IntroductionGaucher disease, a relatively common recessively inherited lysosomal storage disorder, is caused by a deficiency in the enzyme glucocerebrosidase, encoded by the GBA gene. 1 Deficient enzymatic activity of glucocerebrosidase results in the lysosomal accumulation of its substrate glucosylceramide, most prominently in macrophages. Three variants of Gaucher disease are generally distinguished based on the absence (type 1) or presence of central nervous system involvement 1 (types 2 and 3). In the much more common type 1 variant of Gaucher disease, glycosphingolipidladen macrophages, referred to as Gaucher cells, accumulate in the visceral tissues liver, spleen, and bone marrow, inducing a pleiotropic array of symptoms, including hepatosplenomegaly and pancytopenia. In addition, type 1 Gaucher patients often develop bone complications: bone pain and crises, avascular necrosis, and pathologic fractures. 1 Two different types of therapeutic intervention are available for type 1 patients. One relies on chronic intravenous administration of recombinant glucocerebrosidase, denoted enzyme replacement therapy (ERT). 2 Two recombinant enzyme preparations are now registered for ERT in type 1 Gaucher disease: imiglucerase (Cerezyme; Genzyme Corp) and velaglucerase alfa (Vpriv; Shire HGT). 3 A third enzyme, a plant-cellexpressed recombinant glucocerebrosidase, is under clinical development (Taliglucerase; Protalix/Pfizer). 3 The other therapeutic intervention is based on oral administration of the iminosugar N-butyldeoxinojirimycin (Miglustat; Zavesca, Actelion). 4 This compound is thought to effectively lower synthesis of the accumulating metabolite, glucosylceramide, by inhibiting its synthesizing enzyme, glucosylceramide synthase. 5 The clinical responses to ERT are fast and impressive, such as significant corrections in hepatosplenomegaly, improvement of hematologic parameters and reduction of bone marrow infiltration as seen by magnetic resonance imaging. 6 The response to miglustat treatment is less prominent, and its use is authorized for mildly to moderately affected patients who are unsuitable for ERT (EMA) or in whom ERT is not a therapeutic option (FDA). 7 Future use of such small compounds for treating patients with a neuronopathic course of Gaucher disease is appealing given their potential to penetrate the brain (in contrast to recombinant enzyme). 8 The availability of costly therapies has stimulated searches for plasma biomarkers that can assist in clinical management of individual patients. Several circulating protein markers for Gaucher cells have meanwhile been identified (for a review see Aerts et al 9 ). It has been demonstrated that the enzyme chitotriosidase 10 and the chemokine CCL18 11 are produced by Gaucher cells and secreted into the circulation. Both proteins are candidate biomarkers since their plasma concentrations are markedly increased in symptomatic type 1 Gaucher patients and vary This article contains a data supplement.The publication costs of this article were defrayed in part b...

Type 2 diabetes is characterized by excessive lipid storage in skeletal muscle. Excessive intramyocellular lipid (IMCL) storage exceeds intracellular needs and induces lipotoxic events, ultimately contributing to the development of insulin resistance. Lipid droplet (LD)–coating proteins may control proper lipid storage in skeletal muscle. Perilipin 2 (PLIN2/adipose differentiation–related protein [ADRP]) is one of the most abundantly expressed LD-coating proteins in skeletal muscle. Here we examined the role of PLIN2 in myocellular lipid handling and insulin sensitivity by investigating the effects of in vitro PLIN2 knockdown and in vitro and in vivo overexpression. PLIN2 knockdown decreased LD formation and triacylglycerol (TAG) storage, marginally increased fatty-acid (FA) oxidation, and increased incorporation of palmitate into diacylglycerols and phospholipids. PLIN2 overexpression in vitro increased intramyocellular TAG storage paralleled with improved insulin sensitivity. In vivo muscle-specific PLIN2 overexpression resulted in increased LD accumulation and blunted the high-fat diet–induced increase in protein content of the subunits of the oxidative phosphorylation (OXPHOS) chain. Diacylglycerol levels were unchanged, whereas ceramide levels were increased. Despite the increased IMCL accumulation, PLIN2 overexpression improved skeletal muscle insulin sensitivity. We conclude that PLIN2 is essential for lipid storage in skeletal muscle by enhancing the partitioning of excess FAs toward TAG storage in LDs, thereby blunting lipotoxicity-associated insulin resistance.

Glycosphingoid bases are elevated in inherited lysosomal storage disorders with deficient activity of glycosphingolipid catabolizing glycosidases. We investigated the molecular basis of the formation of glucosylsphingosine and globotriaosylsphingosine during deficiency of glucocerebrosidase (Gaucher disease) and α‐galactosidase A (Fabry disease). Independent genetic and pharmacological evidence is presented pointing to an active role of acid ceramidase in both processes through deacylation of lysosomal glycosphingolipids. The potential pathophysiological relevance of elevated glycosphingoid bases generated through this alternative metabolism in patients suffering from lysosomal glycosidase defects is discussed.

