RESUMEN
BACKGROUND AND AIMS: Bacterially derived factors from the gut play a major role in the activation of inflammatory pathways in the liver and in the pathogenesis of alcoholic liver disease. The intestinal brush-border enzyme intestinal alkaline phosphatase (IAP) detoxifies a variety of bacterial pro-inflammatory factors and also functions to preserve gut barrier function. The aim of this study was to investigate whether oral IAP supplementation could protect against alcohol-induced liver disease. METHODS: Mice underwent acute binge or chronic ethanol exposure to induce alcoholic liver injury and steatosis ± IAP supplementation. Liver tissue was assessed for biochemical, inflammatory, and histopathological changes. An ex vivo co-culture system was used to examine the effects of alcohol and IAP treatment in regard to the activation of hepatic stellate cells and their role in the development of alcoholic liver disease. RESULTS: Pretreatment with IAP resulted in significantly lower serum alanine aminotransferase compared to the ethanol alone group in the acute binge model. IAP treatment attenuated the development of alcohol-induced fatty liver, lowered hepatic pro-inflammatory cytokine and serum LPS levels, and prevented alcohol-induced gut barrier dysfunction. Finally, IAP ameliorated the activation of hepatic stellate cells and prevented their lipogenic effect on hepatocytes. CONCLUSIONS: IAP treatment protected mice from alcohol-induced hepatotoxicity and steatosis. Oral IAP supplementation could represent a novel therapy to prevent alcoholic-related liver disease in humans.
Asunto(s)
Fosfatasa Alcalina/administración & dosificación , Suplementos Dietéticos , Hígado Graso Alcohólico/prevención & control , Alanina Transaminasa/sangre , Animales , Técnicas de Cocultivo , Citocinas/análisis , Citocinas/sangre , Etanol , Hígado Graso Alcohólico/sangre , Hígado Graso Alcohólico/enzimología , Femenino , Células Estrelladas Hepáticas/enzimología , Hepatocitos/enzimología , Intestinos/enzimología , Lipogénesis , Lipopolisacáridos/sangre , Hígado/química , Ratones , Ratones Endogámicos C57BL , Permeabilidad , Activador de Tejido Plasminógeno , Triglicéridos/análisisRESUMEN
Metabolic syndrome comprises a cluster of related disorders that includes obesity, glucose intolerance, insulin resistance, dyslipidemia, and fatty liver. Recently, gut-derived chronic endotoxemia has been identified as a primary mediator for triggering the low-grade inflammation responsible for the development of metabolic syndrome. In the present study we examined the role of the small intestinal brush-border enzyme, intestinal alkaline phosphatase (IAP), in preventing a high-fat-diet-induced metabolic syndrome in mice. We found that both endogenous and orally supplemented IAP inhibits absorption of endotoxin (lipopolysaccharides) that occurs with dietary fat, and oral IAP supplementation prevents as well as reverses metabolic syndrome. Furthermore, IAP supplementation improves the lipid profile in mice fed a standard, low-fat chow diet. These results point to a potentially unique therapy against metabolic syndrome in at-risk humans.
Asunto(s)
Fosfatasa Alcalina/metabolismo , Fosfatasa Alcalina/farmacología , Síndrome Metabólico/tratamiento farmacológico , Absorción/efectos de los fármacos , Administración Oral , Fosfatasa Alcalina/administración & dosificación , Fosfatasa Alcalina/genética , Animales , Compuestos Azo , Línea Celular , Cartilla de ADN/genética , Lipopolisacáridos , Hígado/metabolismo , Síndrome Metabólico/etiología , Síndrome Metabólico/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Microvellosidades/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Triglicéridos/metabolismoRESUMEN
The intestinal microbiota plays a pivotal role in maintaining human health and well-being. Previously, we have shown that mice deficient in the brush-border enzyme intestinal alkaline phosphatase (IAP) suffer from dysbiosis and that oral IAP supplementation normalizes the gut flora. Here we aimed to decipher the molecular mechanism by which IAP promotes bacterial growth. We used an isolated mouse intestinal loop model to directly examine the effect of exogenous IAP on the growth of specific intestinal bacterial species. We studied the effects of various IAP targets on the growth of stool aerobic and anaerobic bacteria as well as on a few specific gut organisms. We determined the effects of ATP and other nucleotides on bacterial growth. Furthermore, we examined the effects of IAP on reversing the inhibitory effects of nucleotides on bacterial growth. We have confirmed that local IAP bioactivity creates a luminal environment that promotes the growth of a wide range of commensal organisms. IAP promotes the growth of stool aerobic and anaerobic bacteria and appears to exert its growth promoting effects by inactivating (dephosphorylating) luminal ATP and other luminal nucleotide triphosphates. We observed that compared with wild-type mice, IAP-knockout mice have more ATP in their luminal contents, and exogenous IAP can reverse the ATP-mediated inhibition of bacterial growth in the isolated intestinal loop. In conclusion, IAP appears to promote the growth of intestinal commensal bacteria by inhibiting the concentration of luminal nucleotide triphosphates.
