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1.
J Hepatol ; 73(1): 140-148, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32302728

RESUMO

BACKGROUND & AIMS: Obesity and type 2 diabetes increase hepatocellular carcinoma (HCC) incidence in humans and accelerate diethylnitrosamine (DEN)-induced hepatocarcinogenesis in mice. We investigated whether exercise reduces HCC development in obese/diabetic Alms1 mutant (foz/foz) mice and studied protective mechanisms. METHODS: We measured HCC development in DEN-injected male foz/foz and wild-type (WT) littermates housed with or without an exercise wheel from week 4 until 12 or 24 weeks, and in foz/foz mice pair-fed to WT littermates. We also studied HCC development in DEN-injected Jnk1-/-.foz/foz mice generated by cross breeding, as well as their genetic controls. Dysplastic hepatocytes were identified by glutathione-S-transferase pi form (GST-pi) immunohistochemistry, liver nodules were counted, and HCC was analysed by histopathology. RESULTS: Exercising foz/foz mice maintained similar weight as WT mice up to 10 weeks, but then gained weight and were obese by 24 weeks; a similar body weight profile was obtained by pair-feeding foz/foz mice to WT. At 12 weeks, livers of exercising foz/foz mice exhibited fewer GST-pi positive hepatocytes than sedentary counterparts; by 24 weeks, fewer exercising foz/foz mice developed HCC (15% vs. 64%, p <0.05). Conversely, pair-feeding foz/foz mice failed to reduce HCC incidence. In these insulin-resistant foz/foz mice, exercise failed to activate hepatic AMPK or Akt/mTORC1. Instead, it improved insulin sensitivity, ameliorated steatosis and liver injury, activated p53 to increase p27 expression, and prevented JNK activation. This was associated with suppression of hepatocellular proliferation. DEN-injected Jnk1-/-.foz/foz mice failed to develop liver tumours or HCC at 24 weeks. CONCLUSIONS: Direct effects of exercise dampen proliferation of dysplastic hepatocytes to reduce 3-month dysplastic foci and 6-month incidence of DEN-induced HCC in obese, insulin-resistant mice. The effects of exercise that potentially slow hepatocarcinogenesis include p53-mediated induction of p27 and prevention of JNK activation. LAY SUMMARY: Fatty liver disease commonly occurs alongside obesity and diabetes, contributing to rapidly increasing rates of liver cancer throughout the world. Herein, we show that exercise reduces the incidence and progression of hepatocellular carcinoma in mouse models. The effect of exercise on cancer risk was shown to be independent of changes in weight. Exercise could be a protective mechanism against liver cancer in at-risk individuals.


Assuntos
Carcinogênese , Carcinoma Hepatocelular , Diabetes Mellitus Tipo 2 , Neoplasias Hepáticas , Atividade Motora/fisiologia , Obesidade , Animais , Peso Corporal/fisiologia , Carcinogênese/metabolismo , Carcinogênese/patologia , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/fisiopatologia , Imuno-Histoquímica , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Sistema de Sinalização das MAP Quinases/fisiologia , Camundongos , Camundongos Obesos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Obesidade/metabolismo , Obesidade/fisiopatologia , Condicionamento Físico Animal , Antígeno Nuclear de Célula em Proliferação/metabolismo , Fatores de Proteção , Fatores de Risco , Proteína Supressora de Tumor p53/metabolismo
3.
Biochim Biophys Acta Mol Basis Dis ; 1865(6): 1192-1200, 2019 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-30658161

RESUMO

OBJECTIVE AND BACKGROUND: Activation of sterile inflammation after hepatic ischemia/reperfusion (I/R) culminates in liver injury. The route to liver damage starts with mitochondrial oxidative stress and cell death during early reperfusion. The link between mitochondrial oxidative stress, damage-associate molecular pattern (DAMP) release, and sterile immune signaling is incompletely understood and lacks clinical validation. The aim of the study was to validate this relation in a clinical liver I/R cohort and to limit DAMP release using a mitochondria-targeted antioxidant in I/R-subjected mice. METHODS: Plasma levels of the DAMPs high-mobility group box 1 (HMGB1), mitochondrial DNA, and nucleosomes were measured in 39 patients enrolled in an observational study who underwent a major liver resection with (N = 29) or without (N = 13) intraoperative liver ischemia. Circulating cytokine and neutrophil activation markers were also determined. In mice, the mitochondria-targeted antioxidant MitoQ was intravenously infused in an attempt to limit DAMP release, reduce sterile inflammation, and suppress I/R injury. RESULTS: In patients, HMGB1 was elevated following liver resection with I/R compared to liver resection without I/R. HMGB1 levels correlated positively with ischemia duration and peak post-operative transaminase (ALT) levels. There were no differences in mitochondrial DNA, nucleosome, or cytokine levels between the two groups. In mice, MitoQ neutralized hepatic oxidative stress and decreased HMGB1 release by ±50%. MitoQ suppressed transaminase release, hepatocellular necrosis, and cytokine production. Reconstituting disulfide HMGB1 during reperfusion reversed these protective effects. CONCLUSION: HMGB1 seems the most pertinent DAMP in clinical hepatic I/R injury. Neutralizing mitochondrial oxidative stress may limit DAMP release after hepatic I/R and reduce liver damage.


