Johanna E. M. Groener

Elevated globotriaosylsphingosine is a hallmark of Fabry disease.

Fabry disease is an X-linked lysosomal storage disease caused by deficiency of α-galactosidase A that affects males and shows disease expression in heterozygotes. The characteristic progressive renal insufficiency, cardiac involvement, and neuropathology usually are ascribed to globotriaosylceramide accumulation in the endothelium. However, no direct correlation exists between lipid storage and clinical manifestations, and treatment of patients with recombinant enzymes does not reverse several key signs despite clearance of lipid from the endothelium. We therefore investigated the possibility that globotriaosylceramide metabolites are a missing link in the pathogenesis. We report that deacylated globotriaosylceramide, globotriaosylsphingosine, and a minor additional metabolite are dramatically increased in plasma of classically affected male Fabry patients and plasma and tissues of Fabry mice. Plasma globotriaosylceramide levels are reduced by therapy. We show that globotriaosylsphingosine is an inhibitor of α-galactosidase A activity. Furthermore, exposure of smooth muscle cells, but not fibroblasts, to globotriaosylsphingosine at concentrations observed in plasma of patients promotes proliferation. The increased intima-media thickness in Fabry patients therefore may be related to the presence of this metabolite. Our findings suggest that measurement of circulating globotriaosylsphingosine will be useful to monitor Fabry disease and may contribute to a better understanding of the disorder.

Kupffer cells promote hepatic steatosis via interleukin‐1β–dependent suppression of peroxisome proliferator‐activated receptor α activity

Kupffer cells have been implicated in the pathogenesis of various liver diseases. However, their involvement in metabolic disorders of the liver, including fatty liver disease, remains unclear. The present study sought to determine the impact of Kupffer cells on hepatic triglyceride storage and to explore the possible mechanisms involved. To that end, C57Bl/6 mice rendered obese and steatotic by chronic high‐fat feeding were treated for 1 week with clodronate liposomes, which cause depletion of Kupffer cells. Loss of expression of marker genes Cd68, F4/80, and Clec4f, and loss of Cd68 immunostaining verified almost complete removal of Kupffer cells from the liver. Also, expression of complement components C1, the chemokine (C‐C motif) ligand 6 (Ccl6), and cytokines interleukin‐15 (IL‐15) and IL‐1β were markedly reduced. Importantly, Kupffer cell depletion significantly decreased liver triglyceride and glucosylceramide levels concurrent with increased expression of genes involved in fatty acid oxidation including peroxisome proliferator‐activated receptor alpha (PPARα), carnitine palmitoyltransferase 1A (Cpt1α), and fatty acid transport protein 2 (Fatp2). Treatment of mice with IL‐1β decreased expression of PPARα and its target genes, which was confirmed in primary hepatocytes. Consistent with these data, IL‐1β suppressed human and mouse PPARα promoter activity. Suppression of PPARα promoter activity was recapitulated by overexpression of nuclear factor κB (NF‐κB) subunit p50 and p65, and was abolished upon deletion of putative NF‐κB binding sites. Finally, IL‐1β and NF‐κB interfered with the ability of PPARα to activate gene transcription. Conclusion: Our data point toward important cross‐talk between Kupffer cells and hepatocytes in the regulation of hepatic triglyceride storage. The effect of Kupffer cells on liver triglycerides are at least partially mediated by IL‐1β, which suppresses PPARα expression and activity. (HEPATOLOGY 2010.)

Plasma globotriaosylsphingosine: Diagnostic value and relation to clinical manifestations of Fabry disease

Fabry disease is an X-linked lysosomal storage disorder due to deficiency of alpha-Galactosidase A, causing accumulation of globotriaosylceramide and elevated plasma globotriaosylsphingosine (lysoGb3). The diagnostic value and clinical relevance of plasma lysoGb3 concentration was investigated. All male and adult female patients with classical Fabry disease could be discerned by an elevated plasma lysoGb3. In young pre-symptomatic Fabry heterozygotes, lysoGb3 levels can be normal. Individuals carrying the R112H and P60L mutations, without classical Fabry symptoms, showed no elevated plasma lysoGb3. Multiple regression analysis showed that there is no correlation of plasma lysoGb3 concentration with total disease severity score in Fabry males. However, plasma lysoGb3 concentration did correlate with white matter lesions (odds ratio: 6.1 per 100 nM lysoGb3 increase (95% CI: 1.4-25.9, p=0.015). In females, plasma lysoGb3 concentration correlated with overall disease severity. Furthermore, plasma lysoGb3 level was related to left ventricular mass (19.5+/-5.5 g increase per 10 nM lysoGb3 increase; p=0.001). In addition, it was assessed whether lifetime exposure to lysoGb3 correlates with disease manifestations. Male Fabry patients with a high lysoGb3 exposure (>10,000 U), were moderately or severely affected, only one mildly. Female patients with a low exposure (<1000 U) were asymptomatic or mildly affected. A large proportion of the females with an exposure >1000 U showed disease complications. Plasma lysoGb3 is useful for the diagnosis of Fabry disease. LysoGb3 is an independent risk factor for development of cerebrovascular white matter lesions in male patients and left ventricular hypertrophy in females. Disease severity correlates with exposure to plasma lysoGb3.

