Glucocerebrosidase (GCase), encoded by the GBA gene, degrades the ubiquitous glycosphingolipid glucosylceramide. Inherited GCase deficiency causes Gaucher disease (GD). In addition, carriers of an... Show moreGlucocerebrosidase (GCase), encoded by the GBA gene, degrades the ubiquitous glycosphingolipid glucosylceramide. Inherited GCase deficiency causes Gaucher disease (GD). In addition, carriers of an abnormal GBA allele are at increased risk for Parkinson's disease. GCase undergoes extensive modification of its four N-glycans en route to and inside the lysosome that is reflected in changes in molecular weight as detected with sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Fluorescent activity-based probes (ABPs) that covalently label GCase in reaction-based manner in vivo and in vitro allow sensitive visualization of GCase molecules. Using these ABPs, we studied the life cycle of GCase in cultured fibroblasts and macrophage-like RAW264.7 cells. Specific attention was paid to the impact of 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) supplementation to bicarbonate-buffered medium. Here, we report how HEPES-buffered medium markedly influences processing of GCase, its lysosomal degradation, and the total cellular enzyme level. HEPES-containing medium was also found to reduce maturation of other lysosomal enzymes (α-glucosidase and β-glucuronidase) in cells. The presence of HEPES in bicarbonate containing medium increases GCase activity in GD-patient derived fibroblasts, illustrating how the supplementation of HEPES complicates the use of cultured cells for diagnosing GD. Show less
Glucocerebrosidase (GCase), encoded by the GBA gene, degrades the ubiquitous glycosphingolipid glucosylceramide. Inherited GCase deficiency causes Gaucher disease (GD). In addition, carriers of an... Show moreGlucocerebrosidase (GCase), encoded by the GBA gene, degrades the ubiquitous glycosphingolipid glucosylceramide. Inherited GCase deficiency causes Gaucher disease (GD). In addition, carriers of an abnormal GBA allele are at increased risk for Parkinson's disease. GCase undergoes extensive modification of its four N-glycans en route to and inside the lysosome that is reflected in changes in molecular weight as detected with sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Fluorescent activity-based probes (ABPs) that covalently label GCase in reaction-based manner in vivo and in vitro allow sensitive visualization of GCase molecules. Using these ABPs, we studied the life cycle of GCase in cultured fibroblasts and macrophage-like RAW264.7 cells. Specific attention was paid to the impact of 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) supplementation to bicarbonate-buffered medium. Here, we report how HEPES-buffered medium markedly influences processing of GCase, its lysosomal degradation, and the total cellular enzyme level. HEPES-containing medium was also found to reduce maturation of other lysosomal enzymes (alpha-glucosidase and beta-glucuronidase) in cells. The presence of HEPES in bicarbonate containing medium increases GCase activity in GD-patient derived fibroblasts, illustrating how the supplementation of HEPES complicates the use of cultured cells for diagnosing GD. Show less
Macrophages are key multi-talented cells of the innate immune system and are equipped with receptors involved in damage and pathogen recognition with connected immune response guiding signaling... Show moreMacrophages are key multi-talented cells of the innate immune system and are equipped with receptors involved in damage and pathogen recognition with connected immune response guiding signaling systems. In addition, macrophages have various systems that are involved in the uptake of extracellular and intracellular cargo. The lysosomes in macrophages play a central role in the digestion of all sorts of macromolecules and the entry of nutrients to the cytosol, and, thus, the regulation of endocytic processes and autophagy. Simplistically viewed, two macrophage phenotype extremes exist. On one end of the spectrum, the classically activated pro-inflammatory M1 cells are present, and, on the other end, alternatively activated anti-inflammatory M2 cells. A unique macrophage population arises when lipid accumulation occurs, either caused by flaws in the catabolic machinery, which is observed in lysosomal storage disorders, or as a result of an acquired condition, which is found in multiple sclerosis, obesity, and cardiovascular disease. The accompanying overload causes a unique metabolic activation phenotype, which is discussed here, and, consequently, a unifying phenotype is proposed. Show less
