RESUMEN
Hepatocellular carcinoma (HCC), the fourth leading cause of cancer mortality worldwide, develops almost exclusively in patients with chronic liver disease and advanced fibrosis1,2. Here we interrogated functions of hepatic stellate cells (HSCs), the main source of liver fibroblasts3, during hepatocarcinogenesis. Genetic depletion, activation or inhibition of HSCs in mouse models of HCC revealed their overall tumour-promoting role. HSCs were enriched in the preneoplastic environment, where they closely interacted with hepatocytes and modulated hepatocarcinogenesis by regulating hepatocyte proliferation and death. Analyses of mouse and human HSC subpopulations by single-cell RNA sequencing together with genetic ablation of subpopulation-enriched mediators revealed dual functions of HSCs in hepatocarcinogenesis. Hepatocyte growth factor, enriched in quiescent and cytokine-producing HSCs, protected against hepatocyte death and HCC development. By contrast, type I collagen, enriched in activated myofibroblastic HSCs, promoted proliferation and tumour development through increased stiffness and TAZ activation in pretumoural hepatocytes and through activation of discoidin domain receptor 1 in established tumours. An increased HSC imbalance between cytokine-producing HSCs and myofibroblastic HSCs during liver disease progression was associated with increased HCC risk in patients. In summary, the dynamic shift in HSC subpopulations and their mediators during chronic liver disease is associated with a switch from HCC protection to HCC promotion.
Asunto(s)
Carcinogénesis , Carcinoma Hepatocelular , Células Estrelladas Hepáticas , Neoplasias Hepáticas , Animales , Carcinogénesis/patología , Carcinoma Hepatocelular/patología , Proliferación Celular , Colágeno Tipo I/metabolismo , Receptor con Dominio Discoidina 1/metabolismo , Progresión de la Enfermedad , Células Estrelladas Hepáticas/metabolismo , Células Estrelladas Hepáticas/patología , Factor de Crecimiento de Hepatocito/metabolismo , Hepatocitos , Humanos , Cirrosis Hepática/complicaciones , Neoplasias Hepáticas/patología , Ratones , Miofibroblastos/patologíaRESUMEN
OBJECTIVE: Chemokines are known to play an important role in the pathophysiology of alcoholic hepatitis (AH), a form of acute-on-chronic liver injury frequently mediated by gut derived lipopolysaccharide (LPS). In our study, we hypothesise that chemokine CCL20, one of the most upregulated chemokines in patients with AH, is implicated in the pathogenesis of AH by mediating LPS induced liver injury. DESIGN: CCL20 gene expression and serum levels and their correlation with disease severity were assessed in patients with AH. Cellular sources of CCL20 and its biological effects were evaluated in vitro and in vivo in chronic, acute and acute-on-chronic experimental models of carbon tetrachloride and LPS induced liver injury. RNA interference technology was used to knockdown CCL20 in vivo. RESULTS: CCL20 hepatic and serum levels were increased in patients with AH and correlated with the degree of fibrosis, portal hypertension, endotoxaemia, disease severity scores and short term mortality. Moreover, CCL20 expression was increased in animal models of liver injury and particularly under acute-on-chronic conditions. Macrophages and hepatic stellate cells (HSCs) were identified as the main CCL20 producing cell types. Silencing CCL20 in vivo reduced LPS induced aspartate aminotransferase and lactate dehydrogenase serum levels and hepatic proinflammatory and profibrogenic genes. CCL20 induced proinflammatory and profibrogenic effects in cultured primary HSCs. CONCLUSIONS: Our results suggest that CCL20 upregulation is strongly associated with LPS and may not only represent a new potential biomarker to predict outcome in patients with AH but also an important mediator linking hepatic inflammation, injury and fibrosis in AH.
