RESUMO
Sirtuin 1 (SIRT1) is a class III histone deacetylase that deacetylates histone and nonhistone proteins to regulate gene transcription and protein function. Because SIRT1 regulates very diverse responses such as apoptosis, insulin sensitivity, autophagy, differentiation, and stem cell pluripotency, it has been a challenge to reconcile how it orchestrates such pleiotropic effects. Here we show that SIRT1 serves as an important regulator of Wnt signaling. We demonstrate that SIRT1 loss of function leads to a significant decrease in the levels of all three Dishevelled (Dvl) proteins. Furthermore, we demonstrate that SIRT1 and Dvl proteins complex in vivo and that inhibition of SIRT1 leads to changes in gene expression of Wnt target genes. Finally, we demonstrate that Wnt-stimulated cell migration is inhibited by a SIRT1 inhibitor. Because the three mammalian Dvl proteins serve as key messengers for as many as 19 Wnt ligands, SIRT1-mediated regulation of Dvl proteins may explain the diverse physiological responses observed in different cellular contexts. Previously, SIRT1 had only been shown to mediate the epigenetic silencing of Wnt antagonists. In contrast, here we report that SIRT1 regulates Dvl protein levels and Wnt signaling in several cellular contexts. These findings demonstrate that SIRT1 is a regulator of transient and constitutive Wnt signaling.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Regulação da Expressão Gênica/fisiologia , Fosfoproteínas/metabolismo , Transdução de Sinais/fisiologia , Sirtuína 1/metabolismo , Proteínas Wnt/metabolismo , Western Blotting , Linhagem Celular , Movimento Celular/fisiologia , Primers do DNA/genética , Proteínas Desgrenhadas , Regulação da Expressão Gênica/genética , Humanos , Imunoprecipitação , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Ciclopirox olamine (CPX) is a synthetic antifungal agent clinically used to treat mycoses of the skin and nails. Here, we show that CPX inhibited tumor growth in human breast cancer MDA-MB-231 xenografts. To unveil the underlying mechanism, we further studied the antitumor activity of CPX in cell culture. The results indicate that CPX inhibited cell proliferation and induced apoptosis in human rhabdomyosarcoma (Rh30), breast carcinoma (MDA-MB231) and colon adenocarcinoma (HT-29) cells in a concentration-dependent manner. By cell cycle analysis, CPX induced accumulation of cells in G(1)/G(0) phase of the cell cycle. Concurrently, CPX downregulated cellular protein expression of cyclins (A, B1, D1 and E) and cyclin-dependent kinases (CDK2 and CDK4) and upregulated expression of the CDK inhibitor p21(Cip1), leading to hypophosphorylation of retinoblastoma protein. CPX also downregulated protein expression of Bcl-xL and survivin and enhanced cleavages of Bcl-2. Z-VAD-FMK, a pan-caspase inhibitor, partially prevented CPX-induced cell death, suggesting that CPX-induced apoptosis of cancer cells is mediated at least in part through caspase-dependent mechanism. The results indicate that CPX is a potential antitumor agent.
