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Triacylglycerols: 2. Biosynthesis and Metabolism



All eukaryotic organisms and even a few prokaryotes are able to synthesise triacylglycerols, and in animals, many cell types and organs have this ability, but the liver, intestines and adipose tissue are most active with most of the body stores in the last of these (see our web page on triacylglycerol composition). Within all cell types, even those of the brain, triacylglycerols are stored as cytoplasmic 'lipid droplets' enclosed by a monolayer of phospholipids and hydrophobic proteins such as the perilipins in adipose tissue or oleosins in seeds. These lipid droplets are now treated as distinctive organelles, with their own characteristic metabolic pathways and associated enzymes - no longer boring blobs of fat. However, they are not unique to animals and plants, as Mycobacteria and yeasts have similar lipid inclusions.

The lipid serves as a store of fatty acids for energy, which can be released rapidly on demand, and as a reserve of structural fatty acids and precursors for eicosanoids. However, lipid droplets may also serve as a protective agency to remove any excess of biologically active and potentially harmful lipids such as free fatty acids, oxylipins, diacylglycerols, cholesterol (as cholesterol esters), retinol esters and coenzyme A esters from cells.


2.1.  Biosynthesis of Triacylglycerols

Three main pathways for triacylglycerol biosynthesis are known, the sn-glycerol-3-phosphate and dihydroxyacetone phosphate pathways, which predominates in liver and adipose tissue, and a monoacylglycerol pathway in the intestines. In maturing plant seeds and some animal tissues, a fourth pathway has been recognized in which a diacylglycerol transferase is involved. The most important route to triacylglycerols is the sn-glycerol-3-phosphate or Kennedy pathway, first described by Professor Eugene Kennedy and colleagues in the 1950s, by means of which more than 90% of liver triacylglycerols are produced.

Kennedy pathway of triacylgycerol biosynthesis

In this pathway, the main source of the glycerol backbone has long been believed to be sn-glycerol-3-phosphate produced by the catabolism of glucose (glycolysis) or to a lesser extent by the action of the enzyme glycerol kinase on free glycerol. However, there is increasing evidence that a significant proportion of the glycerol is produced de novo by a process known as glyceroneogenesis via pyruvate. Indeed, this may be the main source in adipose tissue.

Subsequent reactions occur in the endoplasmic reticulum. First, the precursor sn-glycerol-3-phosphate is esterified by a fatty acid coenzyme A ester in a reaction catalysed by a glycerol-3-phosphate acyltransferase (GPAT) at position sn-1 to form lysophosphatidic acid, and this is in turn acylated by an acylglycerophosphate acyltransferase (AGPAT) in position sn-2 to form a key intermediate in the biosynthesis of all glycerolipids - phosphatidic acid. Numerous isoforms of these enzymes are known and they may be expressed and regulated in different ways.

The phosphate group is removed by an enzyme (or family of enzymes) phosphatidic acid phosphohydrolase (PAP or ‘phosphatidate phosphatase’ or ‘lipid phosphate phosphatase’). PAP is also important as it produces sn-1,2-diacylglycerols as essential intermediates in the biosynthesis of phosphatidylcholine and phosphatidylethanolamine. In contrast to the activity responsible for phospholipid biosynthesis in mammals, much of the phosphatase activity leading to triacylglycerol biosynthesis resides in three related cytoplasmic proteins, termed lipin-1, lipin-2 and lipin-3, which were characterized before the nature of their enzymatic activities were determined. The lipins are tissue specific, and each appears to have distinctive expression and functions, but lipin-1 (PAP1) accounts for all the PAP activity in adipose tissue and skeletal muscle. While it occurs mainly in the cytosolic compartment of cells, it is translocated to the endoplasmic reticulum in response to elevated levels of fatty acids within cells. Lipin-1 activity requires Mg2+ ions and is inhibited by N-ethylmaleimide, whereas the membrane-bound activity responsible for synthesising diacylglycerols as a phospholipid intermediate is independent of Mg2+ concentration and is not sensitive to the inhibitor.

Scottish thistlePerhaps surprisingly, lipin-1 has a dual role in that it operates in collaboration with known nuclear receptors as a transcriptional coactivator to modulate lipid metabolism and the expression of genes involved in lipid metabolism. Abnormalities in lipin-1 expression are known to be involved in some human disease states that may lead to the metabolic syndrome. Lipin 2 is a similar phosphatidate phosphohydrolase, which is present in liver and brain and is regulated dynamically by fasting and obesity (in mice), while lipin 3 is found in the gastrointestinal tract and liver.

