[Show abstract][Hide abstract] ABSTRACT: The endothelial dysfunction of Fabry disease results from α-galactosidase A deficiency leading to the accumulation of globotriaosylceramide. Vasculopathy in the α-galactosidase A null mouse is manifested as oxidant-induced thrombosis, accelerated atherogenesis, and impaired arterial reactivity. To better understand the pathogenesis of Fabry disease in humans, we generated a human cell model by using RNA interference. Hybrid endothelial cells were transiently transfected with small interfering RNA (siRNA) specifically directed against α-galactosidase A. Knockdown of α-galactosidase A was confirmed using immunoblotting and globotriaosylceramide accumulation. Endothelial nitric oxide synthase (eNOS) activity was correspondingly decreased by >60%. Levels of 3-nitrotyrosine (3NT), a specific marker for reactive nitrogen species and quantified using mass spectrometry, increased by 40- to 120-fold without corresponding changes in other oxidized amino acids, consistent with eNOS-derived reactive nitrogen species as the source of the reactive oxygen species. eNOS uncoupling was confirmed by the observed increase in free plasma and protein-bound aortic 3NT levels in the α-galactosidase A knockout mice. Finally, 3NT levels, assayed in biobanked plasma samples from patients with classical Fabry disease, were over sixfold elevated compared with age- and gender-matched controls. Thus, 3NT may serve as a biomarker for the vascular involvement in Fabry disease.Kidney International advance online publication, 8 January 2014; doi:10.1038/ki.2013.520.
Kidney International 01/2014; · 8.52 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Plasmalogens represent a unique class of phospholipids. Reduced red blood cell plasmalogen levels in Gaucher disease patients were reported, correlating to total disease burden. The relation between plasmalogen abnormalities in Gaucher disease patients and primary glycosphingolipid abnormalities, malonyldialdehyde levels, an indicator of lipid peroxidation, and the total antioxidant status was further investigated.
Significant reduction of C16:0 and C18:0 plasmalogens in red blood cells of Gaucher disease patients was confirmed. In parallel, a significant increase in the glucosylceramide/ceramide ratio in red blood cell membranes, as well as an average 200-fold increase in plasma glucosylsphingosine levels was observed. Red blood cell malonyldialdehyde levels were significantly increased in patients, whereas their total antioxidant status was significantly reduced.
A negative correlation between plasmalogen species and glucosylceramide, ceramide, glucosylceramide/ceramide ratio, glucosylsphingosine and malonyldialdehyde, significant for the C16:0 species and all the above parameters with the exception of malonyldialdehyde levels, was found along with a positive non-significant correlation with the total antioxidant status.
Our results indicate that increased lipid peroxidation and reduced total antioxidant status exist in Gaucher disease patients. They demonstrate a clear link between plasmalogen levels and the primary glycolipid abnormalities characterizing the disorder and an association with the increased oxidative stress observed in Gaucher disease patients.
Blood Cells Molecules and Diseases 01/2014; · 2.26 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Gaucher disease (GD) and Fabry disease (FD) are two relatively common inherited glycosphingolipidoses caused by deficiencies in the lysosomal glycosidases glucocerebrosidase and alpha-galactosidase A, respectively. For both diseases enzyme supplementation is presently used as therapy. Cells and tissues of GD and FD patients are uniformly deficient in enzyme activity, but the two diseases markedly differ in cell types showing lysosomal accumulation of the glycosphingolipid substrates glucosylceramide and globotriaosylceramide, respectively. The clinical manifestation of Gaucher disease and Fabry disease is consequently entirely different and the response to enzyme therapy is only impressive in the case of GD patients. This review compares both glycosphingolipid storage disorders with respect to similarities and differences. Presented is an update on insights regarding pathophysiological mechanisms as well as recently available biochemical markers and diagnostic tools for both disorders. Special attention is paid to sphingoid bases of the primary storage lipids in both diseases. The value of elevated glucosylsphingosine in Gaucher disease and globotriaosylsphingosine in Fabry disease for diagnosis and monitoring of disease is discussed as well as the possible contribution of the sphingoid bases to (patho)physiology. This article is part of a Special Issue entitled New frontiers in sphingolipid biology.
