RESUMEN
The reactions of intestinal functional parameters to type 2 diabetes at a young age remain unclear. The study aimed to assess changes in the activity of intestinal enzymes, glucose absorption, transporter content (SGLT1, GLUT2) and intestinal structure in young Wistar rats with type 2 diabetes (T2D) and impaired glucose tolerance (IGT). To induce these conditions in the T2D (n = 4) and IGT (n = 6) rats, we used a high-fat diet and a low dose of streptozotocin. Rats fed a high-fat diet (HFD) (n = 6) or a standard diet (SCD) (n = 6) were used as controls. The results showed that in T2D rats, the ability of the small intestine to absorb glucose was higher in comparison to HFD rats (p < 0.05). This was accompanied by a tendency towards an increase in the number of enterocytes on the villi of the small intestine in the absence of changes in the content of SGLT1 and GLUT2 in the brush border membrane of the enterocytes. T2D rats also showed lower maltase and alkaline phosphatase (AP) activity in the jejunal mucosa compared to the IGT rats (p < 0.05) and lower AP activity in the colon contents compared to the HFD (p < 0.05) and IGT (p < 0.05) rats. Thus, this study provides insights into the adaptation of the functional and structural parameters of the small intestine in the development of type 2 diabetes and impaired glucose tolerance in young representatives.
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Diabetes Mellitus Experimental/complicaciones , Diabetes Mellitus Tipo 2/complicaciones , Intolerancia a la Glucosa/complicaciones , Glucosa/farmacocinética , Intestino Delgado/enzimología , Intestino Delgado/metabolismo , Fosfatasa Alcalina/metabolismo , Animales , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Enterocitos/química , Glucano 1,4-alfa-Glucosidasa/metabolismo , Transportador de Glucosa de Tipo 2/análisis , Absorción Intestinal , Masculino , Ratas , Ratas Wistar , Transportador 1 de Sodio-Glucosa/análisis , alfa-Glucosidasas/metabolismoRESUMEN
We report the application of a highly versatile and engineerable novel sensor platform to monitor biologically significant and toxic metal ions in live human Caco-2 enterocytes. The extended conjugation between the fluorescent porphyrin core and metal ions through aromatic phenylphosphonic acid tethers generates a unique turn off and turn on fluorescence and, in addition, shifts in absorption and emission spectra for zinc, cobalt, cadmium and mercury. The reported fluorescent probes p-H8 TPPA and m-H8 TPPA can monitor a wide range of metal ion concentrations via fluorescence titration and also via fluorescence decay curves. Cu- and Zn-induced turn off fluorescence can be differentially reversed by the addition of common chelators. Both p-H8 TPPA and m-H8 TPPA readily pass the mammalian cellular membrane due to their amphipathic character as confirmed by confocal microscopic imaging of living enterocytes.
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Complejos de Coordinación/química , Enterocitos/química , Colorantes Fluorescentes/química , Metales Pesados/análisis , Organofosfonatos/química , Porfirinas/química , Células CACO-2 , Fluorescencia , HumanosRESUMEN
Oleoylethanolamide (OEA), a well-known satiety factor, is produced during feeding in the proximal intestine. Enterocytes sense oleic acid in dietary fat via CD36 and convert it to OEA through NAPE-PLD dependent or independent pathways. The satiety function of OEA is known to involve peroxisome proliferator-activated receptor type-α (PPAR-α). OEA stimulates afferent sensory fibers (possibly those of the vagus nerve) and provoke the recruitment of feeding-controlling circuits in the brain that use oxytocin and histamine as neurotransmitters for regulating satiety. Dysfunction of OEA synthesis by high-fat feeding might contribute to increased weight and obesity. Here, we describe the roles played by OEA in the regulation of energy metabolism and food intake by introducing our preliminary data regarding this lipid mediator, and we briefly outline the biosynthesis and deactivation of OEA.
