RESUMEN
Most patients with alcohol-associated liver disease (ALD) engage in heavy drinking defined as 4 or more drinks per day (56 g) or 8 (112 g) or more drinks per week for women and 5 or more drinks per day (70 g) or 15 (210 g) or more drinks per week for men. Although abstinence from alcohol after diagnosis of ALD improves life expectancy and reduces the risk of decompensation of liver disease, few studies have evaluated whether treatment of alcohol use disorders will reduce progression of liver disease and improve liver-related outcomes. In November 2021, the National Institute of Alcohol Abuse and Alcoholism commissioned a task force that included hepatologists, addiction medicine specialists, statisticians, clinical trialists and members of regulatory agencies to develop recommendations for the design and conduct of clinical trials to evaluate the effect of alcohol use, particularly treatment to reduce or eliminate alcohol use in patients with ALD. The task force conducted extensive reviews of relevant literature on alcohol use disorders and ALD. Findings were presented at one in-person meeting and discussed over the next 16 months to develop the final recommendations. As few clinical trials directly address this topic, the 28 recommendations approved by all members of the task force represent a consensus of expert opinions.
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Consumo de Bebidas Alcohólicas , Ensayos Clínicos como Asunto , Hepatopatías Alcohólicas , Humanos , Hepatopatías Alcohólicas/terapia , Consumo de Bebidas Alcohólicas/efectos adversos , Consenso , Proyectos de Investigación , Alcoholismo/complicaciones , Alcoholismo/terapiaAsunto(s)
Enfermedad del Hígado Graso no Alcohólico/tratamiento farmacológico , United States Food and Drug Administration , Biomarcadores , Ensayos Clínicos como Asunto/normas , Aprobación de Drogas , Humanos , Enfermedad del Hígado Graso no Alcohólico/diagnóstico , Resultado del Tratamiento , Estados Unidos , United States Food and Drug Administration/organización & administración , United States Food and Drug Administration/normasRESUMEN
BACKGROUND & AIMS: The heterodimeric integrin receptor α4ß7 regulates CD4 T cell recruitment to inflamed tissues, but its role in the pathogenesis of non-alcoholic steatohepatitis (NASH) is unknown. Herein, we examined the role of α4ß7-mediated recruitment of CD4 T cells to the intestine and liver in NASH. METHODS: Male littermate F11r+/+ (control) and junctional adhesion molecule A knockout F11r-/- mice were fed a normal diet or a western diet (WD) for 8 weeks. Liver and intestinal tissues were analyzed by histology, quantitative reverse transcription PCR (qRT-PCR), 16s rRNA sequencing and flow cytometry. Colonic mucosa-associated microbiota were analyzed using 16s rRNA sequencing. Liver biopsies from patients with NASH were analyzed by confocal imaging and qRT-PCR. RESULTS: WD-fed knockout mice developed NASH and had increased hepatic and intestinal α4ß7+ CD4 T cells relative to control mice who developed mild hepatic steatosis. The increase in α4ß7+ CD4 T cells was associated with markedly higher expression of the α4ß7 ligand mucosal addressin cell adhesion molecule 1 (MAdCAM-1) in the colonic mucosa and livers of WD-fed knockout mice. Elevated MAdCAM-1 expression correlated with increased mucosa-associated Proteobacteria in the WD-fed knockout mice. Antibiotics reduced MAdCAM-1 expression indicating that the diet-altered microbiota promoted colonic and hepatic MAdCAM-1 expression. α4ß7 blockade in WD-fed knockout mice significantly decreased α4ß7+ CD4 T cell recruitment to the intestine and liver, attenuated hepatic inflammation and fibrosis, and improved metabolic indices. MAdCAM-1 blockade also reduced hepatic inflammation and fibrosis in WD-fed knockout mice. Hepatic MAdCAM-1 expression was elevated in patients with NASH and correlated with higher expression of α4 and ß7 integrins. CONCLUSIONS: These findings establish α4ß7/MAdCAM-1 as a critical axis regulating NASH development through colonic and hepatic CD4 T cell recruitment. LAY SUMMARY: Non-alcoholic steatohepatitis (NASH) is an advanced and progressive form of non-alcoholic fatty liver disease (NAFLD), and despite its growing incidence no therapies currently exist to halt NAFLD progression. Herein, we show that blocking integrin receptor α4ß7-mediated recruitment of CD4 T cells to the intestine and liver not only attenuates hepatic inflammation and fibrosis, but also improves metabolic derangements associated with NASH. These findings provide evidence for the potential therapeutic application of α4ß7 antibody in the treatment of human NASH.