Biochimica et Biophysica Acta 11/2013; · 4.66 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Fetal asphyctic (FA) preconditioning, induced by a brief episode of experimental hypoxia-ischemia, offers neuroprotection to a subsequent more severe asphyctic insult at birth. Extensive cell stress and apoptosis are important contributing factors of damage in the asphyctic neonatal brain. Since ceramide acts as a second messenger for multiple apoptotic stimuli including hypoxia/ischemia, we sought to investigate the possible involvement of the ceramide pathway in endogenous neuroprotection induced by FA preconditioning. Global FA was induced in rats by clamping both uterine and ovarian vasculature for 30 min. FA resulted in acute changes in brain ceramide/sphingomyelin metabolic enzymes, ceramide synthase 1, 2 and 5, acid sphingomyelinase, sphingosine-1-phosphate phosphatase and the ceramide transporter. This observation correlated with an increase in neuronal apoptosis and in astrocyte number. After birth, ceramide and sphingomyelin levels remained high in FA brains, suggesting that a long-term regulation of the ceramide pathway may be involved in the mechanism of tolerance to subsequent, otherwise lethal, asphyctic event.
The Journal of Lipid Research 04/2013; · 4.39 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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] (13)C(5)-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] (13)C(5)-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.
[Show abstract][Hide abstract] ABSTRACT: Transintestinal cholesterol efflux (TICE) provides an attractive target to increase body cholesterol excretion. At present, the cholesterol donor responsible for direct delivery of plasma cholesterol to the intestine is unknown. In this study, we investigated the role of HDL in TICE. ATP-binding cassette protein A1 deficient (Abca1(-/-)) mice that lack HDL and wild-type (WT) mice were intravenously injected with chylomicron-like emulsion particles that contained radiolabeled cholesterol that is liberated in the liver and partly reenters the circulation. Both groups secreted radiolabeled cholesterol from plasma into intestinal lumen and TICE was unaltered between the two mouse models. To further investigate the role of HDL, we injected HDL with radiolabeled cholesterol in WT mice and Abca1(-/-)×Sr-b1(-/-) mice that lack HDL and are also unable to clear HDL via the liver. The intestines of both mice were unable to take up and secrete radiolabeled cholesterol from HDL via TICE. Although a generally accepted major player in the hepatobiliary route-based cholesterol excretion, HDL plays no significant role in TICE in mice.
The Journal of Lipid Research 07/2012; 53(10):2017-23. · 4.39 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Gaucher disease (GD) is the most common lysosomal disorder and is caused by an inherited autosomal recessive deficiency in β-glucocerebrosidase. This enzyme, like other glycohydrolases involved in glycosphingolipid (GSL) metabolism, is present in both plasma membrane (PM) and intracellular fractions. We analyzed the activities of CBE-sensitive β-glucosidase (GBA1) and AMP-DNM-sensitive β-glucosidase (GBA2) in total cell lysates and PM of human fibroblast cell lines from control (normal) subjects and from patients with GD clinical types 1, 2, and 3. GBA1 activities in both total lysate and PM of GD fibroblasts were low, and their relative percentages were similar to those of control cells. In contrast, GBA2 activities were higher in GD cells than in control cells, and the degree of increase differed among the three GD types. The increase of GBA2 enzyme activity was correlated with increased expression of GBA2 protein as evaluated by QRT-PCR. Activities of β-galactosidase and β-hexosaminidase in PM were significantly higher for GD cells than for control cells and also showed significant differences among the three GD types, suggesting the occurrence of cross-talk among the enzymes involved in GSL metabolism. Our findings indicate that the profiles of glycohydrolase activities in PM may provide a valuable tool to refine the classification of GD into distinct clinical types.
[Show abstract][Hide abstract] ABSTRACT: Genome-wide association studies have identified GALNT2 as a candidate gene in lipid metabolism, but it is not known how the encoded enzyme ppGalNAc-T2, which contributes to the initiation of mucin-type O-linked glycosylation, mediates this effect. In two probands with elevated plasma high-density lipoprotein cholesterol and reduced triglycerides, we identified a mutation in GALNT2. It is shown that carriers have improved postprandial triglyceride clearance, which is likely attributable to attenuated glycosylation of apolipoprotein (apo) C-III, as observed in their plasma. This protein inhibits lipoprotein lipase (LPL), which hydrolyses plasma triglycerides. We show that an apoC-III-based peptide is a substrate for ppGalNAc-T2 while its glycosylation by the mutant enzyme is impaired. In addition, neuraminidase treatment of apoC-III which removes the sialic acids from its glycan chain decreases its potential to inhibit LPL. Combined, these data suggest that ppGalNAc-T2 can affect lipid metabolism through apoC-III glycosylation, thereby establishing GALNT2 as a lipid-modifying gene.
