Sphingolipids and their metabolism in physiology and disease

Novel metabolic inputs, outputs and extensions

Studies in the past decade have considerably advanced our understanding of several aspects of sphingolipid metabolism, including the entry of different substrates into sphingolipid metabolic pathways through serine palmitoyltransferase (SPT), the complexity of ceramide species and the exit from sphingolipid metabolism (FIG. 1A). They have also led to the discovery of previously unsuspected enzymes of sphingolipid metabolism.

Among the important novel findings is the demonstration that other substrates can enter sphingolipid metabolism, in addition to the canonically used serine and palmitate. This finding was initiated by the study of human hereditary sensory and autonomic neuropathy (HSAN1), which identified mutations in the genes encoding SPTLC1 and SPTLC2, the key subunits of SPT, as the main cause of this disease. Remarkably, these mutations alter the substrate specificity of SPT such that the mutants favour alanine and glycine over serine compared with wild-type forms of the enzyme⁷. This results in the formation of non-canonical metabolites, the deoxysphingoid and 1-deoxymethyl sphingoid bases, respectively, and their acylated ceramides. Lacking the 1-hydroxyl (1-OH) moiety, both groups result in ‘dead end’ ceramides that cannot be modified at this position (FIG. 1B). These molecules have since been detected as normal products of SPT, albeit at very low levels. It is currently not known whether they have physiological functions, but they have been implicated as the mediators of neurotoxicity in HSAN1.

Additionally, the more recently identified SPTLC3 subunit of SPT confers specificity of the enzyme to myristate (over palmitate) as the fatty acid substrate⁸, whereas small subunits of SPT may favour the use of stearate as the fatty acid substrate⁹ (FIG. 1).

Thus, SPT is able to generate a multitude of sphingoid bases. Accordingly, inspired by molecular genetic nomenclature, SPT can be better dubbed Sag/PmsT to indicate its ability to use alanine and glycine as well as myristate and stearate, in addition to the canonical serine and palmitate, respectively.

These variations in the sphingoid backbone are compounded by variations in the acyl groups through the action of distinct ceramide synthases (CerSs)^10,11. Longer fatty acids are generated by the action of a family of elongases¹² (FIG. 1A). Their combined action then allows a remarkable complexity in the structure of ceramides, which is further compounded by the action of additional hydroxylases and desaturases that act on either the sphingoid base or the fatty acyl group. Thus, ceramide is not a single species but should be considered a family of closely related but distinct molecules with emerging distinct functions (many ceramides instead of a single ceramide; see FIG. 1B for nomenclature)³.

More recent studies have also identified enzymatic activity involved in the ω-acylation of ceramides to form three-chained ceramides and glycosphingolipids in the skin, which regulate epithelial permeability and skin barrier function (see below). The candidate genes include PNPLA1, which encodes patatin-like phospholipase domain-containing 1, which has phospholipase and transacylase functions¹³.

Ceramides can also be acylated at the 1-OH position, and a recent study identified their location in lipid droplets and the pathway leading to their formation involving a previously unrecognized ceramide acyl transferase activity of diacylglycerol O-acyltransferase 1 (DGAT1) and DGAT2 (REF. 14). It was also suggested that DGATs form a complex with CerS and with long-chain-fatty-acid–CoA ligase 5 (ACSL5), which acylates fatty acid substrates required for the action of DGATs. In related studies, the fatty acyl binding protein ACBP has been shown to specifically interact with CerS2 and CerS3 and to channel very-long-chain fatty acids to ceramide synthesis¹⁵. This indicates the important roles of protein complexes in controlling sphingolipid metabolism.

There have also been notable advances in following the fate of the fatty aldehydes as they exit the sphingolipid pathway. These studies defined the dehydrogenase ALDH3A2, CoA synthases ACSL1–6, ACSBG1 and ACSVL1 (also known as VLACS), and reductase TSC13 as the enzymes involved in sequential metabolism of the fatty aldehyde products of sphingolipid clearance¹⁶ (FIG. 1).

Two novel previously unsuspected enzymes were also identified that add to the complexity of sphingolipid metabolism. Sphingomyelin synthase-related (SMSr) was identified as a homologue of SMS but with much reduced activity and with a preference for utilizing phosphatidylethanolamine as a donor of ethanolamine phosphate and thus generating ceramide ethanolamine phosphate rather than sphingomyelin (where phosphatidylcholine serves as the donor of choline phosphate)¹⁷. Mitochondrial sphingomyelinase was identified as a predominantly mitochondrial neutral sphingomyelinase, the functions of which are currently unknown¹⁸.

