Glucosylceramide has a unique and often ambiguous role in mammalian cells. Activation of glucosylceramide synthase, the enzyme that places a glucosyl moiety onto ceramide, is the first pathway-committed step to the production of more complex glycosphingolipids such as lactosylceramide and gangliosides. Alterations in the level of glucosylceramide are noted in cells and tissues in response to cardiovascular disease, diabetes, skin disorders and cancer. Overall, upregulation of glucosylceramide offers cellular protection and primes certain cells for proliferation. However, prolonged overabundance of glucosylceramide is detrimental, as seen in Gaucher disease in humans.
Glucosylceramide (GlcCer) is common to plants, fungi and animals and it is essential for viability of multicellular organisms. Cells in culture can survive a deletion of glucosylceramide synthase (GCS), the gene responsible for the glucosylation ofceramide, whereas embryos cannot.1 GlcCer is the precursor of lactosylceramide (LacCer) and the rest of the neutral and acidic oligo glycosphingolipids. LacCer and several gangliosides have also been shown to function as signaling molecules, however this topic will focus only on the properties of GlcCer and its regulation.
Glucosylceramide Synthesis and Degradation
Ceramide made in the endoplasmic reticulum (ER) is transported by ceramide transport protein CERT to the cytosolic side of the trans-Golgi where it is converted to GlcCer by GCS2 (Fig. 1). GlcCer must cross into the lumen of the Golgi in order to be converted to LacCer and higher order glycosphingolipids. It has been shown that short-chain GlcCer can be pumped into the Golgi by ABC transporters, however it is unclear whether natural GlcCer flips into the Golgi lumen, is pumped into the Golgi by ABC transport proteins, or is shuttled back to the ER by the transport protein FAPP2.3 GlcCer shuttled to the ER has been shown to be transported or flipped into the ER lumen where it enters the trans-Golgi network to undergo transformation to LacCer and GM3.4 GlcCer from the cytosolic leaf of the Golgi may also be pumped or flipped into the lumen.5 The GlcCer that makes it into the Golgi lumen is converted to LacCer by lactosylceramide synthase (LCS).6 Most of the GlcCer that is synthesized is not converted to LacCer, suggesting that GCS and LCS are not functionally coupled. There appears to be a distinct pool of GlcCer that is used for LacCer and downstream oligo glycosphingolipid synthesis, which raises the possibility that the cell utilizes GlcCer for different functions.7,8
The glucose moiety on GlcCer is removed by lysosomal GlcCer-P-glucosidase and by the ER-localized saposin C enzyme to yield ceramide. Deficiencies in these enzymes in humans lead to Gaucher disease. In Gaucher disease, cells cannot degrade GlcCer efficiently and the excess is stored throughout the cell,9 leading to overall increased GlcCer levels. The most pronounced effect occurs in phagocytotic macrophages that participate in ingesting blood cells and apoptotic lymphocytes. These macrophages become engorged with GlcCer and other lipids and release cytokines due to chronic activation.10 Similar to many other diseases, there are differences in severity and clinical manifestations.
Figure 1. Synthesis of glucosylceramide. Ceramide transported from the ER by CERT to the trans-Golgi is glycosylated on the cytosolic leaf by glucosylceramide synthase (GCS). Most of the glucosylceramide (GlcCer) is shuttled back to the endoplasmic reticulum (ER) by transport protein FAPP2 where it will re-enter the Golgi network on the lumenal side. Some GlcCer is transported to the plasma membrane (PM) or endosomes. Alternatively, GlcCer is either flipped or pumped into the Golgi lumen to undergo further modification to lactosylceramide (LacCer) by lactosylceramide synthase (LCS).
Regulation of the rate-limiting enzyme of glycosphingolipid synthesis, GCS, is not well-understood. The protein c-Fos is an established transcription factor, but has also been shown to bind to the ER and activate the biosynthesis of phospholipids.11 Recently, c-Fos was shown to activate GCS, but does not activate GlcCer galactosyltransferase 1 and LacCer sialyltransferase 1. Additionally, c-Fos co-immunoprecipitated with GCS in neuronal PC12 cells.12 GCS transcription may also be regulated in part through Sp1.13,14 Overexpression of a reticulon family member, RTN-1C, sensitized neuroepithelial cells to fenretinide, a synthetic retinoid derivative that induces apoptosis. Di Sano et al15 also showed that RTN-1C interacts with GCS and modulates its activity. Their results indicate that GCS/RTN-1C interaction may mediate signals between Golgi and ER compartments, including the cellular response to apoptotic stimuli. This may occur because RTN-1C also interacts with the proapoptotic protein Bcl-xL.16 GCS may also be regulated by PI3K in natural killer cells.17 The GCS antagonist D-threo-1-phenyl-2-palmitoylamino-3-pyrrolidino-1-propanol (P4),18 used to decrease GlcCer levels, induced tyrosine phosphorylation of PLCy and increased inositol 1,4,5 trisphosphate (a PLCy product).17 In support of this, phosphorylation of PLCy was abolished by the exogenous addition of GlcCer.19
Figure 2. Glucosylceramide is the major precursor of complex glycosphingolipids. Glucosylceramide (GlcCer) is the backbone upon which hundreds of neutral and acidic oli-goglycosphingolipids are formed. Lactosylceramide is Gal|1-4 Gluc|1-1 Ceramide.
