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01-01-2015 | Complex Lipids

The consequences of genetic and pharmacologic reduction in sphingolipid synthesis

Author: Raphael Schiffmann

Published in: Journal of Inherited Metabolic Disease | Issue 1/2015

Abstract

A new therapy based on substrate synthesis reduction in sphingolipidoses is showing promise. The consequences of decreasing sphingolipid synthesis depend on the level at which synthetic blockage occurs and on the extent of the blockage. Complete synthetic blockage may be lethal if it includes all sphingolipids, such as in a global knockout of serine palmitoyltransferase. Partial inhibition of sphingolipid synthetic pathways is usually benign and may have beneficial effects in a number of lysosomal diseases and in more common pathologies, as seen in animal models for atherosclerosis, polycystic kidney disease, diabetes, and asthma. Studies of various forms of sphingolipid synthesis reduction serve to highlight not only the cellular role of these lipids but also the potential risks and therapeutic benefits of pharmacological agents to be used in therapy for human diseases.

Abe A, Gregory S, Lee L et al (2000) Reduction of globotriaosylceramide in fabry disease mice by substrate deprivation. J Clin Invest 105(11):1563–1571PubMedCentralPubMedCrossRef

Aerts JM, Ottenhoff R, Powlson AS et al (2007) Pharmacological inhibition of glucosylceramide synthase enhances insulin sensitivity. Diabetes 56(5):1341–1349PubMedCrossRef

Andersson U, Smith D, Jeyakumar M et al (2004) Improved outcome of N-butyldeoxygalactonojirimycin-mediated substrate reduction therapy in a mouse model of Sandhoff disease. Neurobiol Dis 16(3):506–515PubMedCrossRef

Arthur JR, Wilson MW, Larsen SD, Rockwell HE, Shayman JA, Seyfried TN (2013) Ethylenedioxy-PIP2 oxalate reduces ganglioside storage in juvenile sandhoff disease mice. Neurochem Res 38(4):866–875PubMedCrossRef

Ashe KM, Bangari D, Li L et al (2011) Iminosugar-based inhibitors of glucosylceramide synthase increase brain glycosphingolipids and survival in a mouse model of sandhoff disease. PLoS One 6(6):e21758PubMedCentralPubMedCrossRef

Bietrix F, Lombardo E, van Roomen CP et al (2010) Inhibition of glycosphingolipid synthesis induces a profound reduction of plasma cholesterol and inhibits atherosclerosis development in APOE*3 Leiden and low-density lipoprotein receptor-/- mice. Arterioscler Thromb Vasc Biol 30(5):931–937PubMedCrossRef

Boot RG, Verhoek M, Donker-Koopman W et al (2007) Identification of the non-lysosomal glucosylceramidase as beta-glucosidase 2. J Biol Chem 282(2):1305–1312PubMedCrossRef

Boukhris A, Schule R, Loureiro JL et al (2013) Alteration of ganglioside biosynthesis responsible for complex hereditary spastic paraplegia. Am J Hum Genet 93(1):118–123PubMedCentralPubMedCrossRef

Bremer EG, Hakomori S, Bowen-Pope DF, Raines E, Ross R (1984) Ganglioside-mediated modulation of cell growth, growth factor binding, and receptor phosphorylation. J Biol Chem 259(11):6818–6825PubMed

Breslow DK, Weissman JS (2010) Membranes in balance: mechanisms of sphingolipid homeostasis. Mol Cell 40(2):267–279PubMedCentralPubMedCrossRef

Cabrera-Salazar MA, Deriso M, Bercury SD et al (2012) Systemic delivery of a glucosylceramide synthase inhibitor reduces CNS substrates and increases lifespan in a mouse model of type 2 Gaucher disease. PLoS One 7(8):e43310PubMedCentralPubMedCrossRef

Chatterjee S, Alsaeedi N (2012) Lactosylceramide synthase as a therapeutic target to mitigate multiple human diseases in animal models. Adv Exp Med Biol 749:153–169PubMedCrossRef

Chatterjee S, Kolmakova A, Rajesh M (2008) Regulation of lactosylceramide synthase (glucosylceramide beta1– > 4 galactosyltransferase); implication as a drug target. Curr Drug Targets 9(4):272–281PubMedCrossRef

Chatterjee S, Bedja D, Mishra S, et al (2014) Inhibition of glycosphingolipid synthesis ameliorates atherosclerosis and arterial stiffness in apo E-/- mice and rabbits fed a high fat and cholesterol diet. Circulation doi: 10.1161/CIRCULATIONAHA.113.007559.

