RESUMO
Cholestatic injuries are accompanied by ductular reaction, initiated by proliferation and activation of biliary epithelial cells (BECs), leading to fibrosis. Sortilin (encoded by Sort1) facilitates IL-6 secretion and leukemia inhibitory factor (LIF) signaling. This study investigated the interplay between sortilin and IL-6 and LIF in cholestatic injury-induced ductular reaction, morphogenesis of new ducts, and fibrosis. Cholestatic injury was induced by bile duct ligation (BDL) in wild-type and Sort1-/- mice, with or without augmentation of IL-6 or LIF. Mice with BEC sortilin deficiency (hGFAPcre.Sort1fl/fl) and control mice were subjected to BDL and 3,5-diethoxycarbonyl-1,4-dihydrocollidine diet (DDC) induced cholestatic injury. Sort1-/- mice displayed reduced BEC proliferation and expression of BEC-reactive markers. Administration of LIF or IL-6 restored BEC proliferation in Sort1-/- mice, without affecting BEC-reactive or inflammatory markers. Sort1-/- mice also displayed impaired morphogenesis, which was corrected by LIF treatment. Similarly, hGFAPcre.Sort1fl/fl mice exhibited reduced BEC proliferation, but similar reactive and inflammatory marker expression. Serum IL-6 and LIF were comparable, yet liver pSTAT3 was reduced, indicating that sortilin is essential for co-activation of LIF receptor/gp130 signaling in BECs, but not for IL-6 secretion. hGFAPcre.Sortfl/fl mice displayed impaired morphogenesis and diminished fibrosis after BDL and DDC. In conclusion, sortilin-mediated engagement of LIF signaling in BECs promoted ductular reaction and morphogenesis during cholestatic injury. This study indicates that BEC sortilin is pivotal for the development of fibrosis.
Assuntos
Proteínas Adaptadoras de Transporte Vesicular , Ductos Biliares , Colestase , Células Epiteliais , Fibrose , Animais , Camundongos , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Ductos Biliares/patologia , Proliferação de Células , Colestase/patologia , Colestase/metabolismo , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Interleucina-6/metabolismo , Fator Inibidor de Leucemia/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Transdução de SinaisRESUMO
Drug-induced liver injury is an emerging form of acute and chronic liver disease that may manifest as fatty liver. Amiodarone (AMD), a widely used antiarrhythmic drug, can cause hepatic injury and steatosis by a variety of mechanisms, not all completely understood. We hypothesized that repetitive AMD administration may induce hepatic lipotoxicity not only via effects on the liver but also via effects on adipose tissue. Indeed, repetitive AMD administration induced endoplasmic reticulum (ER) stress in both liver and adipose tissue. In adipose tissue, AMD reduced lipogenesis and increased lipolysis. Moreover, AMD treatment induced ER stress and ER stress-dependent lipolysis in 3T3L1 adipocytes in vitro. In the liver, AMD caused increased expression of genes encoding proteins involved in fatty acid (FA) uptake and transfer (Cd36, Fabp1, and Fabp4), and resulted in increased hepatic accumulation of free FAs, but not of triacylglycerols. In line with this, there was increased expression of hepatic de novo FA synthesis genes. However, AMD significantly reduced the expression of the desaturase Scd1 and elongase Elovl6, detected at mRNA and protein levels. Accordingly, the FA profile of hepatic total lipids revealed increased accumulation of palmitate, an SCD1 and ELOVL6 substrate, and reduced levels of palmitoleate and cis-vaccenate, products of the enzymes. In addition, AMD-treated mice displayed increased hepatic apoptosis. The studies show that repetitive AMD induces ER stress and aggravates lipolysis in adipose tissue while inducing a lipotoxic hepatic lipid environment, suggesting that AMD-induced liver damage is due to compound insult to liver and adipose tissue.NEW & NOTEWORTHY AMD chronic administration induces hepatic lipid accumulation by several mechanisms, including induction of hepatic ER stress, impairment of ß-oxidation, and inhibition of triacylglycerol secretion. Our study shows that repetitive AMD treatment induces not only hepatic ER stress but also adipose tissue ER stress and lipolysis and hepatic accumulation of free fatty acids and enrichment of palmitate in the total lipids. Understanding the toxicity mechanisms of AMD would help devise ways to limit liver damage.
