Sviluppo di un modello genetico di steatosi epatica non alcolica con sovraccarico di ferro per la valutazione della potenzialita' terapeutica di chelanti orali

Nonalcoholic fatty liver disease (NAFLD), the hepatic manifestation of the metabolic syndrome, affects 20-34% of the population, and predisposes to cardiovascular and liver diseases. In NAFLD, steatosis is often associated with inflammation determining steatohepatitis (NASH), characterized by more severe metabolic derangement and possible progression to cirrhosis. Hypocaloric diet and physical exercise have proven beneficial, but no drug therapy is available. Evidence suggest that increased iron stores and mutations in the HFE gene responsible for hereditary hemochromatosis (HH) a) are frequently detected in the metabolic syndrome and NAFLD b) are associated with the progression of liver disease, cardiovascular damage, and with altered release of adipocytokines, involved in the pathogenesis of insulin resistance and altered lipid metabolism c) iron depletion by phlebotomy improves insulin resistance, which underpins NAFLD pathophysiology. Recent data obtained by our group suggest that excess hepatic iron induces insulin resistance in hepatocytes, and iron chelators improve metabolic alterations in rats with steatosis. Preliminary data indicate also that increased activity of iron regulatory protein (IRP)1/aconitase is associated with steatosis. As iron further increases IRP1 aconitase activity, which favors lipogenesis, this mechanism may be involved in iron-promoted steatosis development. Aim of the project is to evaluate the role and mechanisms of hepatic iron accumulation on the progression of liver damage in NAFLD, by means of animal models of NAFLD associated with obesity, insulin resistance, and iron overload, and evaluation of gene expression on histological samples of patients. We will determine whether hepatic iron accumulation worsens the metabolic picture, modifies adipocytokines profile, affects lipogenesis, and influences the progression of liver disease, and whether iron overload interacts with genetic abnormalities of insulin signaling. We will next test whether treatment with an oral iron chelator reverts the pathological phenotype. The study will be subdivided into 3 phases. I) Evaluation of the effect of dietary modulation of iron status on glucose and lipid metabolism, IRP1/aconitase activity, adipokines levels, and liver damage in a mouse model of obesity due to mutation of the leptin gene, characterized by simple uncomplicated hepatic steatosis (Lep ob/ob mice and controls). Iron status will also be manipulated by crossing obese mice with mice with deletion of the HFE gene. Results will be compared to that obtained in histological samples of patients with NAFLD with and without iron overload. Any significant effect of iron status on expression of genes involved in metabolism regulation, apoptosis, and fibrogenesis detected in this animal model will be tested for confirmation in liver samples of patients with NAFLD with and without iron overload. Preliminary data indicate that the effect of iron status on glucose tolerance is markedly enhanced in genetically obese compared to wild-type mice, with iron enriched diet resulting in diabetes development, whereas iron deprived diet improved glucose tolerance compared to normal iron content diet. II) Since genetic factors determining reduced activity of the Insulin receptor (Insr) dependent signaling pathway promote the progression of human NAFLD, and genetic ablation of Insr in hepatocytes causes steatosis and liver failure, we will test the role of Insr haploinsufficiency in accelerating the progression of liver disease. To this purpose, mice with Insr haploinsufficiency will be crossed with obese mice. The effect of iron status will be tested in this model of severe insulin resistance, associated with progressive liver disease. III) Finally, we will be able to evaluate the ability of iron chelators to revert and/or prevent the pathological phenotype in the aforementioned models. Results will be compared to that obtained in patients with NAFLD and hyperferritinemia included in an ongoing clinical trial (ClinicalTrials.gov identifier: NCT00658164) before and after iron depletion therapy. The availability of a new genetic model of NAFLD will allow us to recapitulate NAFLD associated with overweight and hyperferritinemia affecting 5% of the population at increased the risk of metabolic, cardiovascular and hepatic complications, and will clarify the role of mutations of genes involved in insulin signaling and iron metabolism in determining the progression of the disease. The availability of liver samples of patients with and without iron overload, and of paired samples of patients before and after iron depletion will permit to test the relevance of our findings for NAFLD patients. Demonstration of a positive effect of iron depletion hopefully will immediately support a wider adoption of iron depletion, and will refine the optimal dietetic advices based on food iron content for these subjects. Moreover, it could suggest the use of oral iron chelators as potential therapy for patients with progressive NASH. We believe that the described sequence of experiments based on a broad approach, the integrated activity of three experienced RU, the high significance of preliminary data already obtained, the already established collaborations with experienced international groups, are all elements that warrant the achievement of the main project's goals within the planned three year period with a significant chance of success. We also feel that the strong rationale of the project and the novelty of its working hypothesis are important factors that anticipate the scientific impact of its results. The answers that should arise from this research are likely to inform and guide future studies of NAFLD and could provide guidance for better identification and treatment of subjects at higher risk of complications.