Glucosylceramides are required for mycelial growth and full virulence in Penicillium digitatum.

Glucosylceramides (GlcCers) are important lipid components of the membrane systems of eukaryotes. Recent studies have suggested the roles for GlcCers in regulating fungal growth and pathogenesis. In this study, we report the identification and functional characterization of PdGcs1, a gene encoding GlcCer synthase (GCS) essential for the biosynthesis of GlcCers, in Penicilliumdigitatum genome. We demonstrated that the deletion of PdGcs1 in P. digitatum resulted in the complete loss of production of GlcCer (d18:1/18:0 h) and GlcCer (d18:2/18:0 h), a decrease in vegetation growth and sporulation, and a delay in spore germination. The virulence of the PdGcs1 deletion mutant on citrus fruits was also impaired, as evidenced by the delayed occurrence of water soaking lesion and the formation of smaller size of lesion. These results suggest that PdGcs1 is a bona fide GCS that plays an important role in regulating cell growth, differentiation, and virulence of P. digitatum by controlling the biosynthesis of GlcCers.

Ceramides and glucosylceramides are independent antagonists of insulin signaling.

Inhibitors of sphingolipid synthesis protect mice from diet induced-insulin resistance, and sphingolipids such as ceramides and glucosylated-ceramides (e.g., GM3) are putative nutritional intermediates linking obesity to diabetes risk. Herein we investigated the role of each of these sphingolipids in muscle and adipose tissue and conclude that they are independent and separable antagonists of insulin signaling. Of particular note, ceramides antagonize insulin signaling in both myotubes and adipocytes, whereas glucosyceramides are only efficacious in adipocytes: 1) In myotubes exposed to saturated fats, inhibitors of enzymes required for ceramide synthesis enhance insulin signaling, but those targeting glucosylceramide synthase have no effect. 2) Exogenous ceramides antagonize insulin signaling in myotubes, whereas ganglioside precursors do not. 3) Overexpression of glucosylceramide synthase in myotubes induces glucosylceramide but enhances insulin signaling. In contrast, glucosylated ceramides have profound effects in adipocytes. For example, either ganglioside addition or human glucosylceramide synthase overexpression suppresses insulin signaling in adipocytes. These data have important mechanistic implications for understanding how these sphingolipids contribute to energy sensing and the disruption of anabolism under conditions of nutrient oversupply.

Glucosylceramide modulates endolysosomal pH in Gaucher disease.

GlcCer accumulation causes Gaucher disease where GlcCer breakdown is inhibited due to a hereditary deficiency in glucocerebrosidase. Glycolipids are endocytosed and targeted to the Golgi apparatus in normal cells but in Gaucher disease they are mistargeted to lysosomes. To better understand the role of GlcCer in endocytic sorting RAW macrophages were treated with Conduritol B-epoxide to inhibit GlcCer breakdown. Lipid analysis found increases in GlcCer led to accumulation of both triacylglycerol and cholesterol consistent with increased lysosomal pH. Ratio imaging of macrophages using both acridine orange and lysosensor yellow/blue to measure endolysosomal pH revealed increases in Conduritol B-epoxide treated RAW macrophages and Gaucher patient lymphoblasts. Increased endolysosomal pH was restricted to Gaucher lymphoblasts as no significant increases in pH were seen in Fabry, Krabbe, Tay-Sachs and GM1-gangliosidosis lymphoblasts. Substrate reduction therapy utilises inhibitors of GlcCer synthase to reduce storage in Gaucher disease. The addition of inhibitors of GlcCer synthesis to RAW macrophages also led to increases in cholesterol and triacylglycerol and an endolysosomal pH increase of up to 1 pH unit. GlcCer modulation appears specific since glucosylsphingosine but not galactosylsphingosine reversed the effects of GlcCer depletion. Although no acute effects on glycolipid trafficking were observed using bafilomycin A the results are consistent with a multistep model whereby increases in pH lead to altered trafficking via cholesterol accumulation. GlcCer modulates endolysosomal pH in lymphocytes suggesting an important role in normal lysosomes which may be disrupted in Gaucher disease.

Glucocerebroside: an evolutionary advantage for patients with Gaucher disease and a new immunomodulatory agent.

