RESUMEN
Carbamoyl phosphate synthetase-1 (CPS1) is the major mitochondrial urea cycle enzyme in hepatocytes. It is released into mouse and human blood during acute liver injury, where is has a short half-life. The function of CPS1 in blood and the reason for its short half-life in serum are unknown. We show that CPS1 is released normally into mouse and human bile, and pathologically into blood during acute liver injury. Other cytoplasmic and mitochondrial urea cycle enzymes are also found in normal mouse bile. Serum, bile, and purified CPS1 manifest sedimentation properties that overlap with extracellular vesicles, due to the propensity of CPS1 to aggregate despite being released primarily as a soluble protein. During liver injury, CPS1 in blood is rapidly sequestered by monocytes, leading to monocyte M2-polarization and homing to the liver independent of its enzyme activity. Recombinant CPS1 (rCPS1), but not control r-transferrin, increases hepatic macrophage numbers and phagocytic activity. Notably, rCPS1 does not activate hepatic macrophages directly; rather, it activates bone marrow and circulating monocytes that then home to the liver. rCPS1 administration prevents mouse liver damage induced by Fas ligand or acetaminophen, but this protection is absent in macrophage-deficient mice. Moreover, rCPS1 protects from acetaminophen-induced liver injury even when given therapeutically after injury induction. In summary, CPS1 is normally found in bile but is released by hepatocytes into blood upon liver damage. We demonstrate a nonenzymatic function of CPS1 as an antiinflammatory protective cytokine during acute liver injury.
Asunto(s)
Lesión Pulmonar Aguda/metabolismo , Ácidos y Sales Biliares/metabolismo , Carbamoil-Fosfato Sintasa (Amoniaco)/metabolismo , Acetaminofén/metabolismo , Lesión Pulmonar Aguda/enzimología , Adulto , Animales , Bilis/metabolismo , Citocinas/metabolismo , Proteína Ligando Fas/metabolismo , Femenino , Hepatocitos/metabolismo , Humanos , Hígado/metabolismo , Hepatopatías , Macrófagos/metabolismo , Masculino , Ratones , Mitocondrias/metabolismoRESUMEN
Feeding strategies to care for patients who transition from enteral nutrient deprivation while on total parenteral nutrition (TPN) to enteral feedings generally proceed to full enteral nutrition once the gastrointestinal tract recovers; however, an increasing body of literature suggests that a subgroup of patients may actually develop an increased incidence of adverse events, including death. To examine this further, we studied the effects of acute refeeding in a mouse model of TPN. Interestingly, refeeding led to some beneficial effects, including prevention in the decline in intestinal epithelial cell (IEC) proliferation. However, refeeding led to a significant increase in mucosal expression of proinflammatory cytokines, including tumor necrosis factor-α (TNF-α), as well as an upregulation in Toll-like receptor 4 (TLR-4). Refeeding also failed to prevent TPN-associated increases in IEC apoptosis, loss of epithelial barrier function, and failure of the leucine-rich repeat-containing G protein-coupled receptor 5-positive stem cell expression. Transitioning from TPN to enteral feedings led to a partial restoration of the small bowel microbial population. In conclusion, while acute refeeding led to some restoration of normal gastrointestinal physiology, enteral refeeding led to a significant increase in mucosal inflammatory markers and may suggest alternative strategies to enteral refeeding should be considered.
Asunto(s)
Nutrición Enteral/efectos adversos , Homeostasis , Mucosa Intestinal/efectos de los fármacos , Intestino Delgado/efectos de los fármacos , Nutrición Parenteral Total/efectos adversos , Animales , Apoptosis , Proliferación Celular , Citocinas/biosíntesis , Citocinas/metabolismo , Microbioma Gastrointestinal , Mucosa Intestinal/metabolismo , Intestino Delgado/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Receptores Acoplados a Proteínas G/biosíntesis , Receptores Acoplados a Proteínas G/genética , Células Madre , Proteínas de Uniones Estrechas/biosíntesis , Proteínas de Uniones Estrechas/genética , Receptor Toll-Like 4/biosíntesis , Receptor Toll-Like 4/genética , Factor de Necrosis Tumoral alfa/biosíntesisRESUMEN
Total parenteral nutrition (TPN) leads to a shift in small intestinal microbiota with a characteristic dominance of Proteobacteria This study examined how metabolomic changes within the small bowel support an altered microbial community in enterally deprived mice. C57BL/6 mice were given TPN or enteral chow. Metabolomic analysis of jejunal contents was performed by liquid chromatography/mass spectrometry (LC/MS). In some experiments, leucine in TPN was partly substituted with [13C]leucine. Additionally, jejunal contents from TPN-dependent and enterally fed mice were gavaged into germ-free mice to reveal whether the TPN phenotype was transferrable. Small bowel contents of TPN mice maintained an amino acid composition similar to that of the TPN solution. Mass spectrometry analysis of small bowel contents of TPN-dependent mice showed increased concentration of 13C compared with fed mice receiving saline enriched with [13C]leucine. [13C]leucine added to the serosal side of Ussing chambers showed rapid permeation across TPN-dependent jejunum, suggesting increased transmucosal passage. Single-cell analysis by fluorescence in situ hybridization (FISH)-NanoSIMS demonstrated uptake of [13C]leucine by TPN-associated bacteria, with preferential uptake by Enterobacteriaceae Gavage of small bowel effluent from TPN mice into germ-free, fed mice resulted in a trend toward the proinflammatory TPN phenotype with loss of epithelial barrier function. TPN dependence leads to increased permeation of TPN-derived nutrients into the small intestinal lumen, where they are predominately utilized by Enterobacteriaceae The altered metabolomic composition of the intestinal lumen during TPN promotes dysbiosis.
