrolysates (egg albumin, meat, casein, azuki bean, soybean ß-conglycinin, and potato) in the enteroendocrine cell line STC-1. METHODS AND RESULTS: CCK secretions in response to these hydrolysates were measured in the STC-1 cells with or without CaSR antagonist (NPS 2143) treatment. Changes in intracellular calcium concentration ([Ca²âº](i) ) in response to protein hydrolysates were measured in Human embryonic kidney (HEK) 293 cells transfected with CaSR-expression vector. Protein hydrolysates-induced CCK secretions were decreased by CaSR antagonist treatment, except meat hydrolysate-induced secretion. Protein hydrolysates increased [Ca²âº](i) in CaSR-transfected HEK 293 cells. CaSR antagonist treatment suppressed low molecular weight fractions of azuki hydrolysate-induced CCK secretion, but the secretion induced by both low and high molecular weight fractions of ß-conglycinin hydrolysate. Further, CCK secretion induced by peptide fractions (> 500 Da) derived from various protein hydrolysates were also reduced by CaSR antagonist. CONCLUSION: These results demonstrate that CaSR plays a significant role in sensing various dietary peptides in triggering CCK secretion in enteroendocrine cells.
Asunto(s)
Colecistoquinina/metabolismo , Proteínas en la Dieta/farmacología , Células Enteroendocrinas/metabolismo , Hidrolisados de Proteína/farmacología , Receptores Sensibles al Calcio/metabolismo , Aminoácidos/análisis , Aminoácidos/farmacología , Animales , Antígenos de Plantas/farmacología , Calcio/análisis , Calcio/metabolismo , Caseínas/farmacología , Células Cultivadas , Células Enteroendocrinas/efectos de los fármacos , Globulinas/farmacología , Humanos , Carne , Ratones , Peso Molecular , Fragmentos de Péptidos/farmacología , Transportador de Péptidos 1 , Proteínas de Almacenamiento de Semillas/farmacología , Solanum tuberosum/química , Proteínas de Soja/farmacología , Simportadores/antagonistas & inhibidores , para-Aminobenzoatos/farmacologíaRESUMEN
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.
Asunto(s)
Sustitución de Aminoácidos/fisiología , Arginina/fisiología , Ácido Aspártico/fisiología , Activación del Canal Iónico/fisiología , Electricidad Estática , Simportadores/fisiología , Animales , Membrana Celular/metabolismo , Dipéptidos/metabolismo , Fenómenos Electrofisiológicos/fisiología , Femenino , Histidina/fisiología , Concentración de Iones de Hidrógeno , Potenciales de la Membrana/fisiología , Oocitos/metabolismo , Técnicas de Placa-Clamp , Transportador de Péptidos 1 , Protones , ARN Complementario/genética , Conejos , Xenopus laevisRESUMEN
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.
Asunto(s)
Proteínas en la Dieta/farmacocinética , Expresión Génica , Lisina/farmacocinética , Oligopéptidos/farmacocinética , Salmo salar/metabolismo , Simportadores/genética , Animales , Secuencia de Bases , Transporte Biológico/genética , Clonación Molecular , ADN Complementario/análisis , Digestión , Concentración de Iones de Hidrógeno , Absorción Intestinal , Datos de Secuencia Molecular , Oocitos/metabolismo , Transportador de Péptidos 1 , ARN Mensajero/metabolismo , Salmo salar/genética , Simportadores/metabolismo , Distribución Tisular , Xenopus/genética , Xenopus/metabolismoRESUMEN
BACKGROUND: Disease preventing effects gained by garlic consumption have been recognized since early period of history, making commercially available garlic supplements attractive to the general public. Possible pharmacokinetic interactions which could occur between applied drugs and aged garlic extract (AGE) are unknown. AIM: To test in vitro impact of some garlic phytochemicals on P-glycoprotein (Pgp), the most recognized efflux transporter, and the effect of AGE on passive membrane permeability, absorptive and secretory intestinal transporters. METHODS: Rat small intestine and Caco-2 cell monolayers, mounted in side-by-side diffusion chambers were used. RESULTS: Hydrophilic sulphur compounds increased Pgp mediated Rhodamine 123 (Rho123) efflux, whereas the lipophilic ones increased Pgp efflux through rat ileum but not through Caco-2 cell monolayers. Increased activities of secretory (Pgp, multidrug-resistance associated protein 2) and absorptive (monocarboxylate transporter 1, organic anion transporting polypeptide) transporters involved in drug absorption were observed in rat small intestine and Caco-2 cell monolayers in the presence of AGE. Transport of drugs mediated by breast cancer resistance protein and H(+)-oligopeptide transporter 1 was activated in rat intestine but inhibited through Caco-2 cells. Passive membrane permeability of tested compounds remained unaltered through rat small intestine, while significant changes were observed with Caco-2 cell monolayers. CONCLUSIONS: Due to the observed in vitro pharmacokinetic interactions between AGE and investigated cardiovascular, antidiabetic and antiviral drugs, in vivo absorption changes are possible, but the magnitude of change depends on the most profound process involved (influx, efflux, passive diffusion) in compounds permeability.