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Linfocitos T CD4-Positivos/inmunología , Dieta Occidental/efectos adversos , Integrinas/metabolismo , Mucosa Intestinal/inmunología , Hígado/inmunología , Enfermedad del Hígado Graso no Alcohólico/etiología , Enfermedad del Hígado Graso no Alcohólico/inmunología , Animales , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/farmacología , Linfocitos T CD4-Positivos/metabolismo , Moléculas de Adhesión Celular/antagonistas & inhibidores , Moléculas de Adhesión Celular/deficiencia , Moléculas de Adhesión Celular/genética , Moléculas de Adhesión Celular/metabolismo , Modelos Animales de Enfermedad , Microbioma Gastrointestinal/genética , Humanos , Integrinas/antagonistas & inhibidores , Integrinas/inmunología , Hígado/patología , Masculino , Ratones , Ratones Noqueados , Mucoproteínas/antagonistas & inhibidores , Mucoproteínas/metabolismo , Enfermedad del Hígado Graso no Alcohólico/tratamiento farmacológico , Enfermedad del Hígado Graso no Alcohólico/patología , ARN Ribosómico 16S/genética , Receptores de Superficie Celular/deficiencia , Receptores de Superficie Celular/genéticaAsunto(s)
Ensayos Clínicos como Asunto/normas , Cirrosis Hepática/terapia , Enfermedad del Hígado Graso no Alcohólico/terapia , Selección de Paciente , Guías de Práctica Clínica como Asunto , Consenso , Drogas en Investigación/uso terapéutico , Estilo de Vida Saludable , Humanos , Hígado/patología , Cirrosis Hepática/etiología , Cirrosis Hepática/patología , Enfermedad del Hígado Graso no Alcohólico/complicaciones , Enfermedad del Hígado Graso no Alcohólico/patología , Participación de los InteresadosRESUMEN
There is compelling evidence implicating intestinal permeability in the pathogenesis of nonalcoholic steatohepatitis (NASH), but the underlying mechanisms remain poorly understood. Here we examined the role of bile acids (BA) in western diet (WD)-induced loss of colonic epithelial barrier (CEB) function in mice with a genetic impairment in intestinal epithelial barrier function, junctional adhesion molecule A knockout mice, F11r-/- . WD-fed knockout mice developed severe NASH, which was associated with increased BA concentration in the cecum and loss of CEB function. Analysis of cecal BA composition revealed selective increases in primary unconjugated BAs in the WD-fed mice, which correlated with increased abundance of microbial taxa linked to BA metabolism. In vitro permeability assays revealed that chenodeoxycholic acid (CDCA), which was elevated in the cecum of WD-fed mice, increased paracellular permeability, while the BA-binding resin sevelamer hydrochloride protected against CDCA-induced loss of barrier function. Sequestration of intestinal BAs by in vivo delivery of sevelamer to WD-fed knockout mice attenuated colonic mucosal inflammation and improved CEB. Sevelamer also reduced hepatic inflammation and fibrosis, and improved metabolic derangements associated with NASH. Collectively, these findings highlight a hitherto unappreciated role for BAs in WD-induced impairment of the intestinal epithelial barrier in NASH.
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Ácidos y Sales Biliares/metabolismo , Colon/metabolismo , Dieta Occidental/efectos adversos , Enfermedad del Hígado Graso no Alcohólico/metabolismo , Animales , Células CACO-2 , Moléculas de Adhesión Celular/deficiencia , Moléculas de Adhesión Celular/genética , Colon/patología , Modelos Animales de Enfermedad , Humanos , Mucosa Intestinal/efectos de los fármacos , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patología , Hígado/efectos de los fármacos , Hígado/metabolismo , Hígado/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Enfermedad del Hígado Graso no Alcohólico/etiología , Enfermedad del Hígado Graso no Alcohólico/patología , Permeabilidad , Receptores de Superficie Celular/deficiencia , Receptores de Superficie Celular/genética , Sevelamer/administración & dosificaciónRESUMEN
BACKGROUND: Improved knowledge of the molecular pathophysiology and immunopathogenesis of cholestatic liver diseases in recent years has led to an increased interest in developing novel therapies. Patients with cholestatic liver disease often require different approaches to assessment and management of suspected drug-induced liver injury (DILI) compared to those with healthy livers and those with parenchymal liver diseases. At present, there are no regulatory guidelines or society position papers, that systematically address best practices pertaining to detection of DILI in these patients. AIMS: To outline best practices for detection, assessment and management of suspected acute DILI during clinical trials in adults with the cholestatic liver diseases - Primary Biliary Cholangitis (PBC) and Primary Sclerosing Cholangitis (PSC). METHODS: This is one of the several papers developed by the IQ DILI Initiative, which is comprised of members from 16 pharmaceutical companies, in collaboration with DILI experts from academia and regulatory agencies. The contents are the result of an extensive literature review, as well as in-depth discussions among industry, regulatory and academic DILI experts, to achieve consensus recommendations on DILI-related issues occurring during clinical trials for cholestatic liver diseases. RESULTS: Recommended best practices are outlined pertaining to hepatic eligibility criteria, monitoring of liver tests, approach to a suspected DILI signal, and hepatic discontinuation rules. CONCLUSIONS: This paper provides a framework for the approach to detection, assessment and management of suspected acute DILI occurring during clinical trials in adults with cholestatic liver disease.
