1. Structure and Occurrence
Ceramides consist of a long-chain or sphingoid base linked to a fatty acid via an amide bond. They are essential intermediates in the biosynthesis and metabolism of all sphingolipids including the complex sphingolipids in which the terminal primary hydroxyl group is linked to carbohydrate, phosphate, and so forth (sphingomyelin, glycosphingolipids and gangliosides). They are also the primary source of unesterified sphingoid bases and of the important biological mediators sphingosine-1-phosphate and ceramide-1-phosphate. At the last count, 33 different enzymes were known to participate in ceramide metabolism. While ceramides are rarely found as such at greater than trace amounts in tissues other than skin, they can exert important biological effects of their own at these low levels. They are present in membranes where they participate in the formation of raft domains.
Each organism and indeed each tissue may synthesise ceramides in which there are a variety of di- and trihydroxy long-chain bases linked to fatty acids. As discussed in the Introduction to this section, the fatty acids consisting mainly of longer-chain (up to C24 or greater) saturated and monoenoic (mainly (n-9)) components, sometimes with a hydroxyl group in position 2. Other than in certain testicular cells, polyunsaturated fatty acids do not occur. More than 200 structurally distinct molecular species of ceramides have been characterized from mammalian cells. In plants, 2-hydroxy acids predominate sometimes accompanied by small amounts of 2,3-dihydroxy acids. Although small amounts of free ceramides are produced in all tissues as required for the specific biological functions described below, most is converted rapidly to more complex sphingolipids, including sphingomyelin (in animals) and the various glycosylceramides (see the separate web pages). The ceramides in skin are a remarkable exception to this rule, and as such they are discussed separately below.
A shorthand nomenclature simply combines those used conventionally for fatty acids and long-chain bases to denote molecular species of ceramides, including those as components of more complex lipids, e.g. N-palmitoyl-sphingosine is d18:1-16:0. Ceramides containing sphinganine are sometimes termed ‘dihydroceramides’.
2. Ceramide Biosynthesis
Ceramide production is complex and involves at least three pathways. Biosynthesis de novo takes place in the endoplasmic reticulum with palmitoyl-CoA and serine as the precursors for the long-chain base component, which is subsequently converted to ceramide. Biosynthesis of the very specific fatty acids in ceramides involving various chain elongases (ELOVL) requires consideration also, but this is discussed in our web page dealing with saturated fatty acids, although much remains to be learned of how the distinctive fatty acid compositions of ceramides and thence of complex sphingolipids are attained. Alternative routes for ceramide production involve regeneration from complex sphingolipids. For example, in animals in the sphingomyelinase pathway, conversion of sphingomyelin into ceramides (and vice versa) occurs in the plasma membrane, Golgi and mitochondria. Finally, the polar moieties of complex glycosphingolipids can be removed by various hydrolytic enzymes in the lysosomal compartment to recover the ceramides (or their component parts) in a re-cycling/catabolic process. As these biosynthetic or metabolic pathways are located in different organelles, specific pools of ceramide and sphingolipids result with differing biological properties and functions.
Ceramide synthesis de novo: The first of these pathways is described in mechanistic terms in our web pages dealing with sphingoid bases, as important structural features of the latter (double bonds, methyl branches, etc.) are introduced only when they are incorporated into ceramides. In brief in animals, sphinganine is coupled to a long-chain fatty acid to form dihydroceramide by means of one of six ceramide synthases in the endoplasmic reticulum mainly, before the double bond is introduced into position 4 of the sphingoid base. Of these, ceramide synthase 2 is most abundant and is specific for CoA esters of very-long-chain fatty acids (C20 to C26); it is most active in the central nervous system. Ceramide synthase 1 is specific for 18:0 and is located exclusively in brain and skeletal muscle, ceramide synthases 5 and 6 generate 16:0-containing ceramides, and ceramide synthase 3 is responsible for the unusual ceramides of skin and testes.
Each synthase has six membrane-spanning domains and contains a characteristic motif with the specific structures required for catalysis and substrate binding that are essential for its activity, and they have been shown to differ primarily in an 11-residue sequence in a loop between the last two putative transmembrane domains. In addition to separate transcriptional regulation of each of these enzymes, ceramide synthase activity is modulated by many different factors including reversible dimerization, while ceramide synthase 2 has a sphingosine-1-phosphate binding motif and this lipid may inhibits its activity. Acyl-coenzyme A-binding protein (ACBP) facilitates the synthesis of ceramides containing very-long fatty acids and stimulates ceramide synthases 2 and 3 especially.
