[Gene expression in obese patients with non-alcoholic steatohepatitis]. / Expresión génica en pacientes obesos con enfermedad hepática por depósito de grasa.

UNLABELLED: Although the molecular basis for the pathophysiology of non-alcoholic steatohepatitis (NASH) is poorly understood, we evaluate the hepatic gene expression of cytokines, chemokines, cell receptors, growth factors, intracellular transducers and extracellular communication proteins in liver tissue of obese patients (with and without NASH), and we determine the specific intrahepatic gene expression profiles associated with histological severe NASH.Thirty-eight obese patients with BMI > 35 were analyzed, who underwent bariatric surgery. Biopsy specimen samples were snap-frozen in liquid nitrogen. Hepatic gene expression was determined in liver biopsy specimens from 3 groups: a) obese patients without NASH (n = 12); b) patients with NASH without fibrosis (n = 13); and c) patients with NASH and fibrosis (n = 13). Genes were considered to be expressed differentially in NASH only if there was a greater than 2-fold difference in abundance of mRNA when compared with each of the control group. These results were confirmed by real-time PCR. Fourteen genes were differentially expressed (10 overexpressed and 4 underexpressed) in patients with NASH. Genes that were significantly overexpressed included prohibitin, TNF, TNF RI (p55), MCSF, R2-TRAIL, b1-CTGF, FGF, VEGF, and BIGH3OBR. Insulin growth factor-1, insulin growth factor-2, interleukin-2 and tyrosine-receptor were underexpressed in NASH patients. IN CONCLUSION: 1. The obese patients with NASH without fibrosis show an overexpression of proinflammatory and proapoptotic genes. Also, the NASH patients with fibrosis show an overexpression of fibrogenic genes, including the leptin receptor Ob-Rb.2. The up-regulated gene expression of prohibitin suggests mitochondrial dysfunction in NASH patients.

Expresión génica en pacientes obesos con enfermedad hepática por depósito de grasa / No disponible

La fisiopatología de la enfermedad hepática por depósito de grasasólo se conoce de forma parcial. En este trabajo hemos analizadola expresión génica intrahepática de citoquinas, quimioquinas, receptorescelulares, factores de crecimiento, transductores de señalesintracelulares y proteínas de comunicación extracelular en el tejidohepático de sujetos obesos con y sin esteatohepatitis no alcohólica,en un intento de determinar un perfil de expresión génica asociadoa las formas severas de la esteatohepatitis no alcohólica (EHNA).Se analizó un grupo de 38 pacientes obesos con un IMC > 35,que fueron sometidos a cirugía bariátrica. La expresión génica intrahepáticase determinó en el tejido hepático dividiendo a los pacientesen tres grupos: a) pacientes obesos sin datos histológicos sugestivosde EHNA (n = 12); b) pacientes con EHNA sin fibrosis (n =13); y c) pacientes con EHNA y fibrosis (n = 13). Se consideró queexistía una sobreexpresión génica cuando la diferencia en la expresiónera, al menos, de dos veces con respecto al grupo control. Losresultados se confirmaron mediante PCR en tiempo real. Se detectóuna expresión diferencial de 14 genes (10 sobreexpresados y 4infraexpresados). Los genes sobreexpresados incluyeron prohibitina,TNF, TNF RI (p55), MCSF, R2-TRAIL, TGF-b1, CTGF, FGF,VEGF, BIGH3 y ObRb. La expresión de los genes insulin growthfactor-1, insulin growth factor-2, interleuquina-2 y tyrosine-receptorfue menor que en el grupo control.En conclusión:1. Los pacientes obesos con EHNA sin fibrosis muestran unasobreexpresión de genes proinflamatorios y proapoptóticos. Enlos pacientes con EHNA y fibrosis, se observa, además, una sobreexpresiónde genes profibrogénicos, incluyendo el gen del receptorde la leptina.2. La expresión de prohibitiva en los pacientes con EHNA,tanto con fibrosis como sin fibrosis, fue superior que en los controles,lo que sugiere una disfunción mitocondrial en los pacientescon EHNA