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: Obesity and its associated conditions such as type 2 diabetes mellitus are major causes of morbidity and mortality. The iminosugar N-(5-adamantane-1-yl-methoxy-pentyl)-deoxynojirimycin (AMP-DNM) improves insulin sensitivity in rodent models of insulin resistance and type 2 diabetes mellitus. In the current study, we characterized the impact of AMP-DNM on substrate oxidation patterns, food intake, and body weight gain in obese mice. Eight ob/ob mice treated with 100 mg/(kg d) AMP-DNM mixed in the food and 8 control ob/ob mice were placed in metabolic cages during the first, third, and fifth week of the experiment for measurement of substrate oxidation rates, energy expenditure, activity, and food intake. Mice were killed after 6 weeks of treatment. Initiation of treatment with AMP-DNM resulted in a rapid increase in fat oxidation by 129% (P = .05), a decrease in carbohydrate oxidation by 35% (P = .01), and a reduction in food intake by approximately 26% (P < .01) compared with control mice. Treatment with AMP-DNM decreased hepatic triglyceride content by 66% (P < .01) and, in line with the elevated fat oxidation rates, increased hepatic carnitine palmitoyl transferase 1a expression. Treatment with AMP-DNM increased plasma levels of the appetite-regulating peptide YY compared with control mice. Treatment with AMP-DNM rapidly reduces food intake and increases fat oxidation, resulting in improvement of the obese phenotype. These features of AMP-DNM, together with its insulin-sensitizing capacity, make it an attractive candidate drug for the treatment of obesity and its associated metabolic derangements.
Metabolism: clinical and experimental 08/2011; 61(1):99-107. · 3.10 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Parenteral nutrition-associated liver disease is a prevalent and severe complication of long term parenteral nutrition. We present here for the first time data on the presence of ceramide, a bioactive compound involved in a variety of metabolic processes, in different lipid emulsions used in parenteral nutrition. Further research is needed to determine whether this potential harmful bioactive compound is involved in parenteral nutrition-associated liver disease.
Journal of Parenteral and Enteral Nutrition 03/2011; 35(2):270-1. · 2.49 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Fabry disease is treated by two-weekly infusions with α-galactosidase A, which is deficient in this X-linked globotriaosylceramide (Gb3) storage disorder. Elevated plasma globotriaosylsphingosine (lysoGb3) is a hallmark of classical Fabry disease. We investigated effects of enzyme replacement therapy (ERT) on plasma levels of lysoGb3 and Gb3 in patients with classical Fabry disease treated with agalsidase alfa at 0.2mg/kg, agalsidase beta at 0.2mg/kg or at 1.0mg/kg bodyweight. Each treatment regimen led to prominent reductions of plasma lysoGb3 in Fabry males within 3 months (P=0.0313), followed by relative stability later on. Many males developed antibodies against α-galactosidase A, particularly those treated with agalsidase beta. Patients with antibodies tended towards smaller correction in plasma lysoGb3 concentration, whereas treatment with high dose agalsidase beta allowed a reduction comparable to patients without antibodies. Pre-treatment plasma lysoGb3 concentrations of Fabry females were relatively low. In all females and with each treatment regimen, ERT gave reduction or stabilisation of plasma lysoGb3. Our investigation revealed that ERT of Fabry patients reduces plasma lysoGb3, regardless of the recombinant enzyme used. This finding shows that ERT can correct a characteristic biochemical abnormality in Fabry patients.
Biochimica et Biophysica Acta 01/2011; 1812(1):70-6. · 4.66 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Glycosphingolipids are structural membrane components, residing largely in the plasma membrane with their sugar-moieties exposed at the cell's surface. In recent times a crucial role for glycosphingolipids in insulin resistance has been proposed. A chronic state of insulin resistance is a rapidly increasing disease condition in Western and developing countries. It is considered to be the major underlying cause of the metabolic syndrome, a combination of metabolic abnormalities that increases the risk for an individual to develop Type 2 diabetes, obesity, cardiovascular disease, polycystic ovary syndrome and nonalcoholic fatty liver disease. As discussed in this chapter, the evidence for a direct regulatory interaction of glycosphingolipids with insulin signaling is still largely indirect. However, the recent finding in animal models that pharmacological reduction of glycosphingolipid biosynthesis ameliorates insulin resistance and prevents some manifestations of metabolic syndrome, supports the view that somehow glycosphingolipids act as critical regulators, Importantly, since reductions in glycosphingolipid biosynthesis have been found to be well tolerated, such approaches may have a therapeutic potential.