Asunto(s)
Microbioma Gastrointestinal/fisiología , Mucosa Intestinal/metabolismo , Yeyuno/metabolismo , Nutrición Parenteral Total , Sepsis/metabolismo , Animales , Modelos Animales de Enfermedad , Mucosa Intestinal/microbiología , Yeyuno/microbiología , Masculino , Metaboloma , Ratones , Ratones Endogámicos C57BL , Sepsis/microbiologíaRESUMEN
Recent studies suggest a close interaction between epidermal growth factor (EGF) and TLR signaling in the modulation of intestinal epithelial cell (IEC) proliferation; however, how these signaling pathways adjust IEC proliferation is poorly understood. We utilized a model of total parenteral nutrition (TPN), or enteral nutrient deprivation, to study this interaction as TPN results in mucosal atrophy due to decreased IEC proliferation and increased apoptosis. We identified the novel finding of decreased mucosal atrophy in TLR4 knockout (TLR4KO) mice receiving TPN. We hypothesized that EGF signaling is preserved in TLR4KO-TPN mice and prevents mucosal atrophy. C57Bl/6 and strain-matched TLR4KO mice were provided either enteral feeding or TPN. IEC proliferation and apoptosis were measured. Cytokine and growth factor abundances were detected in both groups. To examine interdependence of these pathways, ErbB1 pharmacologic blockade was used. The marked decline in IEC proliferation with TPN was nearly prevented in TLR4KO mice, and intestinal length was partially preserved. EGF was significantly increased, and TNF-α decreased in TLR4KO-TPN versus wild-type (WT)-TPN mice. Apoptotic positive crypt cells were 15-fold higher in WT-TPN versus TLR4KO-TPN mice. Bcl-2 was significantly increased in TLR4KO-TPN mice, while Bax decreased 10-fold. ErbB1 blockade prevented this otherwise protective effect in TLR4KO-sTPN mice. TLR4 blockade significantly prevented TPN-associated atrophy by preserving proliferation and preventing apoptosis. This is driven by a reduction in TNF-α abundance and increased EGF. Potential manipulation of this regulatory pathway may have significant clinical potential to prevent TPN-associated atrophy.
Asunto(s)
Factor de Crecimiento Epidérmico/metabolismo , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patología , Nutrición Parenteral Total/efectos adversos , Receptor Toll-Like 4/metabolismo , Animales , Apoptosis , Atrofia , Proliferación Celular , Factor de Crecimiento Epidérmico/antagonistas & inhibidores , Factor de Crecimiento Epidérmico/genética , Receptores ErbB/antagonistas & inhibidores , Gefitinib , Interferón gamma/metabolismo , Mucosa Intestinal/efectos de los fármacos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Quinazolinas/farmacología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Transducción de Señal , Receptor Toll-Like 4/deficiencia , Receptor Toll-Like 4/genética , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
BACKGROUND: Total parenteral nutrition (TPN), a necessary treatment for patients who cannot receive enteral nutrition, is associated with infectious complications due in part to a loss of intestinal epithelial barrier function (EBF). Using a mouse model of TPN, with enteral nutrient deprivation, we previously demonstrated an increase in mucosal interferon-γ and tumor necrosis factor-α; these cytokine changes are a major mediator driving a reduction in epithelial tight junction (TJ) protein expression. However, the exact ultrastructural changes to the intestinal epithelial barrier have not been previously described. AIM: We hypothesized that TPN dependence results in ultrastructural changes in the intestinal epithelial TJ meshwork. METHODS: C57BL/6 mice underwent internal jugular venous cannulation and were given enteral nutrition or TPN with enteral nutrient deprivation for 7 days. Freeze-fracture electron microscopy was performed on ileal tissue to characterize changes in TJ ultrastructure. EBF was measured using transepithelial resistance and tracer permeability, while TJ expression was measured via Western immunoblotting and immunofluorescence staining. RESULTS: While strand density, linearity, and appearance were unchanged, TPN dependence led to a mean reduction in one horizontal strand out of the TJ compact meshwork to a more basal region, resulting in a reduction in meshwork depth. These findings were correlated with the loss of TJ localization of claudin-4 and tricellulin, reduced expression of claudin-5 and claudin-8, and reduced ex vivo EBF. CONCLUSION: Tight junction ultrastructural changes may contribute to reduced EBF in the setting of TPN dependence.