Assuntos
Neoplasias da Mama/tratamento farmacológico , Piridonas/farmacologia , Animais , Apoptose/efeitos dos fármacos , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Caspase 3/metabolismo , Caspase 7/metabolismo , Inibidores de Caspase , Ciclo Celular/efeitos dos fármacos , Processos de Crescimento Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Ciclopirox , Quinase 2 Dependente de Ciclina/antagonistas & inibidores , Quinase 2 Dependente de Ciclina/biossíntese , Quinase 4 Dependente de Ciclina/antagonistas & inibidores , Quinase 4 Dependente de Ciclina/biossíntese , Inibidor de Quinase Dependente de Ciclina p21/biossíntese , Ciclinas/antagonistas & inibidores , Ciclinas/biossíntese , Humanos , Proteínas Inibidoras de Apoptose , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Proteínas Associadas aos Microtúbulos/biossíntese , Fosforilação/efeitos dos fármacos , Poli(ADP-Ribose) Polimerases/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteína do Retinoblastoma/metabolismo , Survivina , Ensaios Antitumorais Modelo de Xenoenxerto , Proteína bcl-X/biossínteseRESUMO
BACKGROUND: Previously we reported that mice deficient in toll-like receptor 4 (TLR-4) signalling were protected from diet-induced non-alcoholic steatohepatitis (NASH). Another member of the toll-like receptor family, TLR-2, has been shown to play a role in lipid trafficking via uptake of diacylated lipoproteins. However, a role for TLR-2 in NASH has not been elucidated. The objectives of the current study were to examine the influence of dietary fat quality and TLR-2 on NASH pathogenesis. METHODS: Steatohepatitis was induced in male Db, C57BL/6 and TLR-2(-/-) mice by feeding an L-amino acid-defined diet that was deficient in methionine and choline (MCDD). Mice fed the base diet supplemented with methionine and choline (control diet; CD) were used as controls. To determine the role of fat quality, MCDD was enriched with polyunsaturated corn oil (PUFA) or coconut oil that is comprised mostly of saturated fat (SAFA); the total amount of each fat was 112.9 g/kg of diet. After 8 weeks of feeding CD or MCDD, hepatic steatosis, inflammation and necrosis were evaluated in histological sections. Total RNA was extracted from frozen liver samples and mRNA expression of TNFalpha, collagen alpha1, IL-10, peroxisome proliferator-activated receptor-gamma (PPAR-gamma), TLR-4, and CD14, was analyzed via real-time PCR. Protein levels of TLR-2 were analyzed by western blot. RESULTS: Panlobular macrovessicular steatosis and diffuse leukocyte infiltration were noted in PUFA-fed Db mice. Histological scores demonstrated significantly less steatosis, inflammation and necrosis in SAFA-fed mice of all mouse strains. However, compared to wild type mice, hepatocellular damage was notably more severe in TLR-2(-/-) mice. Consistent with histological findings, mRNA expression of TNFalpha was elevated by approximately 3-fold in TLR-2(-/-) mice; PPAR-gamma expression was blunted in this strain compared to wild type. Expression of the matrix protein collagen alphaI was also significantly higher in TLR-2(-/-) mice, indicating a pro-fibrogenic state. Sensitivity to steatohepatitis due to dietary fat or TLR-2 deficiency correlated significantly with alterations in the expression of TLR-4 as well as the co-receptor CD-14. CONCLUSIONS: Our findings suggest that dietary saturated fat plays a protective role against MCDD-induced steatohepatitis, whereas TLR-2 deficiency exacerbated NASH. The mechanism underlying the response to dietary fat and TLR-2 likely involves altered signalling via the TLR-4 pathway.
Assuntos
Deficiência de Colina , Gorduras na Dieta/efeitos adversos , Fígado Gorduroso/etiologia , Metionina/deficiência , Receptor 2 Toll-Like/deficiência , Animais , Colágeno Tipo I/metabolismo , Gorduras Insaturadas na Dieta/efeitos adversos , Modelos Animais de Doenças , Fígado Gorduroso/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , PPAR gama/metabolismo , Transdução de Sinais/fisiologia , Receptor 2 Toll-Like/genética , Receptor 4 Toll-Like/metabolismo , Fator de Necrose Tumoral alfa/metabolismoRESUMO
BACKGROUND: Obese patients display an exaggerated morbidity during sepsis. Since consumption of a western-style diet (WD) is a major factor for obesity in the United States, the purpose of the present study was to examine the influence of chronic WD consumption on hepatic inflammation in mice made septic via cecal ligation and puncture (CLP). Feeding mice diets high in fat has been shown to enhance evidence of TLR signaling and this pathway also mediates the hepatic response to invading bacteria. Therefore, we hypothesized that the combined effects of sepsis and feeding WD on TRL-4 signaling would exacerbate hepatic inflammation. Male C57BL/6 mice were fed purified control diet (CD) or WD that was enriched in butter fat (34.4% of calories) for 3 weeks prior to CLP. Intravital microscopy was used to evaluate leukocyte adhesion in the hepatic microcirculation. To demonstrate the direct effect of saturated fatty acid on hepatocytes, C3A human hepatocytes were cultured in medium containing 100 µM palmitic acid (PA). Quantitative real-time PCR was used to assess mRNA expression of tumor necrosis factor-alpha (TNF-α, monocyte chemotactic protein-1 (MCP-1), intercellular adhesion molecule-1 (ICAM-1), toll-like receptor-4 (TLR-4) and interleukin-8 (IL-8). RESULTS: Feeding WD increased firm adhesion of leukocytes in the sinusoids and terminal hepatic venules by 8-fold six hours after CLP; the increase in platelet adhesion was similar to the response observed with leukocytes. Adhesion was accompanied by enhanced expression of TNF-α, MCP-1 and ICAM-1. Messenger RNA expression of TLR-4 was also exacerbated in the WD+CLP group. Exposure of C3A cells to PA up-regulated IL-8 and TLR-4 expression. In addition, PA stimulated the static adhesion of U937 monocytes to C3A cells, a phenomenon blocked by inclusion of an anti-TLR-4/MD2 antibody in the culture medium. CONCLUSIONS: These findings indicate a link between obesity-enhanced susceptibility to sepsis and consumption of a western-style diet.