Assuntos
Alarminas/sangue , Proteína HMGB1/sangue , Fígado/metabolismo , Traumatismo por Reperfusão/sangue , Idoso , Antioxidantes/farmacologia , Citocinas/sangue , DNA Mitocondrial/sangue , DNA Mitocondrial/genética , Feminino , Humanos , Fígado/irrigação sanguínea , Fígado/efeitos dos fármacos , Masculino , Pessoa de Meia-Idade , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/genética , Mitocôndrias/metabolismo , Compostos Organofosforados/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Traumatismo por Reperfusão/fisiopatologia , Ubiquinona/análogos & derivados , Ubiquinona/farmacologia
4.
Clin Sci (Lond) ; 131(16): 2145-2159, 2017 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-28687713

RESUMO

Background and aims: TLR9 deletion protects against steatohepatitis due to choline-amino acid depletion and high-fat diet. We measured TLR9 in human non-alcoholic steatohepatitis (NASH) livers, and tested whether TLR9 mediates inflammatory recruitment in three murine models of non-alcoholic fatty liver disease (NAFLD). Methods: We assayed TLR mRNA in liver biopsies from bariatric surgery patients. Wild-type (Wt), appetite-dysregulated Alms1 mutant (foz/foz), Tlr9-/-, and Tlr9-/-foz/foz C57BL6/J mice and bone marrow (BM) chimeras were fed 0.2% cholesterol, high-fat, high sucrose (atherogenic[Ath]) diet or chow, and NAFLD activity score (NAS)/NASH pathology, macrophage/neutrophil infiltration, cytokines/chemokines, and cell death markers measured in livers. Results: Hepatic TLR9 and TLR4 mRNA were increased in human NASH but not simple steatosis, and in Ath-fed foz/foz mice with metabolic syndrome-related NASH. Ath-fed Tlr9-/- mice showed simple steatosis and less Th1 cytokines than Wt. Tlr9-/-foz/foz mice were obese and diabetic, but necroinflammatory changes were less severe than Tlr9+/+.foz/foz mice. TLR9-expressing myeloid cells were critical for Th1 cytokine production in BM chimeras. BM macrophages from Tlr9-/- mice showed M2 polarization, were resistant to M1 activation by necrotic hepatocytes/other pro-inflammatory triggers, and provoked less neutrophil chemotaxis than Wt Livers from Ath-fed Tlr9-/- mice appeared to exhibit more markers of necroptosis [receptor interacting protein kinase (RIP)-1, RIP-3, and mixed lineage kinase domain-like protein (MLKL)] than Wt, and ∼25% showed portal foci of mononuclear cells unrelated to NASH pathology. CONCLUSION: Our novel clinical data and studies in overnutrition models, including those with diabetes and metabolic syndrome, clarify TLR9 as a pro-inflammatory trigger in NASH. This response is mediated via M1-macrophages and neutrophil chemotaxis.


Assuntos
Mediadores da Inflamação/metabolismo , Hepatopatia Gordurosa não Alcoólica/metabolismo , Receptor Toll-Like 9/biossíntese , Regulação para Cima/fisiologia , Adiponectina/deficiência , Adulto , Animais , Cirurgia Bariátrica , Biópsia , Células Cultivadas , Citocinas/metabolismo , Dieta Hiperlipídica/efeitos adversos , Modelos Animais de Doenças , Feminino , Deleção de Genes , Hepatócitos/metabolismo , Hepatomegalia/prevenção & controle , Humanos , Fígado/metabolismo , Fígado/patologia , Macrófagos/metabolismo , Síndrome Metabólica/metabolismo , Erros Inatos do Metabolismo/prevenção & controle , Camundongos Knockout , Neutrófilos/metabolismo , Hepatopatia Gordurosa não Alcoólica/patologia , Obesidade/prevenção & controle , RNA Mensageiro/genética , Receptor Toll-Like 9/deficiência , Receptor Toll-Like 9/genética
5.
Exp Cell Res ; 356(1): 48-56, 2017 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-28408319

RESUMO

Dysplastic hepatocytes (DH) represent altered hepatocytes with potential for malignant transformation. To date, most research on pathways to hepatocarcinogenesis has focused on use of "hepatoma" cell lines derived from hepatocellular carcinoma (HCC). We describe a novel technique for deriving/culturing DH and demonstrate their utility for functional studies in vitro, compared to primary hepatocytes (PH) and HCC. PH and DH were prepared by portal vein collagenase perfusion from C57BL/6J mice. DH were subsequently subjected to FACS. HCC from diethylnitrosamine (DEN)-injected mice were mechanically isolated. Cell cycle analyses were performed by flow cytometry and PCNA immunohistochemistry. To establish utility of DH, we studied pathways of p53 turnover, apoptosis and cell proliferation using pfithrin-α (PFT) and nutlin-3. Like PH, DH were minimally proliferative compared to HCC. Only 30±0.03% of DH were in G2/M phase versus 51±0.01% of HCC; this difference corroborated with PCNA-immunostaining of dysplastic nodules from DEN-injected mice. In DH and HCC, nutlin-3 suppressed p53 mRNA, induced p53 and mdm2 activation but paradoxically resulted in increased anti-apoptotic and proliferative activity. Primary murine DH display distinctive biological characteristics compared with PH and HCC. As an intermediate cell type to HCC, they offer a new pathobiologically relevant primary cell culture system with which to interrogate the molecular changes in hepatocarcinogenesis.


Assuntos
Carcinoma Hepatocelular/patologia , Transformação Celular Neoplásica/patologia , Hepatócitos/patologia , Neoplasias Hepáticas Experimentais/patologia , Fígado/patologia , Animais , Apoptose/genética , Ciclo Celular/fisiologia , Proliferação de Células/genética , Células Cultivadas , Dietilnitrosamina , Ativação Enzimática , Imidazóis/farmacologia , Fígado/citologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Piperazinas/farmacologia , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo
6.
J Hepatol ; 66(5): 1037-1046, 2017 05.
Artigo em Inglês | MEDLINE | ID: mdl-28167322

