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Diacylglycerols



1.   Diacylglycerols as Components of Oils and Fats

Diacylglycerols (or "diglycerides") are esters of the trihydric alcohol glycerol in which two of the hydroxyl groups are esterified with long-chain fatty acids. They can exist in three stereochemical forms (see our web document on Triacylglycerols for a discussion of nomenclature). When the stereochemistry is unknown or when the mixture is racemic, sn-1,2- and 2,3-diacylglycerols are sometimes termed α,β-diacylglycerols, while sn-1,3-diacylglycerols may be designated α,α’-diacylglycerols.

Structural formulae of diacylglycerols

α,β-Diacylglycerols are formed as intermediates in the hydrolysis of triacylglycerols (and other glycerolipids) by lipases in animal tissues, and similarly they are generated in seed oils by the action of plant lipases. Their presence is important technologically in commercial seed oils, as small amounts can have a profound influence on the physical properties.

Mixed diacylglycerols are produced commercially by enzymic hydrolysis for use as emulsifiers. In addition, edible oils consisting of 80% 1,3-diacylglycerols, produced by an enzymatic process, are marketed in Japan, Europe and the USA as cooking oils and vegetable oil spreads. It is suggested that they are metabolized in a different way from triacylglycerols with beneficial nutritional effects. When they are digested, the 1/3-monoacylglycerols formed are absorbed into tissues relatively poorly, apparently limiting the accumulation of fats in body tissues. It has also been suggested that they slow the increase in triacylglycerol and cholesterol concentrations in blood after a meal, and that there is increased fat oxidation with an influence on food intake by increasing satiety. Thus, these commercial 1,3-diacylglycerol oils are claimed to be useful anti-obesity agents.

It should be noted that it is easy to generate diacylglycerols artefactually on extracting or storing tissues if inappropriate methods are used. Often, attempts are made to analyse 1,2-/2,3- and 1,3-diacylglycerols separately, but the data may not be meaningful as acyl migration occurs rapidly until an equilibrium mixture is formed that contains about 67% of the 1,3-isomer. All diacylglycerols will isomerize slowly on standing in inert solvents or in the dry state even at low temperatures. They can be recovered from tissues with minimal isomerization when necessary by extracting the tissues with non-alcoholic solvents such as diethyl ether or chloroform, taking care not to heat extracts at any stage. When pure positional isomers are required, it is necessary to chromatograph the partial glycerides on TLC plates coated with silica gel G impregnated with boric acid at a level of 10% of the adsorbent), using a solvent system of chloroform (alcohol-free)-acetone (96:4, v/v).

Routine determination of molecular species of diacylglycerols in oils and fats can be accomplished by various chromatographic methods of which high-temperature gas chromatography was considered the most appropriate until recently, as information on the composition as well as the absolute amount is obtained in this way. Modern mass spectrometric (lipidomics) methodology is now favoured by those who have access to the technology.


2.  sn-1,2-Diacylglycerols in Animal Tissues – Biological Functions

sn-1,2-Diacylglycerols tend to be minor components of most tissues and membranes in quantitative terms, but they are very important in animal cells as they are key intermediates in the formation of many glycerolipids, while they function also in many cellular processes as second messengers, which respond to external stimuli to modulate vital biochemical mechanisms. They are the only one of the three stereoisomers that function in this way, and they are synthesised and metabolized by innumerable enzymes at spatially different cellular locations, each with distinct enzymatic properties and selectivities.

Biosynthesis and metabolism: sn-1,2-Diacylglycerols are key intermediates in the formation of phosphatidylcholine, phosphatidylethanolamine and triacyl-sn-glycerols, and for this purpose, phosphatidate phosphatase is an important enzyme, which converts phosphatidic acid to sn-1,2-diacylglycerol (and is discussed in greater detail in the web pages dealing with these lipids). The reverse reaction in which phosphatidic acid is produced by the action of a diacylglycerol kinase is also of great biological importance (see below).

Biosynthesis of 1,2-diacylglycerols via phosphatidic acid

Most of the phosphatidic acid is generated via the Kennedy pathway with glycerol-3-phosphate as the precursor, but a second mechanism involves the action of a specific phospholipase D on phosphatidylcholine. The latter can also be a direct precursor for diacylglycerol production via the action of phospholipase C (see below), and they are formed as intermediates from monoacylglycerols during the biosynthesis of triacylglycerols by the monoacylglycerol pathway. During the biosynthesis of sphingomyelin, 1,2-sn-diacylglycerols are produced from phosphatidylcholine by an exchange reaction with ceramide catalysed by the sphingomyelin synthases SMS1 and SMS2 in the trans-Golgi and plasma membrane; a related pathway may be important in some pathogenic fungi.