Asunto(s)
Mucosa Intestinal/citología , Nutrición Parenteral Total/efectos adversos , Uniones Estrechas/ultraestructura , Animales , Técnica de Fractura por Congelación , Mucosa Intestinal/efectos de los fármacos , Masculino , Ratones , Ratones Endogámicos C57BL , Microscopía Electrónica/métodos , Uniones Estrechas/efectos de los fármacosRESUMEN
Small intestine luminal nutrient sensing may be crucial for modulating physiological functions. However, its mechanism of action is incompletely understood. We used a model of enteral nutrient deprivation, or total parenteral nutrition (TPN), resulting in intestinal mucosal atrophy and decreased epithelial barrier function (EBF). We examined how a single amino acid, glutamate (GLM), modulates intestinal epithelial cell (IEC) growth and EBF. Controls were chow-fed mice, T1 receptor-3 (T1R3)-knockout (KO) mice, and treatment with the metabotropic glutamate receptor (mGluR)-5 antagonist MTEP. TPN significantly changed the amount of T1Rs, GLM receptors, and transporters, and GLM prevented these changes. GLM significantly prevented TPN-associated intestinal atrophy (2.5-fold increase in IEC proliferation) and was dependent on up-regulation of the protein kinase pAkt, but independent of T1R3 and mGluR5 signaling. GLM led to a loss of EBF with TPN (60% increase in FITC-dextran permeability, 40% decline in transepithelial resistance); via T1R3, it protected EBF, whereas mGluR5 was associated with EBF loss. GLM led to a decline in circulating glucagon-like peptide 2 (GLP-2) during TPN. The decline was regulated by T1R3 and mGluR5, suggesting a novel negative regulator pathway for IEC proliferation not previously described. Loss of luminal nutrients with TPN administration may widely affect intestinal taste sensing. GLM has previously unrecognized actions on IEC growth and EBF. Restoring luminal sensing via GLM could be a strategy for patients on TPN.
Asunto(s)
Ácido Glutámico/metabolismo , Mucosa Intestinal/metabolismo , Nutrición Parenteral Total/métodos , Receptores Acoplados a Proteínas G/metabolismo , Ciencias de la Nutrición Animal , Animales , Atrofia , Proliferación Celular , Modelos Animales de Enfermedad , Regulación hacia Abajo , Células Epiteliales/citología , Epitelio/metabolismo , Alimentos , Péptido 2 Similar al Glucagón/metabolismo , Yeyuno/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Permeabilidad , Piperidinas , Receptor del Glutamato Metabotropico 5/metabolismo , Transducción de Señal , TiazolesRESUMEN
Enteral nutrient deprivation via total parenteral nutrition (TPN) administration leads to local mucosal inflammatory responses, but the underlying mechanisms are unknown. Wild-type (WT) and MyD88(-/-) mice underwent jugular vein cannulation. One group received TPN without chow, and controls received standard chow. After 7 d, we harvested intestinal mucosally associated bacteria and isolated small-bowel lamina propria (LP) cells. Bacterial populations were analyzed using 454 pyrosequencing. LP cells were analyzed using quantitative PCR and multicolor flow cytometry. WT, control mucosally associated microbiota were Firmicutes-dominant, whereas WT TPN mice were Proteobacteria-domiant. Similar changes were observed in MyD88(-/-) mice with TPN administration. UniFrac analysis showed divergent small bowel and colonic bacterial communities in controls, merging toward similar microbiota (but distinct from controls) with TPN. The percentage of LP T regulatory cells significantly decreased with TPN in WT mice. F4/80(+)CD11b(+)CD11c(dull/-) macrophage-derived proinflammatory cytokines significantly increased with TPN. These proinflammatory immunologic changes were significantly abrogated in MyD88(-/-) TPN mice. Thus, TPN administration is associated with significant expansion of Proteobacteria within the intestinal microbiota and increased proinflammatory LP cytokines. Additionally, MyD88 signaling blockade abrogated decline in epithelial cell proliferation and epithelial barrier function loss.
Asunto(s)
Inflamación/patología , Mucosa Intestinal/microbiología , Mucosa Intestinal/patología , Factor 88 de Diferenciación Mieloide/inmunología , Nutrición Parenteral Total/efectos adversos , Animales , Citometría de Flujo , Inflamación/etiología , Inflamación/microbiología , Mucosa Intestinal/inmunología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Microscopía Fluorescente , Membrana Mucosa/microbiología , Membrana Mucosa/patología , Polimorfismo de Longitud del Fragmento de Restricción , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
Tumour necrosis factor-α (TNF-α) has been reported to play a central role in intestinal barrier dysfunction in many diseases; however, the precise role of the TNF-α receptors (TNFRs) has not been well defined using in vivo models. Our previous data showed that enteral nutrient deprivation or total parenteral nutrition (TPN) led to a loss of intestinal epithelial barrier function (EBF), with an associated upregulation of TNF-α and TNFR1. In this study, we hypothesized that TNF-α plays an important role in TPN-associated EBF dysfunction. Using a mouse TPN model, we explored the relative roles of TNFR1 vs. TNFR2 in mediating this barrier loss. C57/BL6 mice underwent intravenous cannulation and were given enteral nutrition or TPN for 7 days. Tumour necrosis factor-α receptor knockout (KO) mice, including TNFR1KO, TNFR2KO or TNFR1R2 double KO (DKO), were used. Outcomes included small intestine transepithelial resistance (TER) and tracer permeability, junctional protein zonula occludens-1, occludin, claudins and E-cadherin expression. In order to address the dependence of EBF on TNF-α further, exogenous TNF-α and pharmacological blockade of TNF-α (Etanercept) were also performed. Total parenteral nutrition led to a loss of EBF, and this was almost completely prevented in TNFR1R2DKO mice and partly prevented in TNFR1KO mice but not in TNFR2KO mice. The TPN-associated downregulation of junctional protein expression and junctional assembly was almost completely prevented in the TNFR1R2DKO group. Blockade of TNF-α also prevented dysfunction of the EBF and junctional protein losses in mice undergoing TPN. Administration of TPN upregulated the downstream nuclear factor-B and myosin light-chain kinase (MLCK) signalling, and these changes were almost completely prevented in TNFR1R2DKO mice, as well as with TNF-α blockade, but not in TNFR1KO or TNFR2KO TPN groups. Tumour necrosis factor-α is a critical factor for TPN-associated epithelial barrier dysfunction, and both TNFR1 and TNFR2 are involved in EBF loss. Nuclear factor-B and MLCK signalling appear to be important downstream mediators involved in this TNF-α signalling process.