Chun L, Junlin Z, Aimin W, Niansheng L, Benmei C, Minxiang L (2011) Inhibition of ceramide synthesis reverses endothelial dysfunction and atherosclerosis in streptozotocin-induced diabetic rats. Diabetes Res Clin Pract 93(1):77–85PubMedCrossRef

Coetzee T, Fujita N, Dupree J et al (1996) Myelination in the absence of galactocerebroside and sulfatide: normal structure with abnormal function and regional instability. Cell 86(2):209–219PubMedCrossRef

Cuzzocrea S, Di Paola R, Genovese T et al (2008) Anti-inflammatory and anti-apoptotic effects of fumonisin B1, an inhibitor of ceramide synthase, in a rodent model of splanchnic ischemia and reperfusion injury. J Pharmacol Exp Ther 327(1):45–57PubMedCrossRef

Dawkins JL, Hulme DJ, Brahmbhatt SB, Auer-Grumbach M, Nicholson GA (2001) Mutations in SPTLC1, encoding serine palmitoyltransferase, long chain base subunit-1, cause hereditary sensory neuropathy type I. Nat Genet 27(3):309–312PubMedCrossRef

Deshmukh GD, Radin NS, Gattone VH 2nd, Shayman JA (1994) Abnormalities of glycosphingolipid, sulfatide, and ceramide in the polycystic (cpk/cpk) mouse. J Lipid Res 35(9):1611–1618PubMed

Eichler FS, Hornemann T, McCampbell A et al (2009) Overexpression of the wild-type SPT1 subunit lowers desoxysphingolipid levels and rescues the phenotype of HSAN1. J Neurosci 29(46):14646–14651PubMedCentralPubMedCrossRef

Elstein D, Hollak C, Aerts JM et al (2004) Sustained therapeutic effects of oral miglustat (Zavesca, N-butyldeoxynojirimycin, OGT 918) in type I Gaucher disease. J Inherit Metab Dis 27(6):757–766PubMedCrossRef

Fan Y, Shi F, Liu J et al (2010) Selective reduction in the sphingomyelin content of atherogenic lipoproteins inhibits their retention in murine aortas and the subsequent development of atherosclerosis. Arterioscler Thromb Vasc Biol 30(11):2114–2120PubMedCentralPubMedCrossRef

Fragaki K, Ait-El-Mkadem S, Chaussenot A et al (2013) Refractory epilepsy and mitochondrial dysfunction due to GM3 synthase deficiency. Eur J Hum Genet 21(5):528–534PubMedCentralPubMedCrossRef

Futerman AH, Hannun YA (2004) The complex life of simple sphingolipids. EMBO Rep 5(8):777–782PubMedCentralPubMedCrossRef

Gable K, Gupta SD, Han G, Niranjanakumari S, Harmon JM, Dunn TM (2010) A disease-causing mutation in the active site of serine palmitoyltransferase causes catalytic promiscuity. J Biol Chem 285(30):22846–22852PubMedCentralPubMedCrossRef

Garofalo K, Penno A, Schmidt BP et al (2011) Oral L-serine supplementation reduces production of neurotoxic deoxysphingolipids in mice and humans with hereditary sensory autonomic neuropathy type 1. J Clin Invest 121(12):4735–4745PubMedCentralPubMedCrossRef

Glaros EN, Kim WS, Quinn CM, Jessup W, Rye KA, Garner B (2008) Myriocin slows the progression of established atherosclerotic lesions in apolipoprotein E gene knockout mice. J Lipid Res 49(2):324–331PubMedCrossRef