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Enfermedad Hepática Inducida por Sustancias y Drogas/diagnóstico , Enfermedad Hepática Inducida por Sustancias y Drogas/terapia , Colestasis/tratamiento farmacológico , Ensayos Clínicos como Asunto , Consenso , Cirrosis Hepática Biliar/tratamiento farmacológico , Adulto , Enfermedad Hepática Inducida por Sustancias y Drogas/patología , Colestasis/patología , Enfermedad Crónica , Ensayos Clínicos como Asunto/estadística & datos numéricos , Industria Farmacéutica/organización & administración , Industria Farmacéutica/normas , Humanos , Hígado/efectos de los fármacos , Hígado/patología , Hígado/fisiopatología , Cirrosis Hepática Biliar/patología , Pruebas de Función Hepática , Sociedades Farmacéuticas/normasRESUMEN
Defining the immune physiology of culture-adapted mesenchymal stromal cells (MSCs) derived from distinct tissue compartments informs their potential utility as pharmaceuticals. Here, we have investigated the comparative immune plasticity of MSCs and hepatic stellate cells (HeSCs) isolated from human and murine bone marrow (BM) and liver, respectively. Although both BM-MSCs and HeSCs share mesenchymal phenotype and overall molecular genetic responses to inflammatory cues, HeSCs differ from BM-MSCs in a meaningful manner. We show that culture-adapted HeSCs express substantially higher levels of hepatocyte growth factor (HGF), matrix metalloproteinase-1, and chemokine (CC motif) ligand 2 (CCL2) than BM-MSCs. Both human BM-MSCs and HeSCs inhibit T-cell proliferation by a shared indoleamine 2,3-dioxygenase (IDO)-dependent mechanism. However, HeSCs are distinct from BM-MSCs by their significant differential expression of HGF, CCL2, IL-8, CCL11, and GMCSF when cocultured with and/or without activated peripheral blood mononuclear cells. We have investigated MSCs and HeSCs derived from murine systems to describe interspecies comparability. Murine BM-MSCs inhibit T-cell proliferation through inducible nitric oxide synthase (iNOS) but not IDO. However, murine HeSCs inhibit T-cell proliferation through a mechanism distinct from either IDO or iNOS. Altogether, these results suggest that although culture-adapted BM-MSCs and HeSCs display a similar phenotype, their secretome and immune plasticity are in part distinct likely mirroring their tissular origins. In addition, the discordance in immune biology between mouse and human sourced HeSC and BM-MSCs speaks to the importance of comparative biology when interrogating rodent systems for human translational insights. Stem Cells 2019;37:1075-1082.
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Antígenos de Diferenciación/inmunología , Células de la Médula Ósea/inmunología , Regulación de la Expresión Génica/inmunología , Células Estrelladas Hepáticas/inmunología , Células Madre Mesenquimatosas/inmunología , Animales , Células de la Médula Ósea/citología , Línea Celular , Células Estrelladas Hepáticas/citología , Humanos , Células Madre Mesenquimatosas/citología , Ratones , Especificidad de la EspecieRESUMEN
BACKGROUND: The last decade has seen a rapid growth in the number of clinical trials enrolling patients with nonalcoholic fatty liver disease and nonalcoholic steatohepatitis (NASH). Due to the underlying chronic liver disease, patients with NASH often require different approaches to the assessment and management of suspected drug-induced liver injury (DILI) compared to patients with healthy livers. However, currently no regulatory guidelines or position papers systematically address best practices pertaining to DILI in NASH clinical trials. AIMS: This publication focuses on best practices concerning the detection, monitoring, diagnosis and management of suspected acute DILI during clinical trials in patients with NASH. METHODS: This is one of several papers developed by the IQ DILI Initiative, comprised of members from 15 pharmaceutical companies, in collaboration with DILI experts from academia and regulatory agencies. This paper is based on extensive literature review, and discussions between industry members with expertise in drug safety and DILI experts from outside industry to achieve consensus on common questions related to this topic. RESULTS: Recommended best practices are outlined pertaining to hepatic inclusion and exclusion criteria, monitoring of liver tests, DILI detection, approach to a suspected DILI signal, causality assessment and hepatic discontinuation rules. CONCLUSIONS: This paper provides a framework for the approach to assessment and management of suspected acute DILI during clinical trials in patients with NASH.