These advances have provided a strong foundation for understanding the biochemistry of sphingolipid metabolism, illustrating its many complexities but also its integration in broader aspects of lipid metabolism and metabolomics in general. Understanding these enzymes and pathways has also opened up new avenues for the development of therapeutics targeting sphingolipid-related diseases (BOX 3; Supplementary information S3 (table)).

Compartmentalization of sphingolipids and their metabolism

An important layer in sphingolipid metabolism is the presence of the multiplicity of enzyme isoforms that are products of distinct genes with specific localization, action and/or substrate specificity. For example, there are at least five distinct ceramidases and five distinct sphingomyelinases that localize to the plasma membrane, endoplasmic reticulum (ER), Golgi, nucleus, lysosome, mitochondria or extracellular space. There are also six different CerSs that show distinct preferences for fatty acids of various lengths (FIG. 1B), thus contributing to the remarkable complexity of ceramides (and their downstream anabolic metabolites). While the CerSs appear to localize primarily to the ER, they have also been specifically noted in the nuclear membrane and in mitochondria.

Given that ceramides, sphingomyelins and some of the glycosphingolipids are hydrophobic (glycosphingolipids with very complex headgroups are much more amphipathic), it is axiomatic that these molecules will reside in the compartments where they are generated (either hydrolytically or synthetically) unless they are specifically transported. Accordingly, specific transporters have been identified for ceramide (ceramide transfer protein (CERT)), S1P (protein spinster homologue 2 (SPNS2)), C1P (C1P transfer protein (CPTP)), and glucosylceramide (phosphatidylinositol-four-phosphate adaptor protein 2 (FAPP2; also known as PH domain-containing family A member 8)) (FIG. 2). Ceramide, as a neutral lipid, appears to readily flip across membrane leaflets, at least in model membranes. This flipping has functional consequences. For example, in the Golgi, ceramide has to flip to serve as the substrate of SMSs in the synthesis of sphingomyelin, as the active site of SMS enzymes faces the lumen of the Golgi¹. By contrast, synthesis of glucosylceramide from ceramide appears to occur on the cytosolic side of the Golgi. This then necessitates the flipping of glucosylceramide in order to serve as a substrate for the synthesis of higher glycosphingolipids (FIG. 2). The exact machineries involved in these flipping events are not known. In addition to ceramide, evidence points to the existence of a small pool of sphingomyelin on the cytosolic side of the plasma membrane that may serve as substrates for neutral sphingomyelinase 2 (nSMase2)¹⁹. In freeze–fracture studies, this pool of sphingomyelin appears to be highly clustered²⁰, but it is not known how the pool is generated (whether by slow flipping of sphingomyelin from the outer leaflet or by mixing of the inner and external leaflets as may occur during endocytosis and exocytosis). Sphingomyelin is also enriched in a subset of secretory vesicles²¹.

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Figure 2

Intracellular compartmentalization and transport of sphingolipids

It is now becoming clear that the cellular actions of bioactive sphingolipids are ‘local’. Therefore, the site of production of these lipids is critical, and it is determined by the location of the specific enzymes involved in the regulatory pathways. For details of the compartmentalization of these processes, please refer to box 1 in REF. 1. Moreover, the metabolic and cellular functions of bioactive lipids are further controlled by specific translocases and lipid transport proteins. A strong case can now be made that ceramides (Cers) generated in the endoplasmic reticulum (ER) can reach the Golgi either by a vesicular route, where they are coupled to the synthesis of glucosylceramide, or by specific translocation via the action of ceramide transfer protein (CERT), which couples ceramides to the synthesis of sphingomyelin (SM)¹⁷⁹. Likewise, the transfer protein phosphatidylinositol-four-phosphate adaptor protein 2 (FAPP2) can transfer glucosylceramide (GlcCer) between Golgi networks and couple it specifically to the synthesis of globosides (globo series of neutral glycosphingolipids) as opposed to the anionic ganglio series¹⁸⁰. Ceramide-1-phosphate (C1P) is the target of the non-vesicular translocation activity of C1P transfer protein (CPTP), which is involved in transferring it from the Golgi to other compartments such as the plasma membrane³⁵. Protein spinster homologue 2 (SPNS2) flips sphingosine-1-phosphate (S1P) across the plasma membrane to deliver it to exocytoplasmic leaflets, where it can access its receptors (a subfamily of G protein-coupled receptors (GPCRs))²². VAP, vesicle-associated membrane protein-associated protein.