Asunto(s)
Insuficiencia Hepática Crónica Agudizada/fisiopatología , Enfermedad Hepática Inducida por Sustancias y Drogas/fisiopatología , Quimiocina CCL20/fisiología , Hepatitis Alcohólica/fisiopatología , Animales , Enfermedad Hepática Inducida por Sustancias y Drogas/etiología , Quimiocina CCL20/análisis , Quimiocina CCL20/sangre , Femenino , Humanos , Lipopolisacáridos/efectos adversos , Masculino , Ratones , Persona de Mediana Edad , ARN Interferente Pequeño , Regulación hacia Arriba/fisiologíaRESUMEN
Increased translocation of intestinal bacteria is a hallmark of chronic liver disease and contributes to hepatic inflammation and fibrosis. Here we tested the hypothesis that the intestinal microbiota and Toll-like receptors (TLRs) promote hepatocellular carcinoma (HCC), a long-term consequence of chronic liver injury, inflammation, and fibrosis. Hepatocarcinogenesis in chronically injured livers depended on the intestinal microbiota and TLR4 activation in non-bone-marrow-derived resident liver cells. TLR4 and the intestinal microbiota were not required for HCC initiation but for HCC promotion, mediating increased proliferation, expression of the hepatomitogen epiregulin, and prevention of apoptosis. Gut sterilization restricted to late stages of hepatocarcinogenesis reduced HCC, suggesting that the intestinal microbiota and TLR4 represent therapeutic targets for HCC prevention in advanced liver disease.
Asunto(s)
Intestinos/microbiología , Hepatopatías/microbiología , Neoplasias Hepáticas Experimentales/microbiología , Receptor Toll-Like 4/fisiología , Animales , Apoptosis/genética , Traslocación Bacteriana , Proliferación Celular , Factor de Crecimiento Epidérmico/metabolismo , Epirregulina , Humanos , Hepatopatías/complicaciones , Neoplasias Hepáticas Experimentales/genética , Neoplasias Hepáticas Experimentales/patología , Ratones , Ratones Endogámicos C57BL , Receptor Toll-Like 4/genética , Células Tumorales CultivadasRESUMEN
Adipose triglyceride lipase (ATGL) catalyzes the first step of triacylglycerol hydrolysis in adipocytes. Abhydrolase domain 5 (ABHD5) increases ATGL activity by an unknown mechanism. Prior studies have suggested that the expression of ABHD5 is limiting for lipolysis in adipocytes, as addition of recombinant ABHD5 increases in vitro TAG hydrolase activity of adipocyte lysates. To test this hypothesis in vivo, we generated transgenic mice that express 6-fold higher ABHD5 in adipose tissue relative to wild-type (WT) mice. In vivo lipolysis increased to a similar extent in ABHD5 transgenic and WT mice following an overnight fast or injection of either a ß-adrenergic receptor agonist or lipopolysaccharide. Similarly, basal and ß-adrenergic-stimulated lipolysis was comparable in adipocytes isolated from ABHD5 transgenic and WT mice. Although ABHD5 expression was elevated in thioglycolate-elicited macrophages from ABHD5 transgenic mice, Toll-like receptor 4 (TLR4) signaling was comparable in macrophages isolated from ABHD5 transgenic and WT mice. Overexpression of ABHD5 did not prevent the development of obesity in mice fed a high-fat diet, as shown by comparison of body weight, body fat percentage, and adipocyte hypertrophy of ABHD5 transgenic to WT mice. The expression of ABHD5 in mouse adipose tissue is not limiting for either basal or stimulated lipolysis.
Asunto(s)
1-Acilglicerol-3-Fosfato O-Aciltransferasa/genética , Tejido Adiposo/metabolismo , Dieta Alta en Grasa/efectos adversos , Lipólisis/genética , Obesidad/genética , Obesidad/prevención & control , 1-Acilglicerol-3-Fosfato O-Aciltransferasa/metabolismo , Animales , Femenino , Expresión Génica , Macrófagos/metabolismo , Ratones , Ratones Transgénicos , Obesidad/etiologíaRESUMEN
Mutations in human CGI-58/ABHD5 cause Chanarin-Dorfman syndrome (CDS), characterized by excessive storage of triacylglycerol in tissues. CGI-58 is an alpha/beta-hydrolase fold enzyme expressed in all vertebrates. The carboxyl terminus includes a highly conserved consensus sequence (HXXXXD) for acyltransferase activity. Mouse CGI-58 was expressed in Escherichia coli as a fusion protein with two amino terminal 6-histidine tags. Recombinant CGI-58 displayed acyl-CoA-dependent acyltransferase activity to lysophosphatidic acid, but not to other lysophospholipid or neutral glycerolipid acceptors. Production of phosphatidic acid increased with time and increasing concentrations of recombinant CGI-58 and was optimal between pH 7.0 and 8.5. The enzyme showed saturation kinetics with respect to 1-oleoyl-lysophosphatidic acid and oleoyl-CoA and preference for arachidonoyl-CoA and oleoyl-CoA. The enzyme showed slight preference for 1-oleoyl lysophosphatidic acid over 1-palmitoyl, 1-stearoyl, or 1-arachidonoyl lysophosphatidic acid. Recombinant CGI-58 showed intrinsic fluorescence for tryptophan that was quenched by the addition of 1-oleoyl-lysophosphatidic acid, oleoyl-CoA, arachidonoyl-CoA, and palmitoyl-CoA, but not by lysophosphatidyl choline. Expression of CGI-58 in fibroblasts from humans with CDS increased the incorporation of radiolabeled fatty acids released from the lipolysis of stored triacylglycerols into phospholipids. CGI-58 is a CoA-dependent lysophosphatidic acid acyltransferase that channels fatty acids released from the hydrolysis of stored triacylglycerols into phospholipids.
