Correlation between enzyme activity and substrate storage in a cell culture model system for Gaucher disease.

Gaucher disease, the most common sphingolipidosis, is caused by a decreased activity of glucosylceramide beta-glucosidase, resulting in the accumulation of glucosylceramide in macrophage-derived cells known as Gaucher cells. Much of the storage material is thought to originate from the turnover of cell membranes, such as phagocytosed red and white blood cells. In this study, an in vitro model of Gaucher disease was developed by treating the murine macrophage cell line J774 with a specific inhibitor of glucosylceramide beta-glucosidase, conduritol B-epoxide, and feeding red blood cell ghosts, in order to mimic the disease state. It was found in this model system that glucosylceramide beta-glucosidase activity could be reduced to about 11-15% of the normal control level before increased storage of glucosylceramide occurred. This in vitro system allows insight into the correlation between enzyme activity and lipid storage as predicted by the theory of residual enzyme activity that was proposed by Conzelmann and Sandhoff.

Toxicity of glucosylsphingosine (glucopsychosine) to cultured neuronal cells: a model system for assessing neuronal damage in Gaucher disease type 2 and 3.

Patients with Gaucher disease have been classified as type 1 nonneuronopathic, type 2 acute neuronopathic, and type 3 chronic neuronopathic phenotypes. Increased quantities of glucocerebroside and glucosylsphingosine (glucopsychosine) are present in the brain of type 2 and type 3 Gaucher patients. Galactosylsphingosine has previously been shown to be neurotoxic in globoid cell leukodystrophy (Krabbe disease). To determine whether glucosylsphingosine is also neurotoxic, we examined its effect on cultured cholinergic neuron-like LA-N-2 cells. When these cells were exposed to 1, 5, or 10 microM glucosylsphingosine for a period of 18 h, they became shriveled, neurite outgrowth was suppressed, and the activities of the lysosomal enzymes glucocerebrosidase, sphingomyelinase, and beta-galactosidase were reduced in a dose-dependent manner. Acetylcholine in cells exposed to glucosylsphingosine also declined. Cells switched to glucosylsphingosine-free medium partially recovered. The data suggest that accumulation of glucosylsphingosine contributes to neuronal dysfunction and destruction in patients with neuronopathic Gaucher disease.

Pergolide protects dopaminergic neurons in primary culture under stress conditions.

Dopamine agonists are an important therapeutic strategy in the treatment of Parkinson's disease. They postpone the necessity for and reduce the required dose of L-3,4-dihydroxyphenylalanine (L-DOPA) medication thus protecting against the development of motor complications and potential oxidative stress due to L-DOPA metabolism. In primary cultures from mouse mesencephalon we show that pergolide, a preferential D(2) agonist enhanced the survival of healthy dopaminergic neurons at low concentrations of 0.001 microM. About 100 fold higher concentrations (0.1 microM) were necessary to partially reverse the toxic effects of 10 microM 1-methyl-4-phenylpyridinium (MPP(+)). Pergolide was equally effective in preventing the reduction of dopamine uptake induced by 200 microM L-DOPA. Furthermore, between 0.001-0.1 microM it also reduced lactate production thus promoting aerobic metabolism. The present findings suggest that pergolide protects dopaminergic neurons under conditions of elevated oxidative stress.

Characterization of two alpha-galactosidase mutants (Q279E and R301Q) found in an atypical variant of Fabry disease.