Transglycosidase Activity of Chitotriosidase IMPROVED ENZYMATIC ASSAY FOR THE HUMAN MACROPHAGE CHITINASE

Chitotriosidase is a chitinase that is massively expressed by lipid-laden tissue macrophages in man. Its enzymatic activity is markedly elevated in serum of patients suffering from lysosomal lipid storage disorders, sarcoidosis, thalassemia, and visceral Leishmaniasis. Monitoring of serum chitotriosidase activity in Gaucher disease patients during progression and therapeutic correction of their disease is useful to obtain insight in changes in body burden on pathological macrophages. However, accurate quantification of chitotriosidase levels by enzyme assay is complicated by apparent substrate inhibition, which prohibits the use of saturating substrate concentrations. We have therefore studied the catalytic features of chitotriosidase in more detail. It is demonstrated that the inhibition of enzyme activity at excess substrate concentration can be fully explained by transglycosylation of substrate molecules. The potential physiological consequences of the ability of chitotriosidase to hydrolyze as well as transglycosylate are discussed. The novel insight in transglycosidase activity of chitotriosidase has led to the design of a new substrate molecule, 4-methylumbelliferyl-(4-deoxy)chitobiose. With this substrate, which is no acceptor for transglycosylation, chitotriosidase shows normal Michaelis-Menten kinetics, resulting in major improvements in sensitivity and reproducibility of enzymatic activity measurements. The novel convenient chitotriosidase enzyme assay should facilitate the accurate monitoring of Gaucher disease patients receiving costly enzyme replacement therapy.

Elevated plasma glucosylsphingosine in Gaucher disease: relation to phenotype, storage cell markers, and therapeutic response.

Gaucher disease, caused by a deficiency of the lysosomal enzyme glucocerebrosidase, leads to prominent glucosylceramide accumulation in lysosomes of tissue macrophages (Gaucher cells). Here we show glucosylsphingosine, the deacylated form of glucosylceramide, to be markedly increased in plasma of symptomatic nonneuronopathic (type 1) Gaucher patients (n = 64, median = 230.7 nM, range 15.6-1035.2 nM; normal (n = 28): median 1.3 nM, range 0.8-2.7 nM). The method developed for mass spectrometric quantification of plasma glucosylsphingosine is sensitive and robust. Plasma glucosylsphingosine levels correlate with established plasma markers of Gaucher cells, chitotriosidase (ρ = 0.66) and CCL18 (ρ = 0.40). Treatment of Gaucher disease patients by supplementing macrophages with mannose-receptor targeted recombinant glucocerebrosidase results in glucosylsphingosine reduction, similar to protein markers of Gaucher cells. Since macrophages prominently accumulate the lysoglycosphingolipid on glucocerebrosidase inactivation, Gaucher cells seem a major source of the elevated plasma glucosylsphingosine. Our findings show that plasma glucosylsphingosine can qualify as a biomarker for type 1 Gaucher disease, but that further investigations are warranted regarding its relationship with clinical manifestations of Gaucher disease.

Treatment of Fabry disease with different dosing regimens of agalsidase: effects on antibody formation and GL-3

Two different enzyme preparations are used for the treatment of Fabry disease patients, agalsidase alpha (Replagal, Shire) and agalsidase beta (Fabrazyme, Genzyme). Therapeutic efficacy of both products has been variable probably due to differences in gender, severity, age and other patient characteristics. We studied the occurrence of alpha-Gal A antibodies and their effect on urinary and plasma globotriaosylceramide (GL-3), plasma chitotriosidase and clinical outcome in 52 patients after 12 months of treatment with either 0.2mg/kg agalsidase alppha (10 males, 8 females) or beta (8 males, 5 females) or 1.0mg/kg agalsidase beta (10 males, 11 females). Antibodies were detected in 18/28 male patients after 6 months. None of the females developed antibodies. Following 12 months of 0.2mg/kg treatment, urinary GL-3 decreased in antibody negative (AB-) but increased in antibody positive (AB+) patients. Treatment with 1.0mg/kg gave a reduction in urinary GL-3 in both AB- and AB+ patients. Levels of plasma GL-3 and chitotriosidase decreased in all patient groups. Twelve months of 0.2mg/kg treatment did not change renal function or left ventricular mass. Further, no change in renal function was seen following 1.0mg/kg treatment and left ventricular mass decreased in both AB- and AB+ patients. In summary, alpha-Gal A antibodies frequently develop in male Fabry disease patients and interfere with urinary GL-3 excretion. Infusion of a dose of 1.0mg/kg results in a more robust decline in GL-3, less impact, if any of antibodies, stable renal function and reduction of LVMass.