The lysosomal storage disease Niemann-Pick type C (NPC) is caused by impaired cholesterol efflux from lysosomes, which is accompanied by secondary lysosomal accumulation of sphingomyelin and... Show moreThe lysosomal storage disease Niemann-Pick type C (NPC) is caused by impaired cholesterol efflux from lysosomes, which is accompanied by secondary lysosomal accumulation of sphingomyelin and glucosylceramide (GlcCer). Similar to Gaucher disease (GD), patients deficient in glucocerebrosidase (GCase) degrading GlcCer, NPC patients show an elevated glucosylsphingosine and glucosylated cholesterol. In livers of mice lacking the lysosomal cholesterol efflux transporter NPC1, we investigated the expression of established biomarkers of lipid-laden macrophages of GD patients, their GCase status, and content on the cytosol facing glucosylceramidase GBA2 and lysosomal integral membrane protein type B (LIMP2), a transporter of newly formed GCase to lysosomes. Livers of 80-week-old Npc1(-/-) mice showed a partially reduced GCase protein and enzymatic activity. In contrast, GBA2 levels tended to be reciprocally increased with the GCase deficiency. In Npc1(-/-) liver, increased expression of lysosomal enzymes (cathepsin D, acid ceramidase) was observed as well as increased markers of lipid-stressed macrophages (GPNMB and galectin-3). Immunohistochemistry showed that the latter markers are expressed by lipid laden Kupffer cells. Earlier reported increase of LIMP2 in Npc1(-/-) liver was confirmed. Unexpectedly, immunohistochemistry showed that LIMP2 is particularly overexpressed in the hepatocytes of the Npc1(-/-) liver. LIMP2 in these hepatocytes seems not to only localize to (endo)lysosomes. The recent recognition that LIMP2 harbors a cholesterol channel prompts the speculation that LIMP2 in Npc1(-/-) hepatocytes might mediate export of cholesterol into the bile and thus protects the hepatocytes. Show less
Glycosphingolipids (GSLs), the main topic of this review, are a subclass of sphingolipids. With their glycans exposed to the extracellular space, glycosphingolipids are ubiquitous components of the... Show moreGlycosphingolipids (GSLs), the main topic of this review, are a subclass of sphingolipids. With their glycans exposed to the extracellular space, glycosphingolipids are ubiquitous components of the plasma membrane of cells. GSLs are implicated in a variety of biological processes including specific infections. Several pathogens use GSLs at the surface of host cells as binding receptors. In addition, lipid-rafts in the plasma membrane of host cells may act as platform for signaling the presence of pathogens. Relatively common in man are inherited deficiencies in lysosomal glycosidases involved in the turnover of GSLs. The associated storage disorders (glycosphingolipidoses) show lysosomal accumulation of substrate(s) of the deficient enzyme. In recent years compounds have been identified that allow modulation of GSLs levels in cells. Some of these agents are well tolerated and already used to treat lysosomal glycosphingolipidoses. This review summarizes present knowledge on the role of GSLs in infection and subsequent immune response. It concludes with the thought to apply glycosphingolipid-lowering agents to prevent and/or combat infections. Show less
Obesity is taking worldwide epidemic proportions, yet effective pharmacological agents with long-term efficacy remain unavailable. Previously, we designed the iminosugar AMP-DNM which potently... Show moreObesity is taking worldwide epidemic proportions, yet effective pharmacological agents with long-term efficacy remain unavailable. Previously, we designed the iminosugar AMP-DNM which potently improves glucose homeostasis by lowering excessive glycosphingolipids. Here we show that AMP-DNM promotes satiety and activates brown adipose tissue (BAT) in obese rodents. Moreover, we demonstrate that the mechanism mediating these favorable actions depends on oral, but not central, administration of AMP-DNM, which ultimately stimulates systemic glucagon-like peptide-1 (GLP1) secretion. We evidence an essential role of brain GLP1 receptors (GLP1r) as AMP-DNM fails to promote satiety and activate BAT in mice lacking the brain GLP1r as well as in mice treated intracerebroventricularly with GLP1r antagonist exendin-9. In conclusion, AMP-DNM markedly ameliorates metabolic abnormalities in obese rodents by restoring satiety and activating BAT through central GLP1r, while improving glucose homeostasis by mechanisms independent of central GLP1r. Show less
Rakhshandehroo, M.; Eijkeren, R.J. van; Gabriel, T.L.; Haar, C. de; Gijzel, S.M.W.; Hamers, N.; ... ; Kalkhoven, E. 2019