Assuntos
Amiodarona/farmacologia , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Ácidos Graxos não Esterificados/metabolismo , Ácidos Graxos/metabolismo , Lipólise/efeitos dos fármacos , Adipócitos/metabolismo , Tecido Adiposo/efeitos dos fármacos , Tecido Adiposo/metabolismo , Amiodarona/metabolismo , Animais , Proteínas de Ligação a Ácido Graxo/metabolismo , Fígado Gorduroso/metabolismo , Metabolismo dos Lipídeos/efeitos dos fármacos , Lipogênese/fisiologia , Fígado/efeitos dos fármacos , Fígado/metabolismo , Hepatopatias/metabolismo , Camundongos , Triglicerídeos/metabolismoRESUMO
Sortilin, a member of the vacuolar protein sorting 10 domain receptor family, traffics newly synthesized proteins from the trans-Golgi network to secretory pathways, endosomes, and cell surface. Sortilin-trafficked molecules, including IL-6 and acid sphingomyelinase (aSMase), mediate cholangiocyte proliferation and liver inflammation, hepatic stellate cell activation, hepatocyte apoptosis, and fibrosis. Based on these sortilin-regulated functions, we investigated its role in biliary damage leading to hepatocellular injury and fibrosis. Sortilin-/- mice displayed impaired inflammation and ductular reaction 3 days after bile duct ligation (BDL), as demonstrated by reduced cholangiocyte proliferation and activation and reduced serum IL-6. Interestingly, liver fibrosis was reduced in Sortilin-/- mice after both BDL and carbon tetrachloride treatment, in line with attenuated in vitro activation of Sortilin-/- hepatic stellate cells. Sortilin-/- hepatic aSMase activity was reduced in the BDL and carbon tetrachloride models and accompanied by reduced in vivo hepatocyte apoptosis. In addition, wild type (WT), but not Sortilin-/- hepatocytes, had increased aSMase-dependent susceptibility to bile acid-induced apoptosis in vitro. Mechanistically, short-term IL-6 neutralization in bile duct-ligated WT mice decreased hepatic inflammation and reactive cholangiocyte-derived cytokines and chemokines, without affecting fibrosis, whereas pharmacological inhibition of aSMase activity was not sufficient to attenuate hepatic fibrosis. Only combined IL-6 and aSMase inhibition significantly reduced fibrosis in bile duct-ligated WT mice. We conclude that sortilin regulates cholestatic liver damage and fibrosis via effects on both aSMase activity and serum IL-6.
Assuntos
Proteínas Adaptadoras de Transporte Vesicular/deficiência , Apoptose , Ductos Biliares/patologia , Colestase/complicações , Hepatócitos/patologia , Cirrose Hepática/patologia , Fígado/lesões , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Animais , Proliferação de Células , Quimiocinas/metabolismo , Colestase/patologia , Células Estreladas do Fígado/metabolismo , Células Estreladas do Fígado/patologia , Hepatócitos/metabolismo , Inflamação/patologia , Interleucina-6/metabolismo , Ligadura , Fígado/metabolismo , Fígado/patologia , Cirrose Hepática/complicações , Cirrose Hepática/metabolismo , Camundongos Endogâmicos C57BL , Testes de Neutralização , Fenótipo , Esfingomielina Fosfodiesterase/metabolismoRESUMO
BACKGROUND & AIMS: Sortilin traffics newly synthesized molecules from the trans-Golgi apparatus along secretory pathways to endosomes, lysosomes or to the cell surface. Sortilin trafficking of acid sphingomyelinase (aSMase) may regulate ceramide levels, a major modulator of insulin signalling. We therefore tested whether sortilin deficiency reduces hepatic and adipose tissue aSMase activity, improving insulin sensitivity in diet-induced obesity (DIO). METHODS: DIO in C57BL/6 (WT) and sortilin(-/-) mice was induced by high-fat diet feeding for 10 weeks. RESULTS: Sortilin(-/-) mice gained less body weight and less visceral fat, despite similar food intake compared to WT type mice and had enhanced glucose uptake in insulin tolerance tests, which was further corroborated by enhanced hepatic pAkt expression. Sortilin deficiency led to attenuated hepatic steatosis, reduced expression of genes involved in lipogenesis, ceramide synthesis and inflammatory cytokine production and reduced activity of ceramide synthase 5/6 (CerS5/6). Sortilin(-/-) mice had reduced hepatic aSMase activity under both steady-state and DIO. Likewise, sortilin(-/-) hepatocytes displayed hypersensitivity to insulin, due to enhanced insulin receptor downstream signalling. In adipose tissue, sortilin(-/-) mice exhibited lower expression of inflammatory cytokines and lower expression and activity of CerS5/6. As in liver, adipose tissue displayed increased insulin signalling, accompanied by attenuated aSMase activity. CONCLUSIONS: Sortilin deficiency induces a beneficial metabolic phenotype in liver and adipose tissue upon DIO, mediated in part by reduced aSMase activity.