Although the molecular basis for the pathophysiology of nonalcoholicsteatohepatitis (NASH) is poorly understood, we evaluatethe hepatic gene expression of cytokines, chemokines, cell receptors,growth factors, intracellular transducers and extracellularcommunication proteins in liver tissue of obese patients (with andwithout NASH), and we determine the specific intrahepatic geneexpression profiles associated with histological severe NASH.Thirty-eight obese patients with BMI > 35 were analyzed, whounderwent bariatric surgery. Biopsy specimen samples were snapfrozenin liquid nitrogen. Hepatic gene expression was determinedin liver biopsy specimens from 3 groups: a) obese patientswithout NASH (n = 12); b) patients with NASH without fibrosis (n= 13); and c) patients with NASH and fibrosis (n = 13). Geneswere considered to be expressed differentially in NASH only ifthere was a greater than 2-fold difference in abundance of mRNAwhen compared with each of the control group. These resultswere confirmed by real-time PCR. Fourteen genes were differentiallyexpressed (10 overexpressed and 4 underexpressed) in patientswith NASH. Genes that were significantly overexpressed includedprohibitin, TNF, TNF RI (p55), MCSF, R2-TRAIL,b1-CTGF, FGF, VEGF, and BIGH3OBR. Insulin growth factor-1,insulin growth factor-2, interleukin-2 and tyrosine-receptor wereunderexpressed in NASH patients.In conclusion:1. The obese patients with NASH without fibrosis show anoverexpression of proinflammatory and proapoptotic genes.Also, the NASH patients with fibrosis show an overexpression offibrogenic genes, including the leptin receptor Ob-Rb.2. The up-regulated gene expression of prohibitin suggestsmitochondrial dysfunction in NASH patients

GNAS1 T393C polymorphism is associated with migraine.

Migraineurs have an interictal sympathetic nervous system (SNS) hypofunctionality and hypersensitivity to adrenergic amines. The GNAS1 T393C polymorphism has been associated with a distinct SNS sensitivity in healthy subjects. We tested GNAS1 T393C variant in two independent sets of subjects. In the case-control subset, 365 migraine patients [194 with aura (MA)] vs. 347 healthy controls were studied. A significant excess of the CC genotype was found in migraneurs (31.2%) as opposed to controls (20.2%; P=0.003). Using a logistic regression model corrected for sex, the CC genotype conferred a general risk for migraine twice [odds ratio (OR) 1.79, 95% confidence interval (CI) 1.27-2.53; P=0.001] higher than CT/TT genotypes. Using parents from 117 migraine families, a marginally significant trend for association could be observed (P=0.025), but the transmission disequilibrium test for alleles maternally transmitted failed to demonstrate familial association. In this subgroup, CC genotype conferred a risk for migraine over twice (OR 2.20; 95% CI 1.14-4.40; P=0.019) higher than TT/TC genotypes. In conclusion, the GNAS1 T393C variant is associated with migraine, which suggests a genetic basis for its higher SNS sensitivity.

Increased expression of Ob-Rb and its relationship with the overexpression of TGF-beta1 and the stage of fibrosis in patients with nonalcoholic steatohepatitis.

AIMS: The main aim of this study was to examine the relationship of the leptin system in steatosis and nonalcoholic steatohepatitis (NASH). The study also analysed the pathogenic role of the leptin system in the development of hepatic fibrosis and its relation with the TGF-beta1 system. MATERIALS AND METHODS: The study included 90 subjects, 55 with NASH and 35 with simple steatosis. Gene expression of leptin, leptin receptor and TGF-beta mRNA was analysed by real-time PCR on liver tissue. Leptin serum levels were determined by RIA. Leptin receptor expression was also assessed by immunohistochemistry. RESULTS: Increased expression was found for leptin receptor mRNA (P=0.0016) and its protein (P<0.05) in patients with NASH, especially those with fibrosis. There was a marked increase in gene expression of TGF-beta1 in patients with NASH (P=0.0002). A strong correlation was demonstrated between leptin receptor gene expression and TGF-beta1 gene expression (P=0.023). No leptin expression was found in the liver tissue. All patients showed a marked hyperleptinemia, which was closely related to the anthropometric characteristics analysed and independent of development or not of NASH. CONCLUSIONS: The results of the study demonstrate for the first time increased leptin receptor expression in liver tissue and its relationship with overexpression of TGF-beta1 and the degree of hepatic fibrosis.

Adiponectin and its receptors in non-alcoholic steatohepatitis.

BACKGROUND: Adiponectin, an adipocyte derived polypeptide, has been shown to alleviate steatosis and inflammation in mice with non-alcoholic fatty liver disease. AIM: In the present study, we wished to define liver expression of adiponectin and its receptors in morbidly obese patients undergoing bariatric surgery. Patients with non-alcoholic steatohepatitis (NASH) or simple steatosis were investigated to test whether dysregulation of this system might be involved in these disorders. PATIENTS AND METHODS: Liver mRNA expression of adiponectin and its recently cloned receptors RI and RII (adipoRI and adipoRII) were analysed by fluorescence based real time polymerase chain reaction in 13 patients with NASH and nine with simple steatosis. Adiponectin and adipoRII protein expression were assessed by immunohistochemistry in a subgroup of patients. RESULTS: Adiponectin and adipoRII mRNA expression were significantly reduced in liver biopsies of patients with NASH compared with simple steatosis while no difference was found in adipoRI mRNA expression. In NASH, adipoRII mRNA expression was negatively correlated with serum aspartate aminotransferase levels, serum alanine aminotransferase levels, and grade of fibrosis. Liver adiponectin protein expression was mainly found in endothelial cells of portal vessels and liver sinusoids whereas adipoRII expression was seen in hepatocytes only. Adiponectin and adipoRII staining were lower in biopsies of subjects with NASH compared with simple steatosis. CONCLUSION: Reduced hepatic expression of adiponectin and adipoRII might be of pathophysiological relevance in non-alcoholic fatty liver diseases.