Hailemariam TK, Huan C, Liu J et al (2008) Sphingomyelin synthase 2 deficiency attenuates NFkappaB activation. Arterioscler Thromb Vasc Biol 28(8):1519–1526PubMedCrossRef

Hannun YA, Obeid LM (2008) Principles of bioactive lipid signalling: lessons from sphingolipids. Nat Rev Mol Cell Biol 9(2):139–150PubMedCrossRef

Hla T, Dannenberg AJ (2012) Sphingolipid signaling in metabolic disorders. Cell Metab 16(4):420–434PubMedCentralPubMedCrossRef

Hojjati MR, Li Z, Jiang XC (2005a) Serine palmitoyl-CoA transferase (SPT) deficiency and sphingolipid levels in mice. Biochim Biophys Acta 1737(1):44–51PubMedCrossRef

Hojjati MR, Li Z, Zhou H et al (2005b) Effect of myriocin on plasma sphingolipid metabolism and atherosclerosis in apoE-deficient mice. J Biol Chem 280(11):10284–10289PubMedCrossRef

Honke K, Hirahara Y, Dupree J et al (2002) Paranodal junction formation and spermatogenesis require sulfoglycolipids. Proc Natl Acad Sci U S A 99(7):4227–4232PubMedCentralPubMedCrossRef

Hornemann T, Richard S, Rutti MF, Wei Y, von Eckardstein A (2006) Cloning and initial characterization of a new subunit for mammalian serine-palmitoyltransferase. J Biol Chem 281(49):37275–37281PubMedCrossRef

Inokuchi J, Mason I, Radin NS (1987) Antitumor activity via inhibition of glycosphingolipid biosynthesis. Cancer Lett 38(1–2):23–30PubMedCrossRef

Inoue M, Fujii Y, Furukawa K et al (2002) Refractory skin injury in complex knock-out mice expressing only the GM3 ganglioside. J Biol Chem 277(33):29881–29888PubMedCrossRef

Jennemann R, Sandhoff R, Wang S et al (2005) Cell-specific deletion of glucosylceramide synthase in brain leads to severe neural defects after birth. Proc Natl Acad Sci U S A 102(35):12459–12464PubMedCentralPubMedCrossRef

Jeyakumar M, Norflus F, Tifft CJ et al (2001) Enhanced survival in Sandhoff disease mice receiving a combination of substrate deprivation therapy and bone marrow transplantation. Blood 97(1):327–329PubMedCrossRef

Karman J, Tedstone JL, Gumlaw NK et al (2010) Reducing glycosphingolipid biosynthesis in airway cells partially ameliorates disease manifestations in a mouse model of asthma. Int Immunol 22(7):593–603PubMedCrossRef

Kumagai T, Sato T, Natsuka S et al (2010) Involvement of murine beta-1,4-galactosyltransferase V in lactosylceramide biosynthesis. Glycoconj J 27(7–9):685–695PubMedCentralPubMedCrossRef

Kurek K, Wiesiolek-Kurek P, Piotrowska DM, Lukaszuk B, Chabowski A, Zendzian-Piotrowska M (2014) Inhibition of ceramide de novo synthesis with myriocin affects lipid metabolism in the liver of rats with streptozotocin-induced type 1 diabetes. Biomed Res Int 2014:980815PubMedCentralPubMedCrossRef

Langeveld M, van den Berg SA, Bijl N et al (2012) Treatment of genetically obese mice with the iminosugar N-(5-adamantane-1-yl-methoxy-pentyl)-deoxynojirimycin reduces body weight by decreasing food intake and increasing fat oxidation. Metabolism 61(1):99–107PubMedCrossRef

Lee SY, Kim JR, Hu Y et al (2012) Cardiomyocyte specific deficiency of serine palmitoyltransferase subunit 2 reduces ceramide but leads to cardiac dysfunction. J Biol Chem 287(22):18429–18439PubMedCentralPubMedCrossRef