RESUMO

BACKGROUND & AIMS: NOD-like receptor protein 3 (NLRP3) inflammasome activation occurs in Non-alcoholic fatty liver disease (NAFLD). We used the first small molecule NLRP3 inhibitor, MCC950, to test whether inflammasome blockade alters inflammatory recruitment and liver fibrosis in two murine models of steatohepatitis. METHODS: We fed foz/foz and wild-type mice an atherogenic diet for 16weeks, gavaged MCC950 or vehicle until 24weeks, then determined NAFLD phenotype. In mice fed an methionine/choline deficient (MCD) diet, we gavaged MCC950 or vehicle for 6weeks and determined the effects on liver fibrosis. RESULTS: In vehicle-treated foz/foz mice, hepatic expression of NLRP3, pro-IL-1ß, active caspase-1 and IL-1ß increased at 24weeks, in association with cholesterol crystal formation and NASH pathology; plasma IL-1ß, IL-6, MCP-1, ALT/AST all increased. MCC950 treatment normalized hepatic caspase 1 and IL-1ß expression, plasma IL-1ß, MCP-1 and IL-6, lowered ALT/AST, and reduced the severity of liver inflammation including designation as NASH pathology, and liver fibrosis. In vitro, cholesterol crystals activated Kupffer cells and macrophages to release IL-1ß; MCC950 abolished this, and the associated neutrophil migration. MCD diet-fed mice developed fibrotic steatohepatitis; MCC950 suppressed the increase in hepatic caspase 1 and IL-1ß, lowered numbers of macrophages and neutrophils in the liver, and improved liver fibrosis. CONCLUSION: MCC950, an NLRP3 selective inhibitor, improved NAFLD pathology and fibrosis in obese diabetic mice. This is potentially attributable to the blockade of cholesterol crystal-mediated NLRP3 activation in myeloid cells. MCC950 reduced liver fibrosis in MCD-fed mice. Targeting NLRP3 is a logical direction in pharmacotherapy of NASH. LAY SUMMARY: Fatty liver disease caused by being overweight with diabetes and a high risk of heart attack, termed non-alcoholic steatohepatitis (NASH), is the most common serious liver disease with no current treatment. There could be several causes of inflammation in NASH, but activation of a protein scaffold within cells termed the inflammasome (NLRP3) has been suggested to play a role. Here we show that cholesterol crystals could be one pathway to activate the inflammasome in NASH. We used a drug called MCC950, which has already been shown to block NLRP3 activation, in an attempt to reduce liver injury in NASH. This drug partly reversed liver inflammation, particularly in obese diabetic mice that most closely resembles the human context of NASH. In addition, such dampening of liver inflammation in NASH achieved with MCC950 partly reversed liver scarring, the process that links NASH to the development of cirrhosis.


Assuntos
Hepatite/prevenção & controle , Compostos Heterocíclicos de 4 ou mais Anéis/uso terapêutico , Cirrose Hepática Experimental/prevenção & controle , Proteína 3 que Contém Domínio de Pirina da Família NLR/antagonistas & inibidores , Hepatopatia Gordurosa não Alcoólica/tratamento farmacológico , Sulfonas/uso terapêutico , Animais , Modelos Animais de Doenças , Feminino , Furanos , Indenos , Interleucina-1beta/sangue , Camundongos , NF-kappa B/fisiologia , Proteína 3 que Contém Domínio de Pirina da Família NLR/fisiologia , Hepatopatia Gordurosa não Alcoólica/complicações , Espécies Reativas de Oxigênio/metabolismo , Sulfonamidas
7.
J Gastroenterol Hepatol ; 31(6): 1210-9, 2016 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-26574916

RESUMO

BACKGROUND AND AIM: While gender differences in hepatocellular carcinoma (HCC) are profound, the mechanism is unclear. Using castration and hormone replacement strategies, we tested whether these gender differences are attributable to testosterone or estradiol/progesterone effects on cell cycle regulators and p53. METHODS: We studied dysplastic liver and HCCs in intact and castrated diethylnitrosamine-injected C57BL/6J male and female mice, with or without hormonal replacement. Effects of sex steroids on proliferation and survival of primary hepatocytes and primary HCC cells were also characterized. RESULTS: Diethylnitrosamine-injected female mice displayed fewer dysplastic foci and slower onset of HCC than male mice, with smaller/more differentiated tumors and fewer metastases. Castration of diethylnitrosamine-injected male mice reduced cyclin E kinase and augmented hepatocyte apoptosis compared with intact male mice; estradiol/progesterone enhanced these effects. In intact female mice, cyclin E kinase activity was less than in males; testosterone administered to ovariectomized female mice upregulated cyclin E, increased cyclin E kinase, and accelerated hepatocarcinogenesis. In vitro, testosterone increased expression of cell cycle regulators (cyclin D1, cyclin E, and cyclin-dependent kinase 2) and reduced p53 and p21, which enhanced hepatocyte viability. In contrast, estradiol both suppressed hepatocyte cell cycle markers, upregulated p53 and reduced viability of hepatocytes and HCC cells. CONCLUSIONS: Testosterone is the positive regulator of hepatocyte cell cycle via cyclin E, while estradiol plays a negative role by effects of p53 and p21. Together, both sex hormones determine the male predominance of gender differences in hepatocarcinogenesis.