Asunto(s)
1-Acilglicerol-3-Fosfato O-Aciltransferasa/metabolismo , Acilcoenzima A/metabolismo , Aciltransferasas/metabolismo , Lisofosfolípidos/metabolismo , 1-Acilglicerol-3-Fosfato O-Aciltransferasa/química , 1-Acilglicerol-3-Fosfato O-Aciltransferasa/genética , 1-Acilglicerol-3-Fosfato O-Aciltransferasa/aislamiento & purificación , Secuencias de Aminoácidos , Animales , Células Cultivadas , Expresión Génica , Humanos , Concentración de Iones de Hidrógeno , Cinética , Metabolismo de los Lípidos/genética , Errores Innatos del Metabolismo Lipídico/enzimología , Errores Innatos del Metabolismo Lipídico/genética , Errores Innatos del Metabolismo Lipídico/metabolismo , Ratones , Posición Específica de Matrices de Puntuación , Unión Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Especificidad por Sustrato , SíndromeRESUMEN
Lipolysis of stored triacylglycerols provides lipid precursors for the assembly of apolipoprotein B (apoB) lipoproteins in hepatocytes. Abhydrolase domain containing 5 (ABHD5) is expressed in liver and facilitates the lipolysis of triacylglycerols. To study the function of ABHD5 in lipoprotein secretion, we silenced the expression of ABHD5 in McA RH7777 cells using RNA interference and studied the metabolism of lipids and secretion of apoB lipoproteins. McA RH7777 cells deficient in ABHD5 secreted reduced amounts of apoB, triacylglycerols, and cholesterol esters. Detailed analysis of liquid chromatography-mass spectrometry data for the molecular species of secreted triacylglycerols revealed that deficiency of ABHD5 significantly reduced secretion of triacylglycerols containing oleate, even when oleate was supplied in the culture medium; the ABHD5-deficient cells partially compensated by secreting higher levels of triacylglycerols containing saturated fatty acids. In experiments tracking the metabolism of [(14)C]oleate, silencing of ABHD5 reduced lipolysis of cellular triacylglycerols and incorporation of intermediates derived from stored lipids into secreted triacylglycerols and cholesterol esters. In contrast, the incorporation of exogenous oleate into secreted triacylglycerols and cholesterol esters was unaffected by deficiency of ABHD5. These findings suggest that ABHD5 facilitates the use of lipid intermediates derived from lipolysis of stored triacylglycerols for the assembly of lipoproteins.