Multiple Functions of Glucosylceramide
Templatefor Higher Order Glycosphingolipids
One of the most important functions of GlcCer is to serve as a template for production of LacCer and complex glycosphingolipids (Fig. 2). Many ofthe higher order oligo glycosphingolipids have distinct signaling functions that are beyond the scope of this topic, but that are required for proper development in humans.
Membrane and Lipid Raft Constituent
GlcCer and other glycosphingolipids have higher affinity for cholesterol compared to the major membrane constituent phosphatidylcholine and they are known to induce formation of liquid-ordered phases.20 Recent analysis of detergent-resistant membrane fractions reveal that in addition to sphingmyelin and cholesterol, glycolipids are also present.21 Furthermore, GlcCer is elevated in the detergent-resistant membranes from Gaucher cells.21 It has been shown that GlcCer, unlike galactosylceramide, is located preferentially on the cytoplasmic surface of cell membranes and therefore likely utilized in lipid rafts at the ER and Golgi.4 Studies have implicated a role for GlcCer in vesicular and glycolipid sorting in the endocytic pathway via lipid rafts. GlcCer on the cytosolic surface of the Golgi appears to aid in sorting membrane proteins to melanosomes and it is believed to be essential for the activity of tyrosinase, a key enzyme in melanin biosynthesis.22 Glycosylphosphatidylinositol-anchored proteins prefer glycosphingolipid-enriched domains and recently it was demonstrated that PKA-RIIa docks to glycosphingolipids in HepG2 cells and affects apical targeting of P-glycoprotein (P-gp).23,24 Treatment with D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP),18 an inhibitor of GCS and LCS, delays the appearance of P-gp, suggesting that GlcCer synthesis is important for Golgi-to-apical transport of P-gp.24 However, conflicting data suggest that the role of glycosphingolipids in P-gp transport may be cell-specific.25 Although the fate of specific proteins may differ, there is a higher concentration of GlcCer in the apical domain of polarized cells such as intestine and kidney epithelial cells.26,27 The directed sorting of GlcCer in brain capillary endothelial cells, which also have continuous tight junctions and polar distributed proteins, suggests that GlcCer aids in the proper function of polarized cell types.28
Cellular Protection in the Skin
The outer layer of our skin is composed of a system of lamellar sheets of lipids in the intracellular space of the stratum corneum. GlcCer constitutes approximately 4% of the total epidermal lipid mass, but is one of the main components of lamellar bodies.29,30 Lamellar bodies are lipid-storage organelles that are exocytosed from keratinocytes. The skin uses this storage function to respond to the environment quickly by altering the level of ceramide, which is an essential lipid for maintaining proper water and electrolyte levels and for skin barrier repair.31 By developing an epidermis-specific GlcCer-|-glucosidase knock-out mouse, Jenneman et al32 showed that lack of GlcCer in the epidermis led to an irregular arrangement of the lamellar bodies and of extracellular lipids in the stratum corneum. In the absence of GlcCer-|-glucosidase, sphingomyelin became the default reservoir for ceramide. It was shown, however, certain subtypes of ceramides necessary to maintain barrier function were lacking. These data provide strong evidence that GlcCer is absolutely essential to provide the correct ceramides needed for barrier function.
Another function of GlcCer in skin is to signal proliferation in keratinocytes. In a study by Uchida et al,33 human keratinocytes treated with exogenous sphingomyelinase initially increased ceramide and, as a consequence, proliferation was stalled. By 24 h, cells began to proliferate parallel to an increase in GlcCer and GCS expression. Cells cotreated with exogenous GlcCer further augmented proliferation, while GCS inhibitor treatment enhanced the antiproliferative effects of sphingomyelinase. Additionally, HaCaT cells engineered to overexpress GCS were resistant to the effects ofsphingomyelinase.33 These data suggest that a balance between ceramide and GlcCer aids in cell differentiation.