Assuntos
Carcinoma Hepatocelular/enzimologia , Transformação Celular Neoplásica/induzido quimicamente , Ciclina E/metabolismo , Quinase 2 Dependente de Ciclina/metabolismo , Terapia de Reposição Hormonal/efeitos adversos , Neoplasias Hepáticas Experimentais/enzimologia , Testosterona/farmacologia , Testosterona/toxicidade , Animais , Apoptose/efeitos dos fármacos , Proteínas Reguladoras de Apoptose/metabolismo , Carcinoma Hepatocelular/induzido quimicamente , Carcinoma Hepatocelular/patologia , Castração , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Transformação Celular Neoplásica/efeitos dos fármacos , Transformação Celular Neoplásica/metabolismo , Células Cultivadas , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Dietilnitrosamina , Estradiol/toxicidade , Terapia de Reposição de Estrogênios/efeitos adversos , Feminino , Hepatócitos/efeitos dos fármacos , Hepatócitos/enzimologia , Hepatócitos/patologia , Neoplasias Hepáticas Experimentais/induzido quimicamente , Neoplasias Hepáticas Experimentais/patologia , Masculino , Camundongos Endogâmicos C57BL , Cultura Primária de Células , Fatores Sexuais , Fatores de Tempo , Proteína Supressora de Tumor p53/metabolismo
8.
J Clin Transl Res ; 2(1): 26-37, 2016 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-30873458

RESUMO

BACKGROUND: There are strong links between obesity, diabetes and hepatocellular carcinoma (HCC), but molecular mechanisms remain unclear. AIM: We tested the proposed involvement of NF-κB, IL-6/STAT3 and Akt/mTORC1 before onset (at 3 months) and at onset (6 months) of accelerated hepatocarcinogenesis in DEN-injected obese and diabetic foz/foz compared to lean wildtype (Wt) mice, and also studied the hepatocyte proliferative response to DNA damage between the obese and lean lines. METHODS: Male foz/foz and Wt littermates fed normal chow were DEN-injected (10mg/kg i.p.) at age 12-15 days. To test the effect of mTOR inhibitor on growth of dysplastic hepatocytes, a separate cohort of DEN-injected foz/foz mice was administered rapamycin (4 mg/kg body weight/day). RESULTS: foz/foz mice developed obesity, hyperinsulinemia, diabetes, adipokine dysregulation and fatty liver, without increased serum or liver TNF-α or serum IL-6. All DEN-injected foz/foz mice developed HCC by 6 mths vs. 0/10 lean Wt. At 3 mths, there were more dysplastic hepatocytes in DEN-injected foz/foz than Wt, with increased liver injury (serum ALT), hepatocyte apoptosis (M30-positive cells) and proliferation (cyclin D1, cyclin E, PCNA), but neither NF-κB nor STAT3 activation. foz/foz livers exhibited upregulation of DNA damage sensors ATM and ATR, with inadequate cell cycle checkpoint controls (CHK1, CHK2, p53, p21). Akt and mTORC1 were highly activated in livers from foz/foz vs. Wt mice. Despite such activation, rapamycin failed to reduce growth of dysplastic hepatocytes. CONCLUSIONS: Accelerated DEN-induced HCC in obese/diabetic mice is linked to enhanced growth of dysplastic hepatocytes that cannot be attributed to NF-κB or IL-6/STAT3 activation, nor to sustained mTORC1 activation. The critical mechanism for obesity-enhanced hepatocarcinogenesis lies in the disconnection between hepatocellular injury with DNA damage, and an unrestrained proliferative response. RELEVANCE FOR PATIENTS: This study supports the epidemiological data linking obesity, diabetes and fatty liver disease with increased risk for developing HCC. The findings also suggest that mTORC1 inhibition may not be beneficial in the prevention of obesity-related hepatocarcinogenesis.

9.
Obesity (Silver Spring) ; 23(9): 1845-55, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-26250514

RESUMO

OBJECTIVE: Adipose inflammation and dysfunction underlie metabolic obesity. Exercise improves glycemic control and metabolic indices, but effects on adipose function and inflammation are less clear. Accordingly, it was hypothesized that exercise improves adipose morphometry to reduce adipose inflammation in hyperphagic obese mice. METHODS: Alms1 mutant foz/foz mice housed in pairs were fed an atherogenic or chow diet; half the cages were fitted with a computer-monitored wheel for voluntary exercise. Insulin-induced AKT-phosphorylation, adipocyte size distribution, and inflammatory recruitment were studied in visceral versus subcutaneous depots, and severity of fatty liver disease was determined. RESULTS: Exercise prevented obesity and diabetes development in chow-fed foz/foz mice and delayed their onset in atherogenic-fed counterparts. Insulin-stimulated phospho-AKT levels in muscle were improved with exercise, but not in adipose or liver. Exercise suppressed adipose inflammatory recruitment, particularly in visceral adipose, associated with an increased number of small adipocyte subpopulations, and enhanced expression of beige adipocyte factor PRDM16 in subcutaneous fat. In atherogenic-fed foz/foz mice liver, exercise suppressed development of nonalcoholic steatohepatitis and related liver fibrosis. CONCLUSIONS: Exercise confers metabo-protective effects in atherogenic-fed hyperphagic mice by preventing early onset of obesity and diabetes in association with enhanced muscle insulin sensitivity, improved adipose morphometry, and suppressed adipose and liver inflammation.


Assuntos
Diabetes Mellitus Experimental/complicações , Inflamação/complicações , Hepatopatia Gordurosa não Alcoólica/terapia , Obesidade/complicações , Condicionamento Físico Animal/métodos , Animais , Camundongos , Camundongos Obesos , Hepatopatia Gordurosa não Alcoólica/complicações
10.
Liver Int ; 35(9): 2174-86, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-25786512