Asunto(s)
Mucosa Intestinal/fisiopatología , Nutrición Parenteral Total/efectos adversos , Receptores Tipo II del Factor de Necrosis Tumoral/fisiología , Receptores Tipo I de Factores de Necrosis Tumoral/fisiología , Factor de Necrosis Tumoral alfa/fisiología , Animales , Etanercept , Inmunoglobulina G/farmacología , Mucosa Intestinal/efectos de los fármacos , Mucosa Intestinal/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Quinasa de Cadena Ligera de Miosina/fisiología , FN-kappa B/fisiología , Receptores del Factor de Necrosis Tumoral , Transducción de Señal , Uniones Estrechas/metabolismoRESUMEN
Epidermal growth factor (EGF) and tumor necrosis factor-α (TNF-α) signaling are critical for effective proliferative and apoptotic actions; however, little is known about the codependency of these signaling pathways in the intestinal epithelium. Because total parenteral nutrition (TPN) is associated with loss of intestinal epithelial cell (IEC) proliferation and increased apoptosis, we utilized a mouse model to explore these transactivation pathways in small bowel epithelium. Mice underwent intravenous cannulation and were given enteral nutrition or TPN for 7 days. Outcomes included IEC proliferation, apoptosis, and survival. To address transactivation or dependence of EGF and TNF on IEC physiology, TNF-α receptor knockout (KO) mice, TNFR1-KO, R2-KO, or R1R2-double KO, were used. Exogenous EGF and pharmacological blockade of ErbB1 were performed in other groups to examine the relevance of the ErB1 pathway. TPN increased IEC TNFR1 and decreased EGF and ErbB1 abundance. Loss of IEC proliferation was prevented by exogenous EGF or blockade of TNFR1. However, EGF action was prevented without effective TNFR2 signaling. Also, blockade of TNFR1 could not prevent loss of IEC proliferation without effective ErbB1 signaling. TPN increased IEC apoptosis and was due to increased TNFR1 signaling. Exogenous EGF or blockade of TNFR1 could prevent increased apoptosis, and both pathways were dependent on effective ErbB1 signaling. Exogenous EGF prevented increased apoptosis in mice lacking TNFR2 signaling. TPN mice had significantly decreased survival vs. controls, and this was associated with the TNFR1 signaling pathway. We concluded that these findings identify critical mechanisms that contribute to TPN-associated mucosal atrophy via altered TNF-α/EGF signaling. It emphasizes the importance of both TNFR1 and TNFR2 pathways, as well as the strong interdependence on an intact EGF/ErbB1 pathway.
Asunto(s)
Apoptosis/fisiología , Proliferación Celular , Factor de Crecimiento Epidérmico/metabolismo , Células Epiteliales/metabolismo , Nutrición Parenteral Total , Transducción de Señal/fisiología , Factor de Necrosis Tumoral alfa/metabolismo , Animales , Factor de Crecimiento Epidérmico/genética , Receptores ErbB/genética , Receptores ErbB/metabolismo , Ratones , Ratones Noqueados , Receptores Tipo I de Factores de Necrosis Tumoral/genética , Receptores Tipo I de Factores de Necrosis Tumoral/metabolismo , Receptores Tipo II del Factor de Necrosis Tumoral/genética , Receptores Tipo II del Factor de Necrosis Tumoral/metabolismo , Factor de Necrosis Tumoral alfa/genéticaRESUMEN
Glial cell line-derived trophic factor (GDNF) is a peptide with pleiotropic survival and growth-promoting effects on neurons. We found that intraspinal injection of a non-replicating herpes simplex virus-based vector coding for GDNF 2 h after blunt trauma to the thoraco-lumbar spinal cord produced sustained improvement in motor behavioral outcomes up to 5 weeks following injury. The improvement in behavior correlated with an increase in synaptophysin and glutamic acid decarboxylase (GAD) in the spinal cord at the level of injury. Addition of recombinant GDNF protein to primary spinal cord neurons in-vitro resulted in enhanced neurite growth and a marked increase in protein levels of GAD65 and GAD67, synapsin I and synaptophysin. GDNF-mediated increases in GAD and the synaptic markers were blocked by the MEK inhibitor UO126, but not by the phosphoinositide 3-kinase inhibitor LY294002. These results suggest that GDNF, acting through the MEK-ERK pathway enhances axonal sprouting, synaptic connectivity, and GABAergic neurotransmission in the spinal cord, that result in improved behavioral outcomes after spinal cord contusion injury.
