Exogenous Liposomal Ceramide-C6 Ameliorates Lipidomic Profile, Energy Homeostasis, and Anti-Oxidant Systems in NASH
Francesca Zanieri,
Ana Levi,
David Montefusco,
Lisa Longato,
Francesco De Chiara,
Luca Frenguelli,
Sara Omenetti,
Fausto Andreola,
Tu Vinh Luong,
Veronica Massey,
Juan Caballeria,
Constantino Fondevila,
Sriram S Shanmugavelandy,
Todd Fox,
Giuseppe Mazza,
Josepmaria Argemi,
Ramon Bataller,
Lauren Ashley Cowart,
Mark Kester,
Massimo Pinzani,
Krista Rombouts
Affiliations
Francesca Zanieri
Department of Experimental and Clinical Medicine and Center of Excellence “DENOthe”, University of Florence, 50134 Florence, Italy
Ana Levi
Institute for Liver & Digestive Health, Royal Free, University College London UCL, London NW3 2PF, UK
David Montefusco
Virginia Commonwealth University, Richmond, VA 23219, USA
Lisa Longato
Institute for Liver & Digestive Health, Royal Free, University College London UCL, London NW3 2PF, UK
Francesco De Chiara
Institute for Liver & Digestive Health, Royal Free, University College London UCL, London NW3 2PF, UK
Luca Frenguelli
Institute for Liver & Digestive Health, Royal Free, University College London UCL, London NW3 2PF, UK
Sara Omenetti
Department of Experimental and Clinical Medicine and Center of Excellence “DENOthe”, University of Florence, 50134 Florence, Italy
Fausto Andreola
Institute for Liver & Digestive Health, Royal Free, University College London UCL, London NW3 2PF, UK
Tu Vinh Luong
Institute for Liver & Digestive Health, Royal Free, University College London UCL, London NW3 2PF, UK
Veronica Massey
Division of Hepatology and Gastroenterology, Departments of Medicine and Nutrition, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA
Liver Transplant Unit, Department of Surgery, Hospital Clinic, University of Barcelona, 08036 Barcelona, Spain
Sriram S Shanmugavelandy
Department of Pharmacology, Penn State University College of Medicine, Hershey, PA 17033, USA
Todd Fox
Department of Pharmacology, Penn State University College of Medicine, Hershey, PA 17033, USA
Giuseppe Mazza
Institute for Liver & Digestive Health, Royal Free, University College London UCL, London NW3 2PF, UK
Josepmaria Argemi
Department of Medicine, Pittsburgh Liver Research Center, University of Pittsburgh, Pittsburgh, PA 15261, USA
Ramon Bataller
Division of Hepatology and Gastroenterology, Departments of Medicine and Nutrition, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA
Lauren Ashley Cowart
Virginia Commonwealth University, Richmond, VA 23219, USA
Mark Kester
Department of Pharmacology, Penn State University College of Medicine, Hershey, PA 17033, USA
Massimo Pinzani
Department of Experimental and Clinical Medicine and Center of Excellence “DENOthe”, University of Florence, 50134 Florence, Italy
Krista Rombouts
Department of Experimental and Clinical Medicine and Center of Excellence “DENOthe”, University of Florence, 50134 Florence, Italy
In non-alcoholic steatohepatitis (NASH), many lines of investigation have reported a dysregulation in lipid homeostasis, leading to intrahepatic lipid accumulation. Recently, the role of dysfunctional sphingolipid metabolism has also been proposed. Human and animal models of NASH have been associated with elevated levels of long chain ceramides and pro-apoptotic sphingolipid metabolites, implicated in regulating fatty acid oxidation and inflammation. Importantly, inhibition of de novo ceramide biosynthesis or knock-down of ceramide synthases reverse some of the pathology of NASH. In contrast, cell permeable, short chain ceramides have shown anti-inflammatory actions in multiple models of inflammatory disease. Here, we investigated non-apoptotic doses of a liposome containing short chain C6-Ceramide (Lip-C6) administered to human hepatic stellate cells (hHSC), a key effector of hepatic fibrogenesis, and an animal model characterized by inflammation and elevated liver fat content. On the basis of the results from unbiased liver transcriptomic studies from non-alcoholic fatty liver disease patients, we chose to focus on adenosine monophosphate activated kinase (AMPK) and nuclear factor-erythroid 2-related factor (Nrf2) signaling pathways, which showed an abnormal profile. Lip-C6 administration inhibited hHSC proliferation while improving anti-oxidant protection and energy homeostasis, as indicated by upregulation of Nrf2, activation of AMPK and an increase in ATP. To confirm these in vitro data, we investigated the effect of a single tail-vein injection of Lip-C6 in the methionine-choline deficient (MCD) diet mouse model. Lip-C6, but not control liposomes, upregulated phospho-AMPK, without inducing liver toxicity, apoptosis, or exacerbating inflammatory signaling pathways. Alluding to mechanism, mass spectrometry lipidomics showed that Lip-C6-treatment reversed the imbalance in hepatic phosphatidylcholines and diacylglycerides species induced by the MCD-fed diet. These results reveal that short-term Lip-C6 administration reverses energy/metabolic depletion and increases protective anti-oxidant signaling pathways, possibly by restoring homeostatic lipid function in a model of liver inflammation with fat accumulation.