Liver transcriptional profile of atherosclerosis-related genes in human nonalcoholic fatty liver disease.

OBJECTIVES AND DESIGN: Epidemiological studies have suggested a role of nonalcoholic fatty liver disease (NAFLD) in the development of cardiovascular disease. We evaluated liver mRNA expression of 84 genes encoding proteins involved in the atherosclerosis pathway in patients with NAFLD proven through biopsy in a case-control design, and examined the putative role of the histological disease severity in the molecular events associated with the atherogenic profile. RESULTS: Nonalcoholic steatohepatitis (NASH), when compared with simple steatosis (SS), significantly increases the expression of TGFB1 (6.8, p<0.005), angiotensin I-converting enzyme (ACE) (2.1, p<0.007), LAMA1 (2.1, p<0.007), SERPINB2 (2.1, p<0.007), CSF2 (2.5, p<0.002), IL1A (2.5, p<0.005), IL3 (2.1, p<0.007), IL4 (2.1, p<0.007), LIF (2.1, p<0.007), and MMP1 (2.1, p<0.007), and decreases the transcript levels of genes involved in the negative regulation of cell-death pathways. A post hoc analysis of liver biopsies of NASH patients who were treated with enalapril monotherapy because of arterial hypertension showed a significant association with lower fibrosis scores in comparison with untreated patients. BIRC3, a severe hypoxia-activated gene, was significantly increased in SS (8.2, p<0.004), when compared with the controls. NASH, but not SS, was also associated with a significant increase in platelet abundance of TGFB1 mRNA. Systems biology analysis revealed highly scored pathways involved in the regulation of programmed cell death, angiogenesis, and immune system, in which TGFB1 was mostly involved. CONCLUSION: NASH, but not SS, may increase atherosclerotic and cardiovascular risk by local overexpression of mediators of atherogenesis, endothelial damage, and regulators of blood pressure; this observation may have therapeutic implications, because ACE inhibitors may improve both cardiovascular outcomes and liver fibrosis. Hepatocyte hypoxia seems to have an important role in the molecular events activated by liver steatosis.

Mitochondrial DNA copy number is modulated by genetic variation in the signal transducer and activator of transcription 3 (STAT3).

The regulation of mitochondrial DNA (mtDNA) copy number not only is critical for the maintenance of the normal mitochondrial function but has a strong clinical significance. A recent report revealed that the signal transducer and activator of transcription 3 (STAT3) is involved in the regulation of the mitochondrial function and is required for the optimal function of the electron transport chain. In this study, we explored whether gene variants in the STAT3 influence the leukocyte mtDNA copy number. Clinical data and blood samples were collected from 179 subjects (aged 52.8 ± 0.9 years). Mitochondrial DNA quantification using nuclear DNA (nDNA) as a reference was carried out by a real-time quantitative polymerase chain reaction method; results are presented as the mtDNA/nDNA ratio. We selected 3 tag single nucleotide polymorphisms showing a minor allele frequency greater than 10% (rs2293152 C/G, rs6503695 C/T, and rs9891119 A/C), representing 24 polymorphic sites of the STAT3 (r(2) > 0.8). We observed a significant association between mtDNA/nDNA ratio and both rs6503695 and rs9891119, adjusted by age and homeostasis model assessment index. The proportion of the total variance of the mtDNA/nDNA ratio accounted for by the rs6503695 and rs9891119 genotypes was 4.7% and 6.53%, respectively. Common variation in the STAT3 may influence mtDNA copy number.

High fat diet-induced liver steatosis promotes an increase in liver mitochondrial biogenesis in response to hypoxia.