Assuntos
Proteínas Adaptadoras de Transporte Vesicular/genética , Fígado Gorduroso/genética , Hepatócitos/metabolismo , Resistência à Insulina/fisiologia , Obesidade/complicações , RNA/genética , Proteínas Adaptadoras de Transporte Vesicular/biossíntese , Proteínas Adaptadoras de Transporte Vesicular/deficiência , Animais , Western Blotting , Modelos Animais de Doenças , Fígado Gorduroso/etiologia , Fígado Gorduroso/metabolismo , Regulação da Expressão Gênica , Hepatócitos/patologia , Metabolismo dos Lipídeos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Obesidade/genética , Obesidade/metabolismo , Reação em Cadeia da Polimerase em Tempo RealRESUMO
Severe neutropenia and protracted thrombocytopenia remain serious clinical problems following cord blood transplantation (CBT) due to the paucity of stem and progenitor cells in the grafts. Administration of ex-vivo expanded megakaryocyte progenitor cells may facilitate platelet production. We propose a novel strategy to expand these rare cells ex-vivo, from a small portion of the cord blood (CB) unit, using fibronectin (FN), a major component of hematopoietic niches, combined with cytokines, including thrombopoietin and the hematopoietic stress-associated acetylcholinesterase readthrough peptide (ARP). Application of multiple gates and high definition flow cytometry enabled clear resolution of expanded hematopoietic stem/precursor cells (HSPC) and megakaryocyte progenitors (Mk-p) and their early subsets while eliminating positively stained non-relevant cells. FN increased viability, expansion of all CD34(+) HSPC populations and Mk-p. The combination of FN + thrombopoietin + ARP maintained and expanded very early myeloid and thrombopoietic precursors, increased the proliferation of megakaryocyte, granulocyte-macrophage and multilineage colony-forming progenitors and supported Mk maturation as measured by ploidy and glycoprotein IIb/IIIa expression by quantiative reverse transcription polymerase chain reaction. This approach, which involves expanding HSPC and Mk precursors from a small portion of the CB unit, without sacrificing the coveted stem cells, may lead to improved cell therapy modalities to facilitate earlier myelopoiesis and platelet production post-CBT.
Assuntos
Sangue Fetal/citologia , Células-Tronco Hematopoéticas/citologia , Células Progenitoras de Megacariócitos/citologia , Acetilcolinesterase/farmacologia , Antígenos CD34/análise , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Ensaio de Unidades Formadoras de Colônias , Citocinas/farmacologia , Fibronectinas/farmacologia , Citometria de Fluxo/métodos , Células-Tronco Hematopoéticas/efeitos dos fármacos , Humanos , Células Progenitoras de Megacariócitos/efeitos dos fármacos , Fragmentos de Peptídeos/farmacologiaRESUMO
Amiodarone is a commonly used antiarrhythmic drug and can cause liver steatosis. We investigated the role of endoplasmic reticulum (ER) stress/unfolded protein response in the pathogenesis of amiodarone-induced steatosis. Amiodarone-induced liver injury was obtained by 1 intraperitoneal injection to wild-type (WT) or C/EBP homologous protein knock-out mice (Ddit3-/-). Amiodarone directly reduced intracellular ATP and Ca2+ in hepatocytes invitro, inducing ER stress and lipid accumulation. In vivo, amiodarone-driven liver damage and lipid accumulation was accompanied by activation of ER stress/unfolded protein response, as demonstrated by up-regulation of genes encoding key ER stress mediators and by phosphorylation of eIF2α. In contrast to WT mice, Ddit3-/- mice were protected from amiodarone-induced ER stress and lipid accumulation. Importantly, amiodarone-induced lipid accumulation was not mediated by de novo hepatic lipogenesis, increased adipose tissue lipolysis or increased hepatic uptake of triglycerides or free fatty acids. Rather, amiodarone strongly increased hepatic mRNA expression of lipid droplet proteins, particularly Cidea and Cidec, in WT, but less so in Ddit3-/- mice, suggesting a link between ER stress and increased triglyceride storage. Moreover, while insulin attenuated amiodarone-induced phosphorylation of hormone sensitive lipase (HSL) in WT, it did not affect pHSL in Ddit3-/-, indicating increased lipolysis and therefore reduced lipid accumulation in these mice. Finally, ER stress attenuation using 2 different pharmacological chaperones reduced lipid accumulation, accompanied by reduced mRNA expression of Cidec. In conclusion, amiodarone-induced ER stress drives liver steatosis and may be considered for therapeutic targeting.