Gaucher disease (GD) is caused by the reduced activity of a lysosomal enzyme, glucocerebrosidase, leading to the accumulation of glucocerebroside (GC). The relatively high prevalence of this disease within an ethnic group is believed to reflect a selective advantage. Treatment with enzyme replacement therapy (ERT) is safe and effective in ameliorating the primary symptoms of the disease, yet there have been reports that some patients on ERT have developed type 2 diabetes or metabolic syndrome, malignancies and central nervous system disorders. A series of animal studies suggest that these complications may be related to the reduction of GC levels by the enzyme administered. GC has been shown to have an immunomodulatory effect through the promotion of dendritic cells, natural killer T cells, and regulatory T cells. The break down of GC to ceramide can underline part of these findings. Clinical trials suggested a beneficial effect of GC in type 2 diabetes or nonalcoholic steatohepatitis. This review of the data from animal models and humans proposes that the increased level of GC may provide an evolutionary advantage for patients with GD. Indirectly, these data support treating symptomatic patients with mild/moderate GD with low-dose ERT and re-evaluating the use of ERT in asymptomatic patients.

Functions of sphingolipid metabolism in mammals--lessons from genetic defects.

Much is known about the pathways that control the biosynthesis, transport and degradation of sphingolipids. During the last two decades, considerable progress has been made regarding the roles this complex group of lipids play in maintaining membrane integrity and modulating responses to numerous signals. Further novel insights have been provided by the analysis of newly discovered genetic diseases in humans as well as in animal models harboring mutations in the genes whose products control sphingolipid metabolism and action. Through the description of the phenotypic consequences of genetic defects resulting in the loss of activity of the many proteins that synthesize, transport, bind, or degrade sphingolipids, this review summarizes the (patho)physiological functions of these lipids.

MRP1 and glucosylceramide are coordinately over expressed and enriched in rafts during multidrug resistance acquisition in colon cancer cells.

Previously we have described a novel multidrug-resistant cell line, HT29(col), which displayed over expression of the multidrug-resistance protein 1 (MRP1) and an altered sphingolipid composition, including enhanced levels of glucosylceramide (GlcCer; Kok JW, Veldman RJ, Klappe K, Koning H, Filipeanu C, Muller M. Int J Cancer 2000;87:172-8). In our study, long-term screening revealed that, during colchicine-induced acquisition of multidrug resistance in a new HT29(col) cell line, increases in GlcCer occurred concomitantly with upregulation of MRP1 expression. Both MRP1 and GlcCer were found enriched in Lubrol-insoluble membrane domains. The expression of MRP1 and GlcCer were tightly correlated, as indicated also by a reversal of both at the later stage of colchicine consolidation. Resistance to colchicine was determined by MRP1, while glucosylceramide synthase (GCS) did not contribute: 1). Resistance was fully inhibited by MK571. 2). GCS expression and activity were not upregulated in HT29(col) cells. 3). Inhibition of GCS did not affect MRP1-mediated efflux function or sensitivity to colchicine. Instead, overall sphingolipid metabolism was upregulated through an increased rate of ceramide biosynthesis. In conclusion, upregulation of MRP1 occurs in concert with upregulation of GlcCer during multidrug-resistance acquisition, and both are enriched in rafts. The increased GlcCer pool does not directly modulate MRP1 function and cell survival.

Glucosylceramides in Colletotrichum gloeosporioides are involved in the differentiation of conidia into mycelial cells.

Glucosylceramides (GlcCer) were extracted from the plant pathogen Colletotrichum gloeosporioides and purified by several chromatographic steps. By using electrospray ionization mass spectrometry and nuclear magnetic resonance, GlcCer from C. gloeosporioides were identified as N-2'-hydroxyoctadecanoyl-1-beta-D-glucopyranosyl-9-methyl-4,8-sphingadienine and N-2'-hydroxyoctadecenoyl-1-beta-D-glucopyranosyl-9-methyl-4,8-sphingadienine. Monoclonal antibodies against these structures were produced and used as tools for the evaluation of the role of GlcCer in the morphological transition of C. gloeosporioides. In the presence of antibodies to GlcCer, the differentiation of conidia into mycelia was blocked. Since GlcCer is present in several plant pathogens, the inhibitory activity of external ligands recognizing these structures may be applicable in other models of fungal infections.

Recently discovered functions of glucosylceramides in plants and fungi.