Mitochondrial DNA (mtDNA) copy number plays a key role in the pathophysiology of metabolic syndrome-related phenotypes, but its role in non-alcoholic fatty liver disease (NAFLD) is not well understood. We evaluated the molecular mechanisms that may be involved in the regulation of liver mtDNA content in a high-fat-induced rat model of NAFLD. In particular, we tested the hypothesis that liver mtDNA copy number is associated with liver expression of HIF-1α. Rats were given either standard chow diet (SCD, n = 10) or high-fat diet (HFD, n = 15) for 20 weeks. Subsequently, mtDNA quantification using nuclear DNA (nDNA) as a reference was carried out using real time quantitative PCR. HFD induced a significant increase in liver mtDNA/nDNA ratio, which significantly correlated with the liver triglyceride content (R: 0.29, P < 0.05). The liver mtDNA/nDNA ratio significantly correlated with the hepatic expression of HIF-1α mRNA (R: 0.37, P < 0.001); liver HIF-1α mRNA was significantly higher in the HFD group. In addition, liver cytochrome c oxidase subunit IV isoform 1 (COX4I1) mRNA expression was also positively correlated with liver mtDNA content. The hepatic expression of mRNA of transcriptional factors that regulate mitochondrial biogenesis, including peroxisome proliferator-activated receptor gamma coactivator-1α (PGC-1α) and PGC-1ß, nuclear respiratory factor-1 (NRF-1), peroxisome proliferator-activated receptor δ and Tfam, was not associated with the liver mtDNA content. Neither hepatocyte apoptosis nor oxidative stress was involved in the HIF-1α-mediated increase in mtDNA copy number. In conclusion, we found that HFD promotes an increase in liver mitochondrial biogenesis in response to hypoxia via HIF-1α, probably to enhance the mitochondrial function as well as to accommodate the metabolic load.

Epigenetic regulation of insulin resistance in nonalcoholic fatty liver disease: impact of liver methylation of the peroxisome proliferator-activated receptor γ coactivator 1α promoter.

UNLABELLED: Insulin resistance (IR) and mitochondrial dysfunction play a central role in the pathophysiology of nonalcoholic fatty liver disease (NAFLD). We hypothesized that genetic factors and epigenetic modifications occurring in the liver contribute to the IR phenotype. We specifically examined whether fatty liver and IR are modified by hepatic DNA methylation of the peroxisome proliferator-activated receptor γ coactivator 1α (PPARGC1A) and mitochondrial transcription factor A (TFAM) promoters, and also evaluated whether liver mitochondrial DNA (mtDNA) content is associated with NAFLD and IR. We studied liver biopsies obtained from NAFLD patients in a case-control design. After bisulfite treatment of DNA, we used methylation-specific polymerase chain reaction (PCR) to assess the putative methylation of three CpG in the PPARGC1A and TFAM promoters. Liver mtDNA quantification using nuclear DNA (nDNA) as a reference was evaluated by way of real-time PCR. Liver PPARGC1A methylated DNA/unmethylated DNA ratio correlated with plasma fasting insulin levels and homeostasis model assessment of insulin resistance (HOMA-IR); TFAM methylated DNA/unmethylated DNA ratio was inversely correlated with insulin levels. PPARGC1A promoter methylation was inversely correlated with the abundance of liver PPARGC1A messenger RNA. The liver mtDNA/nDNA ratio was significantly higher in control livers compared with NAFLD livers. mtDNA/nDNA ratio was inversely correlated with HOMA-IR, fasting glucose, and insulin and was inversely correlated with PPARGC1A promoter methylation. CONCLUSION: Our data suggest that the IR phenotype and the liver transcriptional activity of PPARGC1A show a tight interaction, probably through epigenetic modifications. Decreased liver mtDNA content concomitantly contributes to peripheral IR.

The nuclear receptor PXR gene variants are associated with liver injury in nonalcoholic fatty liver disease.

OBJECTIVE: To explore the contribution of gene variants and derived haplotypes of the pregnane X receptor (NR1I2) to the severity of nonalcoholic fatty liver disease (NAFLD). METHODS: A total of 290 individuals were evaluated in a case-control association study, including 188 NAFLD patients with different stages of disease severity and 102 healthy individuals. Four tag single nucleotide polymorphisms (SNPs; rs12488820 C/T, rs2472671 C/T, rs2461823 A/G, and rs1054191 A/G) encompassing 36 kb in chromosome 3 and representing 33 polymorphic sites (r2>0.8) were genotyped. Four additional SNPs (rs3814055, rs3814057, rs6785049, and rs7643645) were also included because they showed earlier evidence of functionality. RESULTS: Genotypic tests for single SNPs showed that rs7643645 and rs2461823 were significantly associated with disease severity by ordinal multinomial analysis (P<0.0015 and 0.039, respectively). A significant association was also observed under the additive model for both variants (P<0.00038 and 0.012, respectively). Consistent with the analysis of individual markers, we observed that the multimarker composed of rs2461823/A-rs7643645/G was significantly associated with disease severity (P<6.9 x 10(-5), beta: 0.45). In addition, the rs7643645/G variant was significantly associated with ALT level (P<0.026), a surrogate marker of severe liver injury. Finally, in univariate analysis rs7643645/G was significantly associated with fatty liver disease (P<0.04), with an odds ratio of 1.457 (95% confidence interval: 1.018-2.086). CONCLUSION: Our study suggests that pregnane X receptor polymorphisms and related haplotypes may contribute to disease severity in NAFLD by influencing the individual susceptibility to progress to more severe stages of the disease.