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Enfermedad Hepática Inducida por Sustancias y Drogas/terapia , Ensayos Clínicos como Asunto/normas , Manejo de la Enfermedad , Enfermedad del Hígado Graso no Alcohólico/terapia , Guías de Práctica Clínica como Asunto/normas , Enfermedad Hepática Inducida por Sustancias y Drogas/diagnóstico , Enfermedad Hepática Inducida por Sustancias y Drogas/epidemiología , Ensayos Clínicos como Asunto/métodos , Humanos , Pruebas de Función Hepática/métodos , Pruebas de Función Hepática/normas , Enfermedad del Hígado Graso no Alcohólico/diagnóstico , Enfermedad del Hígado Graso no Alcohólico/epidemiologíaRESUMEN
Adiponectin inhibits hepatic stellate cell (HSC) activation and subsequent development of liver fibrosis via multiple mechanisms. Phosphatase and tensin homolog deletion 10 (PTEN) plays a crucial role in suppression of HSC activation, but its regulation by adiponectin is not fully understood. Here, we investigated the effect of adiponectin on PTEN in LX-2 cells, a human cell line and examined the underlying molecular mechanisms involved in adiponectin-mediated upregulation of PTEN activity during fibrosis. PTEN expression was found to be significantly reduced in the livers of mice treated with CCl4, whereas its expression was rescued by adiponectin treatment. The DNA methylation proteins DNMT1, DNMT3A, and DNMT3B are all highly expressed in activated primary HSCs compared to quiescent HSCs, and thus represent additional regulatory targets during liver fibrogenesis. Expression of DNMT proteins was significantly induced in the presence of fibrotic stimuli; however, only DNMT3B expression was reduced in the presence of adiponectin. Adiponectin-induced suppression of DNMT3B was found to be mediated by enhanced miR-29b expression. Furthermore, PTEN expression was significantly increased by overexpression of miR-29b, whereas its expression was markedly reduced by a miR-29b inhibitor in LX-2 cells. These findings suggest that adiponectin-induced upregulation of miR-29b can suppress DNMT3B transcription in LX-2 cells, thus resulting in reduced methylation of PTEN CpG islands and ultimately suppressing the PI3K/AKT pathway. Together, these data suggest a possible new explanation for the inhibitory effect of adiponectin on HSC activation and liver fibrogenesis.
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Adiponectina/metabolismo , Tetracloruro de Carbono/efectos adversos , ADN (Citosina-5-)-Metiltransferasas/genética , Células Estrelladas Hepáticas/citología , Cirrosis Hepática/metabolismo , MicroARNs/genética , Fosfohidrolasa PTEN/genética , Animales , Línea Celular , Proliferación Celular , ADN (Citosina-5-)-Metiltransferasa 1/genética , ADN (Citosina-5-)-Metiltransferasa 1/metabolismo , ADN (Citosina-5-)-Metiltransferasas/metabolismo , Metilación de ADN , ADN Metiltransferasa 3A , Epigénesis Genética , Células Estrelladas Hepáticas/efectos de los fármacos , Células Estrelladas Hepáticas/metabolismo , Humanos , Cirrosis Hepática/inducido químicamente , Cirrosis Hepática/genética , Ratones , Fosfohidrolasa PTEN/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Ratas , Transducción de Señal , Regulación hacia Arriba , ADN Metiltransferasa 3BRESUMEN
Liver fibrosis arises from dysregulated wound healing due to persistent inflammatory hepatic injury. Periostin is a nonstructural extracellular matrix protein that promotes organ fibrosis in adults. Here, we sought to identify the molecular mechanisms in periostin-mediated hepatic fibrosis. Hepatic fibrosis in periostin-/- mice was attenuated as evidenced by significantly reduced collagen fibril density and liver stiffness compared with those in WT controls. A single dose of carbon tetrachloride caused similar acute liver injury in periostin-/- and WT littermates, and we did not detect significant differences in transaminases and major fibrosis-related hepatic gene expression between these two genotypes. Activated hepatic stellate cells (HSCs) are the major periostin-producing liver cell type. We found that in primary rat HSCs in vitro, periostin significantly increases the expression levels and activities of lysyl oxidase (LOX) and lysyl oxidase-like (LOXL) isoforms 1-3. Periostin also induced expression of intra- and extracellular collagen type 1 and fibronectin in HSCs. Interestingly, periostin stimulated phosphorylation of SMAD2/3, which was sustained despite short hairpin RNA-mediated knockdown of transforming growth factor ß (TGFß) receptor I and II, indicating that periostin-mediated SMAD2/3 phosphorylation is independent of TGFß receptors. Moreover, periostin induced the phosphorylation of focal adhesion kinase (FAK) and AKT in HSCs. Notably, siRNA-mediated FAK knockdown failed to block periostin-induced SMAD2/3 phosphorylation. These results suggest that periostin promotes enhanced matrix stiffness in chronic liver disease by activating LOX and LOXL, independently of TGFß receptors. Hence, targeting periostin may be of therapeutic benefit in combating hepatic fibrosis.