In the final step in this pathway, the resultant 1,2-diacyl-sn-glycerol is acylated by diacylglycerol acyltransferases (DGAT), which can utilize a wide range of fatty acyl-CoA esters, to form the triacyl-sn-glycerol. As the glycerol-3-phosphate acyltransferase has the lowest specific activity of these enzymes, this step may be the rate-limiting one. DGAT is the dedicated triacylglycerol-forming enzyme, so it is seen as a target for pharmaceutical intervention in obesity and attendant ailments.

In fact there are two DGAT enzymes, which are structurally and functionally distinct. In animals, DGAT1 is located mainly in the endoplasmic reticulum and is expressed in skeletal muscle, skin and intestine, with lower levels of expression in liver and adipose tissue. Perhaps surprisingly, it is the only one present in the epithelial cells that synthesise milk fat in the mammary gland. Orthologues of this enzyme are present in most eukaryotes, other than yeasts, and it is especially important in plants. Also DGAT1 can utilize a wider range of substrates, including monoacylglycerols, long-chain alcohols (for wax synthesis) and retinol. DGAT2 is the main form of the enzyme in hepatocytes and adipocytes (lipid droplets), although it is expressed much more widely in tissues; it is associated with distinct regions of the endoplasmic reticulum, at the surface of lipid droplets and in mitochondria. Both enzymes are important modulators of energy metabolism, although DGAT2 appears to be especially important in controlling the homeostasis of triacylglycerols in vivo.

In second pathway, dihydroxyacetone-phosphate in peroxisomes or endoplasmic reticulum can be acylated by a specific acyltransferase to form 1-acyl dihydroxyacetone-phosphate, which is reduced by dihydroxyacetone-phosphate oxido-reductase to lysophosphatidic acid, which can then enter the pathway above to triacylglycerols. The precursor dihydroxyacetone-phosphate is important also as part of the biosynthetic route to plasmalogens.

Biosynthesis of triacylglycerols via dihydroxyacetone phosphate

In the enterocytes of intestines after a meal, up to 75% of the triacylglycerols are formed via a monoacylglycerol pathway. 2-Monoacyl-sn-glycerols and free fatty acids released from dietary triacylglycerols by the action of pancreatic lipase within the intestines (see below) are taken up by the enterocytes. There, the monoacylglycerols are first acylated by an acyl coenzyme A:monoacylglycerol acyltransferase with formation of sn-1,2-diacylglycerols mainly as the first intermediate in the process, though some sn-2,3-diacylglycerols (~10%) are also produced (DGAT1 can also acylate monoacylglycerols). Finally, the acyl coenzyme A:diacylglycerol acyltransferase (DGAT1) reacts with the sn-1,2-diacylglycerols only to form triacylglycerols. While enzymes of the monoacylglycerol pathway are present in the liver, the source of the potential monoacylglycerol substrate is not known.

Monoacylglycerol pathway of triacylglycerol biosynthesis

In a fourth biosynthetic pathway, which is less well known, triacylglycerols are synthesised by a transacylation reaction between two racemic diacylglycerols that is independent of acyl-CoA. The reaction was first detected in the endoplasmic reticulum of intestinal micro villus cells and is catalysed by a diacylglycerol transacylase. Both diacylglycerol enantiomers participate in the reaction with equal facility to transfer a fatty acyl group with formation of triacylglycerols and a 2-monoacyl-sn-glycerol. A similar reaction has been observed in seed oils.

Triacylglycerol formation via diacylglycerol transacylases

It has been suggested that this enzyme may function in remodelling triacylglycerols post synthesis, especially in oil seeds, and it is possible that it may be involved in similar processes in the liver and adipose tissue, where extensive hydrolysis/re-esterification is known to occur. There is evidence for selectivity in the biosynthesis of different molecular species in a variety of tissues and organisms, which may be a consequence of the varying biosynthetic pathways. Also in adipose tissue, fatty acids synthesised de novo are utilized in different ways from those from external sources in that they enter positions sn-1 and 2 predominantly, while a high proportion of the oleic acid synthesised in the tissue by desaturation of exogenous stearic acid is esterified to position sn-3.

In prokaryotes, the glycerol-3-phosphate pathway of triacylglycerol biosynthesis only occurs, but in yeast both glycerol-3-phosphate and dihydroxyacetone-phosphate can be the primary precursors and synthesis takes place in cytoplasmic lipid droplets and the endoplasmic reticulum. In plants, the glycerol-3-phosphate pathway is most important, but these process are discussed below in greater detail.

