RESUMEN

Glucose is a major energy source for the entire body, while fructose metabolism occurs mainly in the liver. Fructose consumption has increased over the last decade globally and is suspected to contribute to the increased incidence of non-alcoholic fatty liver disease (NAFLD). NAFLD is a manifestation of metabolic syndrome affecting about one-third of the population worldwide and has progressive pathological potential for liver cirrhosis and cancer through non-alcoholic steatohepatitis (NASH). Here we have reviewed the possible contribution of fructose to the pathophysiology of NAFLD. We critically summarize the current findings about several regulators, and their potential mechanisms, that have been studied in humans and animal models in response to fructose exposure. A novel hypothesis on fructose-dependent perturbation of liver regeneration and metabolism is advanced. Fructose intake could affect inflammatory and metabolic processes, liver function, gut microbiota, and portal endotoxin influx. The role of the brain in controlling fructose ingestion and the subsequent development of NAFLD is highlighted. Although the importance for fructose (over)consumption for NAFLD in humans is still debated and comprehensive intervention studies are invited, understanding of how fructose intake can favor these pathological processes is crucial for the development of appropriate noninvasive diagnostic and therapeutic approaches to detect and treat these metabolic effects. Still, lifestyle modification, to lessen the consumption of fructose-containing products, and physical exercise are major measures against NAFLD. Finally, promising drugs against fructose-induced insulin resistance and hepatic dysfunction that are emerging from studies in rodents are reviewed, but need further validation in human patients.

Asunto(s)

Fructosa/efectos adversos , Enfermedad del Hígado Graso no Alcohólico/etiología , Edulcorantes/efectos adversos , Animales , Dieta , Ejercicio Físico/fisiología , Fructosa/administración & dosificación , Humanos , Incidencia , Hígado/metabolismo , Hígado/fisiopatología , Enfermedad del Hígado Graso no Alcohólico/epidemiología , Enfermedad del Hígado Graso no Alcohólico/fisiopatología , Factores de Riesgo , Edulcorantes/administración & dosificación

RESUMEN

BACKGROUND: Melanocortin 4 receptor (MC4R) is predominantly recognized to mediate energy metabolism and anti-inflammation through the central nervous system. However, the expression of MC4R has recently been identified in rat liver and was shown to be upregulated during acute phase response. This study aims to investigate potential roles of MC4R in liver regeneration. MATERIALS AND METHODS: Rat partial hepatectomy (PH) was performed, and MC4R expression was analyzed at different time points after resection. Sham-operated animals (SH) served as controls. In vitro primary hepatocytes (HCs) were isolated from normal rat liver and stimulated with α-melanocyte-stimulating hormone (MC4R agonist). Real-time polymerase chain reaction, Western blot, and immunofluorescence staining were applied to detect gene expression. RESULTS: Up to 8 h after PH, hepatic messenger RNA of proinflammatory cytokines interleukin 6 and tumor necrosis factor α reached peak values. Between 8 and 72 h after PH, rat liver regeneration was extremely active as assessed by the regeneration indices labeled by Ki-67. Immunofluorescence staining indicated that MC4R was mostly expressed in hepatocyte nuclear factor 4(+) cells (HCs) and upregulated during rat liver regeneration. Concurrently, the expression of hepatic MC4R protein was significantly higher in PH than in SH animals, and phosphorylated extracellular signal-regulated kinase 1/2 was remarkably increased in PH compared with SH animals (P < 0.05, respectively). In vitro experiments showed that the expression of proliferating cell nuclear antigen was significantly higher in HCs treated with α-melanocyte-stimulating hormone than in control HCs, which was correlated to the increase of phosphorylated extracellular signal-regulated kinase 1/2 and reduction of phosphorylated signal transducer and activator of transcription 3 (P < 0.05, respectively). CONCLUSIONS: MC4R is predominantly expressed in HCs and upregulated during rat liver regeneration. In vitro stimulation of HC MC4R is associated with a modulation of extracellular signal-regulated kinase and signal transducer and activator of transcription 3 pathways regulating liver regeneration.