Asunto(s)
Fosfatasa Alcalina/fisiología , Intestinos/microbiología , Adenosina Trifosfato/farmacología , Fosfatasa Alcalina/antagonistas & inhibidores , Fosfatasa Alcalina/genética , Fosfatasa Alcalina/farmacología , Ampicilina/farmacología , Animales , Desoxirribonucleótidos/farmacología , Farmacorresistencia Bacteriana , Enterococcus faecalis/efectos de los fármacos , Escherichia coli/efectos de los fármacos , Heces/microbiología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Morganella morganii/efectos de los fármacos , Fenilalanina/farmacología , Inanición/fisiopatología , Estreptomicina/farmacologíaRESUMEN
OBJECTIVE: To determine the role of intestinal alkaline phosphatase (IAP) in enteral starvation-induced gut barrier dysfunction and to study its therapeutic effect as a supplement to prevent gut-derived sepsis. BACKGROUND: Critically ill patients are at increased risk for systemic sepsis and, in some cases, multiorgan failure leading to death. Years ago, the gut was identified as a major source for this systemic sepsis syndrome. Previously, we have shown that IAP detoxifies bacterial toxins, prevents endotoxemia, and preserves intestinal microbiotal homeostasis. METHODS: WT and IAP-KO mice were used to examine gut barrier function and tight junction protein levels during 48-hour starvation and fed states. Human ileal fluid samples were collected from 20 patients postileostomy and IAP levels were compared between fasted and fed states. To study the effect of IAP supplementation on starvation-induced gut barrier dysfunction, WT mice were fasted for 48 hours +/- IAP supplementation in the drinking water. RESULTS: The loss of IAP expression is associated with decreased expression of intestinal junctional proteins and impaired barrier function. For the first time, we demonstrate that IAP expression is also decreased in humans who are deprived of enteral feeding. Finally, our data demonstrate that IAP supplementation reverses the gut barrier dysfunction and tight junction protein losses due to a lack of enteral feeding. CONCLUSIONS: IAP is a major regulator of gut mucosal permeability and is able to ameliorate starvation-induced gut barrier dysfunction. Enteral IAP supplementation may represent a novel approach to maintain bowel integrity in critically ill patients.
Asunto(s)
Fosfatasa Alcalina/administración & dosificación , Fosfatasa Alcalina/metabolismo , Enfermedad Crítica , Suplementos Dietéticos , Mucosa Intestinal/enzimología , Síndrome de Respuesta Inflamatoria Sistémica/prevención & control , Administración Oral , Animales , Nutrición Enteral , Humanos , Íleon/enzimología , Íleon/inmunología , Inflamación/enzimología , Yeyuno/enzimología , Yeyuno/inmunología , Ratones , Permeabilidad , Inanición , Proteínas de Uniones Estrechas/metabolismo , Regulación hacia ArribaRESUMEN
Uridine diphosphate (UDP) is a proinflammatory nucleotide implicated in inflammatory bowel disease. Intestinal alkaline phosphatase (IAP) is a gut mucosal defense factor capable of inhibiting intestinal inflammation. We used the malachite green assay to show that IAP dephosphorylates UDP. To study the anti-inflammatory effect of IAP, UDP or other proinflammatory ligands (LPS, flagellin, Pam3Cys, or TNF-α) in the presence or absence of IAP were applied to cell cultures, and IL-8 was measured. UDP caused dose-dependent increase in IL-8 release by immune cells and two gut epithelial cell lines, and IAP treatment abrogated IL-8 release. Costimulation with UDP and other inflammatory ligands resulted in a synergistic increase in IL-8 release, which was prevented by IAP treatment. In vivo, UDP in the presence or absence of IAP was instilled into a small intestinal loop model in wild-type and IAP-knockout mice. Luminal contents were applied to cell culture, and cytokine levels were measured in culture supernatant and intestinal tissue. UDP-treated luminal contents induced more inflammation on target cells, with a greater inflammatory response to contents from IAP-KO mice treated with UDP than from WT mice. Additionally, UDP treatment increased TNF-α levels in intestinal tissue of IAP-KO mice, and cotreatment with IAP reduced inflammation to control levels. Taken together, these studies show that IAP prevents inflammation caused by UDP alone and in combination with other ligands, and the anti-inflammatory effect of IAP against UDP persists in mouse small intestine. The benefits of IAP in intestinal disease may be partly due to inhibition of the proinflammatory activity of UDP.