Substrate reduction therapy of glycosphingolipid storage disorders.

SummaryIn the last 15 years enormous progress has been made regarding therapy of type I Gaucher disease, a severely disabling disorder characterized by intralysosomal storage of glucosylceramide in tissue macrophages. Effective enzyme replacement therapy of type I Gaucher disease, based on chronic intravenous administration of mannose-terminated recombinant human glucocerebrosidase, has been available since 1990 and has been applied in several thousand patients without serious adverse effects. An alternative therapeutic approach, so-called substrate reduction therapy, is based on partial reduction of the synthesis of glucosylceramide and hence of subsequent metabolites. Oral administration of an inhibitor of glucosylceramide synthesis (N-butyldeoxynojirimycin, registered in Europe since 2002 as miglustat (Zavesca)), is effective in reversing clinical symptoms in type I Gaucher patients with mild to moderate disease manifestations. The growing long-term experience with substrate reduction therapy indicates that this treatment is also without major adverse effects. Substrate reduction therapy, in conjunction with enzyme replacement therapy, may play an important role in the future clinical management of patients suffering from type I Gaucher disease. Clinical trials are under way that should reveal the value of substrate reduction for maintenance therapy of type I Gaucher disease and for treatment of neuronopathic variants of Gaucher disease, Niemann–Pick disease type C, late-onset Tay–Sachs disease and Sandhoff disease.

Biomarkers in the diagnosis of lysosomal storage disorders: proteins, lipids, and inhibodies

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.

Long-Term Effect of Antibodies against Infused Alpha-Galactosidase A in Fabry Disease on Plasma and Urinary (lyso)Gb3 Reduction and Treatment Outcome

Introduction Enzyme replacement therapy (ERT) with alpha-Galactosidase A (aGal A) may cause antibody (AB) formation against aGal A in males with Fabry disease (FD). Anti agalsidase ABs negatively influence globotriaosylceramide (Gb3) reduction. We investigated the impact of agalsidase AB on Gb3 and lysoGb3 and clinical outcome in Fabry patients on ERT. Methods Adult male and female patients on ERT for at least one year were included. Urinary Gb3 was measured by HPLC, plasma lysoGb3 by LC-ESI-MS/MS and AB with a neutralization assay. Results Of the 59 patients evaluable patients, 0/30 females and 17/29 males developed anti-agalsidase antibodies (AB+). Only 3/17 males had transient (low) titers (tolerized). All AB+ patients developed antibodies during the first year of treatment. Change of agalsidase preparation (or dose) did not induce antibody formation. AB+ males had significant less decline in plasma lysoGb3 compared to AB− males (p = 0.04). Urinary Gb3 levels decreased markedly in AB− but remained comparable to baseline in AB+ males (p<0.01). (Lyso)Gb3 reduction in plasma and urine on ERT was correlated with LVmass reduction in females and development white matter lesions and stroke. Conclusion In male patients antibodies against aGal A remained present up to 10 years of ERT. The presence of these antibodies is associated with a less robust decrease in plasma lysoGb3 and a profound negative effect on urinary Gb3 reduction, which may reflect worse treatment outcome.

Dual-Action Lipophilic Iminosugar Improves Glycemic Control in Obese Rodents by Reduction of Visceral Glycosphingolipids and Buffering of Carbohydrate Assimilation

The lipophilic iminosugar N-[5-(adamantan-1-ylmethoxy)pentyl]-1-deoxynojirimycin (2, AMP-DNM) potently controls hyperglycemia in obese rodent models of insulin resistance. The reduction of visceral glycosphingolipids by 2 is thought to underlie its beneficial action. It cannot, however, be excluded that concomitant inhibition of intestinal glycosidases and associated buffering of carbohydrate assimilation add to this. To firmly establish the mode of action of 2, we developed a panel of lipophilic iminosugars varying in configuration at C-4/C-5 and N-substitution of the iminosugar. From these we identified the l-ido derivative of 2, l-ido-AMP-DNM (4), as a selective inhibitor of glycosphingolipid synthesis. Compound 4 lowered visceral glycosphingolipids in ob/ob mice and ZDF rats on a par with 2. In contrast to 2, 4 did not inhibit sucrase activity or sucrose assimilation. Treatment with 4 was significantly less effective in reducing blood glucose and HbA1c. We conclude that the combination of reduction of glycosphingolipids in tissue and buffering of carbohydrate assimilation by 2 produces a superior glucose homeostasis.