Among other potential routes to the various intermediates, lysophosphatidic acid and phosphatidic acid can be synthesised in mitochondria, but must then be transported to the endoplasmic reticulum before they enter the pathway for triacylglycerol production. 1,2-Diacyl-sn-glycerols are also produced by the action of phospholipase C on phospholipids.

In the glycerol-3-phosphate and other pathways, the starting material is of defined stereochemistry and each of the enzymes catalysing the various steps in the process is distinctive and can have preferences for particular fatty acids (as their coenzyme A esters) and for particular fatty acid combinations in the partially acylated intermediates. It should not be surprising, therefore, that natural triacylglycerols exist in enantiomeric forms with each position of the sn-glycerol moiety esterified by different fatty acids, as discussed in Triacylglycerols - Part 1.

While triacylglycerols are essential for normal physiology, an excessive accumulation in human adipose tissue and other organs results in obesity and other health problems, including insulin resistance, steatohepatitis and cardiomyopathy. Accordingly, there is considerable pharmaceutical interest in drugs that affect triacylglycerol biosynthesis and metabolism.


2.2.  Triacylglycerol Metabolism in the Intestines, Liver and Mammary Gland

Fat comprises up to 40% of the energy intake in the human diet in Western countries, and a high proportion of this is triacylglycerols. The process of fat digestion is begun in the stomach by acid-stable gastric or lingual lipases, the extent of which depending on species but may be important for efficient emulsification. However, this is insignificant in quantitative terms in comparison to the reaction with pancreatic lipase, which occurs in the duodenum. Entry of triacylglycerol degradation products into the duodenum stimulates synthesis of the hormone cholecystokinin and causes the gall bladder to release bile acids, which are strong detergents and act to emulsify the hydrophobic triacylglycerols so increasing the available surface area. In turn, cholecystokinin stimulates the release of the hydrolytic enzyme pancreatic lipase together with a co-lipase, which is essential for the activity of the enzyme. Pancreatic lipase, co-lipase, bile salts and calcium ions act together in a complex at the surface of the emulsified fat droplets to hydrolyse triacylglycerols. The process is regiospecific and results in the release of the fatty acids from the 1 and 3 positions with formation of 2-monoacyl-sn-glycerols. Isomerization of the latter to 1(3)-monoacyl-sn-glycerols occurs to some extent, and these can be degraded completely by the enzyme to glycerol and free fatty acids. Other lipases hydrolyse the phospholipids and other complex lipids in foods at the same time.

Pancreatic lipase action

This process is somewhat different in neonates and young infants, in whom pancreatic lipase is less active but is effectively replaced by lipases in breast milk and by an acid gastric lipase (pH optimum 4-6).

There is evidence that the regiospecific structure of dietary triacylglycerols has an effect on the uptake of particular fatty acids and may influence further the lipid metabolism in humans. In particular, incorporation of palmitic acid into the position sn-2 of milk fat may be of benefit to the human infant (as a source of energy for growth and development), although it increases the atherogenic potential for adults. In addition, 2-monoacylglycerols and 2-oleoylglycerol especially have a signalling function in the intestines by activating a specific G-protein coupled receptor GPR119, sometimes termed the ‘fat sensor’. When stimulated, this causes a reduction in food intake and body weight gain in rats and regulates glucose-stimulated insulin secretion. The free fatty acids released have a similar effect, though by a very different mechanism, via the receptor GPR40. Overall, it has become evident that triacylglycerol metabolism in the intestine has regulatory effects on the secretion of gut hormones and on systemic lipid metabolism and energy balance.

The free fatty acids and 2-monoacyl-sn-glycerols are rapidly taken up by the intestinal cells, from the distal duodenum to the jejunum, via specific carrier molecules but also by passive diffusion. A specific fatty acid binding protein prevents a potentially toxic build-up of unesterified fatty acids and targets them for triacylglycerol biosynthesis. The long-chain fatty acids are converted to the CoA esters and esterified into triacylglycerols by the monoacylglycerol pathway as described above. In contrast, short and medium-chain fatty acids (C12 and below) are absorbed in unesterified form and pass directly into the portal blood stream, where they are transported to the liver to be oxidized.