Glycosphingolipids are ubiquitous membrane lipids of eukaryotic organisms and a few bacteria. Whereas inositol-containing glycosphingolipids are restricted to plants and fungi, galactosylceramide occurs only in fungi and animals. In contrast, glucosylceramide is the unique glycosphingolipid which plants, fungi and animals have in common. However, there are specific differences in the structure of the ceramide backbone of glucosylceramides from these organisms. A comparison of the structural features and the biosynthesis of glucosylceramides from plants, fungi and animals will contribute to our understanding of their functions, which so far have been analysed mainly in animals. The availability of nearly all genes involved in the biosynthesis of glucosylceramides enables the specific manipulation of glycosphingolipid metabolism by techniques of forward and reverse genetics. Application of this approach to unicellular organisms like yeasts, multicellular filamentous fungi, as well as to complex organisms like plants will reveal common and different glucosylceramide functions in these organisms. These glycolipids play a role both in intracellular processes and in cell-to-cell interactions. These interactions may occur between cells of a multicellular organism or between cells of different species, as in host-pathogen interactions.

The fate and function of glycosphingolipid glucosylceramide.

In higher eukaryotes, glucosylceramide is the simplest member and precursor of a fascinating class of membrane lipids, the glycosphingolipids. These lipids display an astounding variation in their carbohydrate head groups, suggesting that glycosphingolipids serve specialized functions in recognition processes. It is now realized that they are organized in signalling domains on the cell surface. They are of vital importance as, in their absence, embryonal development is inhibited at an early stage. Remarkably, individual cells can live without glycolipids, perhaps because their survival does not depend on glycosphingolipid-mediated signalling mechanisms. Still, these cells suffer from defects in intracellular membrane transport. Various membrane proteins do not reach their intracellular destination, and, indeed, some intracellular organelles do not properly differentiate to their mature stage. The fact that glycosphingolipids are required for cellular differentiation suggests that there are human diseases resulting from defects in glycosphingolipid synthesis. In addition, the same cellular differentiation processes may be affected by defects in the degradation of glycosphingolipids. At the cellular level, the pathology of glycosphingolipid storage diseases is not completely understood. Cell biological studies on the intracellular fate and function of glycosphingolipids may open new ways to understand and defeat not only lipid storage diseases, but perhaps other diseases that have not been connected to glycosphingolipids so far.

The roles of ceramide and complex sphingolipids in neuronal cell function.

The roles of sphingolipids, and particularly of the complex glycosphingolipids (GSLs), the gangliosides, have been studied for many years in neurons, glia, and cell lines derived from these tissues, due to their abundance in tissues of neuronal origin. More recently, significant attention has been paid to the simple sphingolipids, particularly ceramide, glucosylceramide (GlcCer), and sphingosine-1-phosphate (S1P), each of which appears to be involved in the regulation of specific aspects of neuronal proliferation, differentiation, survival and apoptosis. In this review, we will summarize studies performed in our laboratory over the past few years using cultured hippocampal neurons in an attempt to define the precise roles of these lipids, and to define their mechanisms of action by identifying down-stream targets with which they interact. We will also discuss work suggesting that complex GSLs, such as gangliosides GM2 and GD3, can also regulate neuronal development, although the down-stream targets with which they interact are less well defined. Our work will be reviewed in light of studies from other laboratories, with particular emphasis on the use of models of sphingolipid storage diseases to determine how these lipids affect neuronal function.

Characterization of glucosylceramides in Pseudallescheria boydii and their involvement in fungal differentiation.

Pseudallescheria boydii is a fungal pathogen that causes disease in immunocompromised patients. Ceramide monohexosides (CMHs) were purified from lipidic extracts of this fungus, showing that, as described for several other species, P. boydii synthesizes glucosylceramides as major neutral glycosphingolipids. CMHs from P. boydii were analyzed by high-performance thin-layer chromatography, gas chromatography coupled to mass spectrometry, fast atom bombardment-mass spectrometry, and nuclear magnetic resonance. These combination of techniques allowed the identification of CMHs from P. boydii as molecules containing a glucose residue attached to 9-methyl-4,8-sphingadienine in amidic linkage to 2-hydroxyoctadecanoic or 2-hydroxyhexadecanoic acids. Antibodies from a rabbit infected with P. boydii recognized CMHs from this fungus. Antibodies to CMH were purified from serum and used in indirect immunofluorescence, which revealed that CMHs are detectable on the surface of mycelial and pseudohyphal but not conidial forms of P. boydii, suggesting a differential expression of glucosylceramides according with morphological phase. We also investigated the influence of antibodies to CMH on growth and germ tube formation in P. boydii. Cultures that were supplemented with these antibodies failed to form mycelium, but the latter was not affected once formed. Similar experiments were performed to evaluate whether antibodies to CMH would influence germ tube formation in Candida albicans, a fungal pathogen that synthesizes glucosylceramide and uses differentiation as a virulence factor. Addition of antiglucosylceramide antibodies to cultures of C. albicans clearly inhibited the generation of germ tubes. These results indicated that fungal CMHs might be involved in the differentiation and, consequently, play a role on the infectivity of fungal cells.

