Objective & design: Nonalcoholic fatty liver disease (NAFLD) is a clinical condition that refers to progressive histological changes ranging from simple steatosis (SS) to nonalcoholic steatohepatitis (NASH). We evaluated the status of cytosine methylation (5mC) of liver mitochondrial DNA (mtDNA) in selected regions of the mtDNA genome, such as D-loop control region, and mitochondrially encoded NADH dehydrogenase 6 ( MT-ND6 ) and cytochrome C oxidase I ( MT-CO1 ), to contrast the hypothesis that epigenetic modifications play a role in the phenotypic switching from SS to NASH. Methods: We studied liver biopsies obtained from patients with NAFLD in a case-control design; 45 patients and 18 near-normal liver-histology subjects. Results: MT-ND6 methylation was higher in the liver of NASH than SS patients (p<0.04) and MT-ND6 methylated DNA/unmethylated DNA ratio was significantly associated with NAFLD activity score (p<0.02). Liver MT-ND6 mRNA expression was significantly decreased in NASH patients (0.26±0.30) versus SS (0.74±0.48), p<0.003, and the protein level was also diminished. The status of liver MT-ND6 methylation in NASH group was inversely correlated with the level of regular physical activity ( R =-0.54, p<0.02). Hepatic methylation levels of D-Loop and MT-CO1 were not associated with the disease severity. DNA (cytosine-5) methyltransferase 1 was significantly upregulated in NASH patients (p<0.002). Ultrastructural evaluation showed that NASH is associated with mitochondrial defects and peroxisome proliferation. Conclusion: Hepatic methylation and transcriptional activity of the MT-ND6 are associated with the histological severity of NAFLD. Epigenetic changes of mtDNA are potentially reversible by interventional programs, as physical activity could modulate the methylation status of MT-ND6.

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.

Insulin resistance (IR) and mitochondrial dysfunction play a central role in the patho-physiology of nonalcoholic fatty liver disease (NAFLD). We hypothesized that genetic fac-tors 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 methyla-tion of the peroxisome proliferator–activated receptor c coactivator 1 a ( 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 puta-tive 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 signif-icantly higher in control livers compared with NAFLD livers. mtDNA/nDNA ratio was inversely correlated with HOMA-IR, fasting glucose, and insulin and was inversely corre-lated 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 concomi-tantly contributes to peripheral IR. (HEPATOLOGY 2010;52:1992-2000)

: Objective.  The major function of the circadian system is the internal cycling of physiological and metabolic events. The present study sought to explore the effect of rotating shift work schedule on leucocyte count and its relationship with risk factors of metabolic syndrome (MS).Design and participants.  From a population‐based design, 1351 men of self‐reported European ancestry were included in a cross‐sectional study: 877 day workers were compared with 474 rotating shift workers. Medical history, health examination including anthropometric and arterial blood pressure measurements, a questionnaire on health‐related behaviours and biochemical determinations was given to all participants.Results.  In comparison with day workers, rotating shift workers had elevated (mean ± SE) body mass index (27.1 ± 0.3 vs. 26.3 ± 0.2, P < 0.0154), waist‐hip ratio (0.95 ± 0.01 vs. 0.93 ± 0.01, P < 0.00024), diastolic arterial blood pressure (78 ± 1 vs. 76 ± 1, P < 0.033), fasting insulin (65.5 ± 2.9 vs. 55.9 ± 1.9 pmol L⁻¹, P < 0.017), Homeostasis Model Assessment index (2.12 ± 0.11 vs. 1.77 ± 0.07, P < 0.0027), triglycerides (1.71 ± 0.1 vs. 1.5 ± 0.1 mmol L⁻¹, P < 0.002), uric acid (292.7 ± 2.8 vs. 282 ± 3.4 μmol L⁻¹, P < 0.01) and leucocyte count (7030 ± 84 vs. 6730 ± 58, P < 0.0094). In multiple regression analysis, leucocyte count was correlated with rotating shift work independently of age, smoking, education and components of MS.Conclusion.  The odds ratio for MS in rotating shift workers compared with day workers was 1.51 (95% CI 1.01–2.25), independently of age and physical activity. Increased leucocyte count, a biological marker of systemic inflammation, was associated with rotating shift work.

