Liver Glycerol Permeability and Aquaporin-9 Are Dysregulated in a Murine Model of Non-Alcoholic Fatty Liver Disease Patrizia Gena 1 , Maria Mastrodonato 2 , Piero Portincasa 3 , Elena Fanelli 1 , Donatella Mentino 2 , Amaia Rodrı´guez 4 , Rau ´ l A. Marinelli 5 , Catherine Brenner 6 , Gema Fru ¨ hbeck 4 , Maria Svelto 1,7 , Giuseppe Calamita 1,7,8 * 1 Department of Biosciences, Biotechnologies and Biopharmaceutics, University of Bari Aldo Moro, Bari, Italy, 2 Department of Biology, University of Bari Aldo Moro, Bari, Italy, 3 Department of Biomedical Sciences and Human Oncology, University of Bari Aldo Moro, Bari, Italy, 4 Metabolic Research Laboratory, Clı ´nica Universidad de Navarra, The Spanish Biomedical Research Centre in Physiopathology of Obesity and Nutrition, Pamplona, Spain, 5 Instituto de Fisiologı ´a Experimental, Consejo Nacional de Investigaciones Cientı ´ficas y Te ´ cnicas, Universidad Nacional de Rosario, Rosario, Argentina, 6 Institut National de la Sante ´ et de la Recherche Me ´ dicale U769, LabEx LERMIT, Universite ´ Paris-Sud, Cha ˆtenay-Malabry, France, 7 Centro di Eccellenza di Genomica in campo Biomedico ed Agrario, Bari, Italy, 8 Network of Apulian Public Research Laboratories ‘‘WAFITECH’’, Bari-Lecce, Italy Abstract One form of liver steatosis, namely Non-Alcoholic Fatty Liver Disease (NAFLD), is a worrisome health problem worldwide characterized by intrahepatic triacylglycerol (TG) overaccumulation. NAFLD is a common feature of metabolic syndrome being often associated with obesity, dyslipidemia and diabetes and mostly closely linked to insulin resistance. The mechanism of NAFLD pathogenesis is object of intense investigation especially regarding complex systems ultimately resulting in excessive TG deposition in hepatocytes. However, scarce is the attention about the relevance of hepatic import of glycerol, the other primary source (as glycerol-3-phosphate) of increased TG in hepatocytes. Obese leptin-deficient (ob/ ob) mice, an animal model of NAFLD, were used to evaluate the functional involvement of Aquaporin-9 (AQP9), the major pathway of liver glycerol entry, in hepatosteatosis. By RT-PCR and qPCR, the level of Aqp9 mRNA in the liver of starved obese mice was comparable with the corresponding control lean littermates. By immunoblotting, the AQP9 protein at the hepatocyte sinusoidal plasma membrane of obese mice was markedly lower (33%) than lean mice, a finding fully confirmed by immunohistochemistry. By stopped-flow light scattering, the liver glycerol permeability of ob/ob mice was significantly lower (53%) than lean mice, a finding consistent with both the observed down-regulation of AQP9 protein and increased level of plasma glycerol characterizing obese mice. In summary, our results suggest implication of AQP9 in liver steatosis. The reduction of hepatocyte AQP9 and, consequently, glycerol permeability might be a defensive mechanism to counteract further fat infiltration in liver parenchyma. Citation: Gena P, Mastrodonato M, Portincasa P, Fanelli E, Mentino D, et al. (2013) Liver Glycerol Permeability and Aquaporin-9 Are Dysregulated in a Murine Model of Non-Alcoholic Fatty Liver Disease. PLoS ONE 8(10): e78139. doi:10.1371/journal.pone.0078139 Editor: Michael Mu ¨ ller, Wageningen University, The Netherlands Received July 3, 2013; Accepted September 11, 2013; Published October 30, 2013 Copyright: ß 2013 Gena et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: Funding from Ministero dell’Istruzione, dell’Universita ` e della Ricerca (PRIN20089SRS2X_003) and Regione Puglia (Rete di Laboratori Pubblici di Ricerca WAFITECH) to GC is gratefully acknowledged. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: giuseppe.calamita@uniba.it Introduction Liver steatosis is characterized by ectopic accumulation of fat (primarily triacylglycerols, TG) in hepatocytes in response to metabolic, toxic and viral insults [1]. The most frequent form, namely the non-alcoholic fatty liver disease (NAFLD), affects subjects who do not abuse alcohol and is recognized as the leading cause of chronic liver disease in adults and children [2], [3], [4]. NAFLD has an estimated prevalence of 20–40% in Western countries [5] and is emerging as health problem also in family practice [6]. NAFLD is frequently associated with another harmful condition, the metabolic syndrome, which encompasses several abnormalities such as insulin resistance or established type 2 Diabetes, increased visceral adiposity, overweight/obesity, dysli- pidemia and blood hypertension [7], characteristics commonly associated with increased cardiovascular risk. The current Western diet, high in saturated fats and fructose, plays a significant role [8]. The most worrisome form of NAFLD is the inflammatory- fibrogenic form, namely non-alcoholic steatohepatitis (NASH) which carries a higher risk of developing liver cirrhosis, and hepatocellular carcinoma [9]. Several studies have been recently focusing on the pathogenetic pathways leading to excess of TG in hepatocytes in NAFLD/ NASH. Dysregulated hepatic fatty acid export, oxidation and desaturation and altered systemic and hepatic insulin sensitivity (insulin resistance) are among the main pathways in NAFLD pathogenesis (for a Review see [1]). Altered glycerol uptake by hepatocytes is also a major intersecting component, however, the underlying mechanism has begun to be understood only recently. In this respect, Aquaporin-9 (AQP9), an aquaporin membrane PLOS ONE | www.plosone.org 1 October 2013 | Volume 8 | Issue 10 | e78139