Advances in experimental medicine and biology 01/2011; 721:99-119. · 1.83 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The chemoenzymatic synthesis of three 1-deoxynojirimycin-type iminosugars is reported. Key steps in the synthetic scheme include a Dibal reduction-transimination-sodium borohydride reduction cascade of reactions on an enantiomerically pure cyanohydrin, itself prepared employing almond hydroxynitrile lyase (paHNL) as the common precursor. Ensuing ring-closing metathesis and Upjohn dihydroxylation afford the target compounds.
[Show abstract][Hide abstract] ABSTRACT: Recent studies have reported that glycosphingolipids (GSLs) might be involved in obesity-induced insulin resistance. Those reports suggested that inhibition of GSL biosynthesis in animals ameliorated insulin resistance accompanied by improved glycemic control and decreased liver steatosis in obese mice. In addition, pharmacologic GSL depletion altered hepatic secretory function. In those studies, ubiquitously acting inhibitors for GSL biosynthesis have been used to inhibit the enzyme Ugcg (UDP-glucose:ceramide glucosyltransferase), catalyzing the first step of the glucosylceramide-based GSL-synthesis pathway. In the present study a genetic approach for selective GSL deletion in hepatocytes was chosen to achieve complete inhibition of GSL synthesis and to avoid possible adverse effects caused by Ugcg inhibitors. Using the Cre/loxP system under control of the albumin promoter, GSL biosynthesis in hepatocytes and their release into the plasma could be effectively blocked. Deletion of GSL in hepatocytes did not change the quantity of bile excretion through the biliary duct. Total bile salt content in bile, feces, and plasma from mutant mice showed no difference as compared to control animals. Cholesterol concentration in liver, bile, feces, and plasma samples remained unaffected. Lipoprotein concentrations in plasma samples in mutant animals reached similar levels as in their control littermates. No alteration in glucose tolerance after intraperitoneal application of glucose and insulin appeared in mutant animals. A preventive effect of GSL deficiency on development of liver steatosis after a high-fat diet was not observed. CONCLUSION: The data suggest that GSL in hepatocytes are not essential for sterol, glucose, or lipoprotein metabolism and do not prevent high-fat diet-induced liver steatosis, indicating that Ugcg inhibitors exert their effect on hepatocytes either independently of GSL or mediated by other (liver) cell types.
[Show abstract][Hide abstract] ABSTRACT: The iminosugar N-(5'-adamantane-1'-yl-methoxy)-pentyl-1-deoxynoijirimycin (AMP-DNM), an inhibitor of the enzyme glucosylceramide synthase catalyzing glycosphingolipid (GSL) biosynthesis, ameliorates diabetes and reduces liver steatosis in ob/ob mice. Because an accumulation of sphingolipids, including sphingomyelin and GSLs, has been reported in atherosclerotic lesions in animal models and in humans, the objective of this study was to determine whether AMP-DNM also exerts beneficial effects on the development of atherosclerosis.
APOE*3 Leiden mice, maintained on a high-cholesterol diet, were treated for up to 18 weeks with AMP-DNM. The iminosugar prevented hyperlipidemia, generated a less atherogenic lipid profile, and induced a dramatic reduction in the development of atherosclerotic lesions. At the highest dose, no lesions were detectable. The effect of AMP-DNM was associated with a decrease in liver cholesterol, an increase in bile secretion, and enhanced excretion of cholesterol in the feces. Similar effects of AMP-DNM were observed in mice deficient for the low-density lipoprotein receptor.
By lowering plasma cholesterol, the iminosugar AMP-DNM dramatically reduces the development of atherosclerosis in APOE*3 Leiden and low-density lipoprotein receptor -/- mice. Thus, targeting GSL synthesis may be a new treatment modality to prevent cardiovascular disease.
[Show abstract][Hide abstract] ABSTRACT: 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.
Journal of Medicinal Chemistry 12/2009; 53(2). · 5.61 Impact Factor