The highly bioactive S1P undergoes transporter-mediated translocation from inside the cell through the action of SPNS2 (FIG. 2) and several ABC transporters²² to the extracellular space, where it can access its S1P receptors. Loss of function of SPNS2 leads to the loss of several actions of S1P that appear to require its flipping to reach its receptors.

It is thus becoming clear that the site of action of bioactive lipids and their metabolism is subjected to targeted regulation by mechanisms controlling their transport and membrane leaflet localization.

Insights from yeast studies

Studies in Saccharomyces cerevisiae have firmly established multiple roles for sphingolipids in the adaptive responses to heat stress, which increases metabolic flux through the de novo pathway, most likely owing to increased uptake of serine. The sequentially generated sphingoid bases, sphingoid base phosphates and ceramides have been implicated in mediating key responses to elevated temperatures, including transient cell cycle arrest, arrest in translation and sequestration of mRNA and regulation of nutrient permeases^42,48,49. In a combined ‘omic’ approach, specific ceramides were implicated in specific transcriptional responses, regulating the expression of genes involved in iron transport, acid sensitivity and RNA metabolism, illustrating the complexity of ceramide functions in yeast⁴⁸. Cell cycle checkpoints mediated by mitosis inhibitor protein kinase Swe1 were attributed to ceramides, in particular C18:1 ceramide^50,51. Additional functions for yeast ceramides include regulation of the diauxic shift and mitochondrial function, the DNA damage response and regulation of responses to reactive oxygen species⁴².

Cell adhesion and migration

A critical role is emerging for bioactive sphingolipids in the regulation of cell adhesion, migration and invasion. Recent studies implicated SK1 and SK2 (the enzyme producing S1P) in filopodia formation, cell migration and invasion in response to different stimuli, including epidermal growth factor, in a mechanism that involves induction of phosphorylation of the ezrin, radixin and moesin (ERM) family protein ezrin⁵², which is known for its function in regulating cellular motility⁵³. The mechanism involves the activation of S1PR2 and implicates the transporting functions of SPNS2 (REF. 52). Remarkably, a recent study using a genome-wide in vivo screen in mice identified SPNS2 as a prominent promoter of metastasis⁵⁴. Also of interest, S1pr2-knockout mice are deaf⁵⁵, similarly to mice deficient for another ERM protein, radixin⁵⁶, as well as to Spns2-knockout mice⁵⁷, suggesting a common pathway involving these three components. Reciprocally, ezrin can be dephosphorylated in a mechanism involving ceramide-mediated activation of the protein phosphatase PP1α⁵⁸. These studies illustrate the mechanistic and functional crosstalk between bioactive sphingolipids (FIG. 3a).

Formation of membrane domains, exosomes and endosomes

Sphingolipid metabolism has been shown to affect membrane organization and dynamics, as well as vesicular transport (FIG. 3b).

Substantial literature supports the existence of specialized membrane domains, especially in the plasma membrane, which are enriched in specific lipid species, including sphingolipids (with an important contribution of ceramides). Although the precise nature and function of these domains, which also include rafts, are still not clear, they could have important roles in regulating the function of membrane receptors. Thus, ceramides could influence cell signalling by contributing to (and potentially promoting) the formation of membrane domains⁵⁹.

Suggesting the role of sphingolipids in membrane dynamics and signalling more broadly, a landmark study identified a role for nSMase and the resultant ceramide in the formation and secretion of exosomes, a specific subtype of secreted vesicles that have been implicated in cell–cell signalling and communication⁶⁰. Subsequently, several studies have disclosed that inhibition of nSMase2 can interfere with the cargo composition of these vesicles, including microRNA (miRNA)⁶¹, prion proteins⁶² and other cargo proteins with important roles in neurodevelopment and neurodegeneration. However, inhibition of SMS2 promotes exosome secretion by neurons and could be linked to the clearance of the neurotoxic amyloid-β by microglia⁶³, suggesting therapeutic possibilities in Alzheimer disease by modulating the metabolism of sphingolipids.

