Pharmacological Inhibition of Glucosylceramide Synthase Enhances Insulin Sensitivity

Johannes Aerts

Roelof Ottenhoff

Andrew Powlson

Aldo Grefhorst

Mireille Serlie+4 authors

^{Department of Medical Biochemistry, Academic Medical Center, University of Amsterdam, Amsterdam, the Netherlands}

^{Macrozyme, Amsterdam, the Netherlands}

^{Department of Clinical Biochemistry, University of Cambridge, Cambridge, U.K.}

^{Department of Pathology, Academic Medical Center, University of Amsterdam, Amsterdam, the Netherlands}

^{Centre for Liver, Digestive, and Metabolic Disease, Academic Hospital Groningen, University of Groningen, Groningen, the Netherlands}

^{Department of Endocrinology and Metabolism, Academic Medical Center, University of Amsterdam, Amsterdam, the Netherlands}

^{Gorlaeus Laboratories, Leiden Institute of Chemistry, Leiden University, Leiden, the Netherlands}

Address correspondence and reprint requests to J.M. Aerts, Department of Medical Biochemistry, Academic Medical Center, University of Amsterdam, Meibergdreef 15, 1105AZ, Amsterdam, Netherlands. ln.avu.cma@strea.m.j

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 that catalyzes 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 tumor necrosis factor-α-induced abnormalities in glycosphingo-lipid concentrations and concomitantly reversed abnormalities in insulin signal transduction. When administered to mice and rats, AMP-DNM significantly reduced glycosphin-golipid but not ceramide concentrations in various tissues. Treatment of ob/ob mice with AMP-DNM normalized their elevated tissue glucosylceramide levels, markedly lowered circulating glucose levels, improved oral glucose tolerance, reduced A1C, 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.

Abstract

Obesity is strongly associated with insulin resistance, but the underlying pathogenic mechanism is still an enigma. The strong correlation between insulin resistance and intramyocellular lipid levels suggests that excessive exposure to lipids or their metabolites, so-called lipotoxicity, may play a crucial role (1-5). The rapid induction of insulin resistance in rodents by infusions with palmitate has directed attention to the sphingolipid ceramide as a potential mediator of insulin resistance (1,4,5). Palmitate is a critical precursor in the synthesis of ceramide, and its enhanced supply inevitably increases sphingolipid formation in tissues (5,6). Increased ceramide concentrations (twofold) have indeed been observed in skeletal muscle from obese insulin-resistant individuals (4). The pivotal role of ceramide in insulin resistance and lipotoxicity has been recently extensively reviewed (5). It is of interest to note that ceramide is also considered a molecular link between inflammation and insulin resistance (7).

Obesity triggers a chronic inflammatory state, and cytokines like tumor necrosis factor (TNF)-α released from either adipocytes or from macrophages infiltrating adipose tissue antagonize insulin action. The well-established induction of insulin resistance by TNF-α is thought to be attributable to its ability to promote sphingolipid biosynthesis, as has been demonstrated at both mRNA and cellular lipid levels (5,7-10). Several investigations with cultured cells have linked excessive ceramide concentrations to disturbed insulin signaling (5). Manipulation of ceramide concentrations in cultured cells was consistently found to affect the insulin signaling pathway downstream of Akt, but conflicting reports exist regarding effects on the insulin receptor, IRS-1, and associated phosphatidylinositol 3-kinase activity (5,9-11). More recently, potentially important roles for protein kinase-Cζ, Jun NH₂-terminal kinase, and IκKβ (Iκ-B kinase-β) in the regulation of insulin signaling have been recognized. Ceramide has been shown to initiate signaling pathways leading to the activation of both Jun NH₂-terminal kinase and IκKβ (5,11,12), processes that would support an insulin-resistant phenotype. Although there are compelling literature reports pointing to direct and indirect antagonistic effects of ceramide on the insulin signaling pathway, an additional role for glycosphingolipid metabolites of ceramide in the development of insulin resistance also has to be considered. Glycosphingolipids are found in specific (detergent-resistant) membrane microdomains in close physical proximity to the insulin receptor, as well as other tyrosine kinase receptors such as the epidermal growth factor receptor (13). A regulatory role for glycosphingolipids in hormone sensitivity was first proposed by Bremer and colleagues (14) who showed that epidermal growth factor–mediated signaling is inhibited by the ganglioside sialosyllactosylceramide (GM3). More recently, Tagami et al. (15) reported that addition of GM3 to cultured adipocytes also suppresses phosphorylation of the insulin receptor and its downstream substrate IRS-1, resulting in reduced glucose uptake. Other observations further substantiate the role of the ganglioside GM3 in responsiveness to insulin. Mutant mice lacking GM3 show an enhanced phosphorylation of the skeletal muscle insulin receptor after ligand binding and are protected from high-fat diet–induced insulin resistance (16). Conversely, GM3 levels are elevated in the muscle of certain obese, insulin-resistant mouse and rat models (15). Inokuchi and colleagues (15) used the ceramide analog 1-phenyl-2-decanoylamino-3-morpholinopropanol (PDMP), an inhibitor of glucosylceramide synthase, to reduce glycosphingolipids in cultured adipocytes. They noted that PDMP counteracted the inhibitory effects of TNF-α on insulin receptor and IRS-1 phosphorylation. More recently, it was reported by the same researchers that high GM3 levels diminished insulin receptor accumulation in detergent-resistant membrane microdomains and insulin-dependent IR internalization (17). Again, glycosphingolipid depletion by incubation of cells with PDMP prevented these abnormalities. However, the observations made with PDMP are difficult to interpret since this compound also inhibits transacylation to 1-O-acylceramide and consequently increases cellular concentrations of ceramide (18).

Based on the present information, it may be conceived that not just ceramide itself but rather its glycosphingo-lipid metabolites are instrumental in the development of insulin resistance. To discriminate between these possibilities, we examined in obese rodents the effect of reduction of glycosphingolipids by N-(5′-adamantane-1′-yl-methoxy)-pentyl-1-deoxynojirimycin (AMP-DNM), a specific inhibitor of glucosylceramide synthase (19). Here we show that pharmacological lowering of glycosphingolipids, without significant reduction of ceramide, dramatically reverses insulin resistance in in vitro and in vivo models.

Glossary

AMP-DNM	N-(5′-adamantane-1′-yl-methoxy)-pentyl-1-deoxynojirimycin
AUC	area under the curve
GM3	sialosyllactosylceramide
HPLC	high-performance liquid chromatography
HRP	horseradish peroxidase
PDMP	1-phenyl-2-decanoylamino-3-morpholinopropanol
TNF	tumor necrosis factor

Glossary

Footnotes

Additional information for this article can be found in an online appendix at http://dx.doi.org/10.2337/db06-1619.

Footnotes

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