Most of the ceramides generated in this way are rapidly utilized for synthesis of complex sphingolipids, especially sphingomyelin and hexosylceramides, to ensure that cellular ceramide concentrations are regulated to control their biological activities. In mammalian cells, most complex glycerolipids are synthesised in the endoplasmic reticulum prior to their transport to their final subcellular locations, but the process is rather different for sphingolipids. Ceramide is synthesised on the cytoplasmic leaflet of the endoplasmic reticulum, but subsequent formation of complex sphingolipids occurs in the Golgi apparatus, and a key cytoplasmic protein, ceramide transporter or 'CERT' (CERamide Trafficking), mediates the transport of ceramide between these organelles in a non-vesicular manner. It has a number of distinct functional domains, including an N-terminal phosphatidylinositol-4-monophosphate (PI(4)P)-binding or Pleckstrin homology (PH) domain, which targets the Golgi apparatus, and a C-terminal ‘START’ domain, which can recognize ceramide species with the natural D-erythro stereochemistry, including dihydroceramide and phytoceramide (but not sphingosine), and holds them within in a long amphiphilic cavity by hydrogen bonding with all three polar atoms of the sphingoid motif. There is also a short peptide motif (FFAT) that recognizes a specific protein in the endoplasmic reticulum. There is sufficient flexibility in the body of the protein to enable transfer of ceramide from the endoplasmic reticulum to the Golgi without free movement through the cytosol.
Very-long-chain ceramides containing 24:0 or 24:1 fatty acids turn over much more rapidly in animal cells than those containing 16:0 or 18:0 fatty acids, because of the more rapid conversion of the former into complex sphingolipids, where they may regulate the levels and perhaps the biological functions of the latter. In contrast, ceramides containing d16:1 and d18:1 sphingoid bases turnover at similar rates so do not affect the flux of ceramides through these pathways. The CERT protein is a major factor in this specificity, as it extracts ceramides from membrane bilayers with a preference for those required for synthesis of complex sphingolipids. Removal of ceramide by this process provides the gradient that enables the process to continue, and prevents an accumulation of ceramide in the endoplasmic reticulum that might otherwise be disruptive to the membrane and even cause cell death. While the transfer process itself is not dependent on ATP, the overall process requires ATP, possibly to keep PI(4)P in a phosphorylated form, and the multiple factors that control the biosynthesis of this lipid must also influence sphingolipid metabolism.
As a neutral lipid, ceramide can flip readily across membrane leaflets, and this is also necessary for the synthesis of sphingomyelin, which occurs on the lumen of the Golgi. The pool of ceramide utilized for synthesis of glycosylceramide is delivered to the Golgi by a separate transport mechanism that also does not require ATP. In addition, some ceramide synthesis occurs in mitochondria although this has the potential to lead to cell death. Regulation of ceramide and subsequent sphingolipid biosynthesis is crucial as an excess of sphingolipids can be toxic, while reduced synthesis can inhibit cell proliferation.
Some ceramides are transported from the liver to other tissues in plasma lipoproteins, but especially subclasses HDL2 and HDL3, i.e. those containing apolipoprotein B. There is a suggestion that transport of ceramides via lipoproteins could be a paracrine mechanism to regulate the metabolism of other cells.
Plants and yeast: Three ceramide synthase isoforms have been identified in Arabidopsis, designated LOH1, LOH2 and LOH3, and again the specificities of these are discussed in more detail in relation to the biosynthesis of long-chain bases. The yeast Saccharomyces cerevisiae has two ceramide synthases designated Lag1 and Lac1 of which Lag1 is responsible for the synthesis of ceramides containing phytosphingosine and is a factor in ageing. Phytosphingolipids derived in this way are especially important for the establishment of a lateral diffusion barrier in the nuclear envelope. In yeasts, ceramide synthase activity is regulated by the Torc2 kinase complex, which controls the steady-state levels of long-chain bases and ceramides, but by mechanisms that are poorly understood. A protein Nvj2p promotes non-vesicular transport of ceramides out of the endoplasmic reticulum in yeast, but there is no CERT protein.
Ceramide regeneration: Ceramides are also produced during the catabolism of other complex sphingolipids, and especially by the action of one or other of the sphingomyelinases or of phospholipase C on sphingomyelin in animal tissues as part of the 'sphingomyelin cycle' (see the web page on this lipid for a more detailed discussion). Many agonists including chemotherapeutic agents, tumor necrosis factor-alpha, 1,25-dihydroxy-vitamin D3, endotoxin, gamma-interferon, interleukins, nerve growth factor, ionizing radiation and heat stimulate hydrolysis of sphingomyelin to produce ceramide. In addition, reversal of the sphingomyelin synthesis reaction may generate ceramide, and some may be produced by operation of the enzyme ceramidase in reverse (see next section). Such reactions are much more rapid than synthesis de novo, so they are of special relevance in relation to the signalling functions of ceramides, especially when they occur at the plasma membrane. For example, in this context, the acid sphingomyelinase may be especially important by generating the ceramides that initiate the train of events that leads to apoptosis (see below).
