CHEMISTRY, OCCURRENCE, BIOLOGY and ANALYSIS
1. Structure and Occurrence
Ceramides consist of a long-chain or sphingoid base linked to a fatty acid via an amide bond. They are rarely found as such at greater than trace levels in tissues, less than 10% of the glucosylceramide content in plants for example, although they can exert important biological effects. Ceramides are formed as the key intermediates in the biosynthesis of all the complex sphingolipids, in which the terminal primary hydroxyl group is linked to carbohydrate, phosphate, etc. Unlike the sphingoid precursors, they are not soluble in water and are located in membranes where they participate in raft formation (see below).
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, the latter consisting mainly of longer-chain (to C24 or greater) saturated and monoenoic (mainly (n-9)) components, sometimes with a hydroxyl group in position 2. Polyunsaturated fatty acids do not occur other than in certain testicular cells. 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. Ceramides are usually converted rapidly to more complex sphingolipids, including sphingomyelin and the various glycosylceramides, and apart from in the skin the precursors never accumulate. Small amounts of ceramides are produced in all tissues as required for the specific biological functions described 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. Skin Ceramides
Exceptionally, the stratum corneum of the skin in which the outer-most layer consists of dead cells 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 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 eleven forms are commonly recognized. They can contain the normal range of longer-chain fatty acids (some with hydroxyl groups in position 2), linked both to dihydroxy bases with trans-double bonds in position 4 or to trihydroxy bases (e.g. formulae 1 and 3 in the figure). In addition, there are O-acyl ceramides in which the long-chain fatty acid component (typically C30) (a) has a terminal hydroxyl group, which may be in the free form or esterified with either linoleate or a 2-hydroxy acid (c); the sphingoid base can be either di- or trihydroxy; the latter is not a common feature in sphingolipids of animal origin, and can include phytosphingosine and the unique 6-hydroxy-4-sphingenine in human epidermis (b) (e.g. formulae 3 and 4 in the figure). Such lipids have been studied in particular detail in the skin of the pig as a convenient experimental model, but they have also been found 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 (epidermis, 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. Natural and synthetic ceramides are now commonly added to cosmetics and other skin care preparations.
The distinctive ceramides in the skin are derived mainly from glucosylceramide (two forms also arise from sphingomyelin), synthesised in specific organelles termed 'lamellar bodies' in the epidermal cells. These organelles must fuse with the apical plasma membrane of the outermost cell layer of the epidermis in order that their lipid and contents can be secreted. It is only then that the final step of hydrolysis of the lipid precursors with generation of ceramides occurs (see next section). This mechanism ensures that ceramides, with their potentially harmful effects, never accumulate within nucleated cells. In diseased skin, there is often an altered lipid composition and organization and 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 and atopic dermatitis.
Some ceramides with a terminal omega-hydroxyl group in the fatty acyl moiety are bound covalently to the proteins of the cornified envelope, especially to involucrin. Recent evidence suggests that ceramides containing O-acyl linoleate in an estolide linkage are acted upon by specific lipoxygenases to form products that ultimately lead to attachment of ceramides to proteins, and this is essential for the barrier function. In essential fatty acid deficiency, the O-acyl linoleate is replaced by oleate with concomitant abnormalities in the cutaneous permeability barrier.
Our web page on waxes describes the non-polar lipids secreted onto skin by the sebaceous glands.
The biosynthesis of ceramides de novo is discussed in greater detail in our web pages dealing with sphingoid bases, as important structural features of the latter are introduced only when they are incorporated into ceramides. In brief, sphinganine is coupled to a long-chain fatty acid to form dihydroceramide by means one of several ceramide synthases, 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) and is most active in the central nervous system, while ceramide synthase 1 is specific for 18:0 and is located exclusively in brain and skeletal muscle. Ceramide synthase 3 is responsible for the unusual ceramides of skin and testes, while ceramide synthases 5 and 6 generate C16 ceramides. Three ceramide synthase genes have been identified in Arabidopsis, LOH1, LOH2 and LOH3, of which LOH2 appears to be specific for the incorporation of palmitic acid.
Ceramides are also produced during the catabolism of other complex sphingolipids, for example 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'. 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 generated 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 importance in relation to the signalling functions of ceramides, especially when they occur at the plasma membrane. The acid sphingomyelinase may be especially important in this context.
Glycosphingolipids can also be hydrolysed by glycosidases to ceramides 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.
Much remains to be learned of how the distinctive fatty acid compositions of ceramides and thence of complex sphingolipids are attained (see the introductory webpage). As discussed in our webpage on long-chain bases, there are specific ceramide synthases that utilize specific fatty acids for ceramide biosynthesis, 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.
Most of the ceramide required for the production of complex lipids is synthesised on the cytoplasmic leaflet of the endoplasmic reticulum, with subsequent metabolism occurring in the Golgi apparatus. A key cytoplasmic protein, ceramide transporter or 'CERT', mediates the transport of ceramide between these organelles in a non-vesicular manner. It has a number of distinct functional domains including a phosphatidylinositol-4-monophosphate-binding domain, which targets the Golgi apparatus, and a further domain believed to catalyse lipid transfer. There is also a short peptide motif that recognizes a specific protein in the endoplasmic reticulum. The CERT protein extracts ceramides only from membrane bilayers with some specificity for those containing C14 to C20 fatty acids, but not those of longer chain length, and delivers them for the synthesis of sphingomyelin, but not for glycosylceramide. The pool of ceramide utilized for synthesis of the latter is delivered to the Golgi by a separate transport mechanism details of which are still uncertain. In addition, some ceramide synthesis occurs in mitochondria although this has the potential to lead to cell death.
