Extensive Glycosphingolipid Depletion in the Liver and Lymphoid Organs of Mice Treated with N-Butyldeoxynojirimycin

Abstract

The imino sugarN-butyldeoxynojirimycin is an inhibitor of the ceramide-specific glucosyltransferase that catalyzes the first step in glycosphingolipid biosynthesis. It results in extensive glycosphingolipid depletion in cells treated in vitro, without causing toxicity. However, we currently do not know the degree to which glycosphingolipids can be depleted in vivo in a mammalian species. We have therefore administeredN-butyldeoxynojirimycin long term to young mice and have found that glycosphingolipid levels are reduced (50–70%) in all tissues examined, without resulting in any overt pathology. When the lymphoid tissues from these mice were examined, they were found to be 50% acellular relative to non-lymphoid tissues. These data implicate a role for glycosphingolipids in the biology of the immune system or indicate an additional as yet unknown activity ofN-butyldeoxynojirimycin. Extensive glycosphingolipid depletion resulting from N-butyldeoxynojirimycin administration is therefore well tolerated in adult mice, and this compound may be in an invaluable tool for probing glycosphingolipid functions in vivo. In addition, this drug may be effective in clinical situations where glycosphingolipid depletion would be desirable, such as the in the treatment of the human glycosphingolipidoses. The imino sugarN-butyldeoxynojirimycin is an inhibitor of the ceramide-specific glucosyltransferase that catalyzes the first step in glycosphingolipid biosynthesis. It results in extensive glycosphingolipid depletion in cells treated in vitro, without causing toxicity. However, we currently do not know the degree to which glycosphingolipids can be depleted in vivo in a mammalian species. We have therefore administeredN-butyldeoxynojirimycin long term to young mice and have found that glycosphingolipid levels are reduced (50–70%) in all tissues examined, without resulting in any overt pathology. When the lymphoid tissues from these mice were examined, they were found to be 50% acellular relative to non-lymphoid tissues. These data implicate a role for glycosphingolipids in the biology of the immune system or indicate an additional as yet unknown activity ofN-butyldeoxynojirimycin. Extensive glycosphingolipid depletion resulting from N-butyldeoxynojirimycin administration is therefore well tolerated in adult mice, and this compound may be in an invaluable tool for probing glycosphingolipid functions in vivo. In addition, this drug may be effective in clinical situations where glycosphingolipid depletion would be desirable, such as the in the treatment of the human glycosphingolipidoses. Glycosphingolipids (GSLs) 1The abbreviations used are: GSL(s), glycosphingolipid(s); PDMP,Dl-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol);NB-DNJ, N-butyldeoxynojirimycin;NB-DGJ, N-butyldeoxygalactonojirimycin; PBS, phosphate-buffered saline; PAGE, polyacrylamide gel electrophoresis; mAb, monoclonal antibody. are ubiquitous components of the plasma membranes of eukaryotic cells where they are believed to mediate a number of biological functions (1Hakomori S J. Biol. Chem. 1990; 265: 18713-18716Abstract Full Text PDF PubMed Google Scholar). Their biosynthesis and catabolism have been extensively studied (2Sandhoff K. van Echten G. Adv. Lipid Res. 1993; 26: 119-142PubMed Google Scholar, 3van Echten G. Sandhoff K. J. Biol. Chem. 1993; 268: 5341-5344Abstract Full Text PDF PubMed Google Scholar, 4Sandhoff K. Kolter T. Trends Cell Biol. 1996; 6: 98-103Abstract Full Text PDF PubMed Scopus (148) Google Scholar), as have the disease states that result from their incomplete catabolism within lysosomes (5Neufeld E.F. Annu. Rev. Biochem. 1991; 60: 257-280Crossref PubMed Scopus (487) Google Scholar). They are exploited as receptors by a number of micro-organisms (6Karlsson K.-A. Annu. Rev. Biochem. 