Li Z, Park TS, Li Y et al (2009) Serine palmitoyltransferase (SPT) deficient mice absorb less cholesterol. Biochim Biophys Acta 1791(4):297–306PubMedCrossRef

Li Z, Fan Y, Liu J et al (2012) Impact of sphingomyelin synthase 1 deficiency on sphingolipid metabolism and atherosclerosis in mice. Arterioscler Thromb Vasc Biol 32(7):1577–1584PubMedCentralPubMedCrossRef

Liu Y, Wada R, Kawai H et al (1999) A genetic model of substrate deprivation therapy for a glycosphingolipid storage disorder. J Clin Invest 103(4):497–505PubMedCentralPubMedCrossRef

Liu J, Huan C, Chakraborty M et al (2009a) Macrophage sphingomyelin synthase 2 deficiency decreases atherosclerosis in mice. Circ Res 105(3):295–303PubMedCentralPubMedCrossRef

Liu J, Zhang H, Li Z et al (2009b) Sphingomyelin synthase 2 is one of the determinants for plasma and liver sphingomyelin levels in mice. Arterioscler Thromb Vasc Biol 29(6):850–856PubMedCentralPubMedCrossRef

Lombardo E, van Roomen CP, van Puijvelde GH et al (2012) Correction of liver steatosis by a hydrophobic iminosugar modulating glycosphingolipids metabolism. PLoS One 7(10):e38520PubMedCentralPubMedCrossRef

Lukina E, Watman N, Arreguin EA et al (2010) A phase 2 study of eliglustat tartrate (Genz-112638), an oral substrate reduction therapy for Gaucher disease type 1. Blood 116(6):893–899PubMedCentralPubMedCrossRef

Marshall J, Ashe KM, Bangari D et al (2010a) Substrate reduction augments the efficacy of enzyme therapy in a mouse model of Fabry disease. PLoS One 5(11):e15033PubMedCentralPubMedCrossRef

Marshall J, McEachern KA, Chuang WL et al (2010b) Improved management of lysosomal glucosylceramide levels in a mouse model of type 1 Gaucher disease using enzyme and substrate reduction therapy. J Inherit Metab Dis 33(3):281–289PubMedCentralPubMedCrossRef

McDonald G, Deepak S, Miguel L et al (2014) Normalizing glycosphingolipids restores function in CD4+ T cells from lupus patients. J Clin Invest 124(2):712–724PubMedCentralPubMedCrossRef

Miyake Y, Kozutsumi Y, Nakamura S, Fujita T, Kawasaki T (1995) Serine palmitoyltransferase is the primary target of a sphingosine-like immunosuppressant, ISP-1/myriocin. Biochem Biophys Res Commun 211(2):396–403PubMedCrossRef

Mullen TD, Hannun YA, Obeid LM (2012) Ceramide synthases at the centre of sphingolipid metabolism and biology. Biochem J 441(3):789–802PubMedCentralPubMedCrossRef

Natoli TA, Smith LA, Rogers KA et al (2010) Inhibition of glucosylceramide accumulation results in effective blockade of polycystic kidney disease in mouse models. Nat Med 16(7):788–792PubMedCentralPubMedCrossRef

Nietupski JB, Pacheco JJ, Chuang WL et al (2012) Iminosugar-based inhibitors of glucosylceramide synthase prolong survival but paradoxically increase brain glucosylceramide levels in Niemann-Pick C mice. Mol Genet Metab 105(4):621–628PubMedCrossRef

Nishie T, Hikimochi Y, Zama K et al (2010) Beta4-galactosyltransferase-5 is a lactosylceramide synthase essential for mouse extra-embryonic development. Glycobiology 20(10):1311–1322PubMedCrossRef

Ohmi Y, Tajima O, Ohkawa Y, Mori A, Sugiura Y, Furukawa K (2009) Gangliosides play pivotal roles in the regulation of complement systems and in the maintenance of integrity in nerve tissues. Proc Natl Acad Sci U S A 106(52):22405–22410PubMedCentralPubMedCrossRef