Glucosylceramide synthase and apoptosis.

Glucosylceramide synthase (GCS) is an enzyme inherent to ceramide metabolism. The enzyme catalyzes the transfer of glucose to ceramide, the first committed step in glycolipid biosynthesis. Known for many years as a branch point enzyme directing synthesis of cerebrosides and gangliosides, GCS has recently been implicated in the cytotoxic response of cancer cells to chemotherapy. With ceramide now occupying a central role in the signaling mechanisms of apoptosis, the position of GCS as sentry is perhaps not unexpected. In particular, it has been recognized that the toxic response of cells to chemotherapy is impaired when GCS activity is elevated and heightened when GCS activity is blocked. Herein we review the control points of ceramide metabolism with special regard to GCS and the cytotoxic response.

Plasma glucosylceramide deficiency as potential risk factor for venous thrombosis and modulator of anticoagulant protein C pathway.

To assess the relationship between venous thrombosis and plasma glucosylceramide (GlcCer) or phosphatidylethanolamine (PE), plasma levels of GlcCer and PE were determined for 70 venous thrombosis patients referred for evaluation and 70 healthy blood donors. The mean GlcCer level, but not the PE level, was lower in patients versus controls (4.9 vs 6.5 microg/mL [P =.0007] and 66 vs 71 microg/mL [P =.48], respectively). As a measure of relative risk, the odds ratio for deep vein thrombosis in subjects with GlcCer levels below the 10th percentile of controls was 5.7 (95% CI, 2.3-14). To assess the influence of glycolipids on anticoagulant response to activated protein C (APC):protein S in modified prothrombin time assays, the effects of depleting endogenous plasma GlcCer by glucocerebrosidase treatment or of adding exogenous purified GlcCer or other neutral glycolipids to plasma were tested. Glucocerebrosidase treatment reduced plasma sensitivity to APC:protein S in parallel with GlcCer reduction. Exogenously added GlcCer and the homologous Glc-containing globotriaosylceramide (Gb3Cer), but not galactosylceramide, dose-dependently prolonged clotting times of normal plasma in the presence, but not absence, of APC:protein S, which suggests that GlcCer or Gb3Cer can enhance protein C pathway anticoagulant activity. In studies using purified proteins, inactivation of factor Va by APC:protein S was enhanced by GlcCer alone and by GlcCer in multicomponent vesicles containing phosphatidylserine and phosphatidylcholine. These results suggest that the neutral glycolipids GlcCer and Gb3Cer may directly contribute to the anticoagulant activity of the protein C pathway and that deficiency of plasma GlcCer may be a risk factor for venous thrombosis. (Blood. 2001;97:1907-1914)

Modulation of carcinoembryonic antigen release by glucosylceramide--implications for HT29 cell differentiation.

Previous work suggested that glucosylceramide (GlcCer) plays a role in the regulation of cell differentiation of HT29 human colon tumor cells. In the present study, we investigated the role of GlcCer in the cellular release of carcinoembryonic antigen (CEA), a marker for cell differentiation. This was done by modulating the intracellular level of the glycolipid, according to two different approaches. The cells were treated with D,L-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP), which resulted in a specific lowering of the cellular GlcCer pool. Alternatively, by exogenous addition of a short-chain analog of the lipid, hexanoyl(C6)-GlcCer, the cellular pool was enhanced. The results demonstrate that PDMP causes an increase in the release of CEA, while exogenous C6-GlcCer suppresses its release. Furthermore, the enhanced release of CEA in the presence of PDMP, could be completely reversed upon exogenous addition of C6-GlcCer. Control experiments reveal that a potential interference of the well-known modulator of cell physiology, ceramide (Cer), can be excluded. Long-term depletion of GlcCer resulted in a change in a morphological feature of differentiation of the cells, i.e. an increase in apical membrane surface with microvilli brush borders, accompanied by an enhanced expression of the cytoskeletal protein villin. These results, together with the observations on modulation of the differentiation marker CEA by GlcCer, provide support for the conclusion that GlcCer interferes with the differentiation of HT29 cells.