RESUMO

BACKGROUND & AIMS: Steatosis accentuates the severity of hepatic ischaemia-reperfusion injury (IRI); 'statins' (HMG-CoA reductase inhibitors) protect the heart and brain against post-ischaemic injury. We tested whether short-term administration of atorvastatin protects fatty livers in obese mice against IRI. METHODS: Mice with dietary or genetic simple steatosis (SS) or non-alcoholic steatohepatitis (NASH) were subjected to 60 min partial hepatic ischaemia/24 h reperfusion. Atorvastatin was injected intravenously (5 mg/kg) 1 h before IRI. Liver injury, Toll-like receptor-4 (TLR4), cytokines/chemokines, iNOS/eNOS expression, eNOS activity and thromboxane B2 (TXB2) production were determined. RESULTS: Ischaemia-reperfusion injury was exaggerated by two- to five-fold in SS and NASH compared with lean liver. Atorvastatin pretreatment conferred 70-90% hepatic protection in all animals. Atorvastatin increased post-ischaemic eNOS mRNA/protein and strikingly enhanced eNOS activity (by phospho-eNOS). It also attenuated microparticle (MP) production, NF-κB activation, significantly dampened post-ischaemic thromboxane B2 production, induction of TNF-α, IL-6, MIP-1a, MCP-1, GM-CSF and vascular cell adhesion molecule-1 (VCAM), with a resultant reduction on macrophage and polymorphonuclear neutrophil recruitment. Up-regulation of HMGB1 and TLR4 after IRI was marked in fatty livers; 1 h pretreatment with atorvastatin reduced HMGB1 and TLR4 expression in all livers. CONCLUSIONS: Acute (1 h) atorvastatin administration is highly hepatoprotective against IRI in NASH, fatty and lean livers. Key mechanisms include suppression of inflammation by prevention of NF-κB activation, microvascular protection via eNOS activation and suppression of TXB2 and MP release. Short-term intravenous statin treatment is a readily available and effective preventive agent against hepatic IRI, irrespective of obesity and fatty liver disease, and merits clinical trials in at-risk patients.


Assuntos
Atorvastatina/administração & dosagem , Quimiocinas/sangue , Citocinas/sangue , Inibidores de Hidroximetilglutaril-CoA Redutases/administração & dosagem , Hepatopatia Gordurosa não Alcoólica/tratamento farmacológico , Traumatismo por Reperfusão/tratamento farmacológico , Animais , Proteína HMGB1 , Fígado/patologia , Masculino , Camundongos , Camundongos Obesos , NF-kappa B/metabolismo , Óxido Nítrico Sintase Tipo III/metabolismo , Tromboxano B2/metabolismo , Receptor 4 Toll-Like/metabolismo , Molécula 1 de Adesão de Célula Vascular/metabolismo
11.
J Lipid Res ; 56(2): 277-85, 2015 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-25520429

RESUMO

Cholesterol crystals form within hepatocyte lipid droplets in human and experimental nonalcoholic steatohepatitis (NASH) and are the focus of crown-like structures (CLSs) of activated Kupffer cells (KCs). Obese, diabetic Alms1 mutant (foz/foz) mice were a fed high-fat (23%) diet containing 0.2% cholesterol for 16 weeks and then assigned to four intervention groups for 8 weeks: a) vehicle control, b) ezetimibe (5 mg/kg/day), c) atorvastatin (20 mg/kg/day), or d) ezetimibe and atorvastatin. Livers of vehicle-treated mice developed fibrosing NASH with abundant cholesterol crystallization within lipid droplets calculated to extend over 3.3% (SD, 2.2%) of liver surface area. Hepatocyte lipid droplets with prominent cholesterol crystallization were surrounded by TNFα-positive (activated) KCs forming CLSs (≥ 3 per high-power field). KCs that formed CLSs stained positive for NLRP3, implicating activation of the NLRP3 inflammasome in response to cholesterol crystals. In contrast, foz/foz mice treated with ezetimibe and atorvastatin showed near-complete resolution of cholesterol crystals [0.01% (SD, 0.02%) of surface area] and CLSs (0 per high-power field), with amelioration of fibrotic NASH. Ezetimibe or atorvastatin alone had intermediate effects on cholesterol crystallization, CLSs, and NASH. These findings are consistent with a causative link between exposure of hepatocytes and KCs to cholesterol crystals and with the development of NASH possibly mediated by NLRP3 activation.


Assuntos
Anticolesterolemiantes/uso terapêutico , Colesterol/metabolismo , Células de Kupffer/efeitos dos fármacos , Células de Kupffer/metabolismo , Animais , Anticolesterolemiantes/farmacologia , Atorvastatina , Azetidinas/farmacologia , Azetidinas/uso terapêutico , Ezetimiba , Feminino , Ácidos Heptanoicos/farmacologia , Ácidos Heptanoicos/uso terapêutico , Inflamassomos/efeitos dos fármacos , Inflamassomos/metabolismo , Fígado/efeitos dos fármacos , Fígado/metabolismo , Camundongos , Camundongos Mutantes , Hepatopatia Gordurosa não Alcoólica/tratamento farmacológico , Pirróis/farmacologia , Pirróis/uso terapêutico
12.
PLoS One ; 9(9): e104376, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25222287

RESUMO

BACKGROUND & AIMS: Ischemia-reperfusion injury (IRI) can cause hepatic failure after liver surgery or transplantation. IRI causes oxidative stress, which injures sinusoidal endothelial cells (SECs), leading to recruitment and activation of Kupffer cells, platelets and microcirculatory impairment. We investigated whether injured SECs and other cell types release microparticles during post-ischemic reperfusion, and whether such microparticles have pro-inflammatory, platelet-activating and pro-injurious effects that could contribute to IRI pathogenesis. METHODS: C57BL6 mice underwent 60 min of partial hepatic ischemia followed by 15 min-24 hrs of reperfusion. We collected blood and liver samples, isolated circulating microparticles, and determined protein and lipid content. To establish mechanism for microparticle production, we subjected murine primary hepatocytes to hypoxia-reoxygenation. Because microparticles express everted phosphatidylserine residues that are the target of annexin V, we analyzed the effects of an annexin V-homodimer (Diannexin or ASP8597) on post-ischemia microparticle production and function. RESULTS: Microparticles were detected in the circulation 15-30 min after post-ischemic reperfusion, and contained markers of SECs, platelets, natural killer T cells, and CD8+ cells; 4 hrs later, they contained markers of macrophages. Microparticles contained F2-isoprostanes, indicating oxidative damage to membrane lipids. Injection of mice with TNF-α increased microparticle formation, whereas Diannexin substantially reduced microparticle release and prevented IRI. Hypoxia-re-oxygenation generated microparticles from primary hepatocytes by processes that involved oxidative stress. Exposing cultured hepatocytes to preparations of microparticles isolated from the circulation during IRI caused injury involving mitochondrial membrane permeability transition. Microparticles also activated platelets and induced neutrophil migration in vitro. The inflammatory properties of microparticles involved activation of NF-κB and JNK, increased expression of E-selectin, P-selectin, ICAM-1 and VCAM-1. All these processes were blocked by coating microparticles with Diannexin. CONCLUSIONS: Following hepatic IRI, microparticles circulate and can be taken up by hepatocytes, where they activate signaling pathways that mediate inflammation and hepatocyte injury. Diannexin prevents microparticle formation and subsequent inflammation.