Subsequently, the triacylglycerols are incorporated into lipoprotein complexes termed chylomicrons in the enterocytes by processes discussed in greater detail in our web page dealing with lipoproteins.. In brief, these consist of a core of triacylglycerols together with some cholesterol esters that is stabilized and rendered compatible with an aqueous environment by a surface film consisting of phospholipids, free cholesterol and one molecule of a truncated form of apoprotein B (48 kDa). These particles are secreted into the lymph and thence into the plasma for transport to the peripheral tissues for storage or structural purposes. Adipose tissue in particular exports appreciable amounts of the enzyme lipoprotein lipase, which binds to the luminal membrane of endothelial cells facing into the blood, where it rapidly hydrolyses the passing triacylglycerols at the cell surface releasing free fatty acids, most of which are absorbed into the adjacent adipocytes and re-utilized for triacylglycerol synthesis within the cell.

A schematic chylomicronThe chylomicrons remnants eventually reach the liver, where the remaining lipids are hydrolysed at the external membranes by a hepatic lipase and absorbed. The fatty acids within the liver can be utilized for a variety of purposes, from oxidation to the synthesis of structural lipids, but a proportion is re-converted into triacylglycerols, and some of this is stored as lipid droplets within the cytoplasm of the cells (see next section). In addition, phosphatidylcholine from the high-density lipoproteins is taken up by the liver, and a high proportion of this is eventually converted to triacylglycerols. Excessive accumulation of storage triacylglycerols is associated with fatty liver, insulin resistance and type 2 diabetes.

Most of the newly synthesised triacylglycerols are exported into the plasma in the form of very-low-density lipoproteins (VLDL), consisting again of a triacylglycerol and cholesterol ester core, surrounded by phospholipids and free cholesterol, together with one molecule of full-length apoprotein B (100 kDa), apoprotein C and sometimes apoprotein E. These particles in turn are transported to the peripheral tissues, where they are hydrolysed and the free acids absorbed. Eventually, the remnants are returned to the liver.

In the mammary gland, triacylglycerols are synthesised in the endoplasmic reticulum and large lipid droplets are produced with a monolayer of phospholipids derived from this membrane. These are transported to the plasma membrane and bud off into the milk with an envelope comprised of the phospholipid membrane to form milk fat globules as food for the newborn. The process is thus very different from that involved in the secretion of triacylglycerol-rich lipoproteins from other organs.


2.3.  Triacylglycerol Synthesis and Catabolism (Lipolysis) in Adipocytes and Lipid Droplets

Adipose tissue and the adipocytes are characterized by accumulations of triacylglycerols, which act as the main energy store for animals and as a reserve of bioactive lipids. They also provide structural components, including cholesterol and retinol, for membrane synthesis and repair. The triacylglycerol droplets are surrounded by a protective monolayer that includes phospholipids, cholesterol and hydrophobic proteins. The phospholipid component of the monolayer consists mainly of phosphatidylcholine and phosphatidylethanolamine with fatty acid compositions distinct from those of the endoplasmic reticulum and plasma membrane. Among the proteins are many that function directly in lipid metabolism, and include lipases, perilipins, caveolins and the Adipose Differentiation Related Protein (ADRP or adipophilin). Similarly, within most other animal cells, even ganglia in the brain, a proportion of the fatty acids taken up from the circulation is converted to triacylglycerols as described above and incorporated into cytoplasmic lipid droplets (also termed 'fat globules', 'oil bodies', 'lipid particles', 'adiposomes', etc). By buffering against fatty acid accumulation that might exceed the capacity of non-adipose cells, they defend them against lipotoxicity. In adipocytes, the lipid droplets can range to up to 200 μm in diameter, while other cell types contain smaller lipid droplets of the order of 50 nm in diameter. Cytosolic lipid droplets with similar metabolic activities are found in the fly Drosophila melanogaster, and in higher plants and yeasts (see below).

Lipid droplet assembly is believed to be initiated in the endoplasmic reticulum, leading to accumulation of diacylglycerols, which attracts perilipins and other proteins and allows lipid droplets to grow in patches of the endoplasmic reticulum. As phospholipid bilayers can support a few mole percent of neutral lipids while retaining their stability, it is suggested that a low level of triacylglycerols diffuse freely within the membrane until saturation is reached and droplets form. Once the nascent lipid droplet emerges, mitochondria, peroxisomes and other organelles contribute further lipids and effect changes in protein composition. In adipocytes, lipid droplets can grow by fusion of smaller droplets, although the mechanism is not fully understood.