Distinct roles for ceramide and glucosylceramide at different stages of neuronal growth.

Sphingolipids (SLs) are important structural and regulatory components of neuronal plasma membranes. Previous studies using fumonisin B1, an inhibitor of the synthesis of ceramide, the precursor of all SLs, demonstrated that ceramide synthesis is required to sustain axonal growth in hippocampal neurons (; ) and dendritic growth in cerebellar Purkinje cells (). We now show that ceramide plays distinct roles at different stages of neuronal development. (1) During axon growth, ceramide must be metabolized to glucosylceramide (GlcCer) to sustain growth. Thus, whereas D-erythro-ceramide, which is metabolized to GlcCer, is able to antagonize the disruptive effects of fumonisin B1 on axon growth, L-threo-ceramide, which is not metabolized to GlcCer, is ineffective. (2) The formation of minor processes from lamellipodia can be stimulated by incubation with short-acyl chain analogs of ceramide that are active in ceramide-mediated signaling pathways, or by generation of endogenous ceramide by incubation with sphingomyelinase. However, GlcCer synthesis is not required for this initial stage of neuronal development. (3) During minor process formation and during axon growth, incubation with high concentrations of ceramide or sphingomyelinase, but not dihydroceramide, induces apoptosis. Together, these observations are consistent with the possibility that minor process formation and apoptosis can be regulated by ceramide-dependent signaling pathways and that the decision whether to enter these diametrically opposed pathways depends on intracellular ceramide concentrations. In contrast, axonal growth requires the synthesis of GlcCer from ceramide, perhaps to support an intracellular transport pathway.

Modulation of renal epithelial cell growth by glucosylceramide. Association with protein kinase C, sphingosine, and diacylglycerol.

Two independent approaches were employed to explore the potential role of endogenous glucosylceramide or a closely related glucosphingolipid in mediating the cellular proliferation of Madin-Darby canine kidney cells. First, cultured cells were depleted of glucosphingolipids by exposure to a glucosylceramide synthase inhibitor, D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol. This agent markedly inhibited cell growth and DNA synthesis in a time- and concentration-dependent manner. Second, cells were grown in the presence of conduritol B epoxide, an inhibitor of glucosylceramide beta-D-glucosidase. Exposure of cells to this inhibitor resulted in the time-dependent accumulation of glucosylceramide with a corresponding increase in cellular proliferation. Alterations in protein kinase C activity were evaluated as a potential mechanism for these effects on growth. Both membrane- and cytosol-associated protein kinase C (PKC) activity declined under conditions of glucosylceramide synthase inhibition and increased under conditions of beta-glucosidase inhibition. The changes in PKC activity were evident after DEAE-cellulose purification. Diacylglycerol levels increased in response to both glucosylceramide synthase and beta-glucosidase inhibition. Ceramide and sphingosine levels changed only in the presence of D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol, increasing due to lack of conversion to glucosylceramide. However, the elevation in endogenous sphingosine was probably insufficient to account for the decrease in PKC, considering the high level of diacylglycerol in the cells. These data demonstrate an association between glucosylceramide levels, PKC activity, and cell growth.

Stratum corneum lipid function.

The stratum corneum contains a complex mixture of polar and nonpolar lipids in its intercellular spaces. These lipids, present in form of multiple lamellae, have been investigated for their role in providing the epidermal barrier to transcutaneous water loss, the selective barrier from the inside to the outside of the organism and partly the process of physiological desquamation. The composition of these lipids varies from species to species, with the body region and the degree of keratinocyte differentiation. The most undifferentiated layers of the epidermis contain typical membrane lipids, phospholipids, while more differentiated layers contain ceramides, cholesterol and free fatty acids. Essential fatty acids are essential for the maintenance of the lamellar structures and epidermal barrier function. Epidermal linoleic and arachidonic acids derive from exogenous sources. Only recently attempts have been made to elucidate the timing and regulation of epidermal fatty acid metabolism. Keratinocytes do not express a low molecular weight fatty acid binding protein like other cells active in lipid metabolism, but may employ alternative ways in fatty acid uptake and metabolism.