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The innate immune response contributes to the development or attenuation of acute and chronic diseases, including cancer. Microbial DNA and mislocalized DNA from damaged host cells can activate different host responses that shape disease outcomes. Here, we show that mice and humans lacking a single allele of the DNA repair protein Ku70 had increased susceptibility to the development of intestinal cancer. Mechanistically, Ku70 translocates from the nucleus into the cytoplasm where it binds to cytosolic DNA and interacts with the GTPase Ras and the kinase Raf, forming a tripartite protein complex and docking at Rab5+Rab7+ early-late endosomes. This Ku70-Ras-Raf signalosome activates the MEK-ERK pathways, leading to impaired activation of cell cycle proteins Cdc25A and CDK1, reducing cell proliferation and tumorigenesis. We also identified the domains of Ku70, Ras, and Raf involved in activating the Ku70 signaling pathway. Therapeutics targeting components of the Ku70 signalosome could improve the treatment outcomes in cancer.
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Neoplasias , Transdução de Sinais , Animais , Humanos , Camundongos , Proliferação de Células , DNA , Sistema de Sinalização das MAP Quinases , Neoplasias/genéticaRESUMO
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.
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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/metabolismoRESUMO
Liver stiffness measurement (LSM) by FibroScan-determined transient elastography is a noninvasive approach to estimate liver fibrosis severity. In non-alcoholic fatty liver disease (NAFLD), advanced liver fibrosis is excluded by normal liver stiffness, but a wide range of cutoffs have been used to predict advanced liver fibrosis or cirrhosis. This may be partly because steatosis (measured by controlled attenuation parameter [CAP]) contributes to liver stiffness and also because LSM fluctuates in NAFLD. In a recent pivotal study, one-third of patients with liver stiffness > 12.0 kPa showed reversal after 4-6 months; these cases did not have advanced liver fibrosis on biopsy. We performed serial FibroScans 6-36 months apart in 73 NAFLD patients, 38 with LSM > 10 kPa at entry. Those who lost ≥ 1 kg of weight (n = 31) significantly reduced liver stiffness (3.6 ± 6.1 vs 0.53 ± 4.1 kPa, P < 0.05) and CAP score (39 ± 63 dB/m of loss vs 24 ± 65 dB/m of gain, P < 0.05) compared with those who did not (n = 29). Patients who reported increased physical activity (n = 25) also reduced liver stiffness (3.6 ± 6 vs 0.35 ± 6 kPa) and CAP (20 ± 71 dB/m of loss vs 32 ± 71 dB/m of gain). Overall, those with improved LSM were significantly more likely to have lost weight and/or improved physical activity. These effects of lifestyle adjustments partly explain why a single measurement of 12.0 kPa is not a reliable cutoff for advanced liver fibrosis in NAFLD. In addition to repeating the study after 6-12 months, documentation of response to lifestyle advice and weight reduction should be determined before assuming any cutoff indicates advanced liver fibrosis. Despite this reservation about diagnostic accuracy, we consider that measurement of liver stiffness and CAP score serve to motivate patients to enact lifestyle modifications that can improve NAFLD severity.