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Grasas de la Dieta/administración & dosificación , Endocannabinoides/química , Ácido Oléico/administración & dosificación , Ácidos Oléicos/química , Animales , Antígenos CD36/metabolismo , Grasas de la Dieta/farmacocinética , Ingestión de Alimentos/efectos de los fármacos , Metabolismo Energético/efectos de los fármacos , Enterocitos/química , Enterocitos/metabolismo , Humanos , Ácido Oléico/farmacocinética , PPAR alfa/metabolismoRESUMEN
In spite of tremendous progress in deciphering the molecular mechanisms involved in intracellular transport in cell culture and in the test tube, many aspects of this process in situ remain unclear. Here, we examined lipid transcytosis in enterocytes in adult rats. Apical clathrin-coated buds and the ER exit sites were not found. After starvation, the Golgi complex was in a non-transporting state and contained many vesicles, but no intercisternal connections and typical the cis-most and the trans-most cisternae. Following the addition of the lipids in the form of chyme, pre-chylomicrons (pre-ChMs) were initially found in the tubules of the smooth SER attached to the basolateral plasmalemma below the belt composed of adhesive junctions (AJ) and always connected with other cisternae. However, the ER exit sites were still absent. Pre-ChMs moved into the cis-most cisterna and were concentrated in cisternal distensions at the trans-side of the Golgi complex. This induced attachment of the cis-most and the trans-most cisternae to the Golgi complex. Post-Golgi carriers fused with the basolateral plasmalemma and delivered ChMs outside. Overloading of enterocytes with lipids resulted in an accumulation of lipid droplets, an increase of the diameter of ChMs, and shift of the Golgi complex to the transporting state with the formation of intercisternal connections, attachment of the cis-most and the trans-most cisternae and disappearance of vesicles. These data are discussed from the functional point of view. In spite of tremendous progress in deciphering the molecular mechanisms involved in intracellular transport in cell culture and in the test tube, many aspects of this process in situ remain unclear. Here, we examined lipid transcytosis in enterocytes in adult rats. Apical clathrin-coated buds and the ER exit sites were not found. After starvation, the Golgi complex was in a non-transporting state and contained many vesicles, but no intercisternal connections and typical the cis-most and the trans-most cisternae. Following the addition of the lipids in the form of chyme, pre-chylomicrons (pre-ChMs) were initially found in the tubules of the smooth SER attached to the basolateral plasmalemma below the belt composed of adhesive junctions (AJ) and always connected with other cisternae. However, the ER exit sites were still absent. Pre-ChMs moved into the cis-most cisterna and were concentrated in cisternal distensions at the trans-side of the Golgi complex. This induced attachment of the cis-most and the trans-most cisternae to the Golgi complex. Post-Golgi carriers fused with the basolateral plasmalemma and delivered ChMs outside. Overloading of enterocytes with lipids resulted in an accumulation of lipid droplets, an increase of the diameter of ChMs, and shift of the Golgi complex to the transporting state with the formation of intercisternal connections, attachment of the cis-most and the trans-most cisternae and disappearance of vesicles. These data are discussed from the functional point of view.
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Enterocitos/citología , Enterocitos/metabolismo , Metabolismo de los Lípidos , Lípidos/química , Transcitosis , Animales , Enterocitos/química , Masculino , Estructura Molecular , Ratas , Ratas Sprague-Dawley , Ratas WistarRESUMEN
BACKGROUND & AIMS: The small intestine (SI) displays regionality in nutrient and immunological function. Following SI tissue loss (as occurs in short gut syndrome, or SGS), remaining SI must compensate, or "adapt"; the capacity of SI epithelium to reprogram its regional identity has not been described. Here, we apply single-cell resolution analyses to characterize molecular changes underpinning adaptation to SGS. METHODS: Single-cell RNA sequencing was performed on epithelial cells isolated from distal SI of mice following 50% proximal small bowel resection (SBR) vs sham surgery. Single-cell profiles were clustered based on transcriptional similarity, reconstructing differentiation events from intestinal stem cells (ISCs) through to mature enterocytes. An unsupervised computational approach to score cell identity was used to quantify changes in regional (proximal vs distal) SI identity, validated using immunofluorescence, immunohistochemistry, qPCR, western blotting, and RNA-FISH. RESULTS: Uniform Manifold Approximation and Projection-based clustering and visualization revealed differentiation trajectories from ISCs to mature enterocytes in sham and SBR. Cell identity scoring demonstrated segregation of enterocytes by regional SI identity: SBR enterocytes assumed more mature proximal identities. This was associated with significant upregulation of lipid metabolism and oxidative stress gene expression, which was validated via orthogonal analyses. Observed upstream transcriptional changes suggest retinoid metabolism and proximal transcription factor Creb3l3 drive proximalization of cell identity in response to SBR. CONCLUSIONS: Adaptation to proximal SBR involves regional reprogramming of ileal enterocytes toward a proximal identity. Interventions bolstering the endogenous reprogramming capacity of SI enterocytes-conceivably by engaging the retinoid metabolism pathway-merit further investigation, as they may increase enteral feeding tolerance, and obviate intestinal failure, in SGS.