1989; 58: 309-350Crossref PubMed Scopus (622) Google Scholar); however, their normal physiological functions remain largely obscure. GSL biosynthesis involves the co-ordinated action of multiple gene products (2Sandhoff K. van Echten G. Adv. Lipid Res. 1993; 26: 119-142PubMed Google Scholar). A key enzyme in this pathway is the ceramide-specific glucosyltransferase, which catalyzes the first step in the GSL biosynthetic pathway, the transfer of glucose to ceramide to form glucosylceramide (GlcCer). The sequential action of glycosyltransferases in the Golgi apparatus converts glucosylceramide into other neutral GSLs and gangliosides (2Sandhoff K. van Echten G. Adv. Lipid Res. 1993; 26: 119-142PubMed Google Scholar). The ceramide-specific glucosyltransferase has been cloned recently and is a unique gene product without substantial homology with other known glycosyltransferase genes (7Ishikawa S. Sakiyama H. Suzuki G. Kazuya I.-P. Hidari J. Hirabayashi Y. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 4638-4643Crossref PubMed Scopus (223) Google Scholar). Mutant B16 melanoma cells lacking this enzyme are not compromised in their growth or morphology, indicating that GSLs are not needed for membrane integrity and do not serve a basic housekeeping function, at least at the level of the single cell (7Ishikawa S. Sakiyama H. Suzuki G. Kazuya I.-P. Hidari J. Hirabayashi Y. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 4638-4643Crossref PubMed Scopus (223) Google Scholar, 8Ichikawa S. Nakajo N. Sakiyama H. Hirabayashi Y. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 2703-2707Crossref PubMed Scopus (142) Google Scholar). One approach to investigate the role of GSLs in intact organisms is to inactivate the glucosyltransferase either by gene disruption techniques or by using a specific inhibitor. It is not currently known the degree to which GSLs can be depleted in vivo in mammalian species without resulting in pathology (9Platt F.M. Butters T.D. Trends Glycosci. Glycotechnol. 1995; 7: 495-511Crossref Scopus (36) Google Scholar). The gene knockout approach would determine whether or not total inhibition of GSL synthesis is compatible with embryonic development. There is circumstantial evidence, such as the lack of human disease states that result from defects in GSL biosynthesis, which suggests that GSL expression may play a critical role in early mammalian development (4Sandhoff K. Kolter T. Trends Cell Biol. 1996; 6: 98-103Abstract Full Text PDF PubMed Scopus (148) Google Scholar). The complete lack of GSL biosynthesis during embryogenesis could therefore be potentially lethal. However, in studies of teleost development using Medaka fish in in vitro culture, extensive GSL inhibition resulting from treatment with the ceramide analogue PDMP did not disrupt development (10Fenderson B.A. Ostrander G.K. Hausken Z. Radin N.S. Hakamori S.-I. Exp. Cell Res. 1992; 198: 362-366Crossref PubMed Scopus (36) Google Scholar). The roles of GSLs during vertebrate development and their functions in the biology of the mature organism therefore remain to be elucidated. The alternative experimental approach which can be taken to explore GSL functions is to use specific inhibitors of enzymes required for the biosynthesis of GSLs. One advantage of this approach is that the consequences of partial GSL depletion can be investigated. An attractive target for enzyme inhibition is the ceramide-specific glucosyltransferase, which catalyzes the first step in the GSL biosynthetic pathway. We have recently identified two inhibitors of GSL biosynthesis that block the action of the ceramide-specific glucosyltransferase (11Platt F.M. Neises G.R. Dwek R.A. Butters T.D. J. Biol. Chem. 1994; 269: 8362-8365Abstract Full Text PDF PubMed Google Scholar, 12Platt F.M. Neises G.R. Karlsson G.B. Dwek R.A. Butters T.D. J. Biol. Chem. 1994; 269: 27108-27114Abstract Full Text PDF PubMed Google Scholar). These inhibitors are the imino sugarsNbutyldeoxynojirimycin (NB-DNJ) andN-butyldeoxygalactonojirimycin (NB-DGJ), a glucose and galactose analogue, respectively. In vitro these analogues result in GSL depletion in a wide range of human and murine cell lines (11Platt F.M. Neises G.R. Dwek R.A. Butters T.D. J. Biol. Chem. 1994; 269: 8362-8365Abstract Full Text PDF PubMed Google Scholar, 12Platt F.M. Neises G.R. Karlsson G.B. Dwek R.A. Butters T.D. J. Biol. Chem. 1994; 269: 27108-27114Abstract Full Text PDF PubMed Google Scholar). However, the extent to which they can reduce GSL levels in vivo is currently unknown, as is the extent to which GSL depletion can be tolerated in an intact adult mammal. The degree to which GSLs depletion is tolerated in vivo is also an important factor when considering GSL biosynthetic inhibitors as potential therapeutic agents for the treatment of the GSL lysosomal storage diseases, where in vivo GSL depletion is the primary clinical objective (9Platt F.M. Butters T.D. Trends Glycosci. Glycotechnol. 