Glycosphingolipids can be hydrolysed by glycosidases to ceramides also (see our web page on monoglycosylceramides, for example) in tissues, but the process tends to be less important in quantitative terms (other than in skin). The key enzymes of sphingolipid metabolism were first characterized from the yeast Saccharomyces cerevisiae, and these were found to be sufficiently similar to the corresponding enzymes in mammals to facilitate their study in the latter.
As discussed above, there are specific ceramide synthases that utilize specific fatty acids for ceramide biosynthesis in animals, and knowledge is slowly being acquired of how these are compartmentalized and regulated within cells. Thus, the synthesis and subsequent catabolism of ceramides involves a complex web of at least 28 distinct enzymes, including six ceramide synthases and five sphingomyelinases, which are all products of different genes. Each of these enzymes may produce distinctive molecular species of ceramides with their own characteristic biological properties. It has been determined that ceramide species containing very-long-chain fatty acids (C24) turnover more rapidly than those containing C16/18 components.
1-O-Acylceramides: In these lipids, which are essential if minor components of lipid droplets, a fatty acid is O-esterified to position 1 of the sphingoid base component of a ceramide. They are formed by the reaction of fatty acyl-CoA esters, produced by the long-chain acyl-CoA synthase ACSL5, with ceramide generated de novo and catalysed by diacylglycerol acyltransferase 2 (DGAT2), a key enzyme in triacylglycerol biosynthesis, on the surface of lipid droplets. It is believed that formation of acylceramides diverts ceramide from a bioactive pool into a storage pool, so limiting ceramide-mediated apoptosis in lipid droplets. In Vernix caseosa, the waxy substance that coats the skin of newborn babies, there is multiplicity of different molecular species of such lipids: C11 to C38 ester-linked (1-O) fatty acids, saturated C12 to C39 amide-linked fatty acids, and C16 to C24 sphingoid bases. Some similar lipids are present in skin (see below).
3. Ceramide Catabolism
In animals, ceramide metabolism and function is controlled in part by the action of ceramidases, which effect hydrolysis to sphingoid bases and free fatty acids, and indeed this is the only route to the formation of unesterified sphingosine. Five such enzymes are known in humans, classified according to their pH optima, i.e. acid (‘ASAH1’), neutral (‘ASAH2’, which differs between humans and animals), and alkaline (three enzymes - ‘ACER1 to ACER3’), with differing cellular locations and fatty acid specificities and with the potential to affect distinct signalling and metabolic events. The acid ceramidase is of particular importance, and aberrations in its synthesis or activity is involved in several human disease states, including the rare autosomal-recessive Farber disease where there is a deficiency in the enzyme so ceramide accumulates; ceramide containing 26:0 in blood is considered to be a biomarker for diagnosis of the disease. ASAH1 is located in the lysosomes and hydrolyses ceramides with small to medium chain fatty acid components (C6 to C18) most efficiently. The neutral ceramidase is located in the plasma membrane and Golgi, especially of intestinal epithelial cells and colorectal tissues, and prefers long-chain components (C16 to C18); it also catalyses the reverse reaction, and this may be a means of ceramide synthesis in mitochondria. ACER1 and ACER2 are found in the endoplasmic reticulum and Golgi, respectively, and they prefer species with very-long-chain acyl groups. ACER3 is present in both the endoplasmic reticulum and Golgi; it has a marked specificity for ceramides, dihydroceramides and phytoceramides linked to unsaturated long-chain fatty acids (18:1, 20:1 or 20:4) in vitro at least. Neutral/alkaline ceramidase activity has also been found in mitochondria and nuclei.
An enzyme broadly similar to the neutral ceramidase has been isolated from plants such as rice, but its specificity is odd in that it does not hydrolyse ceramides containing phytosphingosine. There does not appear to be an equivalent to the acid ceramidase in plants. Ceramidases are also present in lower organisms such as Pseudomonas aeruginosa and slime moulds, where they are secreted proteins rather than integral membrane enzymes. A neutral ceramidase only is found in prokaryotes, including some pathogenic bacteria.
Sphingoid bases released by the action of acid ceramidase can escape from the lysosomes and be re-utilized for ceramide biosynthesis through the action of a ceramide synthase. This has been termed the ‘salvage’ pathway and is important in both quantitative and biological terms. For example, it has been estimated that it contributes from 50 to 90% of sphingolipid biosynthesis. The biological functions of ceramides are discussed below, but there are reasons to believe that ceramides derived from the salvage pathway are spacially and thence functionally distinct from those synthesised de novo. In addition, sphingoid bases released in this way have their own biological functions, which includes utilization for the synthesis of the biologically important metabolite sphingosine-1-phosphate. Therefore, regulation of ceramidase action is central to innumerable biological processes in animals.