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Técnicas de Imagem por Elasticidade/métodos , Elasticidade , Cirrose Hepática/diagnóstico por imagem , Cirrose Hepática/fisiopatologia , Fígado/diagnóstico por imagem , Fígado/fisiopatologia , Hepatopatia Gordurosa não Alcoólica/complicações , Hepatopatia Gordurosa não Alcoólica/diagnóstico por imagem , Exercício Físico , Humanos , Estilo de Vida , Cirrose Hepática/etiologia , Motivação , Educação de Pacientes como Assunto , Pacientes/psicologia , Índice de Gravidade de DoençaRESUMO
BACKGROUND: Inequalities in access to renal services and acute care for rural and remote populations in Australia have been described but not quantified. AIM: To describe: the coverage of renal disease management services in rural and remote Australia; and the characteristics of patients who had an aeromedical retrieval for renal disease by Australia's Royal Flying Doctor Service (RFDS). METHODS: Data from the RFDS, the Australian Bureau of Statistics, and Health Direct were used to estimate provision of renal disease management services by geographic area. RFDS patient diagnostic data were prospectively collected from 2014 to 2018. RESULTS: Many rural and remote areas have limited access to regular renal disease management services. Most RFDS retrievals for renal disease are from regions without such services. The RFDS conducted 1636 aeromedical retrievals for renal disease, which represented 1.6% of all retrievals. Among retrieved patients, there was a higher proportion of men than women (54.6% vs 45.4%, P < 0.01), while indigenous patients (n = 546, 33.4%) were significantly younger than non-indigenous patients (40.9 vs 58.5, P < 0.01). There were significant differences in underlying diagnoses triggering retrievals between genders, with males being more likely than females to be transferred with acute renal failure, calculus of the kidney and ureter, renal colic, obstructive uropathy, and kidney failure (all P < 0.01). Conversely, females were more likely to have chronic kidney disease, disorders of the urinary system, acute nephritic syndrome, tubulo-interstitial nephritis, and nephrotic syndrome (all P < 0.01). CONCLUSION: Aeromedical retrievals for acute care were from rural areas without regular access to renal disease prevention or management services.
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Resgate Aéreo , Serviços de Saúde Rural , Austrália/epidemiologia , Gerenciamento Clínico , Feminino , Acessibilidade aos Serviços de Saúde , Humanos , Rim , Masculino , População RuralAssuntos
Hepatopatia Gordurosa não Alcoólica , Austrália , Hospitais Urbanos , Humanos , Prevalência , Fatores de RiscoRESUMO
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 (Nâ¯=â¯29) or without (Nâ¯=â¯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.
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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/farmacologiaRESUMO
Nonalcoholic steatohepatitis (NASH) arises from a variable interplay between environmental factors and genetic determinants that cannot be completely replicated in animals. Notwithstanding, preclinical models are needed to understand NASH pathophysiology and test mechanism-based therapies. Among several mouse models of NASH, some exhibit the key pathophysiologic as well as histopathologic criteria for human NASH, whereas others may be useful to address specific questions. Models based on overnutrition with adipose restriction/inflammation and metabolic complications, particularly insulin resistance, may be most useful to investigate critical etiopathogenic factors. In-depth pathologic description is required for all models. Some models demonstrate hepatocyte ballooning, which can be confused with microvesicular steatosis, whereas demonstration of an inflammatory infiltrate and pattern of liver fibrosis compatible with human NASH is desirable in models used for pharmacologic testing. When mice with specific genetic strains or mutations that cause overeating consume a diet enriched with fat, modest amounts of cholesterol, and/or simple sugars ("Western diet"), they readily develop obesity with liver disease similar to human NASH, including significant fibrosis. Purely dietary models, such as high-fat/high-cholesterol, Western diet, and choline-deficient, amino acid-defined, are similarly promising. We share concern about using models without weight gain, adipose pathology, or insulin resistance/hyperinsulinemia and with inadequate documentation of liver pathology. NASH-related fibrosis is a key endpoint in trials of possible therapies. When studied for this purpose, NASH models should be reproducible and show steatohepatitis (ideally with ballooning) and at least focal bridging fibrosis, while metabolic factors/disordered lipid partitioning should contribute to etiopathogenesis. Because murine models are increasingly used to explore pharmacologic therapies for NASH, we propose a minimum set of requirements that investigators, drug companies, and journals should consider to optimize their translational value.