1995; 7: 495-511Crossref Scopus (36) Google Scholar, 11Platt F.M. Neises G.R. Dwek R.A. Butters T.D. J. Biol. Chem. 1994; 269: 8362-8365Abstract Full Text PDF PubMed Google Scholar, 12Platt F.M. Neises G.R. Karlsson G.B. Dwek R.A. Butters T.D. J. Biol. Chem. 1994; 269: 27108-27114Abstract Full Text PDF PubMed Google Scholar). In this study we have administered NB-DNJ to young mice using a range of dosing regimes and monitored GSL biosynthesis.NB-DNJ was used, rather than the galactose analogueNB-DGJ, due to the fact that this compound is available in the multigram quantities required for this study. We have found significant GSL depletion in multiple tissues of mice treated withNB-DNJ without any overt toxicity, indicating that partial inhibition of GSL biosynthesis is well tolerated in vivo. The implications these data have for GSL function and the therapeutic administration of GSL inhibitors for treating GSL lysosomal storage disorders are discussed. NB-DNJ was a gift from Searle/Monsanto. Age- and sex-matched (6-week-old female) C57BL/6 mice were fed on a diet of powdered mouse chow (expanded Rat and Mouse Chow 1, ground, SDS Ltd., Witham, Essex, UK) containing NB-DNJ. The diet and compound (both as were at and used within of was available to the The mice were The mice were on mouse for and two were from the and two from the experimental The in the experimental were on a diet for a when two from were The in the treated were fed on a diet for at which all from were In an were on a diet for were by the and at to levels were by compound from of using of and an were with a than of the compound was from the of compound was either by of or by an was with normal to a with NB-DNJ from to of was by an of a with containing of NB-DNJ was by in with the and were in of in at in of and by The was with in by 50% and for The has been (11Platt F.M. Neises G.R. Dwek R.A. Butters T.D. J. Biol. Chem. 1994; 269: 8362-8365Abstract Full Text PDF PubMed Google Scholar). from treated and mice were with for on in the has been (11Platt F.M. Neises G.R. Dwek R.A. Butters T.D. J. Biol. Chem. 1994; 269: 8362-8365Abstract Full Text PDF PubMed Google and was using a on cells were on a of from and two mice were in PBS, in of containing and on for The was by the of of containing The cells were with PBS, in of PBS, and specific and at for The cells were with and GSLs from the cell of at and at An of to was taken for by using a of GSLs were by and their with GSL The cell the were in of to which SDS was and The were to for at for and taken for by was using the UK) and of by and and treatment with UK) Ltd., for at The were used for this study and have been F.M. 1992; PubMed Scopus Google and A was used to and The and have been F.M. 1992; PubMed Scopus Google Scholar). on cells were on a of The of cells with the was by the of on the of the for or on the of the with the the well of the mice treated with the mice were monitored and the on growth The of NB-DNJ the of the study are with the treatment and with the dosing the mice on containing diet than the and The at the of the study were in When the mice were to a diet in glucose lacking the than the The mice at any in the in to mice or of NB-DNJ by The activity and of mice were with the not indicating that the in their diet and were not compromised by levels of NB-DNJ were at all the dosing levels and were found to range in a from at to at levels of levels of NB-DNJ were at of compound on from at least and was by or by The are and the was within for all in a levels of NB-DNJ were at of compound on from at least and was by or by The are and the was within for all a of GSL cell levels were on from treated and mice, using as a for (11Platt F.M. Neises G.R. Dwek R.A. Butters T.D. J. Biol. Chem. 1994; 269: 8362-8365Abstract Full Text PDF PubMed Google Scholar). The cells were by and cell using The in cell was at drug the cell than the to an at in the range of and to 50% at the These data indicate that GSL depletion in vivo in to NB-DNJ and is Cell were in by a into cell treatment of results in