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Moléculas de Adhesión Celular/fisiología , Enfermedad Hepática Inducida por Sustancias y Drogas/patología , Células Estrelladas Hepáticas/patología , Cirrosis Hepática/patología , Proteína-Lisina 6-Oxidasa/metabolismo , Animales , Tetracloruro de Carbono/toxicidad , Células Cultivadas , Enfermedad Hepática Inducida por Sustancias y Drogas/enzimología , Células Estrelladas Hepáticas/enzimología , Cirrosis Hepática/enzimología , Masculino , Ratones , Ratones Endogámicos C57BL , Fosforilación , Ratas , Ratas Sprague-Dawley , Transducción de SeñalRESUMEN
Alcohol consumption promotes loss of intestinal barrier function. However, mechanisms by which ethanol affects the tight junction (TJ), the cellular structure responsible for maintaining the gut epithelial barrier, are not well understood. Three classes of transmembrane proteins comprise TJs: occludin, claudins, and junctional adhesion molecules (JAMs). It has recently been postulated that JAM-A (F11R), the most abundant JAM expressed in intestinal epithelium, regulates "leak" pathway flux, a paracellular route for the nonselective permeation of large solutes. Since transluminal flux of many gut-derived antigens occurs through this pathway, we investigated the role of JAM-A in ethanol-induced disruption of the intestinal epithelial barrier. Using Caco-2 and SK-CO15 monolayers, we found that ethanol induced a dose- and time-dependent decrease in JAM-A protein expression to about 70% of baseline levels. Alcohol also reduced Ras-related protein 2 (Rap2) activity, and enhanced myosin light chain kinase (MLCK) activity, changes consistent with impaired JAM-A signaling. Stable overexpression and shRNA-mediated knockdown of JAM-A were employed to investigate the role of JAM-A in paracellular-mediated flux following alcohol exposure. The paracellular flux of 40-kDa fluorescein isothiocynate (FITC)-dextran following ethanol treatment was decreased by the overexpression of JAM-A; conversely, flux was enhanced by JAM-A knockdown. Thus, we conclude that ethanol-mediated control of JAM-A expression and function contributes to mechanisms by which this chemical induces intestinal epithelial leakiness.
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Moléculas de Adhesión Celular/metabolismo , Etanol/toxicidad , Absorción Intestinal/efectos de los fármacos , Mucosa Intestinal/metabolismo , Receptores de Superficie Celular/metabolismo , Animales , Células CACO-2 , Moléculas de Adhesión Celular/genética , Células HEK293 , Humanos , Mucosa Intestinal/efectos de los fármacos , Masculino , Ratones , Ratones Endogámicos C57BL , Cadenas Ligeras de Miosina/metabolismo , Receptores de Superficie Celular/genética , Proteínas de Unión al GTP rap/metabolismoRESUMEN
Persistent hepatotropic viral infections are a common etiologic agent of chronic liver disease. Unresolved infection can be attributed to nonfunctional intrahepatic CD8+ T-cell responses. In light of dampened CD8+ T-cell responses, liver disease often manifests systemically as immunoglobulin (Ig)-related syndromes due to aberrant B-cell functions. These two opposing yet coexisting phenomena implicate the potential of altered CD4+ T-cell help. Elevated CD4+ forkhead box P3-positive (Foxp3+) T cells were evident in both human liver disease and a mouse model of chemically induced liver injury despite marked activation and spontaneous IgG production by intrahepatic B cells. While this population suppressed CD8+ T-cell responses, aberrant B-cell activities were maintained due to expression of CD40 ligand on a subset of CD4+ Foxp3+ T cells. In vivo blockade of CD40 ligand attenuated B-cell abnormalities in a mouse model of liver injury. A phenotypically similar population of CD4+ Foxp3+, CD40 ligand-positive T cells was found in diseased livers explanted from patients with chronic hepatitis C infection. This population was absent in nondiseased liver tissues and peripheral blood. CONCLUSION: Liver disease elicits alterations in the intrahepatic CD4+ T-cell compartment that suppress T-cell immunity while concomitantly promoting aberrant IgG mediated manifestations. (Hepatology 2017;65:661-677).