Asunto(s)
Terapia Genética/métodos , Factor Neurotrófico Derivado de la Línea Celular Glial/metabolismo , Traumatismos de la Médula Espinal/fisiopatología , Traumatismos de la Médula Espinal/terapia , Transmisión Sináptica/genética , Ácido gamma-Aminobutírico/metabolismo , Análisis de Varianza , Animales , Células Cultivadas , Modelos Animales de Enfermedad , Embrión de Mamíferos , Femenino , Factor Neurotrófico Derivado de la Línea Celular Glial/genética , Factor Neurotrófico Derivado de la Línea Celular Glial/farmacología , Glutamato Descarboxilasa/metabolismo , Proteínas Fluorescentes Verdes/genética , Humanos , Proteínas Asociadas a Microtúbulos/metabolismo , Actividad Motora/efectos de los fármacos , Actividad Motora/genética , Proteínas de Neurofilamentos/metabolismo , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Desempeño Psicomotor/efectos de los fármacos , Desempeño Psicomotor/fisiología , Ratas , Ratas Sprague-Dawley , Recuperación de la Función/genética , Recuperación de la Función/fisiología , Médula Espinal/citología , Traumatismos de la Médula Espinal/genética , Traumatismos de la Médula Espinal/patología , Transmisión Sináptica/efectos de los fármacosRESUMEN
Total parenteral nutrition (TPN) leads to a decline in phosphatidylinositol 3-kinase (PI3K)/phospho-Akt (p-Akt) activity, affecting downstream signaling, reducing epithelial cell (EC) proliferation, and contributing to intestinal mucosal atrophy. We hypothesized that promoting Akt activity would prevent these changes. We used a novel Akt-activating peptide, TCL1 (a head-to-tail dimer of the Akt-binding domain of T-cell lymphoma-1), or an inactive mutant sequence TCL1G conjugated to a transactivator of transcription peptide sequence to promote intracellular uptake. Four groups of mice were studied, enteral nutrition group (control), control mice given a functioning TCL1 (control + TCL1), TPN mice given TCL1G (control peptide, TPN + TCL1G); and TPN mice given TCL1. TPN mice given TCL1G showed a significant decrease in jejunal EC p-Akt (Ser473 and Thr308) abundance, whereas TPN + TCL1 mice showed increased p-Akt (Ser473) abundance. Phosphorylation of beta-catenin and glycogen synthase kinase-3beta (downstream targets of Akt signaling) were also decreased in the TPN + TCL1G group and completely prevented in the TPN + TCL1 group. Use of TCL1 nearly completely prevented the decline in EC proliferation seen in the TPN + TCL1G group, as well as partly returned EC apoptosis levels close to controls. The mammalian target of rapamycin pathway demonstrated a similar reduction in activity in the TPN + TCL1G group that was significantly prevented in the TPN + TCL1 group. These results support a significant loss of PI3K/p-Akt signaling upon replacing enteral nutrition with TPN, and prevention of this loss demonstrates the key importance of PI3K/p-Akt signaling in maintaining gut integrity including EC proliferation and reduction in apoptosis.
Asunto(s)
Mucosa Intestinal/patología , Nutrición Parenteral Total/efectos adversos , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas Proto-Oncogénicas/farmacología , Transducción de Señal/fisiología , Animales , Atrofia/inducido químicamente , Atrofia/patología , Proliferación Celular , Células Epiteliales/efectos de los fármacos , Células Epiteliales/fisiología , Regulación de la Expresión Génica/efectos de los fármacos , Mucosa Intestinal/efectos de los fármacos , Mucosa Intestinal/metabolismo , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Fosforilación , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/genética , Distribución Aleatoria , Transducción de Señal/efectos de los fármacos , Organismos Libres de Patógenos Específicos , Serina-Treonina Quinasas TOR , Transactivadores/farmacología , Proteínas Wnt/genética , Proteínas Wnt/metabolismo , beta Catenina/genética , beta Catenina/metabolismoRESUMEN
Total parenteral nutrition (TPN) leads a loss of epithelial barrier function, decline in epithelial cell (EC) proliferation, and decreased expression of E-cadherin. As a large portion of intracellular beta-catenin is tightly associated with E-cadherin, we hypothesized that the loss of E-cadherin would result in a redistribution of intracellular beta-catenin, and could be a contributing mechanism for this TPN-associated loss of EC proliferation. An assessment of small bowel epithelium was performed in mice given either enteral nutrition (Control) or intravenous nutrition (TPN). TPN significantly down-regulated E-cadherin and beta-catenin expression, and resulted in a loss of a colocalization of these factors. TPN also up-regulated phosphorylated (p)-beta-catenin (Ser31/33,Thr41) and down-regulated (p)-beta-catenin(Ser552) expression. To further address mechanisms driving this, we observed a significant decrease in the abundance of p-AKT and p-GSK3beta expression, and an associated decline in tcf-4 transcription factors (cyclin D1, c-myc and Axin2), as well as a loss of EC proliferation by 7 days. To address whether the mechanism driving these changes was the absence of nutritional factors, glutamine was added to the TPN solution. This resulted in a partial restoration of beta-catenin expression and EC proliferation, suggesting that an alteration in nutrient delivery may affect many of these changes. Based on these findings, the loss of EC proliferation with TPN may well be due to a loss of total beta-catenin, as well as a concomitant change in the differential expression of beta-catenin phosphorylation sites, and a reduction in beta-catenin mediated tcf-4 transcription. This potential pathway may well explain many of the findings of mucosal atrophy associated with TPN.