Assuntos
Dieta/efeitos adversos , Ácidos Graxos/efeitos adversos , Hepatite/metabolismo , Fígado/imunologia , Sepse/fisiopatologia , Animais , Ceco , Quimiocina CCL2/metabolismo , Modelos Animais de Doenças , Ácidos Graxos/administração & dosagem , Humanos , Inflamação/metabolismo , Molécula 1 de Adesão Intercelular/metabolismo , Interleucina-8/metabolismo , Ligadura , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Receptor 4 Toll-Like/metabolismo , Fator de Necrose Tumoral alfa/metabolismoRESUMO
BACKGROUND AND AIMS: Feeding a Western diet (WD) enriched in saturated fat protects against chronic alcoholic hepatitis. However, saturated fat induces lipotoxicity in cultured hepatocytes. The purpose of the present study was to elucidate the influence of WD on acute hepatic injury and healing. METHODS: Male C57BL/6 mice were fed a purified control diet (CD) or WD enriched in palmitate and cholesterol. After 3 weeks, carbon tetrachloride (CCl(4)) was administered (0.1 microL/g, intraperitoneally). Hepatic inflammation and proliferation were assessed by immunostaining for neutrophils and intracellular adhesion molecule-1, and Ki67, respectively. Cytokine expression was analyzed by real-time polymerase chain reaction. Protein levels of peroxisome proliferator-activated receptor-gamma (PPAR-gamma) were assessed by western blotting. RESULTS: Feeding a WD resulted in markedly greater histological evidence of necrosis and enhanced alanine aminotransferase activity (188 +/- 6.2 U/L) compared to CD-fed mice (99.1 +/- 6.3 U/L) by day 2 post-CCl(4). In contrast, WD blunted leukocyte accumulation in necrotic areas and the expression of cytokines (tumor necrosis factor-alpha and interleukin-6) involved in tissue regeneration. Diminished repair was further indexed by lower collagen-alphaI and Ki67 expression in the mice fed a WD. Finally, feeding a WD, as well as the treatment of cultured hepatocytes with palmitic acid, upregulated the expression of PPAR-gamma, which has been previously shown to prevent hepatic repair following CCl(4) exposure. CONCLUSIONS: These findings suggest that impaired healing in WD-fed mice blunted recovery from acute injury and underscored the complex relationship between diet and hepatic injury.