4. Biological Functions of Ceramides
The role of ceramides in the biosynthesis of complex glyco- and phospho-sphingolipids are discussed in other web pages on this site (and see the figure in the introduction to this page), so this topic need not be elaborated here. Ceramides, like other lipid second messengers in signal transduction, are produced rapidly and transiently in response to specific stimuli in order to target specific proteins, for example to activate certain serine/threonine protein kinases or phosphatases. They may also regulate cellular processes by influencing membrane properties. While they can be produced by synthesis de novo for such functions, activation of one of the sphingomyelinases under physiological stress or other agents is a more rapid means of generation in animal tissues at least. In fact, ceramides appear to be formed under all conditions of cellular stress by a multiplicity of activators in eukaryotic organisms. However, it should be noted that ceramides with different fatty acid and long-chain base (molecular species) compositions are formed in different compartments or membranes of the cell by various mechanisms over different time scales and potentially with distinct functions. The biological functions of those ceramides containing medium-chain (up to C14), long-chain (C16 and C18) and very-long-chain (C20 and longer) fatty acids, in particular, may have to be considered separately.
Physical properties: Unsaturation in the sphingoid backbone augments intramolecular hydrogen bonding in the polar region, which permits a close packing of ceramide molecules and a tight intramolecular interaction in membranes. A further important factor in this context is the length of the fatty acyl moiety, as shorter chain ceramides tend to produce a positive curvature in a lipid monolayer, while long-chain molecules have the opposite effect and possess a marked intrinsic negative curvature that facilitates formation of inverted hexagonal phases as well as increasing the order of the acyl chains in bilayers. By their interactions with ion channels, ceramides influence the permeability of membranes and render bilayers and cell membranes permeable to solutes that vary from small- up to protein-size molecules.
While ceramides are minor components of membranes in general, their physical properties ensure that they are concentrated preferentially into lateral liquid-ordered microdomains (a distinct form of 'raft' termed ‘ceramide-rich platforms’), although these effects are again chain-length specific. These domains differ appreciably in composition from those rafts enriched in sphingomyelin and cholesterol, and ceramides containing C12 to C18 fatty acids can in fact displace cholesterol from rafts to modify their physical properties. Ceramides are generated within rafts by the action of acid sphingomyelinase, causing small rafts to merge into larger units and modifying the membrane structure in a manner that is believed to permit oligomerization of specific proteins such as cytokines and death receptors. Ceramides are also essential for the formation and/or secretion of exosomes by facilitating or inducing membrane curvature.
Through the medium of these modified rafts, ceramides are able to function in signal transduction. Specific receptor molecules and signalling proteins are recruited and cluster within such domains, thereby excluding potential inhibitory signals, while initiating and greatly amplifying primary signals. It is believed that ceramide-rich platforms amplify both receptor- and stress-mediated signalling events and thence may influence various disease states. They may also provide an entry route into cells for viral and bacterial pathogens. In contrast, sphingosine, sphingosine-1-phosphate and ceramide-1-phosphate do not facilitate raft formation.
Although ceramides and diacylglycerols have structural similarities, their occurrence, location and behaviour in membranes are different. Ceramides cross synthetic lipid bilayers relatively quickly in vitro, but it is not clear whether they can flip across more complex biological membranes equally readily, especially in the ceramide-rich platforms. Restricted flipping could have important effects upon the signalling role of ceramides in that those generated by different enzymes on each side of a membrane could have distinct functions.
Enzyme activation: In general, ceramides tend to modify intracellular signalling pathways to slow anabolism and promote catabolism. Amongst a wide range of biological functions in relation to cellular signalling, ceramides are especially important in triggering apoptosis, and they have also been implicated in the activation of various protein kinase cascades, dependent on the site of generation. The mechanism of these interactions is the subject of intensive study at present, but in relation to the latter, two intracellular targets for ceramide action of special important have been discovered – at least two protein phosphatases (ceramide-activated protein phosphatases) and a family of protein kinases (ceramide-activated protein kinases). For example, the phosphatase may be involved in the regulation of glycogen synthesis, insulin resistance and response to apoptotic stimuli. Ceramides generated by the action of sphingomyelinase and by synthesis de novo are both important to the process, while ceramidases have contrasting effects in these and other biological effects of ceramides.