Background:Altering circadian rhythmicity results in pathophysiologic changes resembling metabolic syndrome and fat accumulation. Objective:We investigated the role of gene variants and derived haplotypes of the CLOCK transcription factor in obesity and related quantitative metabolic traits. Design:Lean (n = 715) and overweight or obese (n = 391) unrelated subjects aged 34.4 ± 8.6 y were included in a population-based cross-sectional study. Six tag single-nucleotide polymorphisms (SNPs) with a minor (>10%) allele frequency (rs1554483 C/G; rs11932595 A/G; rs4580704 C/G; rs6843722 A/C; rs6850524 C/G, and rs4864548 A/G) encompassing 117 kb of chromosome 4 and representing 115 polymorphic sites (r² > 0.8) were genotyped. Association was tested by PLINK and WHAP software, and multiple testing was controlled by permutation test. Results:The genotype frequencies of 4 tag SNPs—rs1554483, rs6843722, rs6850524, and rs4864548—had significant (empiric P < 0.010, 0.021, 0.021, and 0.010, respectively) associations with overweight or obesity. Haplotype analysis showed that only paired haplotypes, including rs1554483 and rs4864548, had a significant effect on overweight or obesity. Combinations of these SNPs (haplotype block CG and GA) are responsible for the gene effect (GA frequencies: 47% in cases, 41% in controls; empiric P < 0.011). These findings were concurrently observed in a sample of persons from a hospital-based study, and the combined Mantel-Haenszel fixed effect was an odds ratio of 1.82 (95% CI: 1.31, 2.54; P < 0.001) for the paired haplotype, which included CG and GA for rs1554483 and rs4864548. Conclusions:The present study suggests a putative role of the CLOCK polymorphism and related haplotypes in susceptibility to obesity. The haplotype of rs1554483G and rs4864548A was associated with a 1.8-fold risk of overweight or obesity.

We explored the role of transmembrane 6 superfamily member 2 (TM6SF2) rs58542926 C/T nonsynonymous (p.Glu167Lys) variant in genetic susceptibility to nonalcoholic fatty liver disease (NAFLD) and disease severity. A total of 361 individuals (135 control subjects and 226 patients with histologically proven NAFLD) were included in a sample with 97% power for the additive genetic model. A discrete trait analysis of NAFLD showed that rs58542926 was associated with a modest risk of fatty liver (P = 0.038; odds ratio [OR]: 1.37; 95% confidence interval [CI]: 1.02‐1.84); nevertheless, conditioning on patatin‐like phospholipase domain‐containing 3 (PNPLA3)‐rs738409 abolished this effect. We did not observe an interaction between rs738409 and rs58542926 variants on the risk of NAFLD. We observed a significant association of rs58542926 and disease severity (P = 0.027), but not lobular inflammation or fibrosis; rs58542926 was not associated with levels of liver enzymes. An allelic test showed that the T (Lys167) allele was significantly associated with disease progression (P = 0.021; OR, 1.66; 95% CI: 1.08‐2.55). A significant association was found with the histological degree of liver steatosis (β, 0.15; standard error: 0.06; P = 0.0299) that was independent of rs738409. Homozygous carriers of the C (Glu167) allele showed increased risk for cardiovascular disease. TM6SF2 protein expression was decreased markedly in liver of NAFLD patients, compared to controls. In addition, TM6SF2 immunoreactivity was reduced in subjects carrying at least one copy of the T allele, consistent with a difference in liver allele‐specific transcript abundance. Conclusion: rs58542926 is a low‐frequency variant with a modest effect on NAFLD, suggesting that carriers of the T allele are slightly more likely to accumulate fat in the liver and develop nonalcoholic steatohepatitis than those without. TM6SF2 appears to play a significant role in disease biology. (Hepatology 2015;61:515‐525)