Circulating levels and hepatic expression of molecular mediators of atherosclerosis in nonalcoholic fatty liver disease.

OBJECTIVES: We evaluated circulating levels of biomarkers of atherosclerosis (soluble intercellular adhesion molecule: sICAM-1, plasminogen activator inhibitor: PAI-1 and soluble CD40 ligand: sCD40L) in patients with NAFLD proven through biopsy and control subjects, and correlated them with the histological disease severity. We further explored liver protein expression of ICAM-1, CD40 and PAI-1 in patients with different histological forms of NAFLD and control liver biopsies. PATIENTS AND METHODS: We included 215 individuals: 113 patients with NAFLD (simple steatosis n=45 and NASH n=68) and 102 control subjects. Circulating levels of the biomarkers were measured by ELISA. Liver expression of ICAM-1, CD40 and PAI-1 was assessed by immunohistochemistry using monoclonal antihuman antibodies. RESULTS: Patients with NAFLD, in comparison with control subjects, showed significantly higher circulating levels of sICAM-1 (605.3+/-34.6ng/ml vs. 356.5+/-24.6ng/ml, p=5.9 x 10(-6)), PAI-1 (22.8+/-1.7ng/ml vs. 19.0+/-2.1ng/ml, p=0.0149) and sCD40L (1347.5+/-513.7pg/ml vs. 804.5+/-396.1pg/ml, p=0.0229), results expressed as mean+/-SE. sICAM-1 was a strong predictor of histological severity of NAFLD, after adjusting for potential confounders. In addition, patients with NAFLD showed significantly higher liver staining scores for ICAM-1 and PAI-1 than control liver biopsies. ICAM-1 immunoreactivity in lobular inflammatory infiltrate showed high scores in NASH patients; a significant correlation was found between both the degree of liver steatosis and the severity of necroinflammatory activity and liver ICAM-1 expression. CONCLUSIONS: Our study shows that NAFLD is associated with elevated circulating levels and abnormal liver expression of molecular mediators of atherosclerosis. Additionally, ICAM-1 may be involved in liver damage and inflammation.

A nonsynonymous gene variant in the adiponutrin gene is associated with nonalcoholic fatty liver disease severity.

We explored the role of the adiponutrin (PNPLA3) nonsynonymous-rs738409 single nucleotide polymorphism (SNP) in genetic susceptibility to nonalcoholic fatty liver disease (NAFLD) and whether this SNP contributes to the severity of histological disease. Two hundred sixty-six individuals were evaluated in a case-control association study, which included 172 patients with features of NAFLD and 94 control subjects. The rs738409 G allele was significantly associated with NAFLD (P < 0.001; OR 2.8 95%, CI 1.5-5.2), independent of age, sex, body mass index (BMI), and Homeostasis Model Assessment (HOMA) index. When we tested the hypothesis of a relation between the SNP and the histological spectrum of NAFLD, a significant association was observed [chi2 19.9, degree of freedom (df): 2, P < 5 x 10(-5), adjusted for HOMA and BMI]. The degree of liver steatosis, as evaluated by liver biopsy, was significantly associated with the rs738409 G allele. Patients with CC genotype showed a lower steatosis score (14.9% +/- 3.9) in comparison with the CG genotype (26.3% +/- 3.5) and GG genotype (33.3% +/- 4.0) (P < 0.005). The proportion of the total variation attributed to rs738409 genotypes was 5.3% (beta 0.23 +/- 0.07; P < 0.002). Our data suggest that the rs738409 G allele is associated not only with fat accumulation in the liver but also with liver injury, possibly triggered by lipotoxicity.