Ohta E, Ohira T, Matsue K et al (2009) Analysis of development of lesions in mice with serine palmitoyltransferase (SPT) deficiency -Sptlc2 conditional knockout mice. Exp Anim 58(5):515–524PubMedCrossRef

Okada M, Itoh Mi M, Haraguchi M et al (2002) B-series ganglioside deficiency exhibits no definite changes in the neurogenesis and the sensitivity to fas-mediated apoptosis but impairs regeneration of the lesioned hypoglossal nerve. J Biol Chem 277(3):1633–1636PubMedCrossRef

Okuda T, Tokuda N, Numata S et al (2006) Targeted disruption of Gb3/CD77 synthase gene resulted in the complete deletion of globo-series glycosphingolipids and loss of sensitivity to verotoxins. J Biol Chem 281(15):10230–10235PubMedCrossRef

Okuda T, Furukawa K, Nakayama K (2009) A novel, promoter-based, target-specific assay identifies 2-deoxy-D-glucose as an inhibitor of globotriaosylceramide biosynthesis. Febs J 276(18):5191–5202PubMedCrossRef

Osuchowski MF, Johnson VJ, He Q, Sharma RP (2004) Myriocin, a serine palmitoyltransferase inhibitor, alters regional brain neurotransmitter levels without concurrent inhibition of the brain sphingolipid biosynthesis in mice. Toxicol Lett 147(1):87–94PubMedCrossRef

Overkleeft HS, Renkema GH, Neele J et al (1998) Generation of specific deoxynojirimycin-type inhibitors of the non-lysosomal glucosylceramidase. J Biol Chem 273(41):26522–26527PubMedCrossRef

Park TS, Panek RL, Mueller SB et al (2004) Inhibition of sphingomyelin synthesis reduces atherogenesis in apolipoprotein E-knockout mice. Circulation 110(22):3465–3471PubMedCrossRef

Park TS, Panek RL, Rekhter MD et al (2006) Modulation of lipoprotein metabolism by inhibition of sphingomyelin synthesis in ApoE knockout mice. Atherosclerosis 189(2):264–272PubMedCrossRef

Park TS, Rosebury W, Kindt EK, Kowala MC, Panek RL (2008) Serine palmitoyltransferase inhibitor myriocin induces the regression of atherosclerotic plaques in hyperlipidemic ApoE-deficient mice. Pharmacol Res 58(1):45–51PubMedCrossRef

Park JW, Park WJ, Futerman AH (2014) Ceramide synthases as potential targets for therapeutic intervention in human diseases. Biochim Biophys Acta 1841(5):671–681PubMedCrossRef

Peterschmitt MJ, Burke A, Blankstein L et al (2011) Safety, tolerability, and pharmacokinetics of eliglustat tartrate (Genz-112638) after single doses, multiple doses, and food in healthy volunteers. J Clin Pharmacol 51(5):695–705PubMedCrossRef

Pontier SM, Schweisguth F (2012) Glycosphingolipids in signaling and development: from liposomes to model organisms. Dev Dyn 241(1):92–106PubMedCrossRef

Russo SB, Tidhar R, Futerman AH, Cowart LA (2013) Myristate-derived d16:0 sphingolipids constitute a cardiac sphingolipid pool with distinct synthetic routes and functional properties. J Biol Chem 288(19):13397–13409PubMedCentralPubMedCrossRef

Saadat L, Dupree JL, Kilkus J et al (2010) Absence of oligodendroglial glucosylceramide synthesis does not result in CNS myelin abnormalities or alter the dysmyelinating phenotype of CGT-deficient mice. Glia 58(4):391–398PubMedCentralPubMedCrossRef

Schiffmann R (2009) Fabry disease. Pharmacol Ther 122(1):65–77PubMedCrossRef

Shayman JA (2013) The design and clinical development of inhibitors of glycosphingolipid synthesis: will invention be the mother of necessity? Trans Am Clin Climatol Assoc 124:46–60PubMedCentralPubMed

Sheikh KA, Sun J, Liu Y et al (1999) Mice lacking complex gangliosides develop Wallerian degeneration and myelination defects. Proc Natl Acad Sci U S A 96(13):7532–7537PubMedCentralPubMedCrossRef