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. Five such enzymes are known in humans, classified according to their pH optima, i.e. acid (‘ASAH1’), neutral (‘ASAH2’) 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 Farber’s disease where there is a deficiency in the enzyme so ceramide accumulates. ASAH1 is located in the lysosomes, and hydrolyses ceramides with medium chain fatty acid components most efficiently. The neutral ceramidase is located in the plasma membrane and prefers long-chain to very-long-chain components (C16 to >C24); it also catalyses the reverse reaction, although the biological significance of this is not known. ACER1 and ACER2 are found in the endoplasmic reticulum and Golgi, respectively, and they also prefer very-long-chain acyl groups. ACER3 is present in both the endoplasmic reticulum and Golgi. In vitro, it has a marked specificity for ceramides, dihydroceramides and phytoceramides linked to unsaturated long-chain fatty acids (18:1, 20:1 or 20:4). Neutral/alkaline ceramidase activity has also be 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.
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, and indeed this is the only route to the formation of free sphingosine, which can in turn be utilized for the synthesis of the biologically important metabolite sphingosine-1-phosphate. Therefore, regulation of ceramidase action is central to innumerable biological processes in animals.
5. Biological Functions
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. While they can be produced by synthesis de novo, activation of one of the sphingomyelinases under physiological stress or other agents is a more rapids 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 compositions are formed in different compartments or membranes of the cell by a variety of different mechanisms at different times and potentially with distinct functions. In discussing the biological functions of ceramides, it is necessary to consider all of these factors.
Unsaturation in the sphingoid backbone augments intramolecular hydrogen bonding in the polar region, which permits a close packing of the ceramide molecules and a tight intramolecular interaction in membranes. A further important factor in this context is the length of the fatty acyl moiety. Shorter chain ceramides tend to produce a positive curvature in a lipid monolayer, while long-chain molecules have the opposite effect in addition to increasing the order of the acyl chains in bilayers.
While ceramides are minor components of membranes in general, their physical properties ensure that they are concentrated preferentially into lateral liquid-ordered microdomains (a 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 C24 fatty acids can in fact displace cholesterol from rafts. 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. Through the medium of these modified rafts, they are able to function in signal transduction. Specific receptor molecules and signalling proteins 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, ceramide-1-phosphate, sphingosine and sphingosine-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. Ceramides influence the permeability of membranes via interactions with ion channels.
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. 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 – a specific protein phosphatase (ceramide-activated protein phosphatase) 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.
In general, ceramides tend to modify intracellular signalling pathways to slow anabolism and promote catabolism. In particular, 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 actively ‘commits suicide’. It is essential for many aspects of normal development and is required for maintaining tissue homeostasis. Mitochondria are a key site for apoptosis mediated by ceramides, possibly by forming channels in the membrane that enable release of specific mitochondrial proteins. Ceramides with fatty acids of differing chain-lengths are believed to function in different ways. For example, 18:0-ceramide generated by ceramide synthase 1 is reportedly proapoptotic, while 16:0-ceramide generated by ceramide synthase 6 is prosurvival. Similarly, ceramides containing 2-hydroxy acids in keratinocytes appear to be protective against apoptosis.
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 tumor necrosis factor-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, including diabetes and cardiovascular disease. Thus, ceramides synthesised de novo promote apoptosis of pancreatic β-cells in both types 1 and 2 diabetes, promoting insulin resistance and reducing insulin synthesis.
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, dihydroceramides may have separate functions of their own. For example, 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, but in this instance dihydroceramides are equally effective. Dihydroceramides are also believed to be pro-survival under conditions of physiological stress. It seems possible that the latter are ‘safer’ when elevated concentrations of sphingosine-containing ceramides might have deleterious effects.
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.
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. The metabolism of dihydroceramides appears to be particularly important in this context.
In contrast, the ceramide metabolite, sphingosine-1-phosphate, has opposing effects on cell survival and proliferation. As ceramide and sphingosine-1-phosphate are interconvertible via sphingosine as an intermediate, which also has pro-apoptopic activity, the balance between these lipids is obviously of great metabolic importance. It has been termed the ‘sphingolipid-rheostat’.
Drug therapies that influence the relative concentrations of these lipids are generating considerable interest, especially in relation to cancer treatment. 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. Administration of exogenous short-chain ceramides (C2-, C6-, C8-ceramide), encapsulated by nano-technological means, is seen as a promising therapeutic approach to cancer. Ceramides generated by the action of the acid sphingomyelinase may be especially important in inhibiting cancer development. 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 here, but an integrated view is necessary for a full understanding.
Significant changes in ceramide composition have been noted in a number of inflammatory conditions, including irritable bowel syndrome and cystic fibrosis.
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. Certainly there is good evidence that ceramides promote apoptosis in plants, and that this process is in attenuated by ceramide-1-phosphate. Ceramides aggregate in rafts in plant membranes, together with other sphingolipids and sterols, as in animal tissues.
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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James Hutton Institute (and Mylnefield Lipid Analysis), Invergowrie, Dundee (DD2 5DA), Scotland.
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