Characterisation of the First Specific Inhibitor of Ceramide Synthase 1
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Characterisation of the first specific inhibitor of ceramide synthase 1 Xin Ying Lim A thesis submitted for the fulfilment of the requirements for the degree of Doctor of Philosophy (PhD) Prince of Wales Clinical School Faculty of Medicine University of New South Wales May 2018 THE UNIVERSITY OF NEW SOUTH WALES Thesis/Dissertation Sheet Surname or Family name: Lim First name: Xin Ying Abbreviation for degree as given in the University calendar: PhD School: Prince of Wales Clinical School Faculty: Faculty of Medicine Title: Characterisation of the first specific inhibitor of Ceramide Synthase 1 Abstract (348 words): The signalling lipid ceramide has been implicated in the pathogenesis of obesity-related insulin resistance. Ceramide is synthesized by a family of six ceramide synthases in mammals (CerS1-6), each of which preferentially utilises fatty acids of particular acyl chain length (C14-C28). In skeletal muscle (SkM), C18 ceramide, which is synthesized almost exclusively by ceramide synthase 1 (CerS1), has been suggested to mediate systemic insulin resistance in obese humans and rodent models. Therefore, targeted reduction of SkM C18 ceramide may improve detrimental metabolic outcomes induced by a high fat diet (HFD). This thesis describes the characterisation of a new, selective inhibitor of CerS1, P053. P053 inhibits CerS1 with an IC50 of 0.5 micromolar and selectively reduced C18 ceramide in cultured cells. Lipidomic profiling revealed that P053 administration specifically reduced C18 ceramide levels in SkM of mice fed with standard chow or HFD for 4-6 weeks. The reduction of SkM C18 ceramide was coupled to a compensatory increase in C24 ceramides, which are synthesized by CerS2, and loss of triacylglycerols. No other complex lipid species were affected by P053 treatment. At the physiological level, P053 impeded fat deposition induced by a HFD. Despite its effect on adiposity, P053 treatment had no effect on glucose tolerance and insulin sensitivity, assessed by glucose tolerance test and hyperinsulinemic-euglycemic clamps. Unexpectedly, prolonged CerS1 inhibition with P053 improved mitochondrial oxidative capacity in SkM. This effect was associated with significant increases in mitochondrial respiratory chain complex levels, increased activity of TCA cycle and β-oxidation enzymes, and increased mitochondrial respiratory capacity in SkM. Enhanced mitochondrial metabolism may therefore underlie the reduced lipid accretion in mice treated with P053. A significant positive correlation between SkM C18 ceramide and body adiposity was identified. In contrast, SkM C24 ceramides were inversely correlated with adiposity. Thus, P053 may reduce adiposity both directly by reducing SkM C18 ceramide and indirectly through the associated increase in C24 ceramides. In conclusion, we have generated the first selective CerS1 inhibitor. Results from this thesis revealed a potential new role for CerS1 as an endogenous inhibitor of mitochondrial oxidative function in SkM and regulator of whole-body adiposity. Declaration relating to disposition of project thesis/dissertation I hereby grant to the University of New South Wales or its agents the right to archive and to make available my thesis or dissertation in whole or in part in the University libraries in all forms of media, now or here after known, subject to the provisions of the Copyright Act 1968. I retain all property rights, such as patent rights. I also retain the right to use in future works (such as articles or books) all or part of this thesis or dissertation. I also authorise University Microfilms to use the 350 word abstract of my thesis in Dissertation Abstracts International (this is applicable to doctoral theses only). ……………………………………………… ……………………………………..……… ……….……………………...… …………… ……… ….… Signature Witness Date The University recognises that there may be exceptional circumstances requiring restrictions on copying or conditions on use. Requests for restriction for a period of up to 2 years must be made in writing. Requests for a longer period of restriction may be considered in exceptional circumstances and require the approval of the Dean of Graduate Research. FOR OFFICE USE ONLY Date of completion of requirements for Award: I ORIGINALITY STATEMENT ‘I hereby declare that this submission is my own work and to the best of my knowledge it contains no materials previously published or written by another person, or substantial proportions of material which have been accepted for the award of any other degree or diploma at UNSW or any other educational institution, except where due acknowledgement is made in the thesis. Any contribution made to the research by others, with whom I have worked at UNSW or elsewhere, is explicitly acknowledged in the thesis. I also declare that the intellectual content of this thesis is the product of my own work, except to the extent that assistance from others in the project's design and conception or in style, presentation and linguistic expression is acknowledged.’ Signed …………………………………………….............. Date …………………………………………….............. II Abstract The signalling lipid ceramide has been implicated in the pathogenesis of obesity-related insulin resistance. Ceramide is synthesized by a family of six ceramide synthases in mammals (CerS1-6), each of which preferentially utilises fatty acids of particular acyl chain length (C14-C28). In skeletal muscle (SkM), C18 ceramide, which is synthesized almost exclusively by ceramide synthase 1 (CerS1), has been suggested to mediate systemic insulin resistance in obese humans and rodent models. Therefore, targeted reduction of SkM C18 ceramide may improve detrimental metabolic outcomes induced by a high fat diet (HFD). This thesis describes the characterisation of a new, selective inhibitor of CerS1, P053. P053 inhibits CerS1 with an IC50 of 0.5 micromolar and selectively reduced C18 ceramide in cultured cells. Lipidomic profiling revealed that P053 administration specifically reduced C18 ceramide levels in SkM of mice fed with standard chow or HFD for 4-6 weeks. The reduction of SkM C18 ceramide was coupled to a compensatory increase in C24 ceramides, which are synthesized by CerS2, and loss of triacylglycerols. No other complex lipid species were affected by P053 treatment. At the physiological level, P053 impeded fat deposition induced by a HFD. Despite its effect on adiposity, P053 treatment had no effect on glucose tolerance and insulin sensitivity, assessed by glucose tolerance test and hyperinsulinemic-euglycemic clamps. Unexpectedly, prolonged CerS1 inhibition with P053 improved mitochondrial oxidative capacity in SkM. This effect was associated with significant increases in mitochondrial respiratory chain complex levels, increased activity of TCA cycle and β-oxidation enzymes, and increased mitochondrial respiratory capacity in SkM. Enhanced mitochondrial metabolism may therefore underlie the reduced lipid accretion in mice treated with P053. A significant positive correlation between SkM C18 ceramide and body adiposity was identified. In contrast, SkM C24 ceramides were inversely correlated with adiposity. Thus, P053 may reduce adiposity both directly by reducing SkM C18 ceramide and indirectly through the associated increase in C24 ceramides. In conclusion, we have generated the first selective CerS1 inhibitor. Results from this thesis revealed a potential new role for CerS1 as an endogenous inhibitor of mitochondrial oxidative function in SkM and regulator of whole-body adiposity. III Publications and presentations arising from this thesis Publications: • Lim, X. Y., Pickford, R., and Don, A. S. (2016) Assaying Ceramide Synthase Activity In Vitro and in Living Cells Using Liquid Chromatography-Mass Spectrometry. Methods Mol Biol 1376, 11-22 • *Turner, N., *Lim, X. Y., Toop, H. D., Brandon, A., Osborne, B., Taylor, E. N., Fiveash, C. E., Teo, J., McEwen, H. P., Govindaraju, H., Couttas, T. A., Das, A., Kowalski, G. M., Bruce, C. R., Fath, T., Schmitz-Peiffer, C., Cooney, G., Montgomery, M. K., Morris, J. C., Don, A. S. (2018) A selective inhibitor of ceramide synthase 1 reveals a novel role in fat metabolism. Nature Communications (Accepted for publication) *Co-first authors Presentations: • Lim, X. Y., Toop, H., Montgomery, M. K., Turner, N., Morris, J., Don, A. S. (2016) Title: Lipidomic characterization of the first specific inhibitor of Ceramide Synthase 1: Implications for Insulin Resistance. The Australian and New Zealand Metabolomics Conference, 30th March- 1st April, Melbourne, Australia. (oral presentation) • Lim, X. Y., Toop, H., Montgomery, M. K., Turner, N., Morris, J., Don, A. S. (2016) Title: Lipidomic characterization of the first specific inhibitor of Ceramide Synthase 1: Implications for Metabolic diseases. ComBio 2016, 3rd- 7th October, Queensland, Australia. (oral presentation) • Lim, X. Y., Toop, H., Montgomery, M. K., Fiveash, C., Osborne, B., Fath, T., Morris, J., Turner, N., Don, A. S. (2016) Title: Lipidomic characterization of the first specific inhibitor of Ceramide Synthase 1: Implications for Metabolic diseases. 3rd Australian Lipid Meeting, 21st -22nd November, Melbourne, Australia. (poster presentation; best poster prize) • Lim, X. Y., Toop, H., Montgomery, M. K., Morris, J., Turner, N., Don, A. S. (2017) Title: A potent and specific Ceramide Synthase 1 inhibitor: Implications for Metabolic Diseases. Keystone Symposia: Lipidomics and Bioactive Lipids in Metabolism and Disease, 26th February- 2nd March, California, USA. (poster presentation) IV Publications arising