Losartan reduces liver expression of plasminogen activator inhibitor-1 (PAI-1) in a high fat-induced rat nonalcoholic fatty liver disease model.

OBJECTIVE: To evaluate the effect of losartan-an angiotensin II type 1 receptor (AT1R) antagonist- and telmisartan-an AT1R blocker with insulin-sensitizing properties-, on the hepatic expression of plasminogen activator inhibitor-1 (PAI-1) in a rat model of nonalcoholic fatty liver disease (NAFLD). METHODS: Rats were given a high-fat diet (HFD) for 8 weeks and after this period were randomly divided into 3 groups. For 12 weeks along with the same access to HFD, one group (9 rats) received losartan and another group received telmisartan (10 rats), both at 10mg/kg intraperitoneally (ip) every 24h. The third group (8 rats) received saline ip along with the HFD. Finally, a control group (6 rats) was fed with standard chow diet for 20 weeks. RESULTS: Fatty liver was reverted by both losartan and telmisartan. Both drugs had beneficial effects on insulin resistance, reaching statistical significance in telmisartan group. Expression of hepatic mRNA of PAI-1 showed a 42% decrease in losartan-treated rats in comparison with both HFD group and telmisartan-treated rats. To further evaluate this differential effect on PAI-1 expression, we explored the effect of the drugs on liver expression of TNFalpha, PEPCK-C and PPARalpha, and no significant differences were observed. CONCLUSION: These results indicate that AT1R blockers could be eligible drugs for reducing hepatic lipid accumulation in patients with NAFLD. However, only 12 weeks of losartan treatment strongly reduced hepatic PAI-1 gene expression. These differences could provide even more effective options for preventing fatty liver disease and its cardiovascular complications.

A diagnostic model to differentiate simple steatosis from nonalcoholic steatohepatitis based on the likelihood ratio form of Bayes theorem.

OBJECTIVE: To evaluate the performance of a diagnostic model based on a composite index using clinical and laboratory data, including cardiovascular biomarkers, to help practitioners to differentiate patients with simple steatosis from those with nonalcoholic steatohepatitis (NASH). DESIGN AND METHODS: 101 patients with biopsy proven features of nonalcoholic fatty liver disease were included. We investigated the usefulness of 9 biomarkers in predicting the histological disease severity, including routine biochemical tests, C-reactive protein, soluble intercellular adhesion molecule-1 (sICAM-1) and anthropometric evaluation. Receiver operating characteristic (ROC) curves and likelihood ratios (LRs) were used to evaluate the fit of each test. A composite index was calculated as the product of each individual test LR. RESULTS: In a model patient who has all positive tests, the post-test probability for NASH would be 99.5%. CONCLUSION: The capacity of each individual biomarker to independently predict the disease outcome was lower than a composite index constructed after multiplying the LR for each individual test combined into a "multimarker" score.

Genetic variants in STAT3 are associated with nonalcoholic fatty liver disease.

AIMS: To investigate the role of gene variants and derived haplotypes of the STAT3 transcription factor in nonalcoholic fatty liver disease (NAFLD) and their relation with the clinical disease severity. PATIENTS AND METHODS: 108 patients with NAFLD and different stages of clinical disease severity, and a group of 55 healthy individuals were included in a Hospital-based study. We selected 3 tagSNPs showing a minor allele frequency >10% (rs2293152 C/G, rs6503695 C/T, and rs9891119 A/C) encompassing 68.55kb in chromosome 17, representing 24 polymorphic sites (r(2)>0.8). RESULTS: In univariate analysis, there were significant differences in the allele frequency of the rs6503695 and rs9891119 between the healthy individuals and NAFLD patients (empiric P=0.021 and 0.020, respectively). The test results for the multi-marker analysis showed that haplotypes TA and CC of tagSNPs rs6503695, rs9891119 were significantly associated with NAFLD (empiric P=0.035 and 0.015, respectively). When we tested the hypothesis of a relation between the gene variants and the clinical and histological spectrum of NAFLD by multinomial analysis, a significant association was observed with rs9891119 (P=0.02). CONCLUSIONS: Our study suggests a potential role of the STAT3 polymorphisms and their haplotypes in susceptibility to NAFLD and disease severity.