Furthermore, SK1 was implicated in endosome regulation (FIG. 3b), whereby knockdown of SK1 led to defects in endocytic recycling, suggesting an important role for S1P in endocytic membrane trafficking that is likely independent of its role in signalling through S1PRs⁶⁴. Sphingolipids also appear to be involved in the internalization of viruses, as illustrated by the implication of SMS2 in the fusion of the HIV viral envelope with the host cell plasma membrane⁶⁵.

It was also demonstrated that a specific species of sphingomyelin, C18 sphingomyelin, is recognized by p24, a component of the coatomer protein COPI comprising secretion machinery, and this interaction is important for vesicular trafficking⁶⁶. This example highlights the importance of the distinct cellular roles of molecular subspecies of sphingolipids mentioned above.

Finally, the existence of an intricate crosstalk between sphingolipid metabolism and membrane dynamics has been illustrated by cellular studies, demonstrating that a finely tuned mechanism involving the combined action of CERT, SMSs and protein kinase D regulates both sphingomyelin levels and vesicular trafficking from the Golgi⁶⁷.

Implications in DNA damage response

Early studies had demonstrated that many cytotoxic and DNA-damaging agents induced profound changes in the levels of sphingolipids, in particular leading to increased levels of ceramide. Accordingly, enzyme-centric studies identified SK1, alkaline ceramidase 2 (ACER2), nSMase2 and CerSs as specific players in the DNA damage response (DDR) (see also BOXES 1,2). SK1 protein levels are subjected to p53-induced degradation, and this has been implicated in regulating cell survival and inflammatory responses. This connection was supported by an in vivo study demonstrating that Sk1 (also known as Sphk1) knockout protected mice from thymic lymphomas induced by the loss of p53 (REF. 68). By contrast, ACER2 mRNA levels are induced by p53, and this protects cells with wild-type p53 from ceramide-mediated cell death⁶⁹. Similarly, nSMase2 is induced during the DDR and appears to have a role in cell cycle regulation⁷⁰. An analogous role has been shown for the S. cerevisiae protein Isc1 (a homologue of nSMase2)⁵⁰. Additional studies have implicated CerSs as mediators of cytotoxic responses of cancer cells to various chemo therapeutic and stress agents (reviewed in REF. 11; see also BOX 1).

Immune and inflammatory functions

A strong case for S1P in the function of the immune system has been established⁴⁶ (FIG. 3c). This has found a direct application in the treatment of multiple sclerosis with FTY720 (fingolimod), a sphingoid base analogue⁷⁹. Briefly, T cell egress is controlled by a gradient of S1P between the lymph and plasma⁸⁰ that is established by the secretion of S1P from haematopoietic and endothelial cells into plasma⁸¹. S1PR1 expressed on lymphocytes then responds to this gradient, leading to the migration of lymphocytes from lymphoid tissues into the circulation⁸². This process is inhibited by FTY720, which is converted by SK2 into FTY720 phosphate, which then acts to tonically suppress S1PR1, thus leading to the sequestration of T cells in the thymus and lymph nodes. Consistent with these findings, phospholipid phosphatase 3 (LPP3), an S1P phosphatase that hydrolyses S1P, was found to promote lymphocyte egress, presumably by decreasing S1P levels in target organs and thus maintaining a blood (high)– tissue (low) S1P gradient⁸³. In addition, a specific pool of S1P bound to high-density lipoprotein (HDL) has been shown to regulate lymphopoiesis and neuroinflammation not only by regulating delivery of S1P to the blood but also by modulating its receptor signalling pathways⁸⁴.

A connection has also emerged between bioactive sphingolipids and the cPLA2–cyclooxygenase 2 (COX2) pathway, which generates bioactive prostaglandins. Studies have implicated SK1 and S1P in regulating the expression of COX2, although the mechanisms remain unclear. Additionally, C1P and the enzyme that forms it, ceramide kinase, have been implicated in the direct activation of cPLA2 in response to cytokines⁸⁵. These results suggest an important role for C1P and S1P in inflammatory responses. However, it was shown that deletion of SK1 from macrophages is not required for inflammatory responses but did sensitize these cells to autophagy-induced death⁸⁶.

Ongoing studies also suggest roles for aSMase in sustaining inflammation through the production of inflammatory cytokines, especially interleukin-6 (IL-6) and CC-chemokine ligand 5 (CCL5) in response to the action of tumour necrosis factor (TNF) and IL-1 (REF. 87). Clinically, levels of serum aSMase have been shown to predict mortality in patients in intensive care units at risk of developing systemic inflammation⁸⁸, an effect most likely related to the induction of aSMase release by TNF.