The mutant products Q279E ((279)Gln to Glu) and R301Q ((301)Arg to Gln) of the X-chromosomal inherited alpha-galactosidase (EC 3.2.1. 22) gene, found in unrelated male patients with variant Fabry disease (late-onset cardiac form) were characterized. In contrast to patients with classic Fabry disease, who have no detectable alpha-galactosidase activity, atypical variants have residual enzyme activity. First, the properties of insect cell-derived recombinant enzymes were studied. The K(m) and V(max) values of Q279E, R301Q, and wild-type alpha-galactosidase toward an artificial substrate, 4-methylumbelliferyl-alpha-D-galactopyranoside, were almost the same. In order to mimic intralysosomal conditions, the degradation of the natural substrate, globotriaosylceramide, by the alpha-galactosidases was analyzed in a detergent-free-liposomal system, in the presence of sphingolipid activator protein B (SAP-B, saposin B). Kinetic analysis revealed that there was no difference in the degradative activity between the mutants and wild-type alpha-galactosidase activity toward the natural substrate. Then, immunotitration studies were carried out to determine the amounts of the mutant gene products naturally occurring in cells. Cultured lymphoblasts, L-57 (Q279E) and L-148 (R301Q), from patients with variant Fabry disease, and L-20 (wild-type) from a normal subject were used. The 50% precipitation doses were 7% (L-57) and 10% (L-148) of that for normal lymphoblast L-20, respectively. The residual alpha-galactosidase activity was 3 and 5% of the normal level in L-57 and L-148, respectively. The quantities of immuno cross-reacting materials roughly correlated with the residual alpha-galactosidase activities in lymphoblast cells from the patients. Compared to normal control cells, fibroblast cells from a patient with variant Fabry disease, Q279E mutation, secreted only small amounts of alpha-galactosidase activity even in the presence of 10 mM NH(4)Cl. It is concluded that Q279E and R301Q substitutions do not significantly affect the enzymatic activity, but the mutant protein levels are decreased presumably in the ER of the cells.

The glycosphingolipidoses-from disease to basic principles of metabolism.

The glycosphingolipidoses are a set of diseases that are caused by defects in the lysosomal degradation of glycolipids derived from the plasma membrane. By investigating the molecular bases of the diseases, basic principles of storage disease pathology and of membrane digestion were discovered. The generation of mouse models has facilitated the development of new and promising therapeutic strategies for these diseases, most of which are not treatable at present. Lately, the discovery of the importance of glycosphingolipid metabolism for skin development has opened a new and interesting field.

Induction of apoptosis by synthetic ceramide analogues in the human keratinocyte cell line HaCaT.

In contrast to extracellular, long chain ceramides which comprise a structural component of the epidermal water barrier, intracellular ceramides originating from sphingomyelin hydrolysis have been shown to inhibit proliferation and to induce apoptosis in different cell populations. To further elucidate the possible role of intracellular ceramides in human epidermis, two new cell-permeable ceramide analogues, N-thioacetylsphingosine (C2-Cer=S) and 4-dodecanoylamino-decan-5-ol (FS-5), were synthesized and tested for their ability to suppress cell growth and to induce apoptosis in immortalized human keratinocytes. It was shown that the well-investigated ceramide analogue N-acetylsphingosine (C2-Cer=O), as well as the new compound C2-Cer=S inhibited proliferation of HaCaT cells with half-inhibitory concentrations (IC50) of 20 microg/ml and 10 microg/ml, respectively, whereas FS-5 has been potent with an IC50>40 microg/ml. Overall, all three ceramide analogues induced apoptosis in HaCaT cells as assessed by DNA-fragmentation using ELISA technique and in situ nick end labelling, thereby confirming the importance of ceramide signalling in keratinocytes.

Processing of sphingolipid activator proteins and the topology of lysosomal digestion.

Plasma membrane derived glycosphingolipids (GSLs) destined for digestion are internalized through the endocytic pathway and delivered to the lysosomes. There, GSLs are degraded by the action of exohydrolases, which are supported, in the case of GSLs with short oligosaccharide chains, by sphingolipid activator proteins (SAPs). Four of the SAPs, SAP-A to -D (also called saposins) are synthesized from a single precursor protein (pSAP). Intracellular routing of pSAP and of the GM2 activator protein is only in part dependent on mannose-6-phosphate residues. Their endocytosis occurs in a carbohydrate-independent manner. The inherited deficiencies of individual activators, the GM2 activator, SAP-B, and SAP-C, as well as the deficiency of the precursor pSAP give rise to different, neuronal, white matter or visceral sphingolipid storage diseases. The analysis of cultured fibroblasts from corresponding patients suggests a new model for the topology of endocytosis and lysosomal digestion. It supports the hypothesis that endocytosis of plasma membrane-derived lipids occurs via small intraendosomal and intralysosomal vesicles and membrane structures, that are then digested within the lysosomes. In combined activator protein deficient cells nondegradable GSLs on the surface of intralysosomal vesicles protect them against lysosomal digestion. Mice with disrupted genes for activator proteins (SAP precursor -/-, GM2A -/-) as well as disrupted genes for ganglioside GM2 degrading hexosaminidases (HEXA -/-, HEXB -/-) turned out to be useful models for known human diseases whereas double knock out mice (HEXA -/- and HEXB -/-) show a new phenotype of both mucopolysaccharidosis and gangliosidosis.