the of The are reduced with to the this a into cell by gangliosides J. Biol. Chem. Full Text PDF PubMed Google Scholar). The of the of GSLs from from two mice and two treated with NB-DNJ a by in species with from There did not to be a in the gangliosides from cells from the two of mice, were any in the relative of gangliosides in These data that the expression of cell gangliosides is in a as a result of treatment with NB-DNJ. When the from these was by and by could be in the either or of containing from the enzyme mouse was not that inhibition was not at the levels was in determine the degree of GSL depletion in a non-lymphoid was and total by A in GSL levels in the mice treated withNB-DNJ was and the species levels in mice, due to the levels of ceramide available to this biosynthetic pathway, due to inhibition of GSL biosynthesis. of the drug from the diet for in normal GSL levels with in quantities with the and the species levels were to and this was the of NB-DNJ from the GSL depletion was therefore in of mice and was drug The fact that is may indicate long term of the and from NB-DNJ mice were to be a than from from were than the and at they were on than taken from was in to such as and which were by treatment not investigate the of the in lymphoid in to NB-DNJ the were A of were fed on diet and in and monitored tissues reduced in in and in and drug The lymphoid tissues as long as the were treated with to normal of the drug from the diet from the were treated withNB-DNJ and their The drug was from an of for a and their The data are as the for an of mice mice and the drug The of and on NB-DNJ were by The a of cells relative to the in with and the cell were reduced from in to When drug was from the within the relative of and cells within the to normal levels The of cells within the was due to an of and with the in the cells in the to in the cells for of cells in the and in of from and mice with normal in from mice not of NB-DNJ on the of mouse cells of by from was to and mice mice for for drug from diet for in a of by from was to and mice mice for for drug from diet for The from treated with NB-DNJ were also of NB-DNJ treatment in to the which has a of and the mice an of single the from mice and cells There was therefore an in the of single or cells at the of GSLs are not components of mammalian cells at the single cell level S. Nakajo N. Sakiyama H. Hirabayashi Y. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 2703-2707Crossref PubMed Scopus (142) Google Scholar), their functions in of mammalian these functions are to be and are required that GSL vivo as well as in we can GSL biosynthesis in vitro using a number of are required that are well tolerated long term in vivo. There are currently two of GSL biosynthesis inhibitor that have been to which target the ceramide-specific The first of inhibitors were the ceramide analogues of which the compound is PDMP Radin N.S. Chem. 26: PubMed Scopus Google Scholar). of ceramide these the of ceramide by as inhibitors Radin N.S. J. Lipid Res. Full Text PDF Google Scholar). However, the of these their long term administration to (9Platt F.M. Butters T.D. Trends Glycosci. Glycotechnol. 1995; 7: 495-511Crossref Scopus (36) Google Scholar). The of GSL biosynthesis inhibitors are the imino such as the glucose analogue NB-DNJ. The compound not the ceramide-specific glucosyltransferase, and a is required to these with and (11Platt F.M. Neises G.R. Dwek R.A. Butters T.D. J. Biol. Chem. 1994; 269: 8362-8365Abstract Full Text PDF PubMed Google Scholar). The may these ceramide or serve to target these to the membrane where the glucosyltransferase (9Platt F.M. Butters T.D. Trends Glycosci. Glycotechnol. 1995; 7: 495-511Crossref Scopus (36) Google Scholar). The imino have a advantage PDMP and in that they in vitro, at in of Karlsson G.B. Butters T.D. S. Dwek R.A. F.M. J. 1995; PubMed Google Scholar). They therefore have the potential to be well tolerated in vivo. In this study we have whether or not we can GSL depletion in vivo in mice treated withNB-DNJ and whether GSL depletion is in any to the When mice were treated with of levels in the range of were levels level of were in during the of this compound as an when were treated with J. 1994; 7: Google Scholar). The of NB-DNJ are of in mouse relative to human dosing regimes in the mouse to levels in the range required to GSL biosynthesis inhibition (9Platt F.M. Butters T.D. Trends Glycosci. Glycotechnol. 