Skin diseases like atopic dermatitis and psoriasis exhibit hyperproliferation, inflammation and impaired barrier function.34 The mRNA and protein expression of GlcCer-|-glucosidase was shown to be lower in nonlesional skin from patients with psoriatic epidermis compared to control skin, yet was higher in lesional compared with nonlesional skin from psoratic patients.35 These data suggest that flare-ups of certain skin conditions likely occur due to the misregulation of GlcCer. In a clinical study, patients with atopic eczema that ingested GlcCer saw a reduction of transepidermal water loss.36 These results were reiterated in a chronic skin perturbation mouse model that exhibited enhanced skin improvement following dietary supplementation of plant-based GlcCer.37 Although seemingly contradictory, these results are likely due to a systemic anti-inflammatory effect of GlcCer, especially plant-derived GlcCer (see section entitled "Cellular protection in carcinomas").
Cellular Protection in the Cardiovasculature
GlcCer may offer protective effects in the cardiovasculature by serving as an anticoagulant. An analysis of plasma from venous thrombosis patients showed lower GlcCer levels than in healthy blood donors.38 In the same study, plasma from healthy individuals treated with GlcCer |-glucosidase to decrease GlcCer, resulted in a reduction ofplasma sensitivity to activated protein C (APC)/protein S. Exogenously added GlcCer dose-dependently prolonged clotting times in the presence of APC/protein S.38 Furthermore, lipid vesicles containing GlcCer enhanced binding of APC and enhanced factor Va inactivation by protein S-dependent APC.38,39 These results suggest that GlcCer facilitates anticoagulation activity.
Analysis of the glycosphingolipid composition in artery layers from normal and atherosclerotic human aorta show striking differences. The intima layer, which interacts with blood, showed an accumulation ofGlcCer, LacCer and ganglioside GM2 in fatty streaks and atherosclerotic plaques, but not in normal intima.40 Oxidized LDL, which occurs during the progression of atherosclerosis, harbors GlcCer and complex oligo glycosphingolipids. However the oligo glycosphingolipids do not accumulate as GlcCer does, suggesting that GlcCer is synthesized in the artery wall.40 Heart attacks often occur following clot formation from the rupture of an unstable plaque and one of the body’s protective mechanisms is to induce proliferation of smooth muscle cells to form a cap over the plaque. In another study it was demonstrated that LacCer, but not GlcCer derived from plaque intima, exerted an increase in proliferation of cultured human aortic smooth muscle cells.41 Thus, the upregulation ofGlcCer and LacCer is possibly preventing the development of an unstable plaque by the proliferative effect of LacCer and by the anticoagulative effect of GlcCer.
Cellular Protection in the Brain
Cerebrospinal fluid from Alzheimer’s disease patients and in affected brain regions show decreased GCS protein and increased levels of ceramide.42,43 In cell culture experiments, cerebellar granule cells exposed to the GCS inhibitor P4 responded by increasing ceramide and subsequently, viability decreased.42 Experiments in rodents to induce stroke suggest that glycosphingolipids play a role in decreasing the severity of damage. Following middle cerebral artery occlusion in rats, brain ceramide increased with concomitant decrease in GCS activity.44 The stereoisomer of PDMP, L-threo-PDMP, has been used previously to activate GCS in neuronal cells.45 Following permanent occlusion of the left middle cerebral artery, resulting in chronic ischemia, intraperitoneal injection of L-threo-PDMP induced an increase in GlcCer in the ischemic cortex, without changes in sphingomyelin or ceramide. Importantly, the rats showed improved learning and memory compared to artery-occluded rats that did not receive the GCS activator.46 In the case of Alzheimer’s disease, it is unclear whether GlcCer or a downstream glycosphingolipid is the molecule affording protection against cell death. Although it is possible that GlcCer is the molecule protecting ischemic brain areas because it is an anticoagulant,38,39 more evidence would be needed to draw this conclusion.