Asunto(s)

Hepatectomía , Regeneración Hepática/fisiología , Receptor de Melanocortina Tipo 4/metabolismo , Regulación hacia Arriba , Animales , Biomarcadores/metabolismo , Western Blotting , Citocinas/metabolismo , Técnica del Anticuerpo Fluorescente , Masculino , Ratas , Ratas Wistar , Reacción en Cadena en Tiempo Real de la Polimerasa , Transducción de Señal

RESUMEN

Irradiation is known to induce inflammation and affect fat metabolic pathways. The current study investigates hepatic fat accumulation and fatty acid transportation in a rat model of single dose liver irradiation (25-Gy). Rat livers were selectively irradiated in-vivo (25-Gy), sham-irradiated rats served as controls. Hepatic lipids were studied by colorimetric assays in liver and serum. Intracellular lipids, protein and mRNA were studied by Nile red staining, immunohistology, Western Blot analysis and RT-PCR in liver, respectively. Changes in FAT/CD36 expression were studied in-vitro in a human monocyte cell line U937 after irradiation in presence or absence of infliximab (IFX). Nile Red staining of liver cryosections showed a quick (12-48 h) increase in fat droplets. Accordingly, hepatic triglycerides (TG) and free fatty acids (FFA) were elevated. An early increase (3-6 h) in the serum level of HDL-C, TG and cholesterol was measured after single dose irradiation followed by a decrease thereafter. Furthermore, expression of the fat transporter protein FAT/CD36 was increased, immunohistochemistry revealed basolateral and cytoplasmic expression in hepatocytes. Moreover, apolipoprotein-B100, -C3 and enzymes (acetyl-CoA carboxylase, lipoprotein-lipase, carnitine-palmitoyltransferase, malonyl-CoA-decarboxylase) involved in fat metabolism were induced at 12-24 h. Early activation of the NFkß pathway (IκBα) by TNF-α was seen, followed by a significant elevation of serum markers for liver damage (AST and GLDH). TNF-α blockage by anti-TNF-α in cell culture (U937) prevented the increase of FAT/CD36 caused by irradiation. Selective liver irradiation is a model for rapid induction of steatosis hepatis and fat accumulation could be triggered by irradiation-induced inflammatory mediators (e.g. TNF-α).

Asunto(s)

Antígenos CD36/metabolismo , Regulación de la Expresión Génica/efectos de la radiación , Metabolismo de los Lípidos/efectos de la radiación , Hígado/efectos de la radiación , Radioterapia/efectos adversos , Animales , Western Blotting , Modelos Animales de Enfermedad , Hígado Graso/metabolismo , Humanos , Hígado/metabolismo , Masculino , Ratas , Ratas Wistar , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células U937

RESUMEN

Although both alcohol and fructose are particularly steatogenic, their long-term effect in the development of a metabolic syndrome has not been studied in vivo. Consumption of fructose generally leads to obesity, whereas ethanol can induce liver damage in the absence of overweight. Here, Sprague-Dawley rats were fed ad libitum for 28 days on five diets: chow (control), liquid Lieber-DeCarli (LDC) diet, LDC +30%J of ethanol (L-Et) or fructose (L-Fr), and LDC combined with 30%J ethanol and 30%J fructose (L-EF). Body weight (BW) and liver weight (LW) were measured. Blood and liver samples were harvested and subjected to biochemical tests, histopathological examinations, and RT-PCR. Alcohol-containing diets substantially reduced the food intake and BW (≤3rd week), whereas fructose-fed animals had higher LW than controls (P<0.05). Additionally, leukocytes, plasma AST and leptin levels were the highest in the fructose-administered rats. Compared to the chow and LDC diets, the L-EF diet significantly elevated blood glucose, insulin, and total-cholesterol levels (also vs. the L-Et group). The albumin and Quick-test levels were the lowest, whereas ALT activity was the highest in the L-EF group. Moreover, the L-EF diet aggravated plasma triglyceride and reduced HDL-cholesterol levels more than 2.7-fold compared to the sum of the effects of the L-Et and L-Fr diets. The decreased hepatic insulin clearance in the L-EF group vs. control and LDC groups was reflected by a significantly decreased C-peptide:insulin ratio. All diets except the control caused hepatosteatosis, as evidenced by Nile red and H&E staining. Hepatic transcription of insulin receptor substrate-1/2 was mainly suppressed by the L-Fr and L-EF diets. The L-EF diet did not enhance the mitochondrial ß-oxidation of fatty acids (Cpt1α and Ppar-α expressions) compared to the L-Et or L-Fr diet. Together, our data provide evidence for the coaction of ethanol and fructose with a high-fat-diet on dyslipidemia and insulin resistance-accompanied liver damage.