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The membrane lipid glucosylceramide (GlcCer) is continuously formed and degraded. Cells express two GlcCer-degrading β-glucosidases, glucocerebrosidase (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-labeled 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 disease, 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.

Background-Lipotoxicity may be a key contributor to the pathogenesis of cardiac abnormalities in mitochondrial long-chain fatty acid ␤-oxidation (FAO) disorders. Few data are available on myocardial lipid levels and cardiac performance in FAO deficiencies. The purpose of this animal study is to assess fasting-induced changes in cardiac morphology, function, and triglyceride (TG) storage as a consequence of FAO deficiency in a noninvasive fashion. Methods and Results-MRI and proton magnetic resonance spectroscopy ( 1 H-MRS) were applied in vivo in long-chain acyl-CoA dehydrogenase (LCAD) knockout (KO) mice and wild-type (WT) mice (nϭ8 per genotype). Fasting was used to increase the heart's dependency on FAO for maintenance of energy homeostasis. In vivo data were complemented with ex vivo measurements of myocardial lipids. Left ventricular (LV) mass was higher in LCAD KO mice compared with WT mice (PϽ0.05), indicating LV myocardial hypertrophy. Myocardial TG content was higher in LCAD KO mice at baseline (PϽ0.001) and further increased in fasted LCAD KO mice (PϽ0.05). Concomitantly, LV ejection fraction (PϽ0.01) and diastolic filling rate (PϽ0.01) decreased after fasting, whereas these functional parameters did not change in fasted WT mice. Myocardial ceramide content was higher in fasted LCAD KO mice compared with fasted WT mice (PϽ0.05). Conclusions-Using

BACKGROUND Biochemical markers that accurately reflect the severity and progression of disease in patients with Fabry disease and their response to treatment are urgently needed. Globotriaosylsphingosine, also called lysoglobotriaosylceramide (lysoGb3), is a promising candidate biomarker. METHODS We synthesized lysoGb3 and isotope-labeled [5,6,7,8,9] 13C5-lysoGb3 (internal standard). After addition of the internal standard to 25 μL plasma or 400 μL urine from patients with Fabry disease and healthy controls, samples were extracted with organic solvents and the lysoGb3 concentration was quantified by UPLC-ESI-MS/MS (ultraperformance liquid chromatography–electrospray ionization–tandem mass spectrometry). Calibration curves were constructed with control plasma and urine supplemented with lysoGb3. In addition to lysoGb3, lyso-ene-Gb3 was quantified. Quantification was achieved by multiple reaction monitoring of the transitions m/z 786.4 > 282.3 [M+H]+ for lysoGb3, m/z 791.4 > 287.3 [M+H]+ for [5,6,7,8,9] 13C5-lysoGb3, and 784.4 > 280.3 [M+H]+ for lyso-ene-Gb3. RESULTS The mean (SD) plasma lysoGb3 concentration from 10 classically affected Fabry hemizygotes was 94.4 (25.8) pmol/mL (range 52.7–136.8 pmol/mL), from 10 classically affected Fabry heterozygotes 9.6 (5.8) pmol/mL (range 4.1–23.5 pmol/mL), and from 20 healthy controls 0.4 (0.1) pmol/mL (range 0.3–0.5 pmol/mL). Lyso-ene-Gb3 concentrations were 10%–25% of total lysoGb3. The urine concentration of lysoGb3 was 40–480 times lower than in corresponding plasma samples. Lyso-ene-Gb3 concentrations in urine were comparable or even higher than the corresponding lysoGb3 concentrations. CONCLUSIONS This assay for the quantification of lysoGb3 and lyso-ene-Gb3 in human plasma and urine samples will be an important tool in the diagnosis of Fabry disease and for monitoring the effect of enzyme replacement therapy in patients with Fabry disease.

A biomarker is an analyte indicating the presence of a biological process linked to the clinical manifestations and outcome of a particular disease. In the case of lysosomal storage disorders (LSDs), primary and secondary accumulating metabolites or proteins specifically secreted by storage cells are good candidates for biomarkers. Clinical applications of biomarkers are found in improved diagnosis, monitoring disease progression, and assessing therapeutic correction. These are illustrated by reviewing the discovery and use of biomarkers for Gaucher disease and Fabry disease. In addition, recently developed chemical tools allowing specific visualization of enzymatically active lysosomal glucocerebrosidase are described. Such probes, coined inhibodies, offer entirely new possibilities for more sophisticated molecular diagnosis, enzyme replacement therapy monitoring, and fundamental research.