Multilevel involvement of sphingolipids in cancer cell biology

Bioactive sphingolipids have emerged as key regulators of several, if not most, of the key attributes of cancer and cancer cell biology (Supplementary information S3 (table)). On the one hand, S1P has been implicated in tumour cell survival, cancer inflammatory pathways, angiogenesis, resistance to chemotherapy and cancer cell invasion. On the other hand, ceramides have been implicated in cancer cell apoptosis, growth arrest, mediating sensitivity to chemotherapeutics and senescence^89,90. Here, we discuss some of the more mature findings implicating sphingolipids in specific cancers.

A strong case has evolved for a role for SK1 in colon cancer, especially colitis-associated cancer (CAC). These studies were prompted by the finding that SK1 is involved in the regulation of COX2 and cell inflammatory responses⁹¹. Human colon cancer showed upregulation of SK1 in 89% of cases, and this correlated with the high levels of COX2. Higher levels of SK1 in colon cancer in an Asian population correlated with advanced stage and predicted increased mortality⁹². Sk1-knockout mice were partly resistant to chemically induced colitis, and they showed significant attenuation of colon cancer in a model of CAC⁹³. Mechanistic studies defined a role for SK1 in mediating the induction of COX2 in vivo⁹³ as well as in the regulation of the expression of signal transducer and activator of transcription 3 (STAT3), which is a key transducer of pro-inflammatory signals from several types of plasma membrane receptors⁹⁴. Moreover, loss of SK1 also decreased the growth of adenomas in a mouse model of intestinal cancer⁹⁵. In addition to the increase in SK1, S1P lyase is downregulated in colon cancer tissues, further promoting S1P signalling⁹⁶. Interestingly, knockout of SK2 resulted in reciprocal increases in SK1, which were implicated in increased risk of colon cancer in mice⁹⁴. In line with the studies presented above, targeting SK1 with inhibitors showed activity against colon cancer in xenografts⁹⁷ and CAC mouse models⁹⁸. Thus, these studies confirm that the S1P pathway contributes to colon carcinogenesis, likely by regulating synthesis of prostaglandins and other inflammatory mediators.

Interestingly, and in contrast to S1P, ceramide levels have been shown to be reduced in human colon cancer, consistent with the overall opposing functions of these two lipid mediators (FIG. 1A). As such, deletion of NCDase, which augments ceramide levels, protected mice from the development of colon cancer in a carcinogen-induced model of colon cancer⁹⁹. However, it should be noted that the involvement of NCDase appears to be distinct from the role of SK1 in CAC.

The SK1–S1P pathway has also been implicated in several other cancers, including glioblastoma, head and neck cancer, renal cancer and several leukaemias¹⁰⁰. Notably, the cancer-promoting roles of this pathway extend beyond actions on cancer cells (such as protection from apoptosis). For example, S1P has emerged as a key regulator of angiogenesis with widely important implications in vascular biology and cancer^100–102, and SK1 is induced in many cancers in response to hypoxia and in renal cell carcinoma with mutations in the von Hippel–Lindau (VHL) gene. The resultant paracrine increases in S1P in hypoxia have been implicated in angiogenesis and lymphangiogenesis¹⁰³.

Acid ceramidase (ACDase) is another enzyme that has received considerable attention in the context of cancer biology, in particular in the development of therapeutics^104,105. The levels of this enzyme are increased in several cancers, including head and neck cancer, melanoma and prostate cancer, and correlate with the advanced pathology of these tumour types. ACDase serves to attenuate levels of ceramide, and inhibitors of ACDase sensitize cancer cells to chemotherapy and radiotherapy in cell culture and in xenografts¹⁰⁴. A newly developed and potent inhibitor of ACDase synergized with several chemotherapeutics to induce melanoma cell death¹⁰⁶. Higher levels of ACDase correlate with increased aggressiveness of melanoma, whereas the expression of aSMase, which promotes ceramide generation, is inversely correlated with tumour stage¹⁰⁷, indicating a strong correlation between low levels of ceramide and advanced stages of disease in many cancers. However, evaluation of the expression of ACDase in breast cancer showed that it is expressed at higher levels in low-risk, luminal A type breast cancer, and high expression was associated with better prognosis¹⁰⁸. Therefore, whereas ACDase appears to, at least in part, drive malignancy in head and neck cancer, melanoma and prostate cancer, it has rather protective functions in breast cancer.