Indeed, sn-1,2-diacylglycerols can be formed as intermediates in the catabolism of all glycerolipids and during digestion of dietary lipids in the intestines by hydrolysis by lipases. In humans, the lingual lipase hydrolyses the ester bond in the sn-3 position of dietary triacylglycerols preferentially, thus generating mainly sn-1,2-diacylglycerols. On the other hand, pancreatic lipase, produces a racemic mixture of the sn-1,2- and 2,3-isomers on the way to further digestion products such as monoacylglycerols. In adipose tissue, the most active hydrolytic enzyme is the adipose triglyceride lipase, which generates racemic 1,3-diacylglycerols and these are hydrolysed further by the hormone-sensitive lipase.

sn-1,2-Diacylglycerols serve as precursors for the biosynthesis of phosphatidic acid via the action of diacylglycerol kinase (as illustrated above) and of the endocannabinoid 2-arachidonoylglycerol, both of which also have signalling functions in their own right. In particular in mammals, there is a family of at least ten diacylglycerol kinase isoenzymes (in five subfamilies), which are structurally related to the sphingosine kinase, and each of which may have slightly different properties and functions. They may be segregated in distinct cellular organelles and activated by different means; while some are cytosolic, some are associated with membranes and others are located within the nucleus. In the brain different isoenzymes are expressed in different types of neuron, some of which have several isoenzymes. These enzymes have a negative effect on signalling by diacylglycerols by reducing their concentrations in cells, but they can generate phosphatidic acid specifically for its alternative signalling functions in addition to its use as a precursor of other lipids

Signalling: In relation to their signalling function, sn-1,2-diacylglycerols are formed along with the important signalling molecules, the water-soluble inositol phosphates, by the action of the enzyme phospholipase C on phosphatidylinositol and the polyphosphoinositides and especially phosphatidylinositol-4,5-bisphosphate at the plasma membrane (see the web page on these lipids for a more detailed discussion). A family of at least thirteen related enzymes in four sub-families exist with differing subcellular locations and substrate specificities, which are activated by agonists at receptors on membranes. Of these enzymes, a calcium-dependent phosphoinositide-specific phospholipase C (or 'phosphoinositidase C') is especially important. While the response is immediate, it is short-lived. A key feature of the reaction is that the diacylglycerols produced are retained in the membrane in which they are formed. In comparison, the other routes to diacylglycerol production occur more slowly but are of longer duration.

Hydrolysis of phosphatidylinositol to diacylglycerolsby phospholipase C

Other phospholipids can by hydrolysed to sn-1,2-diacylglycerols in the same way, but they may be less relevant from a signalling standpoint. For example, two isoforms of a phospholipase C that is specific for phosphatidylcholine have been identified in natural killer cells at the outer leaflet of the plasma membrane and within the cytoplasm. The fatty acid compositions of the diacylglycerols formed by these various routes then reflect the composition of the parent phospholipids. In particular, those derived from phosphatidylinositols are highly enriched in molecular species containing stearic acid in position sn-1 and arachidonic acid in position sn-2. There is evidence that the diacylglycerols in most cells and organelles must contain polyunsaturated fatty acids to fulfill their function as messengers optimally. However, in the cell nucleus it appears that there are two distinct pools of diacylglycerols with very different compositions produced from phosphatidylinositides (polyunsaturated) and phosphatidylcholine (saturated and monoenoic) by specific stimuli, and these may have different functions. Under basal conditions before stimulation by hormones or neurotransmitters, 1-stearoyl-2-arachidonoyl-sn-glycerol is present at relatively low levels in cells in comparison to other molecular species.

An especially important function of sn-1,2-diacylglycerols, and in particular those derived from phosphatidylinositol, is that they affect vital processes in cell physiology by binding to and activating members of the protein kinase C (PKC) family of enzymes, often acting in concert with the soluble phosphoinositides. The sn-1,2-configuration is essential for this activity. Diacylglycerols appear to function then by increasing the concentration of calcium ions in the cell, which stimulates the translocation of the various iso-enzymes of protein kinase C to the inner face of the plasma membrane. There, the main PCKs (α, β and γ) are bound in a 1:1 ratio to the sn-1,2-diacylglycerols at a highly conserved cysteine-rich ‘C1’ domain, which consists of a sequence of 50 amino acids with a characteristic motif. Other conserved regions such as the ‘C2’ domain assist in membrane recruitment of the kinase by interaction with anionic phospholipids, often by a mechanism triggered by Ca2+. Phosphatidylserine can serve this purpose, but there is a strong preference for phosphatidylinositol 4,5-bisphosphate, mediated by a basic patch distal to the Ca2+ binding site, and this targets these PKC isoenzymes selectively to the plasma membrane. This lipid is doubly important in that only those polyunsaturated diacylglycerol species derived from it are able to bind and activate protein kinase C. A second novel group of PCKs (δ, ε, η, θ) contain tandem C1 domains that bind diacylglycerols and a C2 domain that is not responsive to Ca2+ and does not assist in membrane binding. By exposing small areas of the apolar regions of neighbouring lipids, they improve the hydrophobic interactions with proteins within membranes thereby affecting their activities.