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Perfilación de la Expresión Génica/métodos , Redes Reguladoras de Genes , Intestino Delgado/cirugía , Análisis de Secuencia de ARN/métodos , Análisis de la Célula Individual/métodos , Animales , Reprogramación Celular , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/genética , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Enterocitos/química , Enterocitos/citología , Intestino Delgado/química , Metabolismo de los Lípidos , Masculino , Ratones , Estrés Oxidativo , ARN Nuclear Pequeño/farmacología , Aprendizaje Automático no Supervisado , Regulación hacia ArribaRESUMEN
Bovine colostrum is a rich source of bioactive components which are important in the development of the intestine, in stimulating gut structure and function and in preparing the gut surface for subsequent colonization of microbes. What is not clear, however, is how colostrum may affect the repertoire of receptors and membrane proteins of the intestinal surface and the post-translational modifications associated with them. In the present work, we aimed to characterize the surface receptor and glycan profile of human HT-29 intestinal cells after exposure to a bovine colostrum fraction (BCF) by means of proteomic and glycomic analyses. Integration of label-free quantitative proteomic analysis and lectin array profiles confirmed that BCF exposure results in changes in the levels of glycoproteins present at the cell surface and also changes to their glycosylation pattern. This study contributes to our understanding of how milk components may regulate intestinal cells and prime them for bacterial interaction.
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Calostro/fisiología , Enterocitos/química , Glicómica/métodos , Proteómica/métodos , Animales , Bovinos , Calostro/química , Femenino , Glicoproteínas/análisis , Células HT29 , Humanos , Lectinas/análisis , Polisacáridos/análisis , Receptores de Superficie Celular/análisisRESUMEN
Most orally administered drugs gain access to the systemic circulation by direct passage from the enterocyte layer of the intestinal tract to the mesenteric blood capillaries. Intestinal lymphatic absorption is another pathway that certain drugs may follow to gain access to the systemic circulation after oral administration. Once absorbed, drug diffuses into the intestinal enterocyte and while in transit may associate with fats as they are processed into chylomicrons within the cells. The chylomicron-associated drug is then secreted from the enterocyte into the lymphatic circulation, thus avoiding the hepatic first-pass liver metabolism, and ultimately entering to the systemic circulation for disposition and action. Due to the possibility of parallel and potentially alternative absorptive pathways, mesenteric blood capillary and lymphatic drug exposure are both potential pathways of systemic availability for any individual drug. In this report, an in silico modeling approach was adopted to delineate the salient pharmacokinetic features of lymphatic absorption, and provide further guidance for the rationale design of drugs and drug delivery systems for lymphatic drug transport. The importance of hepatic extraction ratio, absorption lag time, lipoprotein binding, and the influence of competing portal and lymphatic pathways for systemic drug availability were explored using simulations. The degree of hepatic extraction was found to be an essential consideration when examining the influence of lymphatic uptake to overall oral drug bioavailability. Lymphatic absorption could potentially contribute to multiple peaking phenomena and flip flop pharmacokinetics of orally administered drugs.
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Enterocitos/química , Sistema Linfático/química , Preparaciones Farmacéuticas/química , Absorción Fisiológica , Animales , Sistemas de Liberación de Medicamentos , Desarrollo de Medicamentos , Enterocitos/metabolismo , Humanos , Sistema Linfático/metabolismo , Preparaciones Farmacéuticas/administración & dosificación , Preparaciones Farmacéuticas/metabolismoRESUMEN
Adenocarcinomas showing fetal gut-like (enteroblastic) differentiation can arise in a variety of organs and are frequently accompanied by an elevated serum α-fetoprotein (AFP) level. However, no study has investigated fetal gut-like differentiation in gallbladder cancer in detail. Herein, we performed morphological and immunohistochemical analyses of fetal gut-like differentiation in 49 consecutive gallbladder cancer cases. The expression of Sal-like protein 4 (SALL4), an embryonic stem cell marker reported to represent fetal gut-like differentiation, as well as other oncofetal proteins, including glypican-3 (GPC3) and AFP, was assessed. We found 1 case of fetal gut-like adenocarcinoma that coexisted with conventional-type adenocarcinoma. The fetal gut-like adenocarcinoma component revealed diffuse immunoreactivity for SALL4 and partial positivity for AFP, whereas the conventional-type adenocarcinoma component was negative. We also found 2 poorly differentiated adenocarcinomas with hepatoid morphology and 1 clear cell carcinoma, none of which showed SALL4 positivity. In other conventional-type adenocarcinomas, focal immunoreactivity for SALL4 and GPC3 was occasionally observed. The overall positivity rates for SALL4 and GPC3 were 12.2% (6/49) and 16.3% (8/49), respectively. SALL4 and GPC3 expression was not associated with clinicopathological factors, including T category, lymphovascular invasion, and lymph node metastases. In conclusion, fetal gut-like adenocarcinoma was found in 2% of our gallbladder cancer series. We conclude that fetal gut-like adenocarcinoma is a distinct histological subtype of gallbladder cancer, characterized by SALL4 expression.