Assuntos
Dieta , Ácidos Graxos/efeitos adversos , Hepatopatias/metabolismo , Hepatopatias/prevenção & controle , Alanina Transaminase/metabolismo , Animais , Biomarcadores/metabolismo , Western Blotting , Tetracloreto de Carbono , Doença Hepática Induzida por Substâncias e Drogas/complicações , Colágeno Tipo I/metabolismo , Citocinas/metabolismo , Modelos Animais de Doenças , Ácidos Graxos/administração & dosagem , Inflamação/metabolismo , Injeções Intraperitoneais , Molécula 1 de Adesão Intercelular/metabolismo , Antígeno Ki-67/metabolismo , Hepatopatias/genética , Hepatopatias/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Necrose/induzido quimicamente , Neutrófilos/metabolismo , PPAR gama/metabolismo , Reação em Cadeia da PolimeraseRESUMO
The present study addressed the hypothesis that leptin promotes leukocyte trafficking into adipose tissue. Accordingly, male Wistar rats were treated with saline or recombinant rat leptin (1 mg/kg) via the tail vein. Leukocyte trafficking in mesenteric venules was quantified by intravital microscopy. Treatment with leptin resulted in a 3- and 5-fold increases in rolling and firm adhesion, respectively. Compared to vehicle controls, leptin enhanced mRNA levels of IL-6 (8-fold) and MCP-1 (5-fold) in mesenteric adipose tissue (MAT). Similar increases in these markers were observed in mesenteric venules and in liver. Finally, the direct effect of leptin was assessed in C3A hepatocytes treated with leptin for 24 hours (7.8 ng/mL-125 ng/mL). Consistent with observations in vivo, production of ICAM-1, MCP-1, and IL-6 by hepatocytes was increased significantly. These findings support the hypothesis that leptin directly initiates inflammation in the local environment of mesenteric adipose tissue as well as systemically.
Assuntos
Inflamação , Leptina/metabolismo , Tecido Adiposo/metabolismo , Animais , Quimiocina CCL2/metabolismo , Hepatócitos/metabolismo , Humanos , Molécula 1 de Adesão Intercelular/metabolismo , Interleucina-6/metabolismo , Masculino , Modelos Biológicos , Fenótipo , Ratos , Ratos Wistar , Fatores de TempoRESUMO
The worldwide proportion of overweight and obese individuals has increased yearly for more than a decade. Along with rates of obesity, the incidence of co-morbid conditions such as type 2 diabetes, cardiovascular disease and liver disease has also increased. The form of liver disease associated with obesity is termed non-alcoholic steatohepatitis (NASH) due to the histological similarities to livers of chronic alcoholics. NASH has been observed in adult as well as pediatric populations; however, the etiology of this disease is still unknown. This review outlines some of the risk factors commonly associated with NASH and describes molecular mechanisms proposed to underlie disease pathogenesis.
RESUMO
OBJECTIVES: To characterize children at risk for nonalcoholic fatty liver disease (NAFLD) and to explore possible mechanisms underlying the development of NAFLD in Hispanic youth with a body mass index > or =95th percentile. PATIENTS AND METHODS: Hispanic nonoverweight (n = 475) and overweight (n = 517) children, ages 4 to 19 y, were characterized in terms of body composition (dual-energy x-ray absorptiometry), maturation (Tanner stage), diet (24-h recall), physical activity (accelerometry), fitness (maximal oxygen uptake), and biochemical profile (fasting alanine aminotransferase [ALT], glucose, insulin, and lipids; inflammation markers such as adiponectin, leptin, C-reactive protein, and soluble intercellular adhesion molecule-1; and total antioxidants) using standard laboratory techniques. Risk for NAFLD was defined by fasting serum ALT values >97.5th percentile for age- and sex-specific reference values. RESULTS: Fasting serum ALT was elevated in 24% of overweight children and in only 4% of nonoverweight children. Therefore, to identify risk factors associated with elevated ALT, the remaining statistical analysis was restricted to the overweight group. The percentage of overweight children with elevated ALT did not differ by sex, age, or Tanner stage. Weight, body mass index, z score, waist-to-hip ratio, fat-free mass, fat mass, and percent truncal fat mass were higher in the overweight children with elevated ALT. Fasting insulin, glucose, and homeostasis model-insulin resistance were higher in the overweight children with elevated ALT, as were triglycerides, total cholesterol, low-density lipoprotein, thyroid-stimulating hormone, and triiodothyronine. Fasting serum leptin, C-reactive protein, and soluble intercellular adhesion molecule-1 were significantly higher and adiponectin was lower in the overweight children with elevated ALT. CONCLUSIONS: The risk for developing NAFLD was high in the overweight Hispanic children. The proportion of "at risk" children was not influenced by gender, age, or maturation. The risk for elevated ALT was predicted by the severity of obesity, central adiposity, hyperinsulinemia, hypertriglyceridemia, elevated thyroid-stimulating hormone, and systemic inflammation.