As another example, platelet-derived aSMase was implicated in promoting cell adhesion and metastasis of melanoma¹⁰⁹. In addition, aSMase in endothelial cells has been proposed to have a key role in tumour angiogenesis, as it has been shown to mediate sensitivity to antiangiogenic therapy by elevating the levels of ceramide¹¹⁰.

Exciting studies are also pointing towards the usability of sphingolipid levels and the activity of their metabolic enzymes as biomarkers in cancer. A recent metabolomic profiling of pancreatic adenocarcinoma identified changes in sphingolipid levels, including elevation of sphingomyelin, as a dominant component and determinant of a ‘lipogenic’ subtype defined by the investigators as showing distinct metabolism of lipids¹¹¹. Increases in plasma ceramide levels have been shown to be predictive of the response of metastatic tumours to radiation therapy¹¹². Along similar lines, CerS6 emerged as a strong predictor of survival in human colon cancer¹¹³.

These results indicate that specific perturbations in one or more pathways of bioactive sphingolipids can affect various key components of cancer development (for example, growth, migration, angiogenesis and immune signalling) and responses to therapies. These perturbations are emerging as cancer-specific, possibly due to the distinct functions of various enzymes in different tissues and under distinct conditions. Thus, these roles need to be appreciated in this tissue-specific context.

Metabolic functions and connections to diabetes and the cardiovascular system

A strong case is emerging for a role for ceramides in regulating metabolic functions, and changes in levels of ceramide species have been implicated in various metabolic syndromes, such as fatty liver disease¹¹⁴, obesity¹¹⁵ and insulin resistance^47,116. Cell and in vivo studies have demonstrated that increased ceramide levels result in attenuation of insulin action, most likely via inhibition of AKT (FIG. 3a). This inhibitory pathway appears to be particularly engaged by the increased supply of palmitic acid, which can drive ceramide synthesis directly as a precursor substrate. Increased ceramide synthesis in the context of insulin resistance was also shown to be driven by inflammatory agents (for example, agonists of Toll-like receptors, TNF and interleukins)⁴⁷. Attenuation of ceramide generation in vivo, by use of inhibitors of SPT or dihydroceramide desaturase or mice deficient in these enzymes, improves insulin signalling and glucose and lipid metabolism in diabetes and alleviates obesity-associated metabolic changes and cardiac dysfunction^117,118. Furthermore, mice deficient in CerS6 are protected from weight gain and glucose intolerance¹¹⁹ induced by a high-fat diet, and overexpression of ACDase led to a reduction of hepatic steatosis¹²⁰. Similarly to ceramide, other sphingolipids, including sphingomyelin and glucosylceramide, have also been shown to inhibit insulin action¹²¹. Accordingly, Sgms2-knockout mice, in which sphingomyelin levels are reduced, showed increased sensitivity to insulin and resistance to obesity induced by a high-fat diet¹²². Notably, these effects were observed despite an associated increase in the levels of ceramide, suggesting that ceramide is not the primary mediator of insulin signalling inhibition or that in the context of Sgms2 knockout, the elevated ceramide is diverted to other pathways. Interestingly, different metabolic functions were found for SGMS1, deletion of which caused decreased insulin secretion and induced oxidative stress in white adipose tissue, leading to the destruction and dysfunction of white adipocytes¹²³.

Metabolic roles of S1P are also emerging. In mice, deletion of the gene encoding S1P phosphatase 2 (Sgpp2), which degrades S1P, was associated with basal ER stress in pancreatic β-cells and increased sensitivity to a high-fat diet¹²⁴. Consistent with these results, deletion of the gene encoding SK1 was shown to protect against diet-induced hepatic steatosis¹²⁵. In support of the role of ER stress in mediating these metabolic effects of S1P, cell-based studies have demonstrated involvement of S1P signalling in regulating heat shock proteins, the unfolded protein response in the ER and ER stress¹²⁶.

Sphingolipids, and especially ceramide and sphingosine, have been implicated in vascular disease, including atherosclerosis and ischaemic injury. Increased activity of aSMase and levels of ceramides have been linked to atherogenesis; the mechanism possibly involves secreted aSMase that may act on lipoprotein-associated sphingomyelin to convert it to ceramide¹²⁷. Accordingly, attenuation of aSMase was demonstrated to act protectively against retinal ischaemic injury in mice¹²⁸. In support of the general impact of sphingolipid metabolism on vasculature, inhibition of de novo synthesis of sphingolipids with myriocin attenuated myocardial reperfusion injury¹²⁹.