Cellular Protection in the Immune System
The role GlcCer plays in the immune system is not well understood. Decreased GlcCer in macrophages and T-cells appears to shift the cell’s response from "attack" to "self-protect". Isolated peritoneal macrophages from mice treated with N-alkylnojirimycin,47 a GCS inhibitor, showed a decreased capacity to release TNFa or IL-6 in response to whole killed E. coli.48 Renal cell carcinoma cells induce apoptosis in T-cells via RelA degradation as shown in coculture experi-ments.49 Pretreatment with the GCS inhibitor P4 protected T-cells from tumor-induced RelA degradation and subsequent apoptosis.49 Systemic increase in GlcCer appeared to facilitate disease recovery. Diabetic and insulin-resistant rodent models given daily intraperitoneal injections of GlcCer or given inhibitors of GCS responded with increased insulin sensitivity and decreased hyperglycemia.50-52 In Ob/Ob mice, GlcCer injections also led to an increase in IL-10 and periph-eral/intrahepatic natural killer T-cell ratio and a corresponding decrease in serum INF-y.53 Natural killer T-cells are a subset of lymphocytes that lead to the release of either pro-inflammatory or anti-inflammatory chemokines when activated. In one study, mice induced to develop hepatic sclerosis were given intraperitoneal injections of GlcCer. In the experimental arm, GlcCer ameliorated liver damage, measured by decreased transaminase serum levels. Furthermore, there was a decrease in intrahepatic natural killer T and CD8 lymphocytes and a decrease in T(h)1 and T(h)2 cytokines.54 An increase in GlcCer may also prevent certain pathogens from binding to cells, which is the first step of invasion. It was demonstrated that bacteria isolated from patients with cystic fibrosis and pneumonia do not bind to GlcCer, LacCer and several gangliosides, but they do bind to asialo-GM1, asialo-GM2 and fucosylasialo-GM1.55
Ceramide is doubled in human skeletal muscle from patients with insulin-resistance, and patients with Gaucher disease often develop peripheral insulin resistance, possibly through the influence of glycosphingolipids on insulin receptor functioning.56,57 The insulin resistance that develops may occur following the conversion of GlcCer to higher order gangliosides. It has been shown that excess GlcCer is metabolized to neutral glycosphingolipids; and gangliosides such as GM3 were shown to participate in the pathogenesis of insulin resistance.58,59
Cellular Protection in Carcinomas
Cancer cells are more sensitive to the killing effects of increased ceramide. Accordingly, many chemotherapeutic drugs have been developed that work, in part, by increasing ceramide. Some cancer cells become multidrug resistant by increasing the metabolism of ceramide into GlcCer.60 Several studies have shown that decreasing GCS expression using GCS inhibitors in multidrug resistant carcinoma cells enhances the effects of chemotherapeutic drugs.61,62 Blocking GCS in multidrug resistant cancer cells (NCI/ADR-RES) by siRNA also reversed drug resistance, shown by enhanced drug uptake and increased ceramide-induced apoptosis.63,64 Not surprising, the reverse is seen following overexpression of GCS; drug resistance increased 62,65 as did resistance to TNFa-induced apoptosis.65,66 Many researchers in the cancer field are interested in P-gp, an ABC transport protein shown to flip short-chain GlcCer into the Golgi.2 P-gp expression is also elevated in multidrug resistant cancer cells.67 Gouaze et al68 showed that P-gp expression decreased following RNA interference or chemical blockade of GCS; however, overexpression of GCS did not induce P-gp expression.65 In another study, a short-chain analog of ceramide increased P-gp expression, and this effect was due to the cellular conversion of the analog to GlcCer.69 These data suggest a relationship between GicCer, P-gp, and drug resistance in cancer cells. GlcCer has been show to increase proliferation of normal smooth muscle cells and keratinocytes,33,41 and Gaucher disease patients have and increased risk for malignant B and other lymphoid cancers.70,71 It is not a far stretch of the imagination to think that increased GlcCer not only protects cancer cells from chemotherapy, but may actually enhance tumor growth and malignancy. Thus, blocking the conversion of ceramide to GlcCer may be a promising approach to reverse multidrug resistance in cancer cells.
Although upregulation of endogenous GlcCer in cancer cells may protect them from death, ingestion of soy GlcCer appears to have anticancer effects, demonstrated by the suppression of colon cancer growth in mice.72 The apoptotic effects ofsoy GlcCer may be due to the high amount of 68-double bond in the backbone as opposed to mammalian cells that often contain a 54-double bond. Soy GlcCer also has high amounts of a-hydroxy fatty acids compared to mammalian cells.73 In addition to soy GlcCer, GlcCer extracted from malt feed (derived from beer brewing waste) also suppressed growth of skin, lung and liver cancer cells.74 This surprising twist may also be an anticancer avenue to explore.
Because GlcCer is the precursor to hundreds of downstream glycosphingolipids that have unique functions, understanding GlcCer function fully has proved difficult. It is clear, however, that the presence of GlcCer is essential for normal cellular function and viability of multicellular organisms. Additionally, transient increases in GlcCer bestow protection to individual cells and to whole-organisms. For example, GlcCer has been shown to anchor certain proteins to membranes, serve as an anticoagulant in cardiovascular disease and possibly stroke and is essential for the barrier function of our skin. GlcCer is impartial in providing protection, however, because cancer cells also utilize GlcCer to evade our body’s immune response and the effects of chemotherapeutic drugs. In comparison to the beneficial effects oftemporary GlcCer elevation, sustained high levels ofGlcCer lead to adverse effects, as noted in patients with Gaucher disease who have a litany of symptoms depending on severity of the disease. Some groups are developing tissue-specific knock-downs of GlcCer |-glucosidase in an effort to tease apart the complex pathology of Gaucher disease.75 Further advancement in the understanding of glycosphingolipid regulation will be of great benefit. Future studies aimed at increasing GlcCer in diseased tissues or decreasing GlcCer in cancer will likely improve the human condition.