Asunto(s)
Fármacos Cardiovasculares/farmacocinética , Suplementos Dietéticos , Interacciones Alimento-Droga , Ajo/química , Hipoglucemiantes/farmacocinética , Extractos Vegetales , Raíces de Plantas/química , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Animales , Células CACO-2 , Fármacos Cardiovasculares/análisis , Permeabilidad de la Membrana Celular , Suplementos Dietéticos/análisis , Humanos , Hipoglucemiantes/análisis , Absorción Intestinal , Intestino Delgado/metabolismo , Masculino , Transportadores de Ácidos Monocarboxílicos/metabolismo , Proteína 2 Asociada a Resistencia a Múltiples Medicamentos , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/metabolismo , Transportadores de Anión Orgánico/metabolismo , Transportador de Péptidos 1 , Extractos Vegetales/química , Ratas , Ratas Wistar , Simportadores/metabolismo , TranscitosisRESUMEN
5-Aminolevulinic acid (ALA) is a prodrug used in photodynamic therapy, fluorescent diagnosis, and fluorescent-guided resection because it leads to accumulation of the photosensitizer protoporphyrin IX (PpIX) in tumor tissues. ALA has good oral bioavailability, but high oral doses are required to obtain selective PpIX accumulation in colonic tumors because accumulation is also observed in normal gut mucosa. Structural similarities between ALA and GABA led us to test the hypothesis that the H(+)-coupled amino acid transporter PAT1 (SLC36A1) will contribute to luminal ALA uptake. Radiolabel uptake and electrophysiological measurements identified PAT1-mediated H(+)-coupled ALA symport after heterologous expression in Xenopus oocytes. The selectivity of the nontransported inhibitors 5-hydroxytryptophan and 4-aminomethylbenzoic acid for, respectively, PAT1 and the H(+)-coupled di/tripeptide transporter PepT1 (SLC15A1) were examined. 5-Hydroxytryptophan selectively inhibited PAT1-mediated amino acid uptake across the brush-border membrane of the human intestinal (Caco-2) epithelium whereas 4-aminomethylbenzoic acid selectively inhibited PepT1-mediated dipeptide uptake. The inhibitory effects of 5-hydroxytryptophan and 4-aminomethylbenzoic acid were additive, demonstrating that both PAT1 and PepT1 contribute to intestinal transport of ALA. This is the first demonstration of overlap in substrate specificity between these distinct transporters for amino acids and dipeptides. PAT1 and PepT1 expression was monitored by reverse transcriptase-polymerase chain reaction using paired samples of normal and cancer tissue from human colon. mRNA for both transporters was detected. PepT1 mRNA was increased 2.3-fold in cancer tissues. Thus, increased PepT1 expression in colonic cancer could contribute to the increased PpIX accumulation observed. Selective inhibition of PAT1 could enhance PpIX loading in tumor tissue relative to that in normal tissue.
Asunto(s)
Sistemas de Transporte de Aminoácidos/biosíntesis , Ácido Aminolevulínico/farmacocinética , Intestino Delgado/metabolismo , Fotoquimioterapia , Fármacos Fotosensibilizantes/farmacocinética , Profármacos/farmacocinética , Simportadores/biosíntesis , Animales , Transporte Biológico , Células CACO-2 , Humanos , Mucosa Intestinal/metabolismo , Oocitos , Transportador de Péptidos 1 , ARN Mensajero/metabolismo , Xenopus laevisRESUMEN
5'-O-Dipeptide ester prodrugs of antiviral zidovudine (AZT) were designed to target the human intestinal oligopeptide transporter, hPEPT1, and were evaluated for their stability at pH 7.4 in buffer and in human plasma, affinity toward hPEPT1, cytotoxicity, and antiretroviral activity. The dipeptide esters of AZT undergo cyclization in buffer at pH 7.4 to release the parent drug at a rate that depends on the size of the side chains of the peptide carrier; the prodrug is considerably more stable if bulky beta-branched amino acids such as Ile and Val are present, particularly as C-terminal residues. Incubation in human plasma showed that most of the dipeptide esters of AZT release the parent drug through two aminopeptidase-mediated pathways: 1) stepwise cleavage of each of the amino acids and 2) direct cleavage of the dipeptide-drug ester bond. However, the plasma hydrolysis of Gly-Gly-AZT and Phe-Gly-AZT showed only direct cleavage of the dipeptide-drug ester bond. Substrate half-lives in plasma were again remarkably high when hydrophobic beta-branched amino acids (Val, Ile) were present. The esters were also good substrates for the intestinal oligopeptide transporter hPEPT1 in vitro, with Val-Gly-AZT and Val-Ala-AZT presenting the highest affinity toward the transporter (IC(50): 0.20 and 0.15 mM, respectively). The AZT dipeptide esters were assayed against the IIIB and ROD strains of HIV, and their cytotoxicity was evaluated in MT-4 cells. The selectivity index of the prodrugs was two- to threefold higher than that of AZT for all compounds analyzed. These results point to the potential of dipeptide-based carriers for the development of effective antiviral drug-delivery systems. Val-Ala-AZT appears to combine chemical stability with good affinity for the hPEPT1 transporter and an improved cytotoxicity/antiretroviral index relative to AZT.