Gene expression of tumor necrosis factor alpha and TNF-receptors, p55 and p75, in nonalcoholic steatohepatitis patients.

The main objective of this study was to analyze the pathogenic role of the tumor necrosis factor alpha (TNF-alpha) system in the development of nonalcoholic steatohepatitis (NASH). Fifty-two obese patients were studied. We investigated: (1) the expression of mRNA of TNF-alpha and their p55 and p75-receptors by quantitative reverse-transcriptase polymerase chain reaction (RT-PCR) in hepatic and adipose tissues; and (2) the relationship between TNF-alpha, p55, and p75 and the severity of NASH. Obese patients without NASH were the control group. A remarkable increase in the expression of mRNA of TNF-alpha was found in patients with NASH in hepatic tissue (0.65 +/- 0.54) and in peripheral fat (0.43 +/- 0.45); in the control samples, the mRNA expression was 0.28 +/- 0.32, P <.007, and 0.26 +/- 0.22, P <.018, respectively. Furthermore, we found a significant increase in the mRNA levels of p55 receptor (2.42 +/- 1.81 vs. 1.56 +/- 1.17; P <.05); however, the mRNA expression of the p75 receptor was similar in both patients. Those patients with NASH with significant fibrosis presented an increase in the expression of mRNA TNF-alpha in comparison with those with a slight or nonexistent fibrosis. An overexpression of TNF-alpha mRNA is found in the liver and in the adipose tissue of NASH patients. The levels of mRNA-p55 are increased in the liver tissue of NASH patients. This overexpression is more elevated in patients with more advanced NASH. These findings suggest that the TNF-alpha system may be involved in the pathogenesis of NASH.

Are there predictive factors of severe liver fibrosis in morbidly obese patients with non-alcoholic steatohepatitis?

BACKGROUND: Non-alcoholic steatohepatitis (NASH) is a clinicopathological entity characterized by the presence of steatosis and lobular and/or portal inflammation with or without fibrosis. Patients with non-alcoholic fatty liver and fibrosis on liver biopsy have increased liver-related deaths. METHODS: 181 wedge liver biopsies, taken at the time of bariatric surgery from patients with a mean body mass index (BMI) of 47, were studied. In all cases, the liver biopsy was performed without knowledge of the patient's clinical and biochemical data, which were then examined with univariate and multivariate analysis. RESULTS: Diagnosis of NASH was established in 105 patients (91%); 74 patients (70%) showed mild steatosis, 20 (19%) had moderate inflammation and fibrosis, and 11 (10%) had steatosis with severe fibrosis. None of the liver biopsies showed cirrhosis. Age was the only independent predictor of moderate and severe fibrosis (p = 0.001). CONCLUSIONS: Since only age was a predictor of moderate or severe fibrosis, and no clinical or biochemical abnormalities detected slowly progressive hepatic fibrosis, liver biopsy is the only means of detecting progression to more advanced liver disease in a NASH patient.

Two active-site tyrosyl residues of protein TrwC act sequentially at the origin of transfer during plasmid R388 conjugation.

Protein TrwC is the relaxase-helicase responsible for the initiation and termination reactions of DNA processing during plasmid R388 conjugation. Site-directed mutagenesis was used to change to phenylalanine each of a set of four conserved tyrosyl residues in the sequence of the N-terminal relaxation domain of the protein. Simultaneous mutation of both Y18 and Y26 was required to abolish in vitro cleavage and strand-transfer reactions catalyzed by protein TrwC on oligonucleotides containing the nic site. Thus, both Y18 and Y26 could be involved independently in the formation of oligonucleotide-protein covalent complexes that constitute presumed intermediates of these reactions. This hypothesis was confirmed by the observation of Y18 and Y26-specific peptide-oligonucleotide adducts after protease digestion of TrwC and mutant derivatives. Finally mutation Y18F, but not mutation Y26F, abolished nic-cleavage of a supercoiled DNA containing the R388 origin of transfer (oriT). These data allowed the construction of a model for conjugative DNA processing in which Y18 specifically catalyzes the initial cleavage reaction, while Y26 is used for the second strand-transfer reaction, which terminates conjugation. The model suggests a control mechanism that can be effective at each conjugative replication cycle.