Diabetes In Press, published online February 7, 2007 PHARMACOLOGICAL INHIBITION OF GLUCOSYLCERAMIDE SYNTHASE ENHANCES INSULIN SENSITIVITY Received for publication 18 November 2006 and accepted in revised form 1 February 2007. Johannes M. Aerts 1, Roelof Ottenhoff 2, Andrew S. Powlson 3, Aldo Grefhorst 4, Marco van Eijk 2, Peter F. Dubbelhuis 2, Folkert Kuipers 4, Mireille J. Serlie 5, Tom Wennekes 6, Hermen S. Overkleeft 6, Jasswinder K. Sethi 3, Stephen O’Rahilly 3. 1: Department of Medical Biochemistry, Academic Medical Center, University of Amsterdam 2: Macrozyme, Amsterdam 3: Department of Clinical Biochemistry, University of Cambridge. Cambridge UK 4: Centre for Liver, Digestive and Metabolic Disease, Academic Hospital Groningen, University of Groningen 5: Department of Endocrinology and Metabolism, Academic Medical Center, University of Amsterdam 6: Leiden Institute of Chemistry, Gorlaeus Laboratories, Leiden University Running title: Glycosphingolipids and insulin sensitivity Corresponding author: J.M. Aerts Department of Medical Biochemistry Academic Medical Center, University of Amsterdam Meibergdreef 15, 1105AZ, Amsterdam, The Netherlands Email: [email protected] Additional information for this article can be found in an online appendix. Copyright American Diabetes Association, Inc., 2007 Abstract A growing body of evidence implicates ceramide and/or its glycosphingolipid metabolites in the pathogenesis of insulin resistance. We have developed a highly specific small molecule inhibitor of glucosylceramide synthase, an enzyme which catalyses a necessary step in the conversion of ceramide to glycosphingolipids. In cultured 3T3-L1 adipocytes the iminosugar derivative N- (5’- adamantane-1’- yl-methoxy)-pentyl-1-deoxynojirimycin (AMP-DNM), counteracted TNFalpha induced abnormalities in glycosphingolipid concentrations and concomitantly reversed abnormalities in insulin signal transduction. When administered to mice and rats, AMP-DNM significantly reduced glycosphingolipid but not ceramide concentrations in various tissues. Treatment of ob/ob mice with AMP-DNM normalised their elevated tissue glucosylceramide levels, markedly lowered circulating glucose levels, improved oral glucose tolerance, reduced glycated haemoglobin and improved insulin sensitivity in muscle and liver. Similarly beneficial metabolic effects were seen in high-fat fed mice and ZDF rats. These findings provide further evidence that glycosphingolipid metabolites of ceramide may be involved in mediating the link between obesity and insulin resistance and that interference with glycosphingolipid biosynthesis might present a novel approach to the therapy of states of impaired insulin action such as Type 2 diabetes. Introduction additional role for glycosphingolipid metabolites Obesity is strongly associated with insulin of ceramide in the development of insulin resistance but the underlying pathogenic resistance has to be also considered. mechanism is still an enigma. The strong Glycosphingolipids are found in specific correlation between insulin resistance and (detergent-resistant) membrane microdomains in intramyocellular lipid levels suggests that close physical proximity to the insulin receptor, excessive exposure to lipids or their metabolites, as well as other tyrosine kinase receptors such as so called lipotoxicity, may play a crucial role (1- the epidermal growth factor (EGF) receptor (13). 5). The rapid induction of insulin resistance in A regulatory role for glycosphingolipids in rodents by infusions with palmitate, has directed hormone sensitivity was first proposed by attention to the sphingolipid ceramide as a Bremer and coworkers who showed that EGF- potential mediator of insulin resistance (1, 4, 5). mediated signalling is inhibited by the Palmitate is a critical precursor in the synthesis ganglioside GM3 (14). More recently, Tagami et of ceramide and its enhanced supply inevitably al. reported that addition of GM3 to cultured increases sphingolipid formation in tissues (5, 6). adipocytes also suppresses phosphorylation of Increased ceramide concentrations (two-fold) the insulin receptor and its down-stream substrate have indeed been observed in skeletal muscle IRS-1, resulting in reduced glucose uptake (15). from obese insulin-resistant individuals (4). The Other observations further substantiate the role of pivotal role of ceramide in insulin resistance and the ganglioside GM3 in responsiveness to lipotoxicity has recently been extensively insulin. Mutant mice lacking GM3 show an reviewed (5). It is of interest to note that enhanced phosphorylation of the skeletal muscle ceramide is also considered as molecular link insulin receptor after ligand binding and are between inflammation and insulin resistance (7). protected from