Asunto(s)
Apolipoproteínas B/metabolismo , Proteínas Portadoras/fisiología , Esterasas/fisiología , Lipoproteínas/metabolismo , 1-Acilglicerol-3-Fosfato O-Aciltransferasa , Aciltransferasas , Animales , Carcinoma Hepatocelular/metabolismo , Cromatografía Liquida , Lípidos/análisis , Lipoproteínas/genética , Espectrometría de Masas , ARN Interferente Pequeño/farmacología , Ratas , Triglicéridos/metabolismo , Células Tumorales CultivadasRESUMEN
The metabolism of long-chain fatty acids in brain and their incorporation into signaling molecules such as diacylglycerol and LPA and into structural components of membranes, including myelin, requires activation by long-chain acyl-CoA synthetase (ACSL). Because ACSL3 and ACSL6 are the predominant ACSL isoforms in brain, we cloned and characterized these isoforms from rat brain and identified a novel ACSL6 clone (ACSL6_v2). ACSL6_v2 and the previously reported ACSL6_v1 represent splice variants that include exon 13 or 14, respectively. Homologue sequences of both of these variants are present in the human and mouse databases. ACSL3, ACSL6_v1, and ACSL6_v2 with Flag-epitopes at the C-termini were expressed in Escherichia coli and purified on Flag-affinity columns. The three recombinant proteins were characterized. Compared to ACSL4, another brain isoform, ACSL3, ACSL6_v1, and ACSL6_v2 showed similarities in kinetic values for CoA, palmitate, and arachidonate, but their apparent Km values for oleate were 4- to 6-fold lower than for ACSL4. In a direct competition assay with palmitate, all the polyunsaturated fatty acids tested were strong competitors only for ACSL4 with IC50 values of 0.5 to 5 microM. DHA was also strongly preferred by ACSL6_v2. The apparent Km value for ATP of ACSL6_v1 was 8-fold higher than that of ACSL6_v2. ACSL3 and the two variants of ACSL6 were more resistant than ACSL4 to heat inactivation. Despite the high amino acid identity between ACSL3 and ACSL4, rosiglitazone inhibited only ACSL4. Triacsin C, an inhibitor of ACSL1 and ACSL4, also inhibited ACSL3, but did not inhibit the ACSL6 variants. These data further document important differences in the closely related ACSL isoforms and show that amino acid changes near the consensus nucleotide binding site alter function in the two splice variants of ACSL6.
Asunto(s)
Coenzima A Ligasas/química , Proteínas Recombinantes/química , Empalme Alternativo , Secuencia de Aminoácidos , Animales , Encéfalo/enzimología , Encéfalo/metabolismo , Coenzima A Ligasas/antagonistas & inhibidores , Coenzima A Ligasas/genética , Inhibidores Enzimáticos/química , Ácidos Grasos Insaturados/química , Ácidos Grasos Insaturados/metabolismo , Calor , Isoenzimas/antagonistas & inhibidores , Isoenzimas/química , Isoenzimas/genética , Cinética , Datos de Secuencia Molecular , Octoxinol/química , Ratas , Proteínas Recombinantes/antagonistas & inhibidores , Proteínas Recombinantes/genética , Rosiglitazona , Tiazolidinedionas/química , Triazenos/químicaRESUMEN
Long chain fatty acids are converted to acyl-CoAs by acyl-CoA synthetase (fatty acid CoA ligase: AMP forming, E.C. 6.2.1.3; ACS). Escherichia coli has a single ACS, FadD, that is essential for growth when fatty acids are the sole carbon and energy source. Rodents have five ACS isoforms that differ in substrate specificity, tissue expression, and subcellular localization and are believed to channel fatty acids toward distinct metabolic pathways. We expressed rat ACS isoforms 1-5 in an E. coli strain that lacked FadD. All rat ACS isoforms were expressed in E. coli fadD or fadDfadR and had ACS specific activities that were 1.6-20-fold higher than the wild type control strain expressing FadD. In the fadD background, the rat ACS isoforms 1, 2, 3, 4 and 5 oxidized [(14)C]oleate at 5 to 25% of the wild type levels, but only ACS5 restored growth on oleate as the sole carbon source. To ensure that enzymes of beta-oxidation were not limiting, assays of ACS activity, beta-oxidation, fatty acid transport, and phospholipid synthesis were also examined in a fadD fadR strain, thereby eliminating FadR repression of the transporter FadL and the enzymes of beta-oxidation. In this strain, fatty acid transport levels were low but detectable for ACS1, 2, 3, and 4 and were nearly 50% of wild type levels for ACS5. Despite increases in beta-oxidation, only ACS5 transformants were able to grow on oleate. These studies show that although ACS isoforms 1-4 variably supported moderate transport activity, beta-oxidation, and phospholipid synthesis and although their in vitro specific activities were greater than that of chromosomally encoded FadD, they were unable to substitute functionally for FadD regarding growth. Thus, membrane composition and protein-protein interactions may be critical in reconstituting bacterial ACS function.