In situ glucosylceramide synthesis and its pharmacological inhibition analysed in cells by 13C5‐sphingosine precursor feeding and mass spectrometry

Glycosphingolipids (GSLs) fulfil diverse functions in cells. Abnormalities in their metabolism are associated with specific pathologies and, consequently, the pharmacological modulation of GSLs is considered a therapeutic avenue. The accurate measurement of in situ metabolism of GSLs and the modulatory impact of drugs is warranted. Employing synthesised sphingosine and sphinganine containing 13C atoms, we developed a method to monitor the de novo synthesis of glucosylceramide, the precursor of complex GSLs, by the enzyme glucosylceramide synthase (GCS). We show that feeding cells with isotope‐labelled precursor combined with liquid chromatography–mass spectrometry (MS)/MS analysis allows accurate determination of the IC50 values of therapeutically considered inhibitors (iminosugars and ceramide mimics) of GCS in cultured cells. Acquired data were comparable to those obtained with an earlier method using artificial fluorescently labelled ceramide to feed cells.

[1]  H. Oki,et al.  A new brain‐penetrant glucosylceramide synthase inhibitor as potential Therapeutics for Gaucher disease , 2021, Journal of neurochemistry.

[2]  J. Dingemanse,et al.  Assessment of Target Engagement in a First‐in‐Human Trial with Sinbaglustat, an Iminosugar to Treat Lysosomal Storage Disorders , 2020, Clinical and translational science.

[3]  A. Abe,et al.  Optimization of Eliglustat-Based Glucosylceramide Synthase Inhibitors as Substrate Reduction Therapy for Gaucher Disease Type 3. , 2020, ACS chemical neuroscience.

[4]  M. Artola,et al.  Glycosphingolipids and Infection. Potential New Therapeutic Avenues , 2019, Front. Cell Dev. Biol..

[5]  M. Artola,et al.  Glycosphingolipids and lysosomal storage disorders as illustrated by gaucher disease. , 2019, Current opinion in chemical biology.

[6]  K. Sandhoff,et al.  Lysosomal Glycosphingolipid Storage Diseases. , 2019, Annual review of biochemistry.

[7]  G. A. van der Marel,et al.  Functionalized Cyclophellitols Are Selective Glucocerebrosidase Inhibitors and Induce a Bona Fide Neuropathic Gaucher Model in Zebrafish , 2019, Journal of the American Chemical Society.

[8]  M. Artola,et al.  In vivo inactivation of glycosidases by conduritol B epoxide and cyclophellitol as revealed by activity‐based protein profiling , 2019, The FEBS journal.

[9]  O. Morand,et al.  Glucosylceramide synthase inhibition with lucerastat lowers globotriaosylceramide and lysosome staining in cultured fibroblasts from Fabry patients with different mutation types , 2018, Human molecular genetics.

[10]  Y. Hannun,et al.  Probing de novo sphingolipid metabolism in mammalian cells utilizing mass spectrometry[S] , 2018, Journal of Lipid Research.

[11]  G. A. van der Marel,et al.  A Fluorescence Polarization Activity-Based Protein Profiling Assay in the Discovery of Potent, Selective Inhibitors for Human Nonlysosomal Glucosylceramidase , 2017, Journal of the American Chemical Society.

[12]  G. A. van der Marel,et al.  Simultaneous quantitation of sphingoid bases by UPLC-ESI-MS/MS with identical 13C-encoded internal standards. , 2017, Clinica chimica acta; international journal of clinical chemistry.

[13]  S. Sestito,et al.  Combination therapy in a patient with chronic neuronopathic Gaucher disease: a case report , 2017, Journal of Medical Case Reports.

[14]  M. van Eijk,et al.  Accurate quantification of sphingosine-1-phosphate in normal and Fabry disease plasma, cells and tissues by LC-MS/MS with (13)C-encoded natural S1P as internal standard. , 2016, Clinica chimica acta; international journal of clinical chemistry.

[15]  Ying Sun,et al.  CNS-accessible Inhibitor of Glucosylceramide Synthase for Substrate Reduction Therapy of Neuronopathic Gaucher Disease , 2016, Molecular therapy : the journal of the American Society of Gene Therapy.

[16]  S. D. de Jager,et al.  Glucocerebrosidase 1 deficient Danio rerio mirror key pathological aspects of human Gaucher disease and provide evidence of early microglial activation preceding alpha-synuclein-independent neuronal cell death , 2015, Human molecular genetics.

[17]  M. Balwani,et al.  Eliglustat compared with imiglucerase in patients with Gaucher's disease type 1 stabilised on enzyme replacement therapy: a phase 3, randomised, open-label, non-inferiority trial , 2015, The Lancet.