1995; 7: 495-511Crossref Scopus (36) Google Scholar, 11Platt F.M. Neises G.R. Dwek R.A. Butters T.D. J. Biol. Chem. 1994; 269: 8362-8365Abstract Full Text PDF PubMed Google Scholar, 12Platt F.M. Neises G.R. Karlsson G.B. Dwek R.A. Butters T.D. J. Biol. Chem. 1994; 269: 27108-27114Abstract Full Text PDF PubMed Google Scholar). The and from any overt of pathology for the of the which for The was that the growth of mice were reduced relative to the One activity of imino such as NB-DNJ is that they are inhibitors for the and of these enzymes results in and was the when this compound was as a potential therapeutic in J. 1994; 7: Google Scholar). It was therefore that in this at dosing regimes in of known to in mice NB-DNJ by we in the treated The in the drug administration used in this study was that NB-DNJ was with a containing There would therefore be within the the for the and the inhibitor. of drug were the mice this may also have the with administration of these as the in the would be of NB-DNJ to in quantities at with in may be a of and the development of an effective Butters T.D. as Scholar). One potential of the mice have relative to the is that due to glucose is We whether or not this was a factor by the mice to a diet lacking and in this diet mice an in as on the that potential of glucose on the diet is not the these have NB-DNJ could be as an causing reduced or may a in within the treated A in cell gangliosides was in the and of mice, indicating that extensive GSL depletion was depletion in all and depletion was which is with the of action of NB-DNJ as an inhibitor of the first step in GSL biosynthesis. The degree of GSL depletion was as by GSL and cell relative to the We have that cell lines in can be depleted of the cells are treated with NB-DNJ GSL biosynthesis and the inhibitor F.M. Neises G.R. Karlsson G.B. Dwek R.A. Butters T.D. J. Biol. Chem. 1994; 269: 27108-27114Abstract Full Text PDF PubMed Google results in a partial however, is to cells with complete depletion of is on not cells of the as the to to the degree to which inhibitor using and can on the relative to of GSLs. studies are to any biological where is which of is the biological function of When were by and with a of the that a of this The of these and are currently The relative of the for the and the are currently not The cell and the GSL therefore a of GSL depletion (50–70%) on cell that It was found when the cell were studied from cells that been in that was of a in suggests that inhibition is not in at the levels in this study. is considering the for NB-DNJ the ceramide-specific glucosyltransferase and are and respectively. However, the of the two enzyme of NB-DNJ are with the glucosyltransferase with on the of an early Golgi (7Ishikawa S. Sakiyama H. Suzuki G. Kazuya I.-P. Hidari J. Hirabayashi Y. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 4638-4643Crossref PubMed Scopus (223) Google Scholar, H. PubMed Scopus Google Scholar, J. Biol. Chem. 1991; Full Text PDF PubMed Google Scholar, A. van G. J. Cell Biol. 1992; PubMed Scopus Google Scholar, Biochem. J. 1991; PubMed Scopus Google Scholar), is a enzyme J. Biochem. 1995; PubMed Scopus Google Scholar). NB-DNJ therefore not has to the plasma membrane to the glucosyltransferase, has to also the membrane to and in of the two enzymes may result in levels of NB-DNJ in the range the glucosyltransferase not of NB-DNJ required to were not using this dosing is in with in vitro studies in which NB-DNJ activity G.B. Butters T.D. Dwek R.A. F.M. J. Biol. Chem. 1993; 268: Full Text PDF PubMed Google Scholar). the activity of NB-DNJ the GSL biosynthetic pathway and pathway, in these two are by at levels in the range where GSL biosynthesis inhibition not inhibition of and The is a of the of cells and GSLs within are and is when adult mice are studied the of this study not However, when a mouse of disease was of was in the that the compound the and GSL biosynthesis F.M. Neises G.R. G. Dwek R.A. Butters T.D. PubMed Scopus Google Scholar). The lymphoid tissues from mice treated for or withNB-DNJ were with their was not in non-lymphoid tissues such as and where were the of treatment not the were 50% in the of