Asunto(s)
Cadherinas/metabolismo , Mucosa Intestinal/metabolismo , Nutrición Parenteral Total/efectos adversos , beta Catenina/metabolismo , Animales , Atrofia , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice , Cadherinas/genética , Glutamina/metabolismo , Glucógeno Sintasa Quinasa 3/metabolismo , Glucógeno Sintasa Quinasa 3 beta , Humanos , Mucosa Intestinal/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Microvellosidades/metabolismo , Microvellosidades/patología , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Fosforilación , Procesamiento Proteico-Postraduccional , Proteínas Proto-Oncogénicas c-akt/metabolismo , Factores de Transcripción TCF/genética , Factores de Transcripción TCF/metabolismo , Factor de Transcripción 4 , Activación Transcripcional , alfa Catenina/genética , alfa Catenina/metabolismo , beta Catenina/genéticaRESUMEN
Total parenteral nutrition (TPN) results in intestinal mucosal atrophic changes due to an absence of enteral nutrition; however, the mechanisms responsible for this are not fully understood. It has been shown that bone morphogenetic protein (BMP) activation inhibits intestinal epithelial cell (EC) proliferation. Therefore, we hypothesized that the BMP pathway could be upregulated by TPN. To address this, we randomly assigned mice to receive TPN or to be enterally fed (control) for 7 d. Mucosal EC isolates were harvested from the entire length of small intestine for RNA and protein measurements. Full-thickness, mid-small bowel was processed for histological examination. TPN increased the abundance of BMP2, BMP4, and BMP type II receptor at the RNA and protein levels. Phosphorylation of Smad1, Smad5, and Smad8 also was greater in the TPN group than in the control, which helped to confirm activation of this pathway. Interestingly, the TPN and control groups did not differ in the mRNA expression of the extracellular soluble bmp antagonists, noggin, gremlin, chordin, or follistatin. Compared to the control group, the expression of c-Myc (cellular myelocytomatosis) mRNA was lower, whereas the level of p21(WAF1/CIP1) was greater, in the TPN group. Because the BMP family may function through suppression of Wnt-beta-catenin signaling, this pathway was also examined. mRNA expression of Wnt 3, Wnt5a, and the Wnt receptor Lrp5 were lower in the TPN group compared to controls. The results suggest that the BMP signaling pathway may be involved in the development of intestinal mucosal atrophy due to TPN administration.
Asunto(s)
Proteína Morfogenética Ósea 2/genética , Proteína Morfogenética Ósea 4/genética , Proteínas Morfogenéticas Óseas/fisiología , Intestino Delgado/fisiología , Nutrición Parenteral Total , Animales , Receptores de Proteínas Morfogenéticas Óseas de Tipo II/genética , Proteínas Morfogenéticas Óseas/genética , Dipéptidos/farmacología , Regulación de la Expresión Génica , Glutamina/farmacología , Masculino , Ratones , Ratones Endogámicos C57BL , Modelos Animales , Reacción en Cadena de la Polimerasa , ARN/genética , Valores de Referencia , Transducción de SeñalRESUMEN
BACKGROUND: Although serial transverse enteroplasty (STEP) improves function of dilated short bowel, a significant proportion of patients require repeat surgery. To address underlying reasons for unsuccessful STEP, we compared small intestinal mucosal characteristics between initial and repeat STEP procedures in children with short bowel syndrome (SBS). METHODS: Fifteen SBS children, who underwent 13 first and 7 repeat STEP procedures with full thickness small bowel samples at median age 1.5 years (IQR 0.7-3.7) were included. The specimens were analyzed histologically for mucosal morphology, inflammation and muscular thickness. Mucosal proliferation and apoptosis was analyzed with MIB1 and Tunel immunohistochemistry. RESULTS: Median small bowel length increased 42% by initial STEP and 13% by repeat STEP (p=0.05), while enteral caloric intake increased from 6% to 36% (p=0.07) during 14 (12-42) months between the procedures. Abnormal mucosal inflammation was frequently observed both at initial (69%) and additional STEP (86%, p=0.52) surgery. Villus height, crypt depth, enterocyte proliferation and apoptosis as well as muscular thickness were comparable at first and repeat STEP (p>0.05 for all). Patients, who required repeat STEP tended to be younger (p=0.057) with less apoptotic crypt cells (p=0.031) at first STEP. Absence of ileocecal valve associated with increased intraepithelial leukocyte count and reduced crypt cell proliferation index (p<0.05 for both). CONCLUSIONS: No adaptive mucosal hyperplasia or muscular alterations occurred between first and repeat STEP. Persistent inflammation and lacking mucosal growth may contribute to continuing bowel dysfunction in SBS children, who require repeat STEP procedure, especially after removal of the ileocecal valve. LEVEL OF EVIDENCE: Level IV, retrospective study.
