Nicotinamide N-Methyltransferase Expression Decreases in Iron Overload Exacerbating Iron-Induced Hepatotoxicity

Iron overload causes the generation of reactive oxygen species, which can lead to liver failure, particularly when it occurs in conjunction with nonalcoholic fatty liver disease (NAFLD) or viral hepatitis. Previously, we studied the effects of iron deficiency and iron overload on the hepatic transcriptional and metabolomic profile in mice. We found that iron overload induced significant changes in the expression of genes and metabolites involved in glucose and nicotinamide metabolism in both a dietary model, high iron diet, and a genetic model, hemojuvelin knockout mice. In particular, we discovered that the transcript, Nicotinamide N-methyltransferase (NNMT), was significantly downregulated in both models of iron overload. NNMT methylates nicotinamide, a form of vitamin B3, and has recently been shown to play a critical role in hepatic glucose and cholesterol metabolism by stabilizing Sirtuin 1, a nicotinamide-dependent deacetylase. For the current study, we evaluated 63 morbidly obese human subjects who gave written informed consent, validated and approved by the ethical committee of the Hospital Universitari Dr. Josep Trueta (Comitè d'Ètica d'Investigació Clínica, approval number 2009046), for the collection of liver biopsy samples and sera at the time of bariatric surgery after 8 hours of fasting. In these subjects, we found that log hepatic transcript levels of NNMT negatively correlated with measures of body iron stores, including log serum ferritin levels (R=-0.39, p=0.002) and log percent transferrin saturation (R=-0.21, p=0.02). We then performed experiments in primary hepatocytes to evaluate the interaction of iron overload and NNMT. We found that treatment with ferric ammonium citrate significantly decreased NNMT transcript levels in either primary human or mouse hepatocytes. We manipulated NNMT expression in primary mouse hepatocytes using adenoviral vectors and discovered that NNMT knockdown exacerbated iron-induced cytotoxicity and enhanced iron-induced expression of transcriptional markers of oxidative and endoplasmic reticulum stress, while NNMT overexpression protected against iron-induced cytotoxicity. In future studies, we would like to elucidate the mechanisms by which NNMT modulates hepatocyte sensitivity to iron overload. A deeper understanding of the relationship of the pathways involved in maintaining iron and energy homeostasis may reveal new therapeutic targets for patients with iron overload disorders, obesity, and NAFLD.