Scottish thistleThus, protein kinase C enzymes regulated by lipid second-messengers contain one or more membrane-targeting modules, which result in protein kinase activation, typically by relieving autoinhibitory constraints. They are involved in both short- and long-term modifications of normal cellular physiology with more than a hundred substrates identified to date. Of particular importance is the finding that the tumour-promoting phorbol esters mimic the activity of diacylglycerols and activate the same enzymes (phorbol is a plant-derived diterpene that is not metabolized in tissues so is persistent in its activity). Diacylglycerols therefore have a key role in the pathophysiology of cancer and other disease states. In addition, the identification of non-kinase receptors of sn-1,2-diacylglycerols, many but not all of which have the conserved C1 domain, has revealed new and strategic functions in regulating cellular responses and in cytoskeletal remodelling.

In migrating endothelial leader cells, phospholipase C signalling is restricted to the front to generate a diacylglycerol gradient that by interacting with the integrated Ca2+ control system promotes persistent forward migration.

Other functions: Diacylglycerols bind to protein kinase D, a cytosolic serine-threonine kinase that in turn binds to the trans-Golgi membrane network and regulates transport of proteins to the cell surface. Those diacylglycerols generated by the action of sphingomyelin synthase may be important in this context. In the absence of diacylglycerols, protein transport is blocked.

Diacylglycerols accumulate transiently in membranes, where they bind via strong hydrophobic interactions to particular proteins, and then cause changes in the physical properties of the bilayer. As their polar head group is small, they tend to form inverted micellar structures. In practice, this means that they introduce small areas of unstable negative curvature in membranes that facilitate membrane fission or fusion. The fusion of biological membranes is of great importance for the proper functioning of cells, and diacylglycerols in membranes are able to facilitate this process partly via their specific physical properties and partly through activation of certain proteins. While rapid transbilayer movement (or flip-flop) of diacylglycerols can occur to suggest an even distribution between the two leaflets of the plasma membrane, some studies indicate that they flip more slowly across an ordered raft-like bilayer, i.e. one enriched in sphingomyelin and cholesterol, than across a more fluid bilayer composed of unsaturated glycerophospholipids.

In insects, although lipids are stored in the form of triacylglycerols in fat bodies, they are transported in hemolymph (the insect equivalent of plasma) in the form of sn-1,2-diacylglycerols bound to the lipoprotein lipophorin to those tissues where they are required as a source of energy (see our web page on lipoproteins).

Catabolism: Diacylglycerols are hydrolysed efficiently by hormone sensitive lipase in adipose tissue and by diacylglycerol lipases at the plasma membrane and intracellularly in a number of tissues. Of course, they are also removed from cells when they are utilized as intermediates in the synthesis of other lipids.


3.  Diacylglycerol Metabolism in Plants and Microorganisms

In plants and microorganisms, sn-1,2-diacylglycerols are essential intermediates in the biosynthesis of glycerolipids, including phospholipids and the mono- and digalactosyldiacylglycerols. By perturbing membrane structure, diacylglycerols may affect plant enzyme activity indirectly, but there appears to be limited evidence only for the existence of a diacylglycerol signalling pathway in higher plants. Only one enzyme related to the protein kinase C family tends to be found in plants (PKCδ) that might potentially be regulated, but no relevant receptor has been detected. Rather, diacylglycerols generated by the action of non-specific phospholipases C, six forms of which are known in Arabidopsis, are rapidly phosphorylated by diacylglycerol kinases to phosphatidic acid, and this is believed to be the key second messenger in plants. On the other hand, there is at least one phosphatidylinositol-specific phospholipase C and there is evidence that some stress conditions can be alleviated by administering diacylglycerols but not phosphatidic acid. Further, diacylglycerols produced by the action of phospholipase C may participate in remodelling of lipids in membranes, general lipid metabolism and cross-talk with other phospholipid signalling systems.

Diacylglycerols are also formed in bacteria as a by-product of the biosynthesis of lipoteichoic acids and membrane-derived oligosaccharides from phosphatidylglycerol. In addition, phosphatidylcholine-specific phospholipases C have been identified in bacteria such as Bacillus cereus.


References



Lipid listings Credits/disclaimer Updated: June 4th, 2017 Author: William W. Christie LipidWeb icon