Apoptosis: The role of ceramides in the regulation of apoptosis, and cell differentiation, transformation and proliferation has received special attention. Apoptosis is a normal process, which occurs in response to oxidative stress in particular, in which a cell can be considered to actively ‘commit suicide’. It is essential for many aspects of normal development and is required for maintaining tissue homeostasis. There are two pathways - 'extrinsic' initiated in the plasma membrane by ligation of so-called 'death factors', such as the tumor necrosis factor-α (TNF-α), and 'intrinsic' induced by external actions in mitochondria, e.g. by DNA damage, oxidation or radiation injury. Although the mechanism of the ceramide interaction with these pathways is uncertain, it is clear that a cascade of reactions is initiated that culminates in the release of intracellular proteases of the caspase family to promote apoptosis. In dysfunctional mitochondria, one mechanism involves formation of channels in the membrane that enable release of specific mitochondrial proteins that include caspases. Ceramides with fatty acids of differing chain-lengths are believed to function in different ways, and 16:0-ceramide generated by ceramide synthase 6 is especially pro-apoptotic, for example, while ceramides with very-long-chain fatty acids accumulate in necroptosis, a form of apoptosis. On the other hand, ceramides containing 2-hydroxy acids in keratinocytes appear to be protective against apoptosis. Ceramides induce the related process of cellular senescence also.
Failure to properly regulate apoptosis can have catastrophic consequences, and many disease states, including cancer, diabetes, neuropathies, Alzheimer's disease, Parkinson's disease and atherosclerosis, are thought to arise from deregulation of apoptosis. For example, ceramides have been implicated in the actions of TNF-α and in the cytotoxic responses to amyloid Aβ peptide, which are involved in Alzheimer’s disease and neuro-degeneration. In addition, ceramides appear to be involved in many aspects of the biology of aging and of male and female fertility. These effects may hold implications for diseases associated with obesity and insulin resistance, including again diabetes and cardiovascular disease.
Similarly, ceramides are intimately involved in the induction of autophagy, the 'maintenance' process by which cellular proteins and excess or damaged organelles are removed from cells by engulfing them in a membrane-enclosed cellular compartment called the phagosome. In particular, maturing phagosomes are enriched in very-long-chain ceramides. While this process is beneficial in that it aids recycling of cellular nutrients, the presence of excess ceramide can lead to unnecessary apoptosis.
As animals and plants have multiple isoforms of ceramide synthase that are specific for the chain-length of the base and fatty acid, it has been suggested that ceramides containing different fatty acids have distinct roles in cellular physiology. In particular, C16 ceramide appears to be especially important in apoptosis in non-neuronal tissues, while C18 ceramide has growth-arresting properties and may be involved in apoptosis in some carcinomas treated with chemotherapy agents. In addition, a transferase has been identified that transfers the acetyl group from platelet activating factor to sphingosine with a high specificity. The product, N-acetylsphingosine - the simplest of all ceramide molecules, has signalling functions that are distinct from those of the parent lipids or of other ceramides; it does not enter the salvage pathway in cancer cells in vitro and is cytotoxic.
In contrast, the ceramide metabolite, sphingosine-1-phosphate, has opposing effects on cell survival and proliferation. As ceramide and sphingosine-1-phosphate are inter-convertible via sphingosine as an intermediate, which also has pro-apoptopic activity, the balance between these lipids and with ceramide-1-phosphate is obviously of great metabolic importance. It has been termed the ‘sphingolipid-rheostat’.
Cancer: In addition to Farber disease discussed above, drug therapies that influence the relative concentrations of ceramides are generating considerable interest, especially in relation to treatment of breast, ovarian and colorectal cancers. Thus, pathways mediated by ceramide and sphingosine-1-phosphate have been identified in both the development and progression of cancer, with the former acting to suppress tumors by inducing anti-proliferative and apoptotic responses in cancer cells, and the latter functioning to promote tumor growth. Thus, ceramide triggers many different tumor suppressive and anti-proliferative cellular events such as apoptosis, autophagy, senescence, and necroptosis by activating or repressing key effector molecules, while defects in ceramide generation and metabolism in cancer cells contribute to tumor cell survival and their resistance to chemotherapy. However, the effects can depend on the specific cancer, and for example, ceramide synthase 1 (and the product C18 ceramide) is down-regulated in head and neck cancers (gliomas) and up-regulated in breast cancer. Ceramide synthase 2 is believed to be a mediator of chemoresistance and chemosensitivity.
Ceramide glycosyltransferases that catalyse ceramide glycosylation and glycosphingolipid formation play an important role in regulating tumour progression and have a significant correlation with the poor prognosis of cancer patients. In contrast, there is a suggestion that inhibition of the acid ceramidase may be beneficial towards cancer. Lethal mitophagy is an anti-tumorigenic mechanism mediated by ceramide, where cells degrade a sufficient number of mitochondria to kill the cancer cell in an apoptosis-independent manner.
