Transglycosidase activity of chitotriosidase - Improved enzymatic assay for the human macrophage chitinase

Bio-organic Synthesi

Reducing Glycosphingolipid Content in Adipose Tissue of Obese Mice Restores Insulin Sensitivity, Adipogenesis and Reduces Inflammation

Adipose tissue is a critical mediator in obesity-induced insulin resistance. Previously we have demonstrated that pharmacological lowering of glycosphingolipids and subsequently GM3 by using the iminosugar AMP-DNM, strikingly improves glycemic control. Here we studied the effects of AMP-DNM on adipose tissue function and inflammation in detail to provide an explanation for the observed improved glucose homeostasis. Leptin-deficient obese (LepOb) mice were fed AMP-DNM and its effects on insulin signalling, adipogenesis and inflammation were monitored in fat tissue. We show that reduction of glycosphingolipid biosynthesis in adipose tissue of LepOb mice restores insulin signalling in isolated ex vivo insulin-stimulated adipocytes. We observed improved adipogenesis as the number of larger adipocytes was reduced and expression of genes like peroxisome proliferator-activated receptor (PPAR) γ, insulin responsive glucose transporter (GLUT)-4 and adipsin increased. In addition, we found that adiponectin gene expression and protein were increased by AMP-DNM. As a consequence of this improved function of fat tissue we observed less inflammation, which was characterized by reduced numbers of adipose tissue macrophages (crown-like structures) and reduced levels of the macrophage chemo attractants monocyte-chemoattractant protein-1 (Mcp-1/Ccl2) and osteopontin (OPN). In conclusion, pharmacological lowering of glycosphingolipids by inhibition of glucosylceramide biosynthesis improves adipocyte function and as a consequence reduces inflammation in adipose tissue of obese animals

Glucosylated cholesterol in mammalian cells and tissues: formation and degradation by multiple cellular β-glucosidases

The membrane lipid glucosylceramide (GlcCer) is continuously formed and degraded. Cells express two GlcCer-degrading β-glucosidases, GBA and GBA2, located in and outside the lysosome, respectively. Here we demonstrate that through transglucosylation both GBA and GBA2 are able to catalyze in vitro the transfer of glucosyl-moieties from GlcCer to cholesterol, and vice versa. Furthermore, the natural occurrence of 1-O-cholesteryl-β-D-glucopyranoside (GlcChol) in mouse tissues and human plasma is demonstrated using LC-MS/MS and 13C6-labelled GlcChol as internal standard. In cells the inhibition of GBA increases GlcChol, whereas inhibition of GBA2 decreases glucosylated sterol. Similarly, in GBA2-deficient mice GlcChol is reduced. Depletion of GlcCer by inhibition of GlcCer synthase decreases GlcChol in cells and likewise in plasma of inhibitor-treated Gaucher disease patients. In tissues of mice with Niemann-Pick type C, a condition characterized by intralysosomal accumulation of cholesterol, marked elevations in GlcChol occur as well. When lysosomal accumulation of cholesterol is induced in cultured cells, GlcChol is formed via lysosomal GBA. This illustrates that reversible transglucosylation reactions are highly dependent on local availability of suitable acceptors. In conclusion, mammalian tissues contain GlcChol formed by transglucosylation through β-glucosidases using GlcCer as donor. Our findings reveal a novel metabolic function for GlcCer.Bio-organic SynthesisMedical Biochemistr

Common G102S polymorphism in chitotriosidase differentially affects activity towards 4-methylumbelliferyl substrates