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Linfocitos T CD8-positivos/inmunología , Factores de Transcripción Forkhead/metabolismo , Inmunoglobulina G/inmunología , Cirrosis Hepática/inmunología , Cirrosis Hepática/patología , Análisis de Varianza , Animales , Células Cultivadas , Enfermedad Crónica , Modelos Animales de Enfermedad , Ensayo de Inmunoadsorción Enzimática , Ensayo de Immunospot Ligado a Enzimas , Citometría de Flujo , Hepatitis C Crónica/inmunología , Hepatitis C Crónica/patología , Hepatocitos , Humanos , Inmunoglobulina G/metabolismo , Inmunohistoquímica , Masculino , Ratones , Ratones Endogámicos C57BL , Distribución Aleatoria , Estadísticas no Paramétricas , Linfocitos T Reguladores/inmunologíaAsunto(s)
Antivirales/uso terapéutico , Hepatitis B Crónica/tratamiento farmacológico , Hepatitis C Crónica/tratamiento farmacológico , Carga Viral , Activación Viral , Anciano , Quimioterapia Combinada , Guanina/análogos & derivados , Guanina/uso terapéutico , Humanos , Masculino , Persona de Mediana Edad , Simeprevir/uso terapéutico , Sofosbuvir/uso terapéutico , Tenofovir/uso terapéuticoRESUMEN
BACKGROUND & AIMS: There is evidence from clinical studies that compromised intestinal epithelial permeability contributes to the development of nonalcoholic steatohepatitis (NASH), but the exact mechanisms are not clear. Mice with disruption of the gene (F11r) encoding junctional adhesion molecule A (JAM-A) have defects in intestinal epithelial permeability. We used these mice to study how disruption of the intestinal epithelial barrier contributes to NASH. METHODS: Male C57BL/6 (control) or F11r(-/-) mice were fed a normal diet or a diet high in saturated fat, fructose, and cholesterol (HFCD) for 8 weeks. Liver and intestinal tissues were collected and analyzed by histology, quantitative reverse-transcription polymerase chain reaction, and flow cytometry. Intestinal epithelial permeability was assessed in mice by measuring permeability to fluorescently labeled dextran. The intestinal microbiota were analyzed using 16S ribosomal RNA sequencing. We also analyzed biopsy specimens from proximal colons of 30 patients with nonalcoholic fatty liver disease (NAFLD) and 19 subjects without NAFLD (controls) undergoing surveillance colonoscopy. RESULTS: F11r(-/-) mice fed a HFCD, but not a normal diet, developed histologic and pathologic features of severe NASH including steatosis, lobular inflammation, hepatocellular ballooning, and fibrosis, whereas control mice fed a HFCD developed only modest steatosis. Interestingly, there were no differences in body weight, ratio of liver weight:body weight, or glucose homeostasis between control and F11r(-/-) mice fed a HFCD. In these mice, liver injury was associated with significant increases in mucosal inflammation, tight junction disruption, and intestinal epithelial permeability to bacterial endotoxins, compared with control mice or F11r(-/-) mice fed a normal diet. The HFCD led to a significant increase in inflammatory microbial taxa in F11r(-/-) mice, compared with control mice. Administration of oral antibiotics or sequestration of bacterial endotoxins with sevelamer hydrochloride reduced mucosal inflammation and restored normal liver histology in F11r(-/-) mice fed a HFCD. Protein and transcript levels of JAM-A were significantly lower in the intestinal mucosa of patients with NAFLD than without NAFLD; decreased expression of JAM-A correlated with increased mucosal inflammation. CONCLUSIONS: Mice with defects in intestinal epithelial permeability develop more severe steatohepatitis after a HFCD than control mice, and colon tissues from patients with NAFLD have lower levels of JAM-A and higher levels of inflammation than subjects without NAFLD. These findings indicate that intestinal epithelial barrier function and microbial dysbiosis contribute to the development of NASH. Restoration of intestinal barrier integrity and manipulation of gut microbiota might be developed as therapeutic strategies for patients with NASH.