Assuntos
Anexina A5/farmacologia , Micropartículas Derivadas de Células/metabolismo , Traumatismo por Reperfusão/patologia , Animais , Anexina A5/química , Plaquetas/fisiologia , Permeabilidade da Membrana Celular , Movimento Celular , Micropartículas Derivadas de Células/fisiologia , Selectina E/metabolismo , Células Endoteliais/metabolismo , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Molécula 1 de Adesão Intercelular/metabolismo , Fígado/patologia , Masculino , Camundongos Endogâmicos C57BL , Microcirculação/efeitos dos fármacos , NF-kappa B/metabolismo , Neutrófilos/fisiologia , Estresse Oxidativo , Selectina-P/metabolismo , Traumatismo por Reperfusão/tratamento farmacológico , Transdução de Sinais/efeitos dos fármacos , Fator de Necrose Tumoral alfa/farmacologia , Molécula 1 de Adesão de Célula Vascular/metabolismo
13.
J Biol Chem ; 289(28): 19531-7, 2014 Jul 11.
Artigo em Inglês | MEDLINE | ID: mdl-24898253

RESUMO

Transmembrane lipid transporters are believed to establish and maintain phospholipid asymmetry in biological membranes; however, little is known about the in vivo function of the specific transporters involved. Here, we report that developing erythrocytes from mice lacking the putative phosphatidylserine flippase ATP11C showed a lower rate of PS translocation in vitro compared with erythrocytes from wild-type littermates. Furthermore, the mutant mice had an elevated percentage of phosphatidylserine-exposing mature erythrocytes in the periphery. Although erythrocyte development in ATP11C-deficient mice was normal, the mature erythrocytes had an abnormal shape (stomatocytosis), and the life span of mature erythrocytes was shortened relative to that in control littermates, resulting in anemia in the mutant mice. Thus, our findings uncover an essential role for ATP11C in erythrocyte morphology and survival and provide a new candidate for the rare inherited blood disorder stomatocytosis with uncompensated anemia.


Assuntos
Adenosina Trifosfatases/metabolismo , Membrana Eritrocítica/enzimologia , Fosfolipídeos/metabolismo , Desequilíbrio Ácido-Base/genética , Desequilíbrio Ácido-Base/metabolismo , Desequilíbrio Ácido-Base/patologia , Adenosina Trifosfatases/genética , Anemia Hemolítica Congênita/genética , Anemia Hemolítica Congênita/metabolismo , Anemia Hemolítica Congênita/patologia , Animais , Transporte Biológico Ativo , Sobrevivência Celular/fisiologia , Membrana Eritrocítica/genética , Eritrócitos Anormais/metabolismo , Eritrócitos Anormais/patologia , Erros Inatos do Metabolismo/genética , Erros Inatos do Metabolismo/metabolismo , Erros Inatos do Metabolismo/patologia , Camundongos , Camundongos Mutantes , Fosfolipídeos/genética
14.
Liver Int ; 34(7): 1084-93, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-24107103

RESUMO

BACKGROUND & AIMS: Obese Alms1 mutant (foz/foz) NOD.B10 mice develop diabetes and fibrotic NASH when fed high-fat(HF) diet. To establish whether diabetes or obesity is more closely associated with NASH fibrosis, we compared diabetic foz/foz C57BL6/J with non-diabetic foz/foz BALB/c mice. We also determined hepatic cytokines, growth factors and related profibrotic pathways. METHODS: Male and female foz/foz BALB/c and C57BL6/J mice were fed HF or chow for 24 weeks before determining metabolic indices, liver injury, cytokines, growth factors, pathology/fibrosis and matrix deposition pathways. RESULTS: All foz/foz mice were obese. Hepatomegaly, hyperinsulinemia, hyperglycaemia and hypoadiponectinaemia occurred only in foz/foz C57BL6/J mice, whereas foz/foz BALB/c formed more adipose. Serum ALT, steatosis, ballooning, liver inflammation and NAFLD activity score were worse in C57BL6/J mice. In HF-fed mice, fibrosis was severe in foz/foz C57BL6/J, appreciable in WT C57BL6/J, but absent in foz/foz BALB/c mice. Hepatic mRNA expression of TNF-α, IL-12, IL-4, IL-10 was increased (but not IFN-γ, IL-1ß, IL-17A), and IL-4:IFN-γ ratio (indicating Th-2 predominance) was higher in HF-fed foz/foz C57BL6/J than BALB/c mice. In livers of HF-fed foz/foz C57BL6/J mice, TGF-ß was unaltered but PDGFα and CTGF were increased in association with enhanced α-SMA, CD147and MMP activity. CONCLUSIONS: In mice with equivalent genetic/dietary obesity, NASH development is linked to strain differences in hyperinsulinaemia and hyperglycaemia inversely related to lipid partitioning between adipose and liver. Diabetes-mediated CTGF-regulation of MMPs as well as cytokines/growth factors (Th-2 cytokine predominant, PDGFα, not TGF-ß) mobilized in the resultant hepatic necroinflammatory change may contribute to strain differences in NASH fibrosis.