Asunto(s)
Adenocarcinoma/patología , Diferenciación Celular , Enterocitos/patología , Neoplasias de la Vesícula Biliar/patología , Células Madre Neoplásicas/patología , Adenocarcinoma/química , Adenocarcinoma/genética , Anciano , Anciano de 80 o más Años , Análisis Mutacional de ADN , Enterocitos/química , Femenino , Neoplasias de la Vesícula Biliar/química , Neoplasias de la Vesícula Biliar/genética , Glipicanos/análisis , Humanos , Inmunohistoquímica , Masculino , Persona de Mediana Edad , Mutación , Estadificación de Neoplasias , Células Madre Neoplásicas/química , Proteínas Proto-Oncogénicas B-raf/genética , Proteínas Proto-Oncogénicas p21(ras)/genética , Factores de Transcripción/análisis , alfa-Fetoproteínas/análisisRESUMEN
The human riboflavin (RF) transporter-3 (hRFVT-3; product of the SLC52A3 gene) plays an essential role in the intestinal RF absorption process and is expressed exclusively at the apical membrane domain of polarized enterocytes. Previous studies have characterized different physiological/biological aspects of this transporter, but nothing is known about the glycosylation status of the hRFVT-3 protein and role of this modification in its physiology/biology. Additionally, little is known about the residues in the hRFVT-3 protein that interact with the ligand, RF. We addressed these issues using appropriate biochemical/molecular approaches, a protein-docking model, and established intestinal/renal epithelial cells. Our results showed that the hRFVT-3 protein is glycosylated and that glycosylation is important for its function. Mutating the predicted N-glycosylation sites at Asn94 and Asn168 led to a significant decrease in RF uptake; it also led to a marked intracellular (in the endoplasmic reticulum, ER) retention of the mutated proteins as shown by live-cell confocal imaging studies. The protein-docking model used in this study has identified a number of putative substrate-interacting sites: Ser16, Ile20, Trp24, Phe142, Thr314, and Asn315 Mutating these potential interacting sites was indeed found to lead to a significant inhibition in RF uptake and to intracellular (ER) retention of the mutated proteins (except for the Phe142 mutant). These results demonstrate that the hRFVT-3 protein is glycosylated and this glycosylation is important for its function and cell surface expression. This study also identified a number of residues in the hRFVT-3 polypeptide that are important for its function/cell surface expression.
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Enterocitos/metabolismo , Proteínas de Transporte de Membrana/química , Riboflavina/química , Aminoácidos/química , Sitios de Unión , Enterocitos/química , Regulación Enzimológica de la Expresión Génica , Glicosilación , Humanos , Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Membrana/metabolismo , Simulación del Acoplamiento Molecular , Mutación , Unión Proteica , Conformación Proteica , Riboflavina/metabolismo , Especificidad por Sustrato , Propiedades de SuperficieRESUMEN
The neonatal Fc receptor (FcRn) is a major histocompatibility complex class I type molecule that binds to, transports, and recycles immunoglobulin G (IgG) and albumin, thereby protecting them from lysosomal degradation. Therefore, besides the knowledge of FcRn affinity, FcRn protein expression is critical in understanding the pharmacokinetic behavior of Fc-containing biotherapeutics such as monoclonal antibodies. The goal of this investigation was to achieve for the first time a comparative assessment of FcRn distribution across a variety of tissues and species. FcRn was mapped in about 20 tissues including placenta from human and the most frequently used species in non-clinical safety testing of monoclonal antibodies (mouse, rat, cynomolgus monkey). In addition, the FcRn expression pattern was characterized in two humanized transgenic mouse lines (Tg32 and Tg276) expressing human FcRn under different promoters, and in the severe combined immunodeficient (SCID) mouse. Consecutive sections were stained with specific markers, namely, anti-CD68 for macrophages and anti-von Willebrand Factor for endothelial cells. Overall, the FcRn expression pattern was comparable across species and tissues with consistent expression of FcRn in endothelial cells and interstitial macrophages, Kupffer cells, alveolar macrophages, enterocytes, and choroid plexus epithelium. The human FcRn transgenic mouse Tg276 showed a different and much more widespread staining pattern of FcRn. In addition, immunodeficiency and lack of IgG in SCID mice had no negative effect on FcRn expression compared with wild-type mice.