Chitotriosidase (CHIT1) is a chitinase that is secreted by activated macrophages. Plasma chitotriosidase activity reflects the presence of lipid-laden macrophages in patients with Gaucher disease. CHIT1 activity can be conveniently measured using fluorogenic 4-methylumbelliferyl (4MU)-chitotrioside or 4MU-chitobioside as the substrate, however, nonsaturating concentrations have to be used because of apparent substrate inhibition. Saturating substrate concentrations can, however, be used with the newly designed substrate 4MU-deoxychitobioside. We studied the impact of a known polymorphism, G102S, on the catalytic properties of CHIT1. The G102S allele was found to be common in type I Gaucher disease patients in the Netherlands (similar to 24% of alleles). The catalytic efficiency of recombinant Ser102 CHIT1 was similar to 70% that of wild-type Gly102 CHIT1 when measured with 4MU-chitotrioside at a nonsaturating concentration. However, the activity was normal with 4MU-deoxychitobioside as the substrate at saturating concentrations, consistent with predictions from molecular dynamics simulations. In conclusion, interpretation of CHIT1 activity measurements with 4MU-chitotrioside with respect to CHIT1 protein concentrations depends on the presence of Ser102 CHIT1 in an individual, complicating estimation of the body burden of storage macrophages. Use of the superior 4MU-deoxychitobioside substrate avoids such complications because activity towards this substrate under saturating conditions is not affected by the G102S substitution

Common G102S polymorphism in chitotriosidase differentially affects activity towards 4-methylumbelliferyl substrates

Chitotriosidase (CHIT1) is a chitinase that is secreted by activated macrophages. Plasma chitotriosidase activity reflects the presence of lipid-laden macrophages in patients with Gaucher disease. CHIT1 activity can be conveniently measured using fluorogenic 4-methylumbelliferyl (4MU)-chitotrioside or 4MU-chitobioside as the substrate, however, nonsaturating concentrations have to be used because of apparent substrate inhibition. Saturating substrate concentrations can, however, be used with the newly designed substrate 4MU-deoxychitobioside. We studied the impact of a known polymorphism, G102S, on the catalytic properties of CHIT1. The G102S allele was found to be common in type I Gaucher disease patients in the Netherlands (similar to 24% of alleles). The catalytic efficiency of recombinant Ser102 CHIT1 was similar to 70% that of wild-type Gly102 CHIT1 when measured with 4MU-chitotrioside at a nonsaturating concentration. However, the activity was normal with 4MU-deoxychitobioside as the substrate at saturating concentrations, consistent with predictions from molecular dynamics simulations. In conclusion, interpretation of CHIT1 activity measurements with 4MU-chitotrioside with respect to CHIT1 protein concentrations depends on the presence of Ser102 CHIT1 in an individual, complicating estimation of the body burden of storage macrophages. Use of the superior 4MU-deoxychitobioside substrate avoids such complications because activity towards this substrate under saturating conditions is not affected by the G102S substitution

Reducing glycosphingolipid content in adipose tissue of obese mice restores insulin sensitivity, adipogenesis and reduces inflammation

Adipose tissue is a critical mediator in obesity-induced insulin resistance. Previously we have demonstrated that pharmacological lowering of glycosphingolipids and subsequently GM3 by using the iminosugar AMP-DNM, strikingly improves glycemic control. Here we studied the effects of AMP-DNM on adipose tissue function and inflammation in detail to provide an explanation for the observed improved glucose homeostasis. Leptin-deficient obese (Lep(Ob)) mice were fed AMP-DNM and its effects on insulin signalling, adipogenesis and inflammation were monitored in fat tissue. We show that reduction of glycosphingolipid biosynthesis in adipose tissue of Lep(Ob) mice restores insulin signalling in isolated ex vivo insulin-stimulated adipocytes. We observed improved adipogenesis as the number of larger adipocytes was reduced and expression of genes like peroxisome proliferator-activated receptor (PPAR) gamma, insulin responsive glucose transporter (GLUT)-4 and adipsin increased. In addition, we found that adiponectin gene expression and protein were increased by AMP-DNM. As a consequence of this improved function of fat tissue we observed less inflammation, which was characterized by reduced numbers of adipose tissue macrophages (crown-like structures) and reduced levels of the macrophage chemo attractants monocyte-chemoattractant protein-1 (Mcp-1/Ccl2) and osteopontin (OPN). In conclusion, pharmacological lowering of glycosphingolipids by inhibition of glucosylceramide biosynthesis improves adipocyte function and as a consequence reduces inflammation in adipose tissue of obese animals