Assuntos
Fígado Gorduroso/etiologia , Obesidade/complicações , Adolescente , Adulto , Fatores Etários , Composição Corporal , Índice de Massa Corporal , Criança , Pré-Escolar , Dieta , Fígado Gorduroso/sangue , Hispânico ou Latino , Humanos , Atividade Motora , Obesidade/sangue , Obesidade/diagnóstico , Sobrepeso , Risco , Fatores Sexuais , Maturidade SexualRESUMO
C57BL/6 mice were subjected to hindlimb unloading (HU) for a period of 3 wk to determine the possible effects on epithelial wound healing. A standardized corneal epithelial wound was performed, and parameters of the inflammatory response and reepithelialization were analyzed over an observation period of 96 h. Wound closure was significantly retarded in mice during HU with reepithelialization being delayed by approximately 12 h. Both epithelial migration and cell division were significantly depressed and delayed. The inflammatory response to epithelial wounding was also significantly altered during HU. Neutrophils, as detected by the Gr-1 marker, were initially elevated above normal levels before wounding and during the first few hours afterward, but there was a significant reduction in neutrophil response to wounding at times where neutrophil influx and migration in controls were vigorous. A similar pattern was seen with CD11b+CD11c+ cells (monocyte lineage). Langerhans cells are normally resident within the peripheral corneal epithelium. They respond to injury by initially leaving the epithelial site within 6 h and returning to normal levels by 96 h, 2 days after reepithelialization is complete. During HU, this pattern is distinctly different, with Langerhans cell numbers slowly diminishing, reaching a nadir at 96 h, which is significantly below normal. Evidence for systemic effects of HU is provided by findings that collagen deposition within subcutaneous sponges was significantly reduced during HU. In conclusion, HU, a ground-based model simulating some physiological aspects of spaceflight, impairs wound repair of corneas. Multiple factors, both local and systemic, likely contribute to this delayed wound healing.
Assuntos
Epitélio Corneano/imunologia , Epitélio Corneano/fisiopatologia , Elevação dos Membros Posteriores , Cicatrização/fisiologia , Animais , Antígeno CD11b/metabolismo , Antígeno CD11c/metabolismo , Divisão Celular , Movimento Celular/fisiologia , Epitélio Corneano/lesões , Células de Langerhans/citologia , Células de Langerhans/imunologia , Células de Langerhans/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neutrófilos/fisiologia , Simulação de Ausência de PesoRESUMO
Mammalian sirtuin 1 (SIRT1) may control fatty acid homeostasis in liver. However, this possibility and underlying mechanism remain to be established. In this study, we addressed the issues by examining the metabolic phenotypes of SIRT1 heterozygous knockout (SIRT1(+/-)) mice. The study was conducted in the mice on three different diets including a low-fat diet (5% fat wt/wt), mediate-fat diet (11% fat wt/wt), and high-fat diet (HFD, 36% fat wt/wt). On low-fat diet, the mice did not exhibit any abnormality. On mediate-fat diet, the mice exhibited a significant increase in hepatic steatosis with elevated liver/body ratio, liver size, liver lipid (triglyceride, glycerol, and cholesterol) content, and liver inflammation. The hepatic steatosis was deteriorated in the mice by HFD. In the liver, lipogenesis was increased, fat export was reduced, and beta-oxidation was not significantly changed. Body weight and fat content were increased in response to the dietary fat. Fat was mainly increased in sc adipose tissue and liver. Inflammation was also elevated in epididymal fat. Whole body energy expenditure and substrate utilization were reduced. Food intake, locomotor activity, and fat absorption were not changed. These data suggest that a reduction in the SIRT1 activity increases the risk of fatty liver in response to dietary fat. The liver steatosis may be a result of increased lipogenesis and reduced liver fat export. The inflammation may contribute to the pathogenesis of hepatic steatosis as well. A reduction in lipid mobilization may contribute to the hepatic steatosis and low energy expenditure.