Asunto(s)

Dislipidemias , Etanol/efectos adversos , Fructosa/efectos adversos , Resistencia a la Insulina , Hígado , Mitocondrias Hepáticas , Animales , HDL-Colesterol/sangre , Dislipidemias/sangre , Dislipidemias/inducido químicamente , Dislipidemias/patología , Etanol/farmacología , Fructosa/farmacología , Hígado/lesiones , Hígado/metabolismo , Hígado/patología , Masculino , Síndrome Metabólico/sangre , Síndrome Metabólico/inducido químicamente , Síndrome Metabólico/patología , Mitocondrias Hepáticas/metabolismo , Mitocondrias Hepáticas/patología , Ratas , Ratas Sprague-Dawley , Triglicéridos/sangre

RESUMEN

Acute rejection (AR) and acceptance of allograft after liver transplantation (LTx) remain critical issues that need addressing to improve prognosis. We therefore performed rat orthotopic LTx and proteomic analyses to screen for immune response-related biomarkers in sera. Markers identified were validated at the mRNA and/or protein levels, and the molecules of interest were functionally explored. Compared with syngeneic controls, signs of AR as well as spontaneous acceptance were observed in hematoxylin and eosin-stained sections of liver allografts. In accordance with the severity of AR, 30 protein spots displaying significant changes in abundance were identified using two-dimensional differential gel electrophoresis. Ultimately, 14 serum proteins were sequenced and five spots of interest were identified as hemopexin (HPX). Expression of HPX was significantly and inversely associated with the severity of AR at both the mRNA and protein levels. In vitro, Mt-1, Ho-1, Fth, Ifn-γ, and Il-17 transcripts were significantly upregulated in lysates of lymphocytes stimulated with HPX, whereas Il-10 markedly was remarkably downregulated. Interferon-γ, IL-10, and IL-17 proteins in the supernatant of HPX-stimulated lymphocytes were significantly altered in keeping with the mRNA level. Our data facilitated the generation of a proteomic profile to enhance the understanding of rat liver AR. In view of finding that the HPX serum level is negatively associated with the severity of AR of rat liver allograft, we propose that in vitro treatment with HPX regulates cytokine expression in rat lymphocytes.

Asunto(s)

Rechazo de Injerto/metabolismo , Hemopexina/biosíntesis , Trasplante de Hígado , Enfermedad Aguda , Animales , Biomarcadores/sangre , Proliferación Celular/efectos de los fármacos , Citocinas/biosíntesis , Citocinas/genética , Relación Dosis-Respuesta a Droga , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/inmunología , Rechazo de Injerto/genética , Rechazo de Injerto/inmunología , Rechazo de Injerto/patología , Hemopexina/administración & dosificación , Hemopexina/genética , Hemopexina/farmacología , Hígado/metabolismo , Hígado/patología , Activación de Linfocitos/efectos de los fármacos , Prueba de Cultivo Mixto de Linfocitos , Linfocitos/inmunología , Masculino , Proteómica/métodos , ARN Mensajero/genética , Ratas Endogámicas F344 , Ratas Endogámicas Lew , Transcripción Genética