Administration of exogenous ceramides with short-chain acyl moieties (C2-, C6-, C8-ceramide), encapsulated by nano-technological means, is seen as a promising therapeutic approach to cancer. C6-ceramides especially can cause cell death in a number of cancers and they are known to increase the sensitivity to other apoptosis-inducing agents, although the signalling mechanism by which this is accomplished has yet to be determined. Ceramides generated by the action of the acid sphingomyelinase may be especially important in inhibiting cancer development, though a virtual absence of the alkaline sphingomyelinase has been noted in colorectal cancer. A further ceramide metabolite, ceramide-1-phosphate, has anti-apoptosis effects also, as well as being involved in inflammatory responses by activating a specific phospholipase A2. Again, the balance between the precursor and product is of great biological importance. For practical reasons, the metabolism and functions of these two sphingolipids and of ceramides and sphingoid bases are discussed separately in this website, but an integrated view is necessary for a full understanding.
Other disease states: Ceramides are believed to have much less benign effects in many other disease states. Whether ceramide modulation in disease occurs as a side effect in response to a pathological mechanism, such as cell stress or inflammation, or whether they are involved actively in the development of disease does not always appears to be clear. However, significant changes in ceramide levels and composition have been noted in a number of inflammatory conditions, including metabolic diseases, irritable bowel syndrome, asthma, arthritis, multiple sclerosis, retinal degeneration and cystic fibrosis, and the mechanistic implications are under investigation. Ceramides in the circulation may be biomarkers of these diseases. In obesity and dyslipidemia, sphingolipids such as ceramide and its metabolites induce the cellular dysfunctions that underlie diabetes and cardiovascular disease in that they disrupt insulin sensitivity, pancreatic β‑cell function, vascular reactivity and mitochondrial metabolism. Thus, ceramides synthesised de novo promote apoptosis of pancreatic β-cells in both types 1 and 2 diabetes, improving insulin resistance and reducing insulin synthesis. 16:0-Ceramide especially has profound effects upon adipose tissue metabolism, and it has been identified as the main mediator of obesity-derived insulin resistance, together with impaired fatty acid oxidation and hepatic steatosis. Similarly, this ceramide accumulates at the expense of those containing very-long-chain and long-chain ceramides in cystic fibrosis.
Ceramides have cardiotoxic properties and are reported to be drivers of cardiovascular disease. In mice and rats, inhibition or reduction of enzymes involved in ceramide synthesis by pharmacological means prevents the development of diabetes, atherosclerosis, hypertension and heart failure. In cultured cells and isolated tissues, ceramides perturb mitochondrial functions that may contribute to heart disease by inducing cell death and inflammation; by accumulating in mitochondria of cardiomyocytes, they increase their permeability and lead to apoptosis and cell death. In humans, ceramide levels in serum, and in particular an elevated ratio of very-long-chain to long-chain ceramides, are considered to be good biomarkers for adverse outcomes in cardiovascular disease. Here also sphingosine-1-phosphate often has the opposite effect.
There are also reported effects upon neurological diseases, such as Alzheimer's disease, Parkinson's disease and depression, and 18:0-ceramide is reported to be a potential contributor to human aging. In animal models, inhibition of ceramide biosynthesis or promotion of ceramide degradation ameliorates many metabolic disorders. In contrast, there is evidence that increasing the ceramide content of cells can inhibit lipopolysaccharide-stimulated inflammatory responses. A few rare genetic disorders of sphingoid base and ceramide synthesis/catabolism are known in humans, of which the best known is the formation of 1-deoxy-bases and ceramides, discussed here.... Those related to skin ceramides are described briefly below.
Plants: Comparatively little information is available on the role of ceramides in cell signalling in plants, but there are suggestions that sphingolipid catabolic products may be linked to programmed cell death, signal transduction, membrane stability, host-pathogen interactions and stress responses. For example, there is evidence that enhanced synthesis of ceramides with very-long-chain fatty acids and trihydroxy sphingoid bases by ceramide synthases LOH1 and LOH3 promotes cell division and growth, while in contrast, accumulation of the ceramide species C16 fatty acid with a dihydroxy sphingoid base, due to LOH2 overexpression, leads to plant dwarfing and programmed cell death. Ceramides aggregate in rafts in plant membranes, together with other sphingolipids and sterols, as in animal tissues. Similarly, in the yeast S. cerevisiae, widely used as a model organism, it has been reported that ceramide species with different N-acyl chains and sphingoid bases are involved in the regulation of different sets of functionally related genes.