Glycosphingolipid degradation and animal models of GM2-gangliosidoses.

Glycosphingolipids form cell type-specific patterns on the surface of eukaryotic cells. Degradation of glycosphingolipids requires endocytic membrane flow of plasma membrane-derived glycosphingolipids into the lysosomes as the digesting organelles. The inherited deficiencies of lysosomal hydrolases and of sphingolipid activator proteins both give rise to sphingolipid storage diseases. Recent research has focused on the mechanisms leading to selective membrane degradation in the lysosomes and on the mechanism and physiological function of sphingolipid activator proteins. The GM2-degrading system is a paradigm for activator protein-dependent lysosomal degradation. Three polypeptide chains contribute to the in vivo degradation of ganglioside GM2: the alpha- and beta-chains of the beta-hexosaminidases and the GM2 activator. Mouse models of Tay-Sachs disease (alpha-chain deficiency), Sandhoff disease (beta-chain deficiency) and GM2 activator deficiency have been described. While the phenotypes of these variants of GM2-gangliosidoses are only slightly different in humans, the animal models show drastic differences in severity and course of the diseases. The reason for this is the specificity of sialidase, which is different between mouse and human. A double-knockout mouse lacking beta-hexosaminidases A, B and S shows a phenotype of mucopolysaccharidosis and gangliosidosis. A substrate deprivation approach to therapy is discussed with respect to animal models of the GM2-gangliosidoses.

Sphingolipid metabolism. Sphingoid analogs, sphingolipid activator proteins, and the pathology of the cell.

Sphingolipid metabolism and function was investigated using sphingoid analogs, cells from human sphingolipidoses patients, and knockout animals. Treatment of primary cultured murine cerebellar cells with the structurally modified sphingosine base cis-4 methylsphingosine resulted in decreased sphingolipid biosynthesis accompanied by significant morphological changes. Plasma-membrane-derived glycosphingolipids (GSLs) destined for digestion are internalized through the endocytic pathway and delivered to lysosomes. There, GSLs are degraded by the action of exohydrolases, which are supported, in the case of GSLs with short oligosaccharide chains, by sphingolipid activator proteins (SAPs or saposins). The inherited deficiency of activators give rise to sphingolipid storage diseases. The analysis of cultured fibroblasts from corresponding patients suggests a new model for the topology of endocytosis and lysosomal digestion. Mice with disrupted genes for activator proteins and for GM2 degrading hexosaminidases turned out to be useful models for human diseases.

Recent advances in the biochemistry of sphingolipidoses.

Glycosphingolipids are ubiquitous membrane components of eukaryotic cells. They participate in various cell recognition events and can regulate enzymes and receptors within the plasma membrane. Sphingolipidoses are due to an impaired lysosomal digestion of these substances. Glycosphingolipids are degraded by the action of exohydrolases, which are supported, in the case of glycosphingolipids with short oligosaccharide chains, by sphingolipid activator proteins. Five sphingolipid activator proteins are known so far, the GM2-activator and the SAPs, SAP-A to D (also called saposins). Degradation of glycosphingolipids requires endocytic membrane flow of plasma membrane derived glycosphingolipids into the lysosomes. Recent research focused on the topology of this process and on the mechanism and physiological function of sphingolipid activator proteins. Limited knowledge is available about enzymology and topology of glycosphingolipid biosynthesis. Recently, intermediates of this metabolic pathway have been identified as novel signalling molecules. Inhibition of glycosphingolipid biosynthesis has been shown to be beneficial in the animal model of Tay-Sachs disease. Mice with disrupted genes for lysosomal hydrolases and activator proteins are useful models for known human diseases and are valuable tools for the study of glycosphingolipid metabolism, the pathogenesis of sphingolipidoses and novel therapeutic approaches.

Molecular mechanisms of contraction-induced translocation of GLUT4 in isolated cardiomyocytes.