Asunto(s)
Procedimientos Quirúrgicos del Sistema Digestivo/efectos adversos , Mucosa Intestinal/patología , Intestino Delgado/cirugía , Síndrome del Intestino Corto/patología , Apoptosis , Niño , Preescolar , Procedimientos Quirúrgicos del Sistema Digestivo/métodos , Humanos , Inmunohistoquímica , Lactante , Inflamación/patología , Intestino Delgado/patología , Reoperación/efectos adversos , Estudios Retrospectivos , Síndrome del Intestino Corto/cirugíaAsunto(s)
Adaptación Fisiológica , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Proteínas de Unión al Calcio/metabolismo , Proteínas de Homeodominio/metabolismo , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Proteínas de la Membrana/metabolismo , Receptor Notch1/metabolismo , Animales , Masculino , Proteínas Serrate-Jagged , Factor de Transcripción HES-1RESUMEN
BACKGROUND & AIMS: Total parenteral nutrition (TPN), a crucial treatment for patients who cannot receive enteral nutrition, is associated with mucosal atrophy, barrier dysfunction, and infectious complications. Glucagon-like peptide-2 (GLP-2) and epidermal growth factor (EGF) improve intestinal epithelial cell (IEC) responses and attenuate mucosal atrophy in several TPN models. However, it remains unclear whether these 2 factors use distinct or overlapping signaling pathways to improve IEC responses. We investigated the interaction of GLP-2 and EGF signaling in a mouse TPN model and in patients deprived of enteral nutrition. METHODS: Adult C57BL/6J, IEC-Egfrknock out (KO) and IEC-pik3r1KO mice receiving TPN or enteral nutrition were treated with EGF or GLP-2 alone or in combination with reciprocal receptor inhibitors, GLP-2(3-33) or gefitinib. Jejunum was collected and mucosal atrophy and IEC responses were assessed by histologic, gene, and protein expression analyses. In patients undergoing planned looped ileostomies, fed and unfed ileum was analyzed. RESULTS: Enteral nutrient deprivation reduced endogenous EGF and GLP-2 signaling in mice and human beings. In the mouse TPN model, exogenous EGF or GLP-2 attenuated mucosal atrophy and restored IEC proliferation. The beneficial effects of EGF and GLP-2 were decreased upon Gefitinib treatment and in TPN-treated IEC-EgfrKO mice, showing epidermal growth factor-receptor dependency for these IEC responses. By contrast, in TPN-treated IEC-pi3kr1KO mice, the beneficial actions of EGF were lost, although GLP-2 still attenuated mucosal atrophy. CONCLUSIONS: Upon enteral nutrient deprivation, exogenous GLP-2 and EGF show strong interdependency for improving IEC responses. Understanding the differential requirements for phosphatidylinositol 3-kinase/phosphoAKT (Ser473) signaling may help improve future therapies to prevent mucosal atrophy.
RESUMEN
Intestinal resident macrophages (MÏs) regulate gastrointestinal homeostasis via production of an anti-inflammatory cytokine interleukin (IL)-10. Although a constant replenishment by circulating monocytes is required to maintain the pool of resident MÏs in the colonic mucosa, the homeostatic regulation of MÏ in the small intestine (SI) remains unclear. Here, we demonstrate that direct stimulation by dietary amino acids regulates the homeostasis of intestinal MÏs in the SI. Mice that received total parenteral nutrition (TPN), which deprives the animals of enteral nutrients, displayed a significant decrease of IL-10-producing MÏs in the SI, whereas the IL-10-producing CD4(+) T cells remained intact. Likewise, enteral nutrient deprivation selectively affected the monocyte-derived F4/80(+) MÏ population, but not non-monocytic precursor-derived CD103(+) dendritic cells. Notably, in contrast to colonic MÏs, the replenishment of SI MÏs and their IL-10 production were not regulated by the gut microbiota. Rather, SI MÏs were directly regulated by dietary amino acids. Collectively, our study highlights the diet-dependent, microbiota-independent regulation of IL-10-producing resident MÏs in the SI.
Asunto(s)
Interleucina-10/metabolismo , Intestino Delgado/metabolismo , Macrófagos/metabolismo , Membrana Mucosa/metabolismo , Aminoácidos/metabolismo , Alimentación Animal , Animales , Antígenos CD/metabolismo , Antígeno CD11b/metabolismo , Linfocitos T CD4-Positivos/citología , Quimiocina CCL2/metabolismo , Citocinas/metabolismo , Microbioma Gastrointestinal , Homeostasis , Cadenas alfa de Integrinas/metabolismo , Mucosa Intestinal/metabolismo , Intestino Delgado/microbiología , Macrófagos/microbiología , Masculino , Ratones , Ratones Endogámicos C57BL , Monocitos/metabolismo , Membrana Mucosa/microbiología , Receptores CCR2/metabolismoRESUMEN
OBJECTIVE: To investigate the effect of nutrient withdrawal on human intestinal epithelial barrier function (EBF). We hypothesized that unfed mucosa results in decreased EBF. This was tested in a series of surgical small intestinal resection specimens. DESIGN: Small bowel specifically excluding inflamed tissue, was obtained from pediatric patients (aged 2 days to 19 years) undergoing intestinal resection. EBF was assessed in Ussing chambers for transepithelial resistance (TER) and passage of fluorescein isothiocyanate (FITC)-dextran (4 kD). Tight junction and adherence junction proteins were imaged with immunofluorescence staining. Expression of Toll-like receptors (TLR) and inflammatory cytokines were measured in loop ileostomy takedowns in a second group of patients. RESULTS: Because TER increased with patient age (P < .01), results were stratified into infant versus teenage groups. Fed bowel had significantly greater TER versus unfed bowel (P < .05) in both age populations. Loss of EBF was also observed by an increase in FITC-dextran permeation in enteral nutrient-deprived segments (P < .05). Immunofluorescence staining showed marked declines in intensity of ZO-1, occludin, E-cadherin, and claudin-4 in unfed intestinal segments, as well as a loss of structural formation of tight junctions. Analysis of cytokine and TLR expression showed significant increases in tumor necrosis factor (TNF)-α and TLR4 in unfed segments of bowel compared with fed segments from the same individual. CONCLUSION: EBF declined in unfed segments of human small bowel. This work represents the first direct examination of EBF from small bowel derived from nutrient-deprived humans and may explain the increased incidence of infectious complications seen in patients not receiving enteral feeds.