Dihydroceramides: The biological function of ceramides in animal tissues may usually require the presence of the 4,5-double bond in the long-chain base, although the trans conformation may not be essential in that synthetic ceramide containing a cis-4,5-double bond is an equally potent inducer of apoptosis at least. On the other hand, the dihydroceramides in which sphinganine is the sphingoid base are now known to accumulate to a far greater extent in tissues than had previously been thought, and they have some distinct and separate functions from those of the more conventional ceramides. Dihydroceramides regulate autophagy by a mechanism distinct from that of ceramides per se, in particular autophagy-induced death of cancer cells, but they are believed to be pro-survival under conditions of physiological stress. It seems possible that the former are 'safer' when elevated concentrations of sphingosine-containing ceramides might have deleterious effects. In addition, dihydroceramides are involved in the regulation of such diverse processes as production of reactive oxygen species in mitochondria, and the activity of cytochrome P450s and phosphatases. They have been implicated in the progression of a number of disease states, e.g. non-alcoholic fatty liver disease, diabetes, ischemia/reperfusion injury, and neurodegenerative diseases. Dihydroceramides appear to be particularly important in the yeast S. cerevisiae.
5. Skin Ceramides
The skin forms the protective barrier between the internal tissues of the host and the hostile external environment, which can include chemicals, ultraviolet light, mechanical damage and pathogenic microorganisms, while preventing the loss of water and electrolytes. Exceptionally, the stratum corneum of the skin in which the outer-most layer consists of dead cells or corneocytes (non-nucleated cells without cytoplasmic organelles) contains relatively high levels of ceramides (as much as 50% of the total lipids), including O-acylceramides. These are present mainly in the extracellular domains (interstices) and are accompanied by nearly equimolar amounts of cholesterol, and free fatty acids. This ratio is believed to be essential for the normal organization of the tissue into the membrane structures that are responsible for the functioning of the epidermal barrier. Ceramides exist both in the free form and linked by ester bonds to structural proteins. The lipid organization in the membranes of skin is different from that of other biological membranes in that two lamellar phases are present, which form crystalline lateral phases mainly, with repeat distances of approximately 6 and 13 nm. Small sub-domains of lipids in a liquid phase may also exist.
Some of these skin ceramides have distinctive structures not seen in other tissues, and many different forms are commonly recognized. They can contain the normal range of longer-chain fatty acids (a), e.g. formula 1 in the figure, some with hydroxyl groups in position 2 (a*), e.g. formula 2, linked both to dihydroxy bases with trans-double bonds in position 4 or to trihydroxy bases. In addition, there are O-acyl ceramides in which a unique very-long-chain fatty acid component (typically C30 or C32) has a terminal hydroxyl group, and this may be in the free form or esterified with linoleate (c), e.g. formulae 3 and 4; the sphingoid base can be either di- (b) or trihydroxy (b*), e.g. formula 4; the latter is not a common feature in sphingolipids of animal origin, and can include both phytosphingosine and the unique 6-hydroxy-4-sphingenine in human epidermis. Ceramides of type 1 in which the 1-O-hydroxyl group of the sphingoid base is acylated by a very-long-chain fatty acid are also present (1‑O‑acylceramides - illustrated above); these comprise 5% of the total ceramides in the epidermis of mice and humans and comprise as much as 700 molecular species. In all, 15 classes of free ceramides and 3 classes of covalently bound ceramides with up to 1700 distinct molecular species have been identified. Such lipids were first studied in detail in the skin of the pig as a convenient experimental model, but they now been characterized in humans and rats. In addition, several molecular forms of glucosylceramide, based on similar ceramide structures, have been characterized in skin, and these are also essential for its proper function.
Depending on the particular layer of the skin (keratinocytes, stratum corneum, etc.), the lipid composition can vary. These lipids have an obvious role in the barrier properties of the skin, limiting loss of water and solutes and at the same time preventing ingress of harmful substances. As the aliphatic chains in the ceramides and the fatty acids are mainly non-branched long-chain saturated compounds with a high melting point and a small polar head group, the lipid chains are mostly in a solid crystalline or gel state, which exhibits low lateral diffusional properties and low permeability at physiological temperatures. There is a report that the stratum corneum layer of the skin has a water permeability only one thousandth that of other biomembranes, for example. Natural and synthetic ceramides are now commonly added to cosmetics and other skin care preparations.
Most steps in the biosynthesis of ceramides linked to ω-O-acylated fatty acids occur in the endoplasmic reticulum of keratinocytes. First, fatty acid synthesis of very-long-chain (ultra-long-chain) acyl-CoA de novo must take place, requiring the chain-elongation enzymes ELOVL1 and ELOVL4. Desaturation can occur, and importantly oxidation in the 2 (α) and terminal (ω) positions. The ω-hydroxylation step requires an enzyme of the cytochrome P450 family, designated CYP4F22, of the kind involved in the synthesis of hydroxy-eicosatetraenoic acids (HETE). Mutations are a cause of lamellar ichthyosis, and knockout mice deficient in the equivalent enzyme were found to die within 8 hours of birth.