Isolated adult rat ventricular cardiomyocytes were used to investigate the effects of contractile activity on 3-O-methylglucose transport on the translocation of the insulin-responsive glucose transporter GLUT4, and the possible activation of intermediates of the insulin signaling cascade. When elicited by field stimulation, contraction at 1 Hz did not significantly affect the adenosine triphosphate (ATP) content of cardiac cells, even after 60 min. At 5 Hz, a stable ATP level was observed until 15 minutes with a rapid decline at later time points. Stimulation of cardiomyocytes at 5 Hz for 5 minutes induced a 2-3 fold increase of 3-O-methylglucose transport with no additional stimulation in the presence of insulin (10[-7] M). Subcellular fractionation and immunoblotting analysis of GLUT4 distribution indicated that both contraction and insulin induced an identical increase (8-9-fold) of GLUT4 in the plasma membrane with a concomitant decrease (one third) in the microsomal fraction. Treatment of cardiomyocytes with wortmannin produced a complete inhibition of insulin- and contraction-induced glucose uptake. However, immunoprecipitation of insulin receptor substrate-1 (IRS-1) showed that the p85 regulatory subunit of phosphatidylinositol-3 kinase did not associate with IRS-1 upon contraction but with a marked stimulated association in response to insulin. These data suggest the existence of identical insulin- and contraction-recruitable GLUT4 pool. Contraction-induced signaling may use a limited part of the insulin-signaling cascade, possibly involving IRS-2. We further suggest that insulin resistance at the level of IRS-1 will not affect contraction-regulated glucose uptake by the heart.

Bcl-2 antagonizes apoptotic cell death induced by two new ceramide analogues.

Ceramides which arise in part from the breakdown of sphingomyelin comprise a class of antiproliferative lipids and have been implicated in the regulation of programmed cell death better known as apoptosis. In the present study, two new synthetic ceramide analogues, N-thioacetylsphingosine and FS-5, were used in Molt 4 cells to induce cell death. Besides their cytotoxic effects at concentrations > or = 14 microM the data obtained clearly show that both analogues induced apoptosis at concentrations below this critical concentration as assessed by trypan blue exclusion and cleavage of the death substrate poly-(ADP-ribose) polymerase (PARP). Additional experiments in bcl-2-transfected Molt 4 cells revealed that the apoptotic but not the lytic effects of the analogues were antagonized by the apoptosis inhibitor Bcl-2. Furthermore, neither N-thio-acetylsphingosine nor FS-5 induced PARP cleavage in bcl-2-transfected Molt 4 cells indicating that the induction of apoptotic cell death by cell permeable ceramides is not due to unspecific disturbance of the cell membrane.

Molecular analysis of insulin resistance in isolated ventricular cardiomyocytes of obese Zucker rats.

Isolated ventricular cardiomyocytes obtained from lean and genetically (fa/fa) obese Zucker rats were used to correlate alterations of insulin-induced glucose transport activation and GLUT-4 translocation to possible defects of the insulin signaling cascade. Maximal stimulation with insulin was found to produce an unaltered translocation of GLUT-4 to the plasma membrane (4.2- and 3.7-fold increase for lean and obese rats, respectively). However, a largely reduced sensitivity of 3-O-methylglucose transport could be detected in obese rats at physiological doses of insulin (completely unresponsive at 8 x 10(-11) M compared with 3-fold stimulation of glucose transport in lean controls). Tyrosine phosphorylation of the insulin receptor beta-subunit and the insulin receptor substrate 1 (IRS-1) was stimulated identically in cardiomyocytes from both lean and obese rats. Labeling of cells with [33P]orthophosphate revealed a marked increase in the serine and/or threonine phosphorylation of IRS-1 in the obese group (370% of lean controls), with a concomitant reduction in IRS-1 abundance (30-40%). The reduced sensitivity of glucose transport at 8 x 10(-11) M insulin was then found to correlate to a completely blunted response of IRS-1-associated phosphatidylinositol 3-kinase activity in cardiomyocytes from obese rats. Those data show that cardiac insulin resistance of obesity involves defective insulin signaling at low concentrations of the hormone, whereas GLUT-4 translocation is fully operative in the isolated cell. It is suggested that hyperphosphorylation of IRS-1 may significantly contribute to the pathogenesis of insulin resistance in the heart.