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Antígenos de Histocompatibilidad Clase I/análisis , Receptores Fc/análisis , Animales , Plexo Coroideo/química , Plexo Coroideo/metabolismo , Células Endoteliales/química , Células Endoteliales/metabolismo , Enterocitos/química , Enterocitos/metabolismo , Epitelio/química , Epitelio/metabolismo , Antígenos de Histocompatibilidad Clase I/biosíntesis , Humanos , Macrófagos del Hígado/química , Macrófagos del Hígado/metabolismo , Macaca fascicularis , Macrófagos/química , Macrófagos/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones SCID , Ratones Transgénicos , Ratas , Ratas Wistar , Receptores Fc/biosíntesisRESUMEN
Niemann-Pick C1 like-1 (NPC1L1) mediates cholesterol absorption at the apical membrane of enterocytes through a yet unknown mechanism. Bean, pea, and lentil proteins are naturally hydrolyzed during digestion to produce peptides. The potential for pulse peptides to have high binding affinities for NPC1L1 has not been determined. In this study , in silico binding affinities and interactions were determined between the N-terminal domain of NPC1L1 and 14 pulse peptides (5≥ amino acids) derived through pepsin-pancreatin digestion. Peptides were docked in triplicate to the N-terminal domain using docking program AutoDock Vina, and results were compared to those of ezetimibe, a prescribed NPC1L1 inhibitor. Three black bean peptides (-7.2 to -7.0kcal/mol) and the cowpea bean dipeptide Lys-Asp (-7.0kcal/mol) had higher binding affinities than ezetimibe (-6.6kcal/mol) for the N-terminal domain of NPC1L1. Lentil and pea peptides studied did not have high binding affinities. The common bean peptide Tyr-Ala-Ala-Ala-Thr (-7.2kcal/mol), which can be produced from black or navy bean proteins, had the highest binding affinity. Ezetimibe and peptides with high binding affinities for the N-terminal domain are expected to interact at different locations of the N-terminal domain. All high affinity black bean peptides are expected to have van der Waals interactions with SER130, PHE136, and LEU236 and a conventional hydrogen bond with GLU238 of NPC1L1. Due to their high affinity for the N-terminal domain of NPC1L1, black and cowpea bean peptides produced in the digestive track have the potential to disrupt interactions between NPC1L1 and membrane proteins that lead to cholesterol absorption.
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Ezetimiba/metabolismo , Proteínas de la Membrana/metabolismo , Péptidos/metabolismo , Colesterol/metabolismo , Simulación por Computador , Enterocitos/química , Enterocitos/metabolismo , Ezetimiba/química , Humanos , Absorción Intestinal , Proteínas de la Membrana/química , Proteínas de Transporte de Membrana , Pisum sativum/química , Péptidos/química , Phaseolus/química , Unión ProteicaRESUMEN
Monolayered epithelia are composed of tight cell assemblies that ensure polarized exchanges. EpCAM, an unconventional epithelial-specific cell adhesion molecule, is assumed to modulate epithelial morphogenesis in animal models, but little is known regarding its cellular functions. Inspired by the characterization of cellular defects in a rare EpCAM-related human intestinal disease, we find that the absence of EpCAM in enterocytes results in an aberrant apical domain. In the course of this pathological state, apical translocation towards tricellular contacts (TCs) occurs with striking tight junction belt displacement. These unusual cell organization and intestinal tissue defects are driven by the loss of actomyosin network homoeostasis and contractile activity clustering at TCs, yet is reversed by myosin-II inhibitor treatment. This study reveals that adequate distribution of cortical tension is crucial for individual cell organization, but also for epithelial monolayer maintenance. Our data suggest that EpCAM modulation protects against epithelial dysplasia and stabilizes human tissue architecture.
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Células Epiteliales/química , Epitelio/química , Actomiosina/química , Actomiosina/genética , Actomiosina/metabolismo , Adolescente , Fenómenos Biomecánicos , Células CACO-2 , Polaridad Celular , Niño , Preescolar , Diarrea Infantil/genética , Diarrea Infantil/metabolismo , Enterocitos/química , Enterocitos/metabolismo , Molécula de Adhesión Celular Epitelial/química , Molécula de Adhesión Celular Epitelial/genética , Molécula de Adhesión Celular Epitelial/metabolismo , Células Epiteliales/citología , Células Epiteliales/metabolismo , Epitelio/metabolismo , Femenino , Humanos , Lactante , Síndromes de Malabsorción/genética , Síndromes de Malabsorción/metabolismo , Masculino , Uniones Estrechas/química , Uniones Estrechas/genética , Uniones Estrechas/metabolismoRESUMEN
sn-2-Monoacylglycerol (2-MAG) and sn-1(3)-monoacylglycerol [1(3)-MAG] are important but yet little studied intermediates in lipid metabolism. The current study compared the metabolic fate of 2-MAG and 1(3)-MAG in isolated caecal enterocytes and hepatocytes of brown trout (Salmo trutta). 1(3)-Oleoyl [9,10-3H(N)]-glycerol and 2-Oleoyl [9,10-3H(N)]-glycerol were prepared by pancreatic lipase digestion of triolein [9,10-3H(N)]. The 1(3)-MAG and 2-MAG were efficiently absorbed by enterocytes and hepatocytes at similar rates. The 2-MAG was quickly resynthesized into TAG through the monoacylglycerol acyltransferase (EC: 2.3.1.22, MGAT) pathway in both tissues, whereas 1(3)-MAG was processed into TAG and phospholipids at a much slower rate, suggesting 2-MAG was the preferred substrates for MGAT. Further analysis showed that 1(3)-MAG was synthesized into 1,3-DAG, but there were no accumulation of 1,3-DAG in either enterocytes or hepatocytes, which contrasts that of mammalian studies. Some of the 1(3)-MAG may be acylated to 1,2(2,3)-DAG and then utilized for TAG synthesis. Alternatively, 1(3)-MAG can be hydrolyzed to free fatty acid and glycerol, and re-synthesized into TAG through the glycerol-3-phosphate (Gro-3-P) pathway. The overall data suggested that the limiting step of the intracellular 1(3)-MAG metabolism is the conversion of 1(3)-MAG itself.