Assuntos
Fígado Gorduroso/imunologia , Fígado Gorduroso/metabolismo , Inflamação/fisiopatologia , Metabolismo dos Lipídeos/fisiologia , Sirtuína 1/genética , Sirtuína 1/metabolismo , Animais , Western Blotting , Metabolismo Energético/genética , Metabolismo Energético/fisiologia , Fígado Gorduroso/genética , Genótipo , Inflamação/genética , Metabolismo dos Lipídeos/genética , Fígado/imunologia , Fígado/metabolismo , Fígado/patologia , Imageamento por Ressonância Magnética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
BACKGROUND/AIMS: Sepsis remains a leading cause of death in critically ill patients. Because endotoxemia is viewed as a key mediator of sepsis-induced inflammation, administration of bacterial endotoxin (LPS) is often used to simulate sepsis in experimental animals. This study tests the hypothesis that LPS is a critical determinant of the hepatic microvascular dysfunction in mice made septic by cecal ligation and puncture (CLP). METHODS: Intravital videomicroscopy was used to quantify sinusoidal perfusion, and platelet and leukocyte adhesion in terminal hepatic venules (THV) and sinusoids in LPS-sensitive and LPS-insensitive mice subjected to CLP or LPS (i.p.). mRNA expression of TLR-2, TLR-4, MyD-88, and Ly-96 was also assessed. RESULTS: While LPS-sensitive mice responded to both CLP and LPS challenges with elevated leukocyte and platelet adhesion in THV and sinusoids, and a reduced sinusoidal perfusion density, LPS-insensitive mice exhibited comparable blood cell adhesion and sinusoidal malperfusion following CLP, but not LPS. Hepatic mRNA of MyD-88 and TLR-2 was elevated in the CLP and LPS groups. Endotoxin was not detectable in the blood of LPS-sensitive mice after CLP, but was elevated after LPS administration. CONCLUSIONS: These findings do not support a major role for LPS in the hepatic microvascular disturbances associated with polymicrobial sepsis.
Assuntos
Lipopolissacarídeos/farmacologia , Circulação Hepática/efeitos dos fármacos , Hepatopatias/etiologia , Sepse/etiologia , Animais , Plaquetas/citologia , Adesão Celular , Modelos Animais de Doenças , Endotoxinas/sangue , Leucócitos/citologia , Hepatopatias/microbiologia , Camundongos , Microcirculação/efeitos dos fármacos , Microcirculação/fisiopatologia , Microscopia de VídeoRESUMO
BACKGROUND/AIMS: Studies in animal models and humans suggest a link between endotoxemia and non-alcoholic steatohepatitis. Since Kupffer cells are responsible for clearing endotoxin and are activated via endotoxin interaction with Toll-like receptor 4 (TLR-4), we examined the relationship between hepatic TLR-4 expression and Kupffer cell content during the genesis of steatohepatitis. METHODS: Male C57BL/6, C3H/HouJ and TLR-4 mutant C3H/HeJ mice were fed control or methionine/choline-deficient diet (MCDD). In one group of C57BL/6 mice, Kupffer cells were depleted by weekly intraperitoneal injections of clodronate liposomes. After 3 weeks, serum ALT activity and portal endotoxin levels were measured. Real-time PCR was used to examine mRNA expression of TLR-4, TLR-2, CD14, MD-2, TGFbeta, TNFalpha, CD36, PPAR-alpha, liver fatty acid binding protein (L-FABP) and collagen alpha1. RESULTS: We observed histological evidence typical of steatohepatitis, portal endotoxemia and enhanced TLR-4 expression in wild type mice fed MCDD. In contrast, injury and lipid accumulation markers were significantly lower in TLR-4 mutant mice. Destruction of Kupffer cells with clodronate liposomes blunted histological evidence of steatohepatitis and prevented increases in TLR-4 expression. CONCLUSIONS: These findings demonstrate the importance of TLR-4 signaling and underscore a direct link between TLR-4 and Kupffer cells in the pathogenesis of steatohepatitis.