[18]  H. Overkleeft,et al.  Synthesis of a Panel of Carbon‐13‐Labelled (Glyco)Sphingolipids , 2015 .

[19]  H. Overkleeft,et al.  Identification and development of biphenyl substituted iminosugars as improved dual glucosylceramide synthase/neutral glucosylceramidase inhibitors. , 2014, Journal of medicinal chemistry.

[20]  S. Sonnino,et al.  Membrane domains and the "lipid raft" concept. , 2012, Current medicinal chemistry.

[21]  Alfred H. Merrill,et al.  Sphingolipid and Glycosphingolipid Metabolic Pathways in the Era of Sphingolipidomics , 2011, Chemical reviews.

[22]  M. Langeveld,et al.  Glycosphingolipids and insulin resistance. , 2011, Advances in experimental medicine and biology.

[23]  B. Bembi,et al.  Eye Movement Impairment Recovery in a Gaucher Patient Treated with Miglustat , 2010, Neurology research international.

[24]  H. Overkleeft,et al.  Glycosphingolipids — Nature, Function, and Pharmacological Modulation , 2010 .

[25]  M. Langeveld,et al.  Modulation of glycosphingolipid metabolism significantly improves hepatic insulin sensitivity and reverses hepatic steatosis in mice , 2009, Hepatology.

[26]  P. Dubbelhuis,et al.  Reducing Glycosphingolipid Content in Adipose Tissue of Obese Mice Restores Insulin Sensitivity, Adipogenesis and Reduces Inflammation , 2009, PloS one.

[27]  R. Schiffmann,et al.  Randomized, controlled trial of miglustat in Gaucher's disease type 3 , 2008, Annals of neurology.

[28]  L. Bour,et al.  Potential efficacy of enzyme replacement and substrate reduction therapy in three siblings with Gaucher disease type III , 2008, Journal of Inherited Metabolic Disease.

[29]  Y. Hannun,et al.  The sphingolipid salvage pathway in ceramide metabolism and signaling. , 2008, Cellular signalling.

[30]  J. Shayman,et al.  A specific and potent inhibitor of glucosylceramide synthase for substrate inhibition therapy of Gaucher disease. , 2007, Molecular genetics and metabolism.

[31]  P. Alfonso,et al.  Neurologic Improvement in a Type 3 Gaucher Disease Patient Treated with Imiglucerase/Miglustat Combination , 2007, Epilepsia.

[32]  S. O’Rahilly,et al.  Pharmacological Inhibition of Glucosylceramide Synthase Enhances Insulin Sensitivity , 2007, Diabetes.

[33]  C. Hollak,et al.  HPLC for simultaneous quantification of total ceramide, glucosylceramide, and ceramide trihexoside concentrations in plasma. , 2007, Clinical chemistry.

[34]  S. Sonnino,et al.  Sphingolipid Uptake by Cultured Cells COMPLEX AGGREGATES OF CELL SPHINGOLIPIDS WITH SERUM PROTEINS AND LIPOPROTEINS ARE RAPIDLY CATABOLIZED* , 2005 .

[35]  Alfred H. Merrill,et al.  De Novo Sphingolipid Biosynthesis: A Necessary, but Dangerous, Pathway* , 2002, The Journal of Biological Chemistry.

[36]  J. Aerts,et al.  Low-dose N-butyldeoxynojirimycin (OGT 918) for type I Gaucher disease. , 2002, Blood cells, molecules & diseases.

[37]  Reuben.,et al.  Glycosphingolipids in Cultured Human Skin Fibroblasts , 2002 .

[38]  R. Dwek,et al.  Novel oral treatment of Gaucher's disease with N-butyldeoxynojirimycin (OGT 918) to decrease substrate biosynthesis , 2000, The Lancet.

[39]  Y. Hirabayashi,et al.  Expression cloning of a cDNA for human ceramide glucosyltransferase that catalyzes the first glycosylation step of glycosphingolipid synthesis. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[40]  R. Dwek,et al.  N-butyldeoxynojirimycin is a novel inhibitor of glycolipid biosynthesis. , 1994, The Journal of biological chemistry.

[41]  J. Goldblatt,et al.  Clinical phenotype of Gaucher disease in relation to properties of mutant glucocerebrosidase in cultured fibroblasts. , 1991, Biochimica et biophysica acta.

[42]  S. Sonnino,et al.  Recycling of glucosylceramide and sphingosine for the biosynthesis of gangliosides and sphingomyelin in rat liver. , 1990, The Biochemical journal.

[43]  W. J. Dyer,et al.  A rapid method of total lipid extraction and purification. , 1959, Canadian journal of biochemistry and physiology.