high-fat diet induced insulin Obesity triggers a chronic inflammatory state, resistance (16). Conversely, GM3 levels are and cytokines like TNF-alpha released from elevated in the muscle of certain obese, insulin either adipocytes or from macrophages resistant mouse and rat models (15). Inokuchi infiltrating adipose tissue antagonize insulin and coworkers employed the ceramide-analogue action. The well-established induction of insulin 1-phenyl-2-decanoylamino-3- resistance by TNF-alpha is thought to be morpholinopropanol (PDMP), an inhibitor of attributable to its ability to promote sphingolipid glucosylceramide synthase, to reduce biosynthesis, as has been demonstrated at both glycosphingolipids in cultured adipocytes. They mRNA and cellular lipid levels (5, 7, 8-10). noted that PDMP counteracted the inhibitory Several investigations with cultured cells have effects of TNF-alpha on IR and IRS-1 linked excessive ceramide concentrations to phosphorylation (15). More recently it was disturbed insulin signalling (5). Manipulation of reported by the same researchers that high GM3 ceramide concentrations in cultured cells was levels diminished IR accumulation in detergent- consistently found to affect the insulin signalling resistant membrane microdomains and insulin- pathway downstream of AKT, but conflicting dependent IR internalization (17). Again reports exist regarding effects on the insulin glycosphingolipid depletion by incubation of receptor, IRS-1 and associated PI 3-kinase cells with PDMP prevented these abnormalities. activity (5, 9-11). More recently potentially However, the observations made with PDMP are important roles for PKCzeta, JNK and IDKE in difficult to interpret since this compound also the regulation of insulin signalling have been inhibits transacylation to 1-O-acylceramide and recognized. Ceramide has been shown to initiate consequently increases cellular concentrations of signalling pathways leading to the activation of ceramide (18). Based on the present information both JNK and IDKE (5, 11, 12), processes that it may be conceived that not just ceramide itself would support an insulin resistant phenotype. but rather its glycosphingolipid metabolites are Although there are compelling literature reports instrumental in the development of insulin pointing to direct and indirect antagonistic effects resistance. To discriminate between these of ceramide on the insulin signalling pathway, an possibilities we examined in obese rodents the effect of reduction of glycosphingolipids by N- glycolipids were derivatised on line for 30 min (5’-adamantane-1’-yl-methoxy)-pentyl-1- with O-phtaldehyde. Analysis was performed deoxynojirimycin (AMP-DNM), a specific using an HPLC system (Waters Associates, inhibitor of glucosylceramide synthase (19). Here Milford, MA) and a Hypersil BDS C18 3µ, 150 x we show that pharmacological lowering of 4.6 mm reverse phase column (Alltech). glycosphingolipids, without significant reduction Ganglioside composition was determined by of ceramide, dramatically reverses insulin analysis of the acidic glycolipid fraction obtained resistance in in vitro and in vivo models. after Folch extraction. Gangliosides were quantified by densitometry after HPTLC METHODS separation, spraying with resorcinol reagent and Animals. Experimental procedures were all heating at 105 °C for 10 min. Alternatively, approved by the appropriate Ethics Committee gangliosides were quantified following HPLC for Animal Experiments. C57Bl/6J and ob/ob separation of oligosaccharides released from mice (C57Bl/6J background) were obtained from glycolipids by ceramide glycanase digestion (24). Harlan (Horst, The Netherlands) and ZDF Activity of glucosylceramide synthase and (ZDF/GMi-fa/fa) rats from Charles River glucocerebrosidase in living cells was determined Laboratories (Wilmington, USA). Animals were using as substrates fluorescently labelled NBD- fed a commercially available lab chow (RMH-B, ceramide and NBD-glucosylceramide, Hope Farms BV, Woerden, The Netherlands). To respectively (25). IC50 values of AMP-DNM for induce glucose intolerance in C57Bl/6J mice, various enzymes were determined by exposing animals were fed with high fat diet (16.4 % cells or enzyme preparations to an appropriate protein, 25.5 % carbohydrates and 58.0% fat) for range of iminosugar concentrations. Lactase, 4 weeks. The iminosugar AMP-DNM was mixed maltase and sucrase were determined with in the food or when indicated administered by homogenates of freshly isolated rat intestine oral gavage two times daily. using assay conditions described earlier (26). Plasma and tissue sampling. Blood samples were Debranching enzyme (alpha-1, 6-glucosidase) collected by either tail vein or retroorbital plexus was measured with an erythrocyte