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Enterocitos/metabolismo , Monoglicéridos/metabolismo , Trucha/metabolismo , Absorción Fisiológica , Animales , Vías Biosintéticas , Ciego/citología , Ciego/metabolismo , Enterocitos/química , Hepatocitos/metabolismo , Hidrólisis , Lipasa/metabolismo , Especificidad por SustratoRESUMEN
Doublecortin-like kinase 1 (DCLK1) is a microtubule-associated kinase. In murine intestine, DCLK1 marks tuft cells with characteristic microvilli, features of neuroendocrine cells and also quiescent stem cell-like properties. The occurrence and pathological role of DCLK1-positive cells in human intestinal mucosa is unknown. We analysed DCLK1 expression in healthy duodenal, jejunal and colorectal mucosa samples (n = 35), and in duodenal specimens from patients with coeliac disease (n = 20). The samples were immunohistochemically double-stained with DCLK1, and synaptophysin, chromogranin A and Ki-67. Ultrastructure of DCLK1-expressing duodenal cells was assessed using correlative light and electron microscopy. DCLK1 expression was seen in about 1% of epithelial cells diffusely scattered through the intestinal epithelium. Electron microscopy showed that the duodenal DCLK1-positive cells had short apical microvilli similar to neighbouring enterocytes and cytoplasmic granules on the basal side. DCLK1-positive cells were stained with synaptophysin. The number of DCLK1-positive cells was decreased in villus atrophy in coeliac disease. Our findings indicate that in human intestinal epithelium, DLCK1-positive cells form a subpopulation of non-proliferating neuroendocrine cells with apical brush border similar to that in enterocytes, and their number is decreased in untreated coeliac disease.
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Duodeno/citología , Enterocitos/química , Enterocitos/clasificación , Mucosa Intestinal/citología , Intestino Grueso/citología , Péptidos y Proteínas de Señalización Intracelular/análisis , Yeyuno/citología , Proteínas Serina-Treonina Quinasas/análisis , Adulto , Anciano , Enfermedad Celíaca/patología , Cromogranina A/análisis , Gránulos Citoplasmáticos/ultraestructura , Quinasas Similares a Doblecortina , Enterocitos/ultraestructura , Femenino , Humanos , Inmunohistoquímica , Antígeno Ki-67/análisis , Masculino , Microscopía , Microvellosidades/ultraestructura , Persona de Mediana Edad , Sinaptofisina/análisis , Adulto JovenRESUMEN
The protective effect of different polyphenols, catechin (Cat), quercetin (Qc) (flavonoids), gallic acid (GA), caffeic acid (CfA), chlorogenic acid (ChA) (phenolic acids), and capsaicin (Cap), against H2O2-induced oxidative stress was evaluated in rat enterocytes using Attenuated Total Reflectance-Fourier Transform Infrared (ATR-FTIR) Spectroscopy and Fourier Transform Infrared Microspectroscopy (FTIRM), and results were compared to standard lipid peroxidation techniques: conjugated dienes (CD) and Thiobarbituric Acid Reactive Substances (TBARS). Analysis of ATR-FTIR and FTIRM spectral data allowed the simultaneous evaluation of the effects of H2O2 and polyphenols on lipid and protein oxidation. All polyphenols showed a protective effect against H2O2-induced oxidative stress in enterocytes, when administered before or after H2O2. Cat and capsaicin showed the highest protective effect, while phenolic acids had weaker effects and Qc presented a mild prooxidative effect (IR spectral profile of biomolecules between control and H2O2-treated cells) according to FTIR analyses. These results demonstrated the viability to use infrared spectroscopy to evaluate the oxidant and antioxidant effect of molecules in cell systems assays.