Ceramides are first synthesized by ceramide synthase 3 (SERS3), which has a high specificity for very-long chain fatty acids (>C26) with incorporation of the ω-hydroxy fatty acid. This is acylated with linoleate by the action of an unusual enzyme related to the phospholipase A family, PNPLA1, which catalyses esterification by first releasing linoleate from triacylglycerols in the skin while acting as an acyltransferase to link the linoleate directly to the ω-hydroxyl moiety of the ultra-long chain fatty acid. PNPLA1 is unique among phospholipases in that it is involved in the metabolism of sphingolipids rather than glycerophospholipids and catalyses transacylation rather than hydrolysis. This process is vital for proper skin barrier function and keratinocyte differentiation, as mice with defective triacylglycerol metabolism are unable to synthesis ω‑O‑acylceramides and have an impaired skin barrier. Mutations in the human PNPLA1 gene are believed to be the cause of autosomal recessive disease congenital ichthyosis.
The resulting ceramides are converted to the complex sphingolipids sphingomyelin and especially glucosylceramide, which are transferred with the aid of ATP-binding cassette (ABC) transporters together with degradative enzymes into the stratum corneum via specific organelles termed 'lamellar bodies.' These organelles must fuse with the apical plasma membrane of the outermost cell layer of the epidermis in order that their contents can be secreted. It is only then that the final step of hydrolysis of the lipid precursors occurs in the extracellular spaces of the stratum corneum, i.e. ceramides are generated from sphingomyelin by the action of acid sphingomyelinase and from glucosylceramides by β-glucocerebrosidase. This mechanism ensures that ceramides, with their potentially harmful biological activities, never accumulate within nucleated cells.
Eventually, ceramides with a terminal ω-hydroxyl group in the fatty acyl moiety are bound covalently to the proteins of the cornified envelope, especially to involucrin. Recent evidence suggests that in the first step of this process ceramides containing O-acyl linoleate in an estolide linkage are acted upon by specific lipoxygenases (12R-LOX and eLOX3) to form hepoxilin-like products, which may have specific functions in skin (see the web page on these oxylipins for a discussion of their biosynthesis in skin). The high polarity of the main isomer, 9R,10S,13R-trihydroxy-11E-octadecenoate (a triHOME), is believed to facilitate its hydrolysis, initiating a procedure that leads to attachment of the free ω-hydroxyl groups of the very-long-chain fatty acid components of ceramides and glucosylceramides via ester bonds to glutamate residues of proteins exposed on the surface of corneocytes by means of a transglutaminase (TGase). Ultimately, the covalently bound glucosylceramides and ceramides are further degraded until only the long-chain ω-hydroxy acid remains attached to protein. These proteolipid structures, i.e. the corneocyte lipid envelope, form a complete lipid monolayer over the surface of each cell and are essential to the function of the epidermal water barrier; they act as a template or scaffold to direct the assembly of the extruded lipids into lamellar bilayers.
An alternative or additional mechanism has been proposed for the final steps in which the linoleate residue attached to the ω‑O‑acylceramide is oxidized by 12R-LOX and eLOX3 and a NAD+-dependent dehydrogenation to a highly reactive 9,10-trans-epoxy,11E-ene,13-keto intermediate, which rather than being hydrolysed is able to link non-enzymatically by the Michael addition reaction to cysteine or histidine residues in proteins of the corneocyte envelope, or by formation of a Schiff base and eventually a pyrrole derivative with a lysine residue (see our web page on reactive aldehydes, for discussion of such reactions). This appears to offer a more convincing explanation for the role of linoleate in this process.
In essential fatty acid deficiency, the O-acyl linoleate is replaced by oleate with concomitant abnormalities in the cutaneous permeability barrier. In diseased skin, there is often an altered lipid composition and organization with impaired barrier properties. Thus, diminished levels of ceramide in the epidermis, reflecting altered sphingolipid metabolism especially in relation to the esterified and non-esterified omega-hydroxy-ceramides and trihydroxy bases, have been implicated in such skin disorders as psoriasis, ichthyosis and atopic dermatitis. For example, tri-HOME accumulate in atopic dermatitis. Much less emphasis in research has been placed upon the signalling functions of ceramides in skin, but there is increasing evidence for their involvement in the regulation of the same metabolic events as in other tissues, with implications for health.
Our web page on waxes describes the non-polar lipids secreted onto skin by the sebaceous glands.
The analysis of ceramides presents no particular problems. They can be isolated by adsorption chromatography (TLC and HPLC), and further analysed by HPLC or GC after conversion to less polar derivatives. Nowadays, modern mass spectrometric methods are increasingly being used for the purpose. One widely used method for analysis of molecular species of sphingomyelin involves their hydrolysis with phospholipase C to ceramides to simplify the technical problems.
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