RESUMEN

AIM: To explore lipocalin-2 (LCN-2) expression and its possible role and mechanism(s) of production in rat models of diet-inducible fatty liver. METHODS: Fatty liver was triggered in male Sprague-Dawley rats fed either with liquid Lieber-DeCarli (LDC) or LDC + 70% cal fructose (L-HFr) diet for 4 or 8 wk. Chow-nourished animals served as controls. Hepatic expression of LCN-2 and other metabolic and inflammatory mediators was assessed by quantitative reverse transcription polymerase chain reaction and Western blotting. Serum LCN-2, fasting leptin, and lipid profile were evaluated via Enzyme-Linked Immunosorbent Assay, Radioimmunoassay, and colorimetric assays, respectively. The localization of LCN-2 in the liver was detected by using immunofluorescence staining. Furthermore, HE stain was used to evaluate hepatic fat degeneration and inflammation. RESULTS: Both LDC-fed and L-HFr-fed rat histologically featured fatty liver. In the liver, mRNA transcriptions of Mcp-1, a2-m, Il-8 and Glut5 were increased in the L-HFr group at both time points (P < 0.001), while the transcription of Tlr4, Inos, and Tnf-α was significantly up-regulated at week 4. Interestingly, hepatic Lcn-2 expression was 90-fold at week 4 and 507-fold at week 8 higher in L-HFr-subjected rats vs control (P < 0.001). In contrast to HDL-cholesterol, systemic levels of LCN-2, fasting leptin and triglycerides were elevated in the L-HFr regimen (P < 0.001). Moreover, protein expression of hepatic LCN-2, CD14, phospho-MAPK, caspase-9, cytochrome c and 4-hydroxynonenal was increased in the L-HFr group. Conversely, the hepatic expression of PGC-1α (a mitochondrial-biogenic protein) was reduced in the L-HFr category at week 8. The localization of LCN-2 in the liver was predominantly restricted to MPO⁺ granulocytes. CONCLUSION: Fructose diet up-regulates hepatic LCN-2 expression, which correlates with the increased indicators of oxidative stress and mitochondrial dysfunction. The LCN-2 may be involved in liver protection.

Asunto(s)

Alimentación Animal , Hígado Graso/metabolismo , Fructosa/metabolismo , Lipocalinas/metabolismo , Animales , Apoptosis , Quimiocina CCL2/metabolismo , Colorimetría , Ensayo de Inmunoadsorción Enzimática , Regulación de la Expresión Génica , Transportador de Glucosa de Tipo 5/metabolismo , Inflamación , Interleucina-8/metabolismo , Leptina/metabolismo , Peroxidación de Lípido , Lipocalina 2 , Hígado/metabolismo , Masculino , Estrés Oxidativo , Radioinmunoensayo , Ratas , Ratas Sprague-Dawley , Receptor Toll-Like 4/metabolismo

RESUMEN

Decreased serum and increased hepatic iron uptake is the hallmark of acute-phase (AP) response. Iron uptake is controlled by iron transport proteins such as transferrin receptors (TfRs) and lipocalin 2 (LCN-2). The current study aimed to understand the regulation of iron uptake in primary culture hepatocytes in the presence/absence of AP mediators. Rat hepatocytes were stimulated with different concentrations of iron alone (0.01, 0.1, 0.5 mM) and AP cytokines (interleukin 6 [IL-6], IL-1ß, tumor necrosis factor α) in the presence/absence of iron (FeCl3: 0.1 mM). Hepatocytes were harvested at different time points (0, 6, 12, 24 h). Total mRNA and proteins were extracted for reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blot. A significant iron uptake was detected with 0.1 mM iron administration with a maximum (133.37 ± 4.82 µg/g of protein) at 24 h compared with control and other iron concentrations. This uptake was further enhanced in the presence of AP cytokines with a maximum iron uptake (481 ± 25.81 µg/g of protein) after concomitant administration of IL-6 + iron to cultured hepatocytes. Concomitantly, gene expression of LCN-2 and ferritin subunits (light- and heavy-chain ferritin subunits) was upregulated by iron or/and AP cytokines with a maximum at 24 h both at mRNA and protein levels. In contrast, a decreased TfR1 level was detected by IL-6 and iron alone, whereas combination of iron and AP cytokines (mainly IL-6) abrogated the downregulation of TfR1. An increase in LCN-2 release into the supernatant of cultured hepatocytes was observed after addition of iron/AP cytokines into the medium. This increase in secretion was further enhanced by combination of IL-6 + iron. In conclusion, iron uptake is tightly controlled by already present iron concentration in the culture. This uptake can be further enhanced by AP cytokines, mainly by IL-6.