The for this in was unknown and as the mice were of immune as they were and in a and normal lymphoid at the level of when not In in to 50% of the of was an in the of cells with and was a in the of The was also 50% acellular and an in with the of single cells or relative to normal with the of cells We currently do not the for these these are the result of of biosynthesis, they result from reduced GSL biosynthesis, as the pathway is not at the levels However, we a due to a or to NB-DNJ The indicate that the in lymphoid within of treatment and are by The as long as the drug is in the However, within of of NB-DNJ from the the their normal and were in and from The of lymphoid tissues is with cell within normal by which are the in within the could reduce the available of which would reduce the of the lymphoid disruption of normal could also be as reduced cell within lymphoid tissues. Cell within the could also reduce cell However, as of the in the in as of an immune would be that this could have an on immune the mice have not as yet been with and the fact that they are not to is that they are not The in the could reduced to the of in cell of or of single cells from the studies be required to the for the in treatment and also to determine whether or not these result from GSL depletion or an activity of this The in as a result of NB-DNJ has implications in of the potential of this drug for the treatment of disease (9Platt F.M. Butters T.D. Trends Glycosci. Glycotechnol. 1995; 7: 495-511Crossref Scopus (36) Google Scholar, 11Platt F.M. Neises G.R. Dwek R.A. Butters T.D. J. Biol. Chem. 1994; 269: 8362-8365Abstract Full Text PDF PubMed Google Scholar, 12Platt F.M. Neises G.R. Karlsson G.B. Dwek R.A. Butters T.D. J. Biol. Chem. 1994; 269: 27108-27114Abstract Full Text PDF PubMed Google Scholar). is a of this disease and is used as a disease that is monitored when clinical 1992; PubMed Scopus Google Scholar). NB-DNJ in a in the may be reduced in of an on lysosomal clinical therefore be used as of therapeutic as may be an due to this activity of NB-DNJ on lymphoid tissues. The of extensive GSL depletion in the lymphoid and of a normal treated with NB-DNJ the for using NB-DNJ as an in vivo tool for GSL levels in vivo to GSL the fact that mice with to GSL depletion are not compromised by the depletion GSL depletion can be without causing pathology. It may well be the that a critical level which the biology can which There are potential cells could for GSL for on to mediate S. T. Sakiyama H. Hirabayashi Y. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google or by other There have been two in which GSL species can potentially function for a GSL species that is from knockout in mice in the that the mouse has levels of in as this biosynthetic pathway It is that species may be of in the mice, the of the in not found in rather than the has T. N. J. A. Suzuki K. Suzuki K. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar). The from studies in which the consequences of a mouse for has been which these mice of gangliosides K. A. K. S. S. N. K. H. K. S. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: PubMed Scopus Google Scholar). There is in these mice, and is that the gangliosides and could for the gangliosides that are in these studies the potential biological to this of GSL species. in of the degree of by an extensively depleted cell could GSLs into which could have GSL to mediate their may be to this as all GSL species have been of the GSLs are due to incomplete inhibition of the of these GSLs on the plasma membrane may the of biological studies are required to levels can GSL biosynthesis to the where pathology It has been that GSL is not to cells the a critical level and to pathology. has been in studies of mouse of the GSL storage diseases, and disease S. A. H. Suzuki K. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: PubMed Scopus Google Scholar, K. S. A. Y. A. H. Sandhoff K. Suzuki K. 1995; PubMed Scopus Google Scholar). with the data in this this would that GSLs can be depleted or by mammalian cells within without resulting in In GSL levels can be extensively depleted long vivo without overt toxicity. GSL biosynthesis inhibitors such as NB-DNJ can be in of GSL storage and also used as in vivo for probing GSL functions in mature and We and for and for