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Moléculas de Adhesión Celular/deficiencia , Dieta Alta en Grasa/efectos adversos , Enfermedad del Hígado Graso no Alcohólico/genética , Receptores de Superficie Celular/deficiencia , Animales , Colesterol , Dieta Alta en Grasa/métodos , Carbohidratos de la Dieta , Modelos Animales de Enfermedad , Disbiosis/complicaciones , Disbiosis/genética , Fructosa , Microbioma Gastrointestinal/genética , Mucosa Intestinal/metabolismo , Mucosa Intestinal/microbiología , Hígado/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Enfermedad del Hígado Graso no Alcohólico/microbiología , Enfermedad del Hígado Graso no Alcohólico/patología , Permeabilidad , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
The CCAAT/enhancer-binding protein (C/EBP) homologous protein (CHOP), a major transcriptional regulator of endoplasmic reticulum (ER) stress-mediated apoptosis, is implicated in lipotoxicity-induced ER stress and hepatocyte apoptosis in non-alcoholic fatty liver disease (NAFLD). We have previously demonstrated that the glucagon-like peptide-1 (GLP-1) agonist, liraglutide, protects steatotic hepatocytes from lipotoxicity-induced apoptosis by improved handling of free fatty acid (FFA)-induced ER stress. In the present study, we investigated whether CHOP is critical for GLP-1-mediated restoration of ER homeostasis and mitigation of hepatocyte apoptosis in a murine model of NASH (non-alcoholic steatohepatitis). Our data show that despite similar caloric intake, CHOP KO (CHOP(-/-)) mice fed a diet high in fat, fructose, and cholesterol (HFCD) for 16 weeks developed more severe histological features of NASH compared with wild-type (WT) controls. Severity of NASH in HFCD-fed CHOP(-/-) mice correlated with significant decrease in peroxisomal ß-oxidation, and increased de novo lipogenesis and ER stress-mediated hepatocyte apoptosis. Four weeks of liraglutide treatment markedly attenuated steatohepatitis in HFCD-fed WT mice by improving insulin sensitivity, and suppressing de novo lipogenesis and ER stress-mediated hepatocyte apoptosis. However, in the absence of CHOP, liraglutide did not improve insulin sensitivity, nor suppress peroxisomal ß-oxidation or ER stress-mediated hepatocyte apoptosis. Taken together, these data indicate that CHOP protects hepatocytes from HFCD-induced ER stress, and has a significant role in the mechanism of liraglutide-mediated protection against NASH pathogenesis.
Asunto(s)
Liraglutida/farmacología , Enfermedad del Hígado Graso no Alcohólico/metabolismo , Enfermedad del Hígado Graso no Alcohólico/prevención & control , Factor de Transcripción CHOP/metabolismo , Animales , Apoptosis/efectos de los fármacos , Glucemia/metabolismo , Células Cultivadas , Colesterol/metabolismo , Dieta Alta en Grasa/efectos adversos , Carbohidratos de la Dieta/administración & dosificación , Carbohidratos de la Dieta/efectos adversos , Modelos Animales de Enfermedad , Estrés del Retículo Endoplásmico/efectos de los fármacos , Exenatida , Hepatocitos/efectos de los fármacos , Hepatocitos/metabolismo , Resistencia a la Insulina , Metabolismo de los Lípidos/efectos de los fármacos , Hígado/efectos de los fármacos , Hígado/metabolismo , Hígado/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Enfermedad del Hígado Graso no Alcohólico/patología , Péptidos/farmacología , Sustancias Protectoras/farmacología , Factor de Transcripción CHOP/deficiencia , Factor de Transcripción CHOP/genética , Ponzoñas/farmacologíaRESUMEN
Moderate macrovesicular steatosis (>30%), which is present in almost 50% of livers considered for transplantation, increases the risk of primary graft dysfunction. Our previously published data showed that glial cell line-derived neurotrophic factor (GDNF) is protective against high-fat diet (HFD)-induced hepatic steatosis in mice. Hence, we hypothesized that perfusion of steatotic livers with GDNF may reduce liver fat content before transplantation. Livers from 8 weeks of regular diet (RD) and of HFD-fed mice were perfused ex vivo for 4 hours with either vehicle, GDNF, or a previously described defatting cocktail. The liver's residual fat was quantified colorimetrically using a triglyceride (TG) assay kit and by Oil Red O (ORO) and Nile red/Hoechst staining. Liver tissue injury was assessed by using a lactate dehydrogenase (LDH) activity assay. In vitro induction of lipolysis in HepG2 cells was assessed by measuring glycerol and free fatty acid release. ORO staining showed significantly more steatosis in livers from HFD-fed mice compared with RD-fed mice (P < 0.001). HFD livers perfused with GDNF had significantly less steatosis than those not perfused (P = 0.001) or perfused with vehicle (P < 0.05). GDNF is equally effective in steatotic liver defatting compared to the defatting cocktail; however, GDNF induces less liver damage than the defatting cocktail. These observations were consistent with data obtained from assessment of liver TG content. Assessment of liver injury revealed significant hepatocyte injury in livers perfused with the control defatting cocktail but no evidence of injury in livers perfused with either GDNF or vehicle. In vitro, GDNF reduced TG accumulation in HepG2 cells and stimulated increased TG lipolysis. In conclusion, GDNF can decrease mice liver fat content to an acceptable range and could be a potential defatting agent before liver transplantation.
