Ceramides as Lipotoxic Mediators of Non-Alcoholic Fatty Liver Diseases and Non-Alcoholic Steatohepatitis

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ABSTRACT Non-alcoholic fatty liver disease (NAFLD) and subsequent progression to non-alcoholic steatohepatitis (NASH) are hepatic manifestations of the metabolic syndrome that represent an unmet therapeutic need. This fellowship application proposes to evaluate the role of a class of lipotoxic fat molecules, termed ceramides, in driving hepatic steatosis and fibrosis. Despite prior studies determining ceramides may be amongst the most deleterious fat-derived metabolites that accumulate in obesity, detailed genetic studies directly manipulating ceramide levels in pre-clinical models of NASH have yet to be performed, lending the research proposed herein novel. Specifically, mechanisms of ceramides within hepatocytes will be probed. Preliminary data demonstrate a single double bond within the sphingosine backbone of these lipids as necessary for these liver pathologies to manifest in mice. Genetically engineered mice lacking dihydroceramide desaturase 1 (DES1), which inserts this double bond, show blunted ceramide levels with a concomitant increase in the dihydroceramides lacking the unique structural feature. When evaluated in preclinical NASH and fibrosis models, whole body DES1 knockout animals are protected from hepatic injury. In Aim 1 hepatocyte specific DES1 knockout animals will be evaluated in models of NASH. In Aim 2, ceramides will be elevated through genetic manipulation of acid ceramidase (ASAH1), an enzyme that degrades ceramides. Whole body and hepatocyte specific depletion of ASAH1 will be evaluated on the basis of ceramide accumulation and NASH progression. Based upon RNA sequencing data indicating a close relationship between ceramide concentrations, fibrosis and endoplasmic reticulum (ER) stress, Aim 3 will evaluate the role of ceramide- coupled ER stress in NASH both in vivo and in vitro. This series of gain and loss of ceramide studies will provide insight into the therapeutic potential of ceramide-lowering interventions and mechanistic insight into how ceramides participate in NASH related metabolic perturbation and histopathology. The proposed research will also greatly enrich my graduate training through acquisition of skills detailed in the Sponsor/Co-Sponsor statement (i.e., histological evaluation of NASH, mass spectrometry-based analysis of ceramide levels, mentorship-guided training in experimental design and statistical analysis). The combination of the proposed research studies and the guidance of my Sponsor and Co-Sponsor will yield valuable information regarding NASH disease progression, novel insight into ceramide mechanisms of action, and be a critical step in the development of my independent research career.