Assuntos
Proteínas de Ligação a DNA/genética , Diabetes Mellitus Tipo 2/etiologia , Dieta Hiperlipídica/efeitos adversos , Cirrose Hepática/etiologia , Hepatopatia Gordurosa não Alcoólica/etiologia , Análise de Variância , Animais , Proteínas de Ciclo Celular , Citocinas/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Feminino , Imunofluorescência , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Fígado/metabolismo , Fígado/patologia , Cirrose Hepática/patologia , Masculino , Metaloproteinases da Matriz/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Hepatopatia Gordurosa não Alcoólica/patologia , Especificidade da Espécie
15.
J Gastroenterol Hepatol ; 29(3): 435-41, 2014 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-24199670

RESUMO

Coffee is one of the most popular beverages in the world. Several studies consistently show that coffee drinkers with chronic liver disease have a reduced risk of cirrhosis and a lower incidence of hepatocellular carcinoma regardless of primary etiology. With the increasing prevalence of non-alcoholic fatty liver disease (NAFLD) worldwide, there is renewed interest in the effect of coffee intake on NAFLD severity and positive clinical outcomes. This review gives an overview of growing epidemiological and clinical evidence which indicate that coffee consumption reduces severity of NAFLD. These studies vary in methodology, and potential confounding factors have not always been completely excluded. However, it does appear that coffee, and particular components other than caffeine, reduce NAFLD prevalence and inflammation of non-alcoholic steatohepatitis. Several possible mechanisms underlying coffee's hepatoprotective effects in NAFLD include antioxidative, anti-inflammatory, and antifibrotic effects, while a chemopreventive effect against hepatocarcinogenesis seems likely. The so-far limited data supporting such effects will be discussed, and the need for further study is highlighted.


Assuntos
Café , Fígado Gorduroso/prevenção & controle , Anti-Inflamatórios , Antioxidantes , Carcinoma Hepatocelular/prevenção & controle , Quimioprevenção , Humanos , Cirrose Hepática/prevenção & controle , Neoplasias Hepáticas/prevenção & controle , Hepatopatia Gordurosa não Alcoólica , Prevalência , Prognóstico , Índice de Gravidade de Doença
16.
Obesity (Silver Spring) ; 21(6): 1189-99, 2013 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-23666886

RESUMO

BACKGROUND: Alms1 mutant (foz/foz) mice develop hyperphagic obesity, diabetes, metabolic syndrome, and fatty liver (steatosis). High-fat (HF) feeding converts pathology from bland steatosis to nonalcoholic steatohepatitis (NASH) with fibrosis, which leads to cirrhosis in humans. OBJECTIVE: We sought to establish how dietary composition contributes to NASH pathogenesis. DESIGN AND METHODS: foz/foz mice were fed HF diet or chow 24 weeks, or switched HF to chow after 12 weeks. Serum ALT, NAFLD activity score (NAS), fibrosis severity, neutrophil, macrophage and apoptosis immunohistochemistry, uncoupling protein (UCP)2, ATP, NF-κB activation/expression of chemokines/adhesion molecules/fibrogenic pathways were determined. RESULT: HF intake upregulated liver fatty acid and cholesterol transporter, CD36. Dietary switch expanded adipose tissue and decreased hepatomegaly by lowering triglyceride, cholesterol ester, free cholesterol and diacylglyceride content of liver. There was no change in lipogenesis or fatty acid oxidation pathways; instead, CD36 was suppressed. These diet-induced changes in hepatic lipids improved NAS, reduced neutrophil infiltration, normalized UCP2 and increased ATP; this facilitated apoptosis with a change in macrophage phenotype favoring M2 cells. Dietary switch also abrogated NF-κB activation and chemokine/adhesion molecule expression, and arrested fibrosis by dampening stellate cell activation. CONCLUSION: Reversion to a physiological dietary composition after HF feeding in foz/foz mice alters body weight distribution but not obesity. This attenuates NASH severity and fibrotic progression by suppressing NF-κB activation and reducing neutrophil and macrophage activation. However, adipose inflammation persists and is associated with continuing apoptosis in the residual fatty liver disease. Taken together, these findings indicate that other measures, such as weight reduction, may be required to fully reverse obesity-related NASH.


Assuntos
Dieta , Fígado Gorduroso/dietoterapia , Cirrose Hepática/dietoterapia , Fígado/patologia , Obesidade/dietoterapia , Tecido Adiposo/metabolismo , Animais , Apoptose/fisiologia , Antígenos CD36/genética , Antígenos CD36/metabolismo , Colesterol/metabolismo , Dieta Hiperlipídica/efeitos adversos , Gorduras na Dieta/administração & dosagem , Modelos Animais de Doenças , Fígado Gorduroso/complicações , Células Estreladas do Fígado/metabolismo , Inflamação/dietoterapia , Inflamação/patologia , Canais Iônicos/genética , Canais Iônicos/metabolismo , Metabolismo dos Lipídeos , Lipogênese/fisiologia , Fígado/metabolismo , Cirrose Hepática/patologia , Masculino , Metaloproteinase 2 da Matriz/genética , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/genética , Metaloproteinase 9 da Matriz/metabolismo , Camundongos , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , NF-kappa B/genética , NF-kappa B/metabolismo , Hepatopatia Gordurosa não Alcoólica , Obesidade/complicações , Obesidade/patologia , Inibidor Tecidual de Metaloproteinase-1/genética , Inibidor Tecidual de Metaloproteinase-1/metabolismo , Inibidor Tecidual de Metaloproteinase-2/genética , Inibidor Tecidual de Metaloproteinase-2/metabolismo , Triglicerídeos/metabolismo , Proteína Desacopladora 2 , Regulação para Cima
17.
J Gastroenterol Hepatol ; 28(9): 1545-54, 2013 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-23574010

