Epigenetic regulation of the Insulin-Resistance in Human NAFLD: Role of Hepatic DNA Methylation of Master Genes that Control Mitochondrial Function.

SOOKOIAN S; ROSSELLI MS; CASTANO GO; BURGUEÑO A,; PIROLA CJ

Boston, MA, USA

Congreso; AASLD: The Liver Meeting 2010--AASLD's 61st Annual Meeting; 2010

American Association for the Study of Liver Diseases

Insulin resistance (IR) and mitochondrial dysfunction are critical factors involved in the pathophysiology of nonalcoholic fatty liver disease (NAFLD). We hypothesized that both genetic and epigenetic factors in the liver tissue of NAFLD-affected patients contribute to the IR phenotype. To address this hypothesis we selected the peroxisome proliferative activated receptor gamma, coactivator 1 alpha (PPARGC1A), which altered signaling lead to glucose intolerance, IR and diabetes, and mitochondrial transcription factor A (Tfam), which is involved in the maintenance of the mitochondrial genome. We examined specifically whether fatty liver and IR are modified by epigenetic factors like hepatic promoter DNA methylation. We further evaluated whether liver mtDNA copy number plays a role in the NAFLD-related phenotypes. After bisulfite treatment of genomic DNA from liver tissues, we used methylation-specific PCR to assess the putative DNA methylation of three CpG in the promoters of PPARGC (-513, -519 and –615) and Tfam (-433, –442 and –499). Liver mtDNA quantification using nuclear DNA (nDNA) as a reference was carried out by a real-time quantitative PCR method. We studied liver biopsies obtained from adult NAFLD patients in a case-control design. Control liver specimens were obtained by percutaneous liver biopsy. Results: Liver PPARGC promoter methylated DNA/unmethylated DNA ratio was significantly correlated with plasma fasting insulin levels (R=0.51, p<0.01) and HOMA-IR (R=0.58, p<0.003); liver Tfam promoter methylated DNA/unmethylated DNA ratio was inversely correlated with fasting insulin (R= -0.49, p<0.04). Interestingly, we found that PPARGC promoter methylation negatively correlated with the abundance of liver PPARGC mRNA (R=-0.602, p<0.004). The liver mtDNA/nDNA ratio (mean±SD) was significantly (p< 0.01) higher in the control liver (110.6±89.5, n=10) in comparison with NAFLD (60.5±39.9, n=62). mtDNA/nDNA ratio was inversely correlated with HOMA-IR (R: -0.38, p<0.01), fasting glucose (R: -0.24, p<0.03) and fasting insulin (R: -0.35, p<0.02). In conclusion, this is the first demonstration that changes in hepatic DNA methylation may play a role in the pathogenesis of the IR. In addition, we demonstrated that liver PPARGC methylation controls liver PPARGC mRNA expression. Moreover, NAFLD patients have a lower hepatic mtDNA copy number, and the liver mtDNA content is inversely correlated with IR. Finally, despite our findings cannot establish a cause/effect relation between liver steatosis and IR, they may have direct clinical translation, as epigenetic changes are potentially reversible with any intervention, including pharmacological manipulation.