Oxidative Stress Is Activated by Free Fatty Acids in Cultured Human Hepatocytes Giorgio Soardo, M.D., 1 Debora Donnini, M.D., 1 Liana Domenis, M.D., 1 Cristiana Catena, M.D., 1 Daniele De Silvestri, M.D., 1 Dario Cappello, M.D., 1 Alessia Dibenedetto, M.D., 1 Alessia Carnelutti, M.D., 1 Vincenzo Bonasia, M.D., 1 Claudio Pagano, M.D., 2 and Leonardo A. Sechi, M.D. 1 Abstract Background: Nonalcoholic fatty liver disease (NAFLD) is strongly associated to oxidative stress, metabolic syndrome, and cardiovascular risk. Hepatocytes overloaded with fatty acids (FA) could generate substances that interfere with endothelial function, providing a potential explanation for this association. We have inves- tigated the response of cultured human hepatoblastoma cells (Hep-G2) that were exposed to FA by measuring markers of oxidative stress and thrombosis and expression of the insulin receptor. Methods: Hep-G2 cells were conditioned with a mixture of FA with or without N-acetyl-L-cysteine (NAC), glutathione (GSH), or adiponectin (ADN). After 7 days, we measured intracellular GSH (iGSH), nitric oxide (NO), malondialdehyde (MDA), and tissue plasminogen inhibitor-1 (PAI-1). Real-time polymerase chain reac- tion (PCR) was used to determine gene expression of inducible nitric oxide synthase (iNOS) and insulin receptor (INS-R). Results: Exposure to FA decreased iGSH and NO levels in Hep-G2 cells and increased MDA and PAI-1 pro- duction. Gene expression of iNOS and INS-R in Hep-G2 cells was decreased by exposure to FA. Co-incubation with NAC and GSH prevented the change of iNOS mRNA levels, but not of INS-R; co-incubation with ADN restored the gene expression of INS-R, but not of i-NOS. ADN prevented also the FA-induced increase in MDA in cultured human endothelial cells. Conclusion: Exposure to FA activates oxidative stress and production of prothrombotic markers and decreases expression of insulin receptors in cultured human hepatocytes. These effects of FA are partially prevented by ADN and might contribute to the increased cardiovascular risk in patients with NAFLD and metabolic syndrome. Introduction N onalcoholic fatty liver disease (NAFLD) identifies a continuum of pathologic conditions that includes simple steatosis, nonalcoholic steatohepatitis (NASH), and liver cirrhosis. 1,2 NAFLD is a common cause of chronic liver disease and affects approximately 15%–30% of the general population and 70%–90% of persons with obesity and type 2 diabetes. 3,4 Abdominal obesity, hyperglycemia, increased blood pressure, dyslipidemia, and insulin resistance are the hallmarks of the metabolic syndrome and they are fre- quently associated with NAFLD. 5,6 The prevalence of car- diovascular disease is increased in patients with NAFLD independent of other classical risk factors, 7,8 and observa- tional studies demonstrate that cardiovascular events are the leading cause of death in these patients. 9,10 Recent evidence indicates that NAFLD is also associated with preclinical atherosclerosis. 11,12 The biological mechanisms linking NAFLD with athero- sclerotic complications are presently unclear, although some authors have suggested insulin resistance and dyslipidemia as possible factors. 13,14 Another possible mechanism linking NAFLD and atherosclerosis may be activation of oxidative stress that could sustain both the liver and vascular dam- age. 3,4,6 Past studies demonstrated that oxidative stress is increased in patients with NAFLD 15,16 and a decreased ac- tivity of endothelial nitric oxide synthase (NOS) has been shown in rodents with experimentally induced steatosis. 17 In vitro models are useful tools for investigation of the cellular mechanisms leading to activation of oxidative stress 1 Liver and Hypertension Unit, Department of Pathology and Experimental and Clinical Medicine, University of Udine, Udine, Italy. 2 Endocrine-Metabolic Laboratory, Department of Medical and Surgical Sciences, University of Padua, Padua, Italy. METABOLIC SYNDROME AND RELATED DISORDERS Volume 9, Number 5, 2011 Ó Mary Ann Liebert, Inc. Pp. 397–401 DOI: 10.1089/met.2010.0140 397