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Antioxidantes/farmacología , Enterocitos/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Polifenoles/farmacología , Animales , Antioxidantes/química , Supervivencia Celular/efectos de los fármacos , Enterocitos/química , Enterocitos/citología , Peróxido de Hidrógeno/toxicidad , Intestinos/citología , Peroxidación de Lípido/efectos de los fármacos , Polifenoles/química , Análisis de Componente Principal , Ratas , Ratas Sprague-Dawley , Espectroscopía Infrarroja por Transformada de FourierRESUMEN
Transporter proteins expressed in the gastrointestinal tract play a major role in the oral absorption of some drugs, and their involvement may lead to drug-drug interaction (DDI) susceptibility when given in combination with drugs known to inhibit gut wall transporters. Anticipating such liabilities and predicting the magnitude of the impact of transporter proteins on oral drug absorption and DDIs requires quantification of their expression in human intestine, and linking these to data obtained through in vitro experiments. A quantitative targeted absolute proteomic method employing liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) together with a quantitative concatenation (QconCAT) strategy to provide proteotypic peptide standards has been applied to quantify ATP1A1 (sodium/potassium-ATPase; Na/K-ATPase), CDH17 (human peptide transporter 1; HPT1), ABCB1 (P-glycoprotein; P-gp), ABCG2 (breast cancer resistance protein; BCRP), ABCC2 (multidrug resistance-associated protein 2; MRP2) and SLC51A (Organic Solute Transporter subunit alpha; OST-α), in human distal jejunum (n=3) and distal ileum (n=1) enterocyte membranes. Previously developed selected reaction monitoring (SRM) schedules were optimised to enable quantification of the proteotypic peptides for each transporter. After harvesting enterocytes by calcium chelation elution and generating a total membrane fraction, the proteins were subjected to proteolytic digestion. To account for losses of peptides during the digestion procedure, a gravimetric method is also presented. The linearity of quantifying the QconCAT from an internal standard (correlation coefficient, R(2)=0.998) and quantification of all target peptides in a pooled intestinal quality control sample (R(2)≥ 0.980) was established. The assay was also assessed for within and between-day precision, demonstrating a <15% coefficient of variation for all peptides across 3 separate analytical runs, over 2 days. The methods were applied to obtain the absolute abundances for all targeted proteins. In all samples, Na/K-ATPase, HPT1, P-gp and BCRP were detected above the lower limit of quantitation (i.e., >0.2 fmol/µg membrane protein). MRP2 abundance could be quantified in distal jejunum but not in the distal ileum sample. OST-α was not detected in 2 out of 3 jejunum samples. This study highlights the utility of a QconCAT strategy to quantify absolute transporter abundances in human intestinal tissues.
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Cromatografía Liquida , Íleon/química , Yeyuno/química , Proteínas de Transporte de Membrana/análisis , Proteómica/métodos , Espectrometría de Masas en Tándem , Calibración , Membrana Celular/química , Cromatografía Liquida/normas , Enterocitos/química , Humanos , Íleon/citología , Yeyuno/citología , Modelos Lineales , Proteína 2 Asociada a Resistencia a Múltiples Medicamentos , Proteómica/normas , Estándares de Referencia , Reproducibilidad de los Resultados , Espectrometría de Masas en Tándem/normasRESUMEN
BACKGROUND: Mesenteric ischemia-reperfusion induces gut mucosal damage. Intestinal mucosal wounds are repaired by epithelial restitution. Although many different molecular mechanisms have been shown to affect cell metabolism under oxidative conditions, these molecular mechanisms and metabolic phenotypes are not well understood. Nuclear magnetic resonance (NMR) spectroscopic data can be used to study metabolic phenotypes in biological systems. Pattern recognition with multivariate analysis is one chemometric technique. The purpose of this study was to visualize, using a chemometric technique to interpret NMR data, different degrees of oxidant injury in rat small intestine (IEC-6) cells exposed to H2O2. METHODS: Oxidant stress was induced by H2O2 in IEC-6 cells. Cell restitution and viability were assessed at different H2O2 concentrations and time points. Cells were harvested for pattern recognition analysis of (1)H-NMR data. RESULTS: Cell viability and restitution were significantly suppressed by H2O2 in a dose-dependent manner compared with control. Each class was clearly separated into clusters by partial least squares discriminant analysis, and class variance was greater than 90% from 2 factors. CONCLUSION: Pattern recognition of NMR spectral data using a chemometric technique clearly visualized the differences of oxidant injury in IEC-6 cells under oxidant stress.