Background: Natural killer T (NKT) cells in adipose tissue (AT) contribute to whole body energy homeostasis. Results: Inhibition of the glucosylceramide synthesis in adipocytes impairs iNKT cell... Show moreBackground: Natural killer T (NKT) cells in adipose tissue (AT) contribute to whole body energy homeostasis. Results: Inhibition of the glucosylceramide synthesis in adipocytes impairs iNKT cell activity. Conclusion: Glucosylceramide biosynthesis pathway is important for endogenous lipid antigen activation of iNKT cells in adipocytes.Significance: Unraveling adipocyte-iNKT cell communication may help to fight obesity-induced AT dysfunction.Overproduction and/or accumulation of ceramide and ceramide metabolites, including glucosylceramides, can lead to insulin resistance. However, glucosylceramides also fulfill important physiological functions. They are presented by antigen presenting cells (APC) as endogenous lipid antigens via CD1d to activate a unique lymphocyte subspecies, the CD1d-restricted invariant (i) natural killer T (NKT) cells. Recently, adipocytes have emerged as lipid APC that can activate adipose tissue-resident iNKT cells and thereby contribute to whole body energy homeostasis. Here we investigate the role of the glucosylceramide biosynthesis pathway in the activation of iNKT cells by adipocytes.UDP-glucose ceramide glucosyltransferase (Ugcg), the first rate limiting step in the glucosylceramide biosynthesis pathway, was inhibited via chemical compounds and shRNA knockdown in vivo and in vitro. beta-1,4-Galactosyltransferase (B4Galt) 5 and 6, enzymes that convert glucosylceramides into potentially inactive lactosylceramides, were subjected to shRNA knock down. Subsequently, (pre)adipocyte cell lines were tested in co-culture experiments with iNKT cells (IFN gamma and 114 secretion).Inhibition of Ugcg activity shows that it regulates presentation of a considerable fraction of lipid self-antigens in adipocytes. Furthermore, reduced expression levels of either B4Galt5 or -6, indicate that B4Galt5 is dominant in the production of cellular lactosylceramides, but that inhibition of either enzyme results in increased iNKT cell activation. Additionally, in vivo inhibition of Ugcg by the aminosugar AMP-DNM results in decreased iNKT cell effector function in adipose tissue.Inhibition of endogenous glucosylceramide production results in decreased iNKT cells activity and cytokine production, underscoring the role of this biosynthetic pathway in lipid self-antigen presentation by adipocytes. Show less
Tol, M.J.; Lienden, M.J.C. van der; Gabriel, T.L.; Hagen, J.J.; Scheij, S.; Veenendaal, T.; ... ; Eijk, M.C. van 2018
Impaired function of NPC1 or NPC2 lysosomal proteins leads to the intracellular accumulation of unesterified cholesterol, the primary defect underlying Niemann-Pick type C (NPC) disease. In... Show moreImpaired function of NPC1 or NPC2 lysosomal proteins leads to the intracellular accumulation of unesterified cholesterol, the primary defect underlying Niemann-Pick type C (NPC) disease. In addition, glycosphingolipids (GSLs) accumulate in lysosomes as well. Intralysosomal lipid accumulation triggers the activation of a set of genes, including potential biomarkers. Transcript levels of Gpnmb have been shown to be elevated in various tissues of an NPC mouse model. We speculated that Gpnmb could serve as a marker for visceral lipid accumulation in NPC disease. We report that Gpnmb expression is increased at protein level in macrophages in the viscera of Npc1nih/nih mice. Interestingly, soluble Gpnmb was also found to be increased in murine and NPC patient plasma. Exposure of RAW264.7 macrophages to the NPC-phenotype-inducing drug U18666A also upregulated Gpnmb expression. Inhibition of GSL synthesis with the glucosylceramide synthase (GCS) inhibitor N-butyl-1-deoxynojirimycin prevented U18666A-induced Gpnmb induction and secretion. In summary, we show that Gpnmb is upregulated in NPC mice and patients, most likely due to GSL accumulation. Show less
Kramer, G.; Wegdam, W.; Donker-Koopman, W.; Ottenhoff, R.; Gaspar, P.; Verhoek, M.; ... ; Eijk, M.C. van 2016
Gaucher disease is caused by inherited deficiency of lysosomal glucocerebrosidase. Proteome analysis of laser-dissected splenic Gaucher cells revealed increased amounts of glycoprotein... Show moreGaucher disease is caused by inherited deficiency of lysosomal glucocerebrosidase. Proteome analysis of laser-dissected splenic Gaucher cells revealed increased amounts of glycoprotein nonmetastatic melanoma protein B (gpNMB). Plasma gpNMB was also elevated, correlating with chitotriosidase and CCL18, which are established markers for human Gaucher cells. In Gaucher mice, gpNMB is also produced by Gaucher cells. Correction of glucocerebrosidase deficiency in mice by gene transfer or pharmacological substrate reduction reverses gpNMB abnormalities. In conclusion, gpNMB acts as a marker for glucosylceramide-laden macrophages in man and mouse and gpNMB should be considered as candidate biomarker for Gaucher disease in treatment monitoring. Show less