RESUMO

BACKGROUND AND AIM: By array-comparative genomic hybridization, we demonstrated cyclin E as one of seven genes associated with hepatocellular carcinoma (HCC) development in Ku70 DNA repair-deficient mice. We therefore explored the hypothesis that during hepatocarcinogenesis, cyclin E kinase can overcome the inhibitory effects of p53 and establish whether abnormal miRNA(mi-R)-34, a co-regulator of cyclin E and p53, can account for their interactions as "drivers" of HCC. METHODS: Dysplastic hepatocytes (DNs) and HCCs were generated from diethylnitrosamine (DEN)-injected C57BL/6 male mice at 3-12 months. RESULTS: Cyclin E/cdk2 was barely expressed in normal liver, but was readily detected in dysplastic hepatocytes, localizing to glutathione-S transferase pi-form positive cells dissected by laser-dissection. Cyclin E kinase activity preceded cyclin D1, proliferating cell nuclear antigen expression in DNs and HCCs despite maximal p53 and p21 expression. We confirmed that cyclin E, rather than cyclin D1, is the proliferative driver in hepatocarcinogenesis by immunoprecipitation experiments demonstrating preferential binding of p21 to cyclin D1, allowing cyclin E-mediated "escape" from G1/S checkpoint. We then showed cyclin E was responsible for regulating wild-type p53 by knockdown experiments in primary HCC cells; cyclin E-knockdown increased p53 and p21, diminished anti-apoptotic Bcl-XL and reduced cell viability. Conversely, blocking p53 augmented cyclin E, Bcl-XL expression and increased proliferation. Physiological interactions between cyclin E/p53/p21 were confirmed in primary hepatocytes. miR-34a,c were upregulated in dysplastic murine, human liver and HCCs compared with normal liver, and appeared to be linked to cyclin E/p53. CONCLUSION: Upregulation of functionally active cyclin E via miR34 with loss of p53 function is associated with cell-cycle checkpoint failure increasing proliferative drive that favors hepatocarcinogenesis.


Assuntos
Transformação Celular Neoplásica/patologia , Ciclina E/fisiologia , Pontos de Checagem da Fase G1 do Ciclo Celular/fisiologia , Hepatócitos/patologia , Neoplasias Hepáticas Experimentais/patologia , Animais , Apoptose , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Proliferação de Células , Transformação Celular Neoplásica/metabolismo , Ciclina E/biossíntese , Quinase 2 Dependente de Ciclina , Dietilnitrosamina , Técnicas de Silenciamento de Genes , Hepatócitos/metabolismo , Humanos , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Neoplasias Hepáticas Experimentais/induzido quimicamente , Neoplasias Hepáticas Experimentais/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , MicroRNAs , Células Tumorais Cultivadas , Proteína Supressora de Tumor p53/biossíntese , Proteína Supressora de Tumor p53/fisiologia
18.
J Hepatol ; 59(1): 144-52, 2013 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-23500152

RESUMO

BACKGROUND & AIMS: We have recently showed that hyperinsulinemia promotes hepatic free cholesterol (FC) accumulation in obese, insulin-resistant Alms1 mutant (foz/foz) mice with NASH. Here we tested whether cholesterol-lowering drugs reduce stress-activated c-Jun N-terminal kinase (JNK) activation, hepatocyte injury/apoptosis, inflammation, and fibrosis in this metabolic syndrome NASH model. METHODS: Female foz/foz and WT mice were fed HF (0.2% cholesterol) 16 weeks, before adding ezetimibe (5 mg/kg), atorvastatin (20 mg/kg), or both to diet, another 8 weeks. Hepatic lipidomic analysis, ALT, liver histology, Sirius Red morphometry, hepatic mRNA and protein expression and immunohistochemistry (IHC) for apoptosis (M30), macrophages (F4/80), and polymorphs (myeloperoxidase) were determined. RESULTS: In mice with NASH, ezetimibe/atorvastatin combination normalized hepatic FC but did not alter saturated free fatty acids (FFA) and had minimal effects on other lipids; ezetimibe and atorvastatin had similar but less profound effects. Pharmacological lowering of FC abolished JNK activation, improved serum ALT, apoptosis, liver inflammation/NAFLD activity score, designation as "NASH", macrophage chemotactic protein-1 expression, reduced macrophage and polymorph populations, and liver fibrosis. CONCLUSIONS: Cholesterol lowering with ezetimibe/atorvastatin combination reverses hepatic FC but not saturated FFA accumulation. This dampens JNK activation, ALT release, hepatocyte apoptosis, and inflammatory recruitment, with reversal of steatohepatitis pathology and liver fibrosis. Ezetimibe/statin combination is a potent, mechanism-based treatment that could reverse NASH and liver fibrosis.


Assuntos
Anticolesterolemiantes/uso terapêutico , Complicações do Diabetes/tratamento farmacológico , Fígado Gorduroso/tratamento farmacológico , Síndrome Metabólica/tratamento farmacológico , Adiponectina/sangue , Animais , Anticolesterolemiantes/administração & dosagem , Atorvastatina , Azetidinas/administração & dosagem , Azetidinas/uso terapêutico , Colesterol/metabolismo , Complicações do Diabetes/metabolismo , Complicações do Diabetes/patologia , Quimioterapia Combinada , Ezetimiba , Fígado Gorduroso/complicações , Fígado Gorduroso/metabolismo , Feminino , Ácidos Heptanoicos/administração & dosagem , Ácidos Heptanoicos/uso terapêutico , Insulina/sangue , Fígado/efeitos dos fármacos , Fígado/metabolismo , Fígado/patologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Síndrome Metabólica/complicações , Síndrome Metabólica/metabolismo , Camundongos , Camundongos Mutantes , Camundongos Obesos , Hepatopatia Gordurosa não Alcoólica , Pirróis/administração & dosagem , Pirróis/uso terapêutico
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