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Extractos Celulares/análisis , Enterocitos/química , Espectroscopía de Resonancia Magnética , Estrés Oxidativo , Reconocimiento de Normas Patrones Automatizadas/métodos , Protones , Animales , Línea Celular , Supervivencia Celular/efectos de los fármacos , Análisis Discriminante , Enterocitos/efectos de los fármacos , Peróxido de Hidrógeno/toxicidad , Análisis de los Mínimos Cuadrados , Estrés Oxidativo/efectos de los fármacos , Ratas , Factores de Tiempo , Cicatrización de Heridas/efectos de los fármacosRESUMEN
Previous studies have shown that high-fat diet (HFD) enhances adaptation if provided immediately following small bowel resection (SBR). The purpose of this study was to determine if HFD could further enhance villus growth after resection-induced adaptation had already taken place. C57/Bl6 mice underwent a 50 % proximal SBR or sham operation and were then provided a standard rodent liquid diet (LD) ad lib. After a typical period of adaptation (7 days), SBR and sham-operated mice were randomized to receive either LD or HFD (42 % kcal fat) for an additional 7 days. Mice were then harvested, and small intestine was collected for analysis. Adaptation occurred in both SBR groups; however, the SBR/HFD had significantly increased villus height compared to SBR/LD. Reverse transcription-polymerase chain reaction of villus enterocytes showed a marked increase in CD36 expression in the SBR/HFD group compared with SBR/LD mice. While exposure to increased enteral fat alone did not affect villus morphology in sham-operated mice, HFD significantly increased villus growth in the setting of resection-induced adaptation, supporting the clinical utility of enteral fat in augmenting adaptation. Increased expression of CD36 suggests a possible mechanistic role in dietary fat metabolism and villus growth in the setting of short gut syndrome.
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Adaptación Fisiológica , Dieta Alta en Grasa , Íleon/cirugía , Mucosa Intestinal/anatomía & histología , Yeyuno/cirugía , Animales , Apolipoproteínas B/genética , Composición Corporal , Antígenos CD36/análisis , Antígenos CD36/genética , Proteínas Portadoras/genética , Proliferación Celular , Enterocitos/química , Enterocitos/metabolismo , Mucosa Intestinal/metabolismo , Masculino , Ratones Endogámicos C57BL , ARN Mensajero/metabolismoRESUMEN
The developmental toxicity of nanostructured materials, as well as their impact on the biological barriers, represents a crucial aspect to be assessed in a nanosafety policy framework. Nanosized metal oxides have been demonstrated to affect Xenopus laevis embryonic development, with nZnO specifically targeting the digestive system. To study the mechanisms of the nZnO-induced intestinal lesions, we tested two different nominally sized ZnO nanoparticles (NPs) at effective concentrations. Advanced microscopy techniques and molecular marker analyses were applied in order to describe the NP-epithelial cell interactions and the mechanisms driving NP toxicity and translocation through the intestinal barrier. We attributed the toxicity to NP-induced cell oxidative damage, the small-sized NPs being the more effective. This outcome is sustained by a marked increase in anti-oxidant genes' expression and high lipid peroxidation level in the enterocytes, where disarrangement of the cytoskeleton and cell junctions' integrity were evidenced. These events led to diffuse necrotic changes in the intestinal barrier, and trans- and paracellular NP permeation through the mucosa. The uptake routes, leading NPs to cross the intestinal barrier and reach secondary target tissues, have been documented. nZnOs embryotoxicity was confirmed to be crucially mediated by the NPs' reactivity rather than their dissolved ions. The ZnO NPs' ability to overwhelm the intestinal barrier must be taken into high consideration for a future design of safer ZnO NPs.
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Uniones Intercelulares/metabolismo , Mucosa Intestinal/metabolismo , Nanopartículas del Metal/química , Óxido de Zinc/farmacocinética , Animales , Endocitosis , Enterocitos/química , Enterocitos/metabolismo , Femenino , Mucosa Intestinal/citología , Mucosa Intestinal/efectos de los fármacos , Mucosa Intestinal/patología , Larva/metabolismo , Masculino , Nanopartículas del Metal/toxicidad , Microvellosidades/metabolismo , Necrosis/inducido químicamente , Necrosis/patología , Estrés Oxidativo/efectos de los fármacos , Xenopus laevis , Óxido de Zinc/química , Óxido de Zinc/toxicidadRESUMEN
The primary function of the small intestine is digesting and absorbing nutrients from consumed food. Because of this, the small intestine is often thought of as a nutrient thoroughfare-enterocytes taking up nutrients on the apical side and then secreting nutrients from the basolateral side. The small intestine is not commonly thought of as a lipid storage organ; however, when meals and diets containing high amounts of fat are consumed, some dietary fat is stored in cytoplasmic lipid droplets (CLDs). The balance between storage and secretion of dietary fat by enterocytes is important in determining the physiological fate of dietary fat, including regulating blood lipid concentrations and energy balance. The existence of CLDs within enterocytes has likely evolved for three important physiological functions: (i) to allow the small intestine to efficiently absorb large amounts of energy dense fat, (ii) to control the rate of dietary fat entering circulation, and (iii) to alleviate lipotoxicity to enterocytes induced by high concentrations of free fatty acids, especially when a high fat meal is consumed. The purpose of this chapter is to provide methods for imaging CLDs in enterocytes and assessing different aspects of dietary fat absorption.