Asunto(s)
Hígado Graso/terapia , Factor Neurotrófico Derivado de la Línea Celular Glial/uso terapéutico , Trasplante de Hígado/métodos , Disfunción Primaria del Injerto/prevención & control , Triglicéridos/metabolismo , Animales , Enfermedad Hepática Inducida por Sustancias y Drogas/etiología , Enfermedad Hepática Inducida por Sustancias y Drogas/metabolismo , Colorimetría , Dieta Alta en Grasa/efectos adversos , Modelos Animales de Enfermedad , Hígado Graso/etiología , Factor Neurotrófico Derivado de la Línea Celular Glial/efectos adversos , Supervivencia de Injerto/efectos de los fármacos , Células Hep G2 , Hepatocitos/metabolismo , Humanos , Lipólisis/efectos de los fármacos , Masculino , Ratones , Ratones Endogámicos C57BL , Perfusión , Ratas , Triglicéridos/análisisRESUMEN
Glial cell line-derived neurotrophic factor (GDNF) protects against high-fat diet (HFD)-induced hepatic steatosis in mice, however, the mechanisms involved are not known. In this study we investigated the effects of GDNF overexpression and nanoparticle delivery of GDNF in mice on hepatic steatosis and fibrosis and the expression of genes involved in the regulation of hepatic lipid uptake and de novo lipogenesis. Transgenic overexpression of GDNF in liver and other metabolically active tissues was protective against HFD-induced hepatic steatosis. Mice overexpressing GDNF had significantly reduced P62/sequestosome 1 protein levels suggestive of accelerated autophagic clearance. They also had significantly reduced peroxisome proliferator-activated receptor-γ (PPAR-γ) and CD36 gene expression and protein levels, and lower expression of mRNA coding for enzymes involved in de novo lipogenesis. GDNF-loaded nanoparticles were protective against short-term HFD-induced hepatic steatosis and attenuated liver fibrosis in mice with long-standing HFD-induced hepatic steatosis. They also suppressed the liver expression of steatosis-associated genes. In vitro, GDNF suppressed triglyceride accumulation in Hep G2 cells through enhanced p38 mitogen-activated protein kinase-dependent signaling and inhibition of PPAR-γ gene promoter activity. These results show that GDNF acts directly in the liver to protect against HFD-induced cellular stress and that GDNF may have a role in the treatment of nonalcoholic fatty liver disease.
Asunto(s)
Dieta Alta en Grasa , Hígado Graso/metabolismo , Hígado Graso/prevención & control , Factor Neurotrófico Derivado de la Línea Celular Glial/metabolismo , Hígado/metabolismo , PPAR gamma/metabolismo , Animales , Antígenos CD36/genética , Antígenos CD36/metabolismo , Hígado Graso/etiología , Hígado Graso/patología , Factor Neurotrófico Derivado de la Línea Celular Glial/administración & dosificación , Factor Neurotrófico Derivado de la Línea Celular Glial/genética , Factor Neurotrófico Derivado de la Línea Celular Glial/uso terapéutico , Células Hep G2 , Humanos , Hígado/patología , Ratones , Ratones Transgénicos , Nanopartículas/administración & dosificación , Nanopartículas/uso terapéutico , PPAR gamma/genética , Transducción de Señal/fisiología , Triglicéridos/metabolismoRESUMEN
Cadmium (Cd) is present in food at low levels and accumulates in humans throughout life because it is not effectively excreted. Cd from smoking or occupational exposure shows adverse effects on health, but the mechanistic effect of Cd at low dietary intake levels is poorly studied. Epidemiology studies found that nonalcoholic fatty liver disease (NAFLD), common in U.S. adults, is associated with Cd burden. In cell studies, we found that environmental low-dose Cd oxidized proteins and stimulated inflammatory signaling. However, little is known about low-dose Cd effects on liver function and associated metabolic pathways in vivo. We investigated effects of low-level Cd exposure on liver gene transcripts, metabolites, and associated metabolic pathways and function after challenging mice with Cd (10 mg/l) by drinking water. Results showed liver Cd in treated mice was similar to adult humans without occupational or smoking exposures and 10-fold higher than control mouse values. Pathway analysis of significantly altered liver genes and metabolites mapped to functional pathways of lipid metabolism, cell death and mitochondrial oxidative phosphorylation. These are well-recognized pathways associated with NAFLD. Cd-treated mice had higher liver enzymes in plasma and a trend toward fat accumulation in liver. To verify low-dose Cd-induced stimulation of cell death pathways, phosphorylation of c-Jun N-terminal kinase (JNK) was examined in cultured hepatic cells. Consistent with mouse liver data, low-dose Cd stimulated JNK activation. Together, the results show that low-dose Cd exposure causes liver function changes consistent with a role in NAFLD and possibly also nonalcoholic steatohepatitis.