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Modelos Animais de Doenças , Hepatopatia Gordurosa não Alcoólica , Animais , Progressão da Doença , Cirrose Hepática , CamundongosRESUMO
BACKGROUND: Liver regeneration following partial hepatectomy (PHx) is a complicated process involving multiple organs and several types of signaling networks. The bile acid-activated metabolic pathways occupy an auxiliary yet important chapter in the entire biochemical story. PHx is characterized by rapid but transient bile acid overload in the liver, which constitutes the first wave of proliferative signaling in the remnant hepatocytes. Bile acids trigger hepatocyte proliferation through activation of several nuclear receptors. Following biliary passage into the intestines, enterocytes reabsorb the bile acids, which results in the activation of farnesoid X receptor (FXR), the consequent excretion of fibroblast growth factor (FGF)19/FGF15, and its release into the enterohepatic circulation. FGF19/FGF15 subsequently binds to its cognate receptor, fibroblast growth factor receptor 4 (FGFR4) complexed with ß-klotho, on the hepatocyte membrane, which initiates the second wave of proliferative signaling. Because some bile acids are toxic, the remnant hepatocytes must resolve the potentially detrimental state of bile acid excess. Therefore, the hepatocytes orchestrate a bile acid detoxification and elimination response as a protective mechanism in concurrence with the proliferative signaling. The response in part results in the excretion of (biotransformed) bile acids into the canalicular system, causing the bile acids to end up in the intestines. RELEVANCE FOR PATIENTS: Recently, FXR agonists have been shown to promote regeneration via the gut-liver axis. This type of pharmacological intervention may prove beneficial for patients with hepatobiliary tumors undergoing PHx. In light of these developments, the review provides an in-depth account of the pathways that underlie post-PHx liver regeneration in the context of bile acid homeostasis in the liver and the gut-liver signaling axis.
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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 â¼25% 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.
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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/genéticaRESUMO
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.
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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/metabolismoRESUMO
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.
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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 , SulfonamidasRESUMO
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.
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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.
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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/metabolismoRESUMO
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.
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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/complicaçõesRESUMO
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/metabolismoRESUMO
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 terapêuticoRESUMO
Peroxisome proliferator-activated receptor alpha (PPARα) ligands have been reported to suppress cancer growth. However, the role of PPARα in hepatocarcinogenesis remains unclear. We investigated the functional significance of PPARα in HCC. PPARα-knockout (PPARα-/-) mice were more susceptible to diethylnitrosamine (DEN)-induced HCC at 6 months compared with wild-type (WT) littermates (80% versus 43%, P < 0.05). In resected HCCs, TUNEL-positive apoptotic cells were significantly less in PPARα-/- mice than in WT mice (P < 0.01), commensurate with a reduction in cleaved caspase-3 and caspase-7 protein expression. Ki-67 staining showed increased cell proliferation in PPARα-/- mice (P < 0.01), with concomitant up-regulation of cyclin-D1 and down-regulation of p15. Moreover, ectopic expression of PPARα in HCC cells significantly suppressed cell proliferation and induced apoptosis. The anti-tumorigenic function of PPARα was mediated via NF-κB as evidenced by inhibition of NF-κB promoter activity, diminution of phosphor-p65, phosphor-p50 and BCL2 levels, and enhancing IkBα protein. Chromatin immunoprecipitation analysis confirmed PPARαdirectly binds to the IkBα promoter. In conclusion, PPARα deficiency enhances susceptibility to DEN-initiated HCC. PPARα suppresses tumor cell growth by inhibiting cell proliferation and inducing cell apoptosis via direct targeting IκBα and NF-κB signaling pathway.
Assuntos
Carcinoma Hepatocelular/prevenção & controle , Transformação Celular Neoplásica/metabolismo , Neoplasias Hepáticas/prevenção & controle , Fígado/metabolismo , NF-kappa B/metabolismo , PPAR alfa/metabolismo , Transdução de Sinais , Animais , Apoptose , Proteínas Reguladoras de Apoptose/metabolismo , Sítios de Ligação , Carcinoma Hepatocelular/induzido quimicamente , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Proliferação de Células , Transformação Celular Neoplásica/patologia , Dietilnitrosamina , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Células Hep G2 , Humanos , Proteínas I-kappa B/genética , Proteínas I-kappa B/metabolismo , Fígado/patologia , Neoplasias Hepáticas/induzido quimicamente , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Inibidor de NF-kappaB alfa , PPAR alfa/deficiência , PPAR alfa/genética , Regiões Promotoras Genéticas , Fatores de Tempo , TransfecçãoRESUMO
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.