From a clinical perspective, a very compelling case is developing for the utility of measuring serum sphingolipids (mostly ceramides) as indicators of metabolic disorders and atherosclerotic cardiovascular diseases. For example, plasma levels of C24 and C26 ceramides as well as deoxy-C24 ceramide have been associated with diabetic neuropathy¹³⁰. Elevated C18:0 and C18:1 ceramides were also shown to be strong predictors of major adverse cardiovascular effects in healthy subjects¹³¹. Similarly, C18:1 ceramide was found to be a key predictor of high amounts of necrosis after coronary angiography procedures¹³². Another study identified C24:1 ceramide and sphingomyelin as being strongly associated with cardiovascular mortality¹³³. Higher levels of plasma C22:0 and C24:0 ceramides have also been shown to predict less improvement in verbal memory in response to exercise in patients with coronary artery disease in which these skills decline¹³⁴. In addition to the use of ceramides as biomarkers, serum S1P levels inversely correlated with atherosclerotic disease¹³⁵, whereas deoxysphingolipids have been shown to serve as a biomarker in diabetes progression¹³⁶.

Sphingolipids as drivers of neurodevelopment and neurodegeneration

Genetic defects in various enzymes of sphingolipid metabolism reveal important roles for these enzymes in brain development and health (reviewed in REF. 6). These compelling conclusions have come from converging studies using both mouse knockouts and genetic tracing of human disease. These include defects in fatty acid 2-hydroxylase (FA2H)¹³⁷, ACER3 (REF. 138), SPTSSB (a small subunit of SPT)¹³⁹, CerS1 (REF. 140) and CerS2 (REFS ^141,142) (Supplementary information S3 (table)). These recent studies suggest that key sphingolipid metabolites are critical for normal brain development and function. In most cases, the sphingolipid species that may cause neural dysfunction are not known. However, in a recent study of Cers1 knockout, neuronal production of C18:1 ceramides and other sphingolipids was found to be impaired, leading to the accumulation of sphingoid bases and neurodegeneration. These effects could be corrected by overexpression of CerS2 (REF. 143), suggesting that sphingoid bases are the candidate mediators of neuronal injury.

In addition, deficiency of nSMase2 was shown to decrease pathology associated with Alzheimer disease in mice¹⁴⁴, whereas deletion of Asah2, which encodes NCDase, attenuated brain damage associated with traumatic brain injury¹⁴⁵.

Taken together, these studies begin to define several causal roles for sphingolipids in normal brain functions and as mediators of neuronal damage and neurodegenerative disorders.

Skin-specific sphingolipids and their functions

Skin is characterized by the presence of unusual sphingolipids based on ultra-long fatty acyl chains as well as fatty acyl groups linked on the ω-end of the N-linked fatty acids (thereby generating a three-chain rather than a two-chain molecule). Loss of function of several enzymes involved in sphingolipid metabolism leads to various forms of skin and hair defects that are often fatal — loss of CerS3 (REF. 146), elongation of very-long-chain fatty acids protein 4 (ELOVL4), CYP4F22 (the ω-hydroxylase)¹⁴⁷, glucosylceramide synthase (UGCG)¹⁴⁸ or PNPLA1 (REF. 13) leads to defects in skin epithelium organization and function, whereas loss of ELOVL3 and ELOVL4 (REFS ^149,150), CerS4 (REF. 151) or ACER1 (REF. 152) results in hair loss phenotypes. At least for some of these enzymes, the defects resulting from their deficiency appear to be mechanical in nature, in that the skin fails to form an impermeable barrier owing to the loss of its specialized, long-chain ceramides.

The authors thank the members of their laboratories and M. Airola, C. Luberto, C. Clarke, D. Canals, C. Senkal, C. Rhein and F. Velazquez for helpful discussions. The authors are very grateful for the contribution of M. Hernandez for assembling table 1 and supplementary information tables S2 and S3. Due to space limitations, the authors have striven to reference the more recent studies pertinent to the presentation while directing the readers to more in-depth targeted reviews. The authors apologize for the multitude of sphingolipid investigators whose works they could not cite in this Review.