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Uniones Adherentes/metabolismo , Nutrición Enteral , Mucosa Intestinal/fisiopatología , Intestino Delgado/fisiopatología , Nutrición Parenteral/efectos adversos , Uniones Estrechas/metabolismo , Adolescente , Biomarcadores/metabolismo , Niño , Preescolar , Citocinas/metabolismo , Femenino , Humanos , Lactante , Recién Nacido , Mucosa Intestinal/metabolismo , Intestino Delgado/metabolismo , Modelos Lineales , Masculino , Permeabilidad , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Receptores Toll-Like/metabolismo , Adulto JovenRESUMEN
Total parenteral nutrition (TPN) is commonly used clinically to sustain patients; however, TPN is associated with profound mucosal atrophy, which may adversely affect clinical outcomes. Using a mouse TPN model, removing enteral nutrition leads to decreased crypt proliferation, increased intestinal epithelial cell (IEC) apoptosis and increased mucosal tumor necrosis factor alpha (TNF-α) expression that ultimately produces mucosal atrophy. Upregulation of TNF-α signaling plays a central role in mediating TPN-induced mucosal atrophy without intact epidermal growth factor receptor (EGFR) signaling. Currently, the mechanism and the tissue-specific contributions of TNF-α signaling to TPN-induced mucosal atrophy remain unclear. ADAM17 is an ectodomain sheddase that can modulate the signaling activity of several cytokine/growth factor receptor families, including the TNF-α/TNF receptor and ErbB ligand/EGFR pathways. Using TPN-treated IEC-specific ADAM17-deficient mice, the present study demonstrates that a loss of soluble TNF-α signaling from IECs attenuates TPN-induced mucosal atrophy. Importantly, this response remains dependent on the maintenance of functional EGFR signaling in IECs. TNF-α blockade in wild-type mice receiving TPN confirmed that soluble TNF-α signaling is responsible for downregulation of EGFR signaling in IECs. These results demonstrate that ADAM17-mediated TNF-α signaling from IECs has a significant role in the development of the proinflammatory state and mucosal atrophy observed in TPN-treated mice.
Asunto(s)
Proteínas ADAM/genética , Mucosa Intestinal/patología , Nutrición Parenteral Total/efectos adversos , Transducción de Señal , Factor de Necrosis Tumoral alfa/inmunología , Proteínas ADAM/inmunología , Proteína ADAM17 , Animales , Apoptosis , Atrofia/inmunología , Atrofia/patología , Proliferación Celular , Citocinas/inmunología , Receptores ErbB/inmunología , Femenino , Técnicas de Inactivación de Genes , Humanos , Mucosa Intestinal/citología , Mucosa Intestinal/inmunología , Mucosa Intestinal/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Factor de Transcripción STAT3/inmunologíaRESUMEN
BACKGROUND: Injectable fat emulsions (FEs) are a clinically dependable source of essential fatty acids (FA). ω-6 FA is associated with an inflammatory response. Medium-chain triglycerides (MCT, ω-3 FA), fish oil, and olive oil are reported to decrease the inflammatory response. However, the effect of these lipids on the gastrointestinal tract has not been well studied. To address this, we used a mouse model of parenteral nutrition (PN) and hypothesized that a decrease in intestinal inflammation would be seen when either fish oil and MCT or olive oil were added. METHODS: Three FEs were studied in adult C57BL/6 mice via intravenous cannulation: standard soybean-based FE (SBFE), 80% olive oil -supplemented FE (OOFE), or a combination of a soybean oil, MCT, olive oil, and fish oil emulsion (SMOF). PN was given for 7 days, small bowel mucosa-derived cytokines, animal survival rate, epithelial cell (EC) proliferation and apoptosis rates, intestinal barrier function and mucosal FA composition were analyzed. RESULTS: Compared to the SBFE and SMOF groups, the best survival, highest EC proliferation and lowest EC apoptosis rates were observed in the OOFE group; and associated with the lowest levels of tumor necrosis factor-α, interleukin-6, and interleukin-1ß expression. Jejunal FA content showed higher levels of eicosapentaenoic and docosapentaenoic acid in the SMOF group and the highest arachidonic acid in the OOFE group. CONCLUSION: The study showed that PN containing OOFE had beneficial effects to small bowel health and animal survival. Further investigation may help to enhance bowel integrity in patients restricted to PN.