Simpson MA, Cross H, Proukakis C et al (2004) Infantile-onset symptomatic epilepsy syndrome caused by a homozygous loss-of-function mutation of GM3 synthase. Nat Genet 36(11):1225–1229PubMedCrossRef

Takamiya K, Yamamoto A, Furukawa K et al (1996) Mice with disrupted GM2/GD2 synthase gene lack complex gangliosides but exhibit only subtle defects in their nervous system. Proc Natl Acad Sci U S A 93(20):10662–10667PubMedCentralPubMedCrossRef

Ternes P, Brouwers JF, van den Dikkenberg J, Holthuis JC (2009) Sphingomyelin synthase SMS2 displays dual activity as ceramide phosphoethanolamine synthase. J Lipid Res 50(11):2270–2277PubMedCentralPubMedCrossRef

Togayachi A, Kozono Y, Ikehara Y et al (2010) Lack of lacto/neolacto-glycolipids enhances the formation of glycolipid-enriched microdomains, facilitating B cell activation. Proc Natl Acad Sci U S A 107(26):11900–11905PubMedCentralPubMedCrossRef

Vacaru AM, Tafesse FG, Ternes P et al (2009) Sphingomyelin synthase-related protein SMSr controls ceramide homeostasis in the ER. J Cell Biol 185(6):1013–1027PubMedCentralPubMedCrossRef

Wennekes T, Meijer AJ, Groen AK et al (2010) Dual-action lipophilic iminosugar improves glycemic control in obese rodents by reduction of visceral glycosphingolipids and buffering of carbohydrate assimilation. J Med Chem 53(2):689–698PubMedCrossRef

Worgall TS (2011) Sphingolipid synthetic pathways are major regulators of lipid homeostasis. Adv Exp Med Biol 721:139–148PubMedCrossRef

Yamashita T, Wada R, Sasaki T et al (1999) A vital role for glycosphingolipid synthesis during development and differentiation. Proc Natl Acad Sci U S A 96(16):9142–9147PubMedCentralPubMedCrossRef

Yamashita T, Hashiramoto A, Haluzik M et al (2003) Enhanced insulin sensitivity in mice lacking ganglioside GM3. Proc Natl Acad Sci U S A 100(6):3445–3449PubMedCentralPubMedCrossRef

Yamashita T, Allende ML, Kalkofen DN, Werth N, Sandhoff K, Proia RL (2005a) Conditional LoxP-flanked glucosylceramide synthase allele controlling glycosphingolipid synthesis. Genesis 43(4):175–180PubMedCrossRef

Yamashita T, Wu YP, Sandhoff R et al (2005b) Interruption of ganglioside synthesis produces central nervous system degeneration and altered axon-glial interactions. Proc Natl Acad Sci U S A 102(8):2725–2730PubMedCentralPubMedCrossRef

Yoshikawa M, Go S, Takasaki K et al (2009) Mice lacking ganglioside GM3 synthase exhibit complete hearing loss due to selective degeneration of the organ of Corti. Proc Natl Acad Sci U S A 106(23):9483–9488PubMedCentralPubMedCrossRef

Zhao H, Przybylska M, Wu IH et al (2007) Inhibiting glycosphingolipid synthesis improves glycemic control and insulin sensitivity in animal models of type 2 diabetes. Diabetes 56(5):1210–1218PubMedCrossRef

Zimran A, Elstein D (2003) Gaucher disease and the clinical experience with substrate reduction therapy. Philos Trans R Soc Lond B Biol Sci 358(1433):961–966PubMedCentralPubMedCrossRef

Title: The consequences of genetic and pharmacologic reduction in sphingolipid synthesis
Author: Raphael Schiffmann
Publication date: 01-01-2015
Publisher: Springer Netherlands
Published in: Journal of Inherited Metabolic Disease / Issue 1/2015
Print ISSN: 0141-8955
Electronic ISSN: 1573-2665
DOI: https://doi.org/10.1007/s10545-014-9758-8

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