Asunto(s)

Reacción de Fase Aguda/metabolismo , Citocinas/farmacología , Hepatocitos/metabolismo , Hierro/farmacocinética , Animales , Apoferritinas/biosíntesis , Células Cultivadas , Relación Dosis-Respuesta a Droga , Combinación de Medicamentos , Hepatocitos/efectos de los fármacos , Interleucina-6/farmacología , Hierro/administración & dosificación , Hierro/farmacología , L-Lactato Deshidrogenasa/metabolismo , Lipocalina 2 , Lipocalinas/metabolismo , Masculino , ARN Mensajero/genética , Ratas , Ratas Wistar , Receptores de Transferrina/biosíntesis , Regulación hacia Arriba/efectos de los fármacos

RESUMEN

OBJECTIVES: FoxP3 expression is a marker for Tregs which are known to be involved in tumor immunity. We aimed to evaluate FoxP3 promoter demethylation in human colorectal cancer (CRC) and rat intrahepatic cholangiocarcinoma (ICC). DESIGN AND METHODS: Bisulfite-treated genomic DNA templates of shock frozen paired samples were studied from 13 anonymous CRC patients and from 10 male rats (n=6 ICC induced by thioacetamide and n=4 age-matched controls). Real-time PCR was carried out using a LightCycler 480 system. Human FoxP3 and CD3 promoter demethylations were estimated using previously described assays; and rat FoxP3 promoter demethylation using a newly developed assay. RESULTS: A significant 3.5-fold increase of the demethylation in FoxP3 promoter region was found in human CRC and rat ICC (P<0.05). The average frequency of cells with FoxP3 demethylation in patients suffering from CRC was 0.26% in normal tissue and 0.92% in tumor tissue (n=11 paired samples). Although, no significant difference was found between the mean frequency of CD3 demethylation in normal tissue (4.80%, n=6) and in tumor tissue (4.14%, n=6) from CRC patients, the ratio of demethylated CD3/FoxP3 promoter areas was significantly lower in tumor specimens (P<0.05). Using our novel assay, we found a significant increase in mean frequencies of cells with FoxP3 demethylation in rats with ICC (7.42%, n=6) in comparison to controls (2.14%, n=4). CONCLUSION: FoxP3 seems to be an interesting biomarker for immune response to epithelial tumors. Functional consequences from the increase of Tregs remain to be demonstrated. Further studies with outcome data are necessary.

Asunto(s)

Biomarcadores de Tumor/metabolismo , Colangiocarcinoma/metabolismo , Neoplasias Colorrectales/metabolismo , Metilación de ADN , ADN de Neoplasias/metabolismo , Factores de Transcripción Forkhead/metabolismo , Proteínas de Neoplasias/metabolismo , Regiones Promotoras Genéticas , Adulto , Anciano , Animales , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/inmunología , Complejo CD3/genética , Complejo CD3/inmunología , Complejo CD3/metabolismo , Colangiocarcinoma/genética , Colangiocarcinoma/inmunología , Colangiocarcinoma/patología , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/inmunología , Neoplasias Colorrectales/patología , ADN de Neoplasias/genética , ADN de Neoplasias/inmunología , Femenino , Factores de Transcripción Forkhead/genética , Factores de Transcripción Forkhead/inmunología , Humanos , Masculino , Persona de Mediana Edad , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/inmunología , Ratas , Linfocitos T Reguladores/inmunología , Linfocitos T Reguladores/metabolismo , Linfocitos T Reguladores/patología