Some of the surface proteins on lipid droplets can extend long helical hairpins of hydrophobic peptides deep into the lipid core. For example, perilipins constitute a family of at least five phosphorylated proteins that bind to droplets in animals and share a common region, the so-called ‘PAT’ domain, named for the three original members of the family that include perilipin and ADRP. Proteins related evolutionarily to these are found in more primitive organisms, including insects, slime moulds and fungi, but not in the nematode Caenorhabditis elegans. In mammals, perilipin A (or 'PLIN1' or more accurately the splice variant 'PLIN1a') is a well-established regulator of lipolysis in adipocytes, and it is believed to be involved in the formation of the large lipid droplets in white adipose tissue. The perilipins PLIN1 and PLIN2 have functions in triacylglycerol metabolism in tissues other than adipocytes, and PLIN2 in particular is the main perilipin in hepatocytes; PLIN5 operates in tissues that oxidize fatty acids such as the heart. Other surface proteins of lipid droplets are enzymes intimately involved in triacylglycerol metabolism, although there is a suggestion that cytoplasmic droplets may act as a storage organelle for hydrophobic proteins whose function is elsewhere in the cell.

cartoonOn the basis of profiling of the surface proteins and phospholipids, lipid droplets in cells are now considered to be complex, metabolically active organelles that function in the supply of fatty acids for various purposes, including membrane trafficking and possibly in the recycling of both simple and complex lipids. For example, within the liver, triacylglycerols are stored as lipid droplets in the cytoplasm adjacent to the endoplasmic reticulum where a triacylglycerol hydrolase can effect lipolysis to di- and monoacylglycerols that are more soluble in the membrane, which they are able to cross. They are then available for re-synthesis into triacylglycerols by luminally oriented acyltransferases before assembly into nascent lipoprotein complexes. Similar organelles can be found in most eukaryotic cells and in bacteria, and they provide a reservoir not only for triacylglycerols but also for eicosanoids, esterified cholesterol and in some specialized cells of retinol esters, for example. This process is especially important in adipose tissue, which is the major energy-dense store of lipids in animals.

Lipolysis: When fatty acids are required by other tissues for energy or other purposes, they are released from the triacylglycerols by the sequential actions of three enzymes, i.e. adipose triacylglycerol lipase (ATGL), hormone-sensitive lipase (HSL) and monoacylglycerol lipase. Simplistically, ATGL hydrolyses triacylglycerols to diacylglycerols, which are hydrolysed by HSL to monoacylglycerols before these are hydrolysed by the monoacylglycerol lipase. Perilipin (PLIN1) has been described as "the gatekeeper of the adipocyte lipid storehouse". Thus, the lipolytic process is regulated by perilipin, which acts as a barrier to lipolysis in non-stimulated cells, but on stimulation as during fasting is phosphorylated by the cAMP-protein kinase. This changes its shape and reduces its hydrophobicity, and in the process activates lipolysis. An isoform, perilipin A, is the main regulatory factor in white adipose tissue.

The adipose triacylglycerol lipase was discovered relatively recently and its properties are now being revealed. It is structurally related to the plant acyl-hydrolases in that it has a patatin-like domain in the NH2-terminal region (patatin is a non-specific acyl-hydrolase in potato) and is located on the surface of the lipid droplet both in the basal and activated states. This lipase is specific for triacylglycerols and yields diacylglycerols and free fatty acids as the main products, with low activity only towards diacylglycerols, and none to monoacylglycerols, retinol esters and cholesterol esters (although it also has transacylase and phospholipase activities). Adipose triacylglycerol lipase can be activated at the same time as hormone-sensitive lipase and is now believed to be rate limiting for the first step in triacylglycerol hydrolysis. Regulation of the enzymatic activity is a complex process, and for example, a lipid droplet protein designated ‘CGI-58’ or ‘ABHD5’, is known to be an important activating factor. In the resting state this protein binds to perilipin, but on phosphorylation of the latter, it dissociates and interacts with adipose triacylglycerol lipase to activate triacylglycerol hydrolysis. Mutations in adipose triacylglycerol lipase or CGI-58 are believed to be responsible for a syndrome in humans known as ‘neutral lipid storage disease’.

Hormone-sensitive lipase is regulated by the action of the hormones insulin and noradrenaline by a mechanism that ultimately involves phosphorylation of the enzyme by cAMP-protein kinase (as with perilipin), thereby increasing its activity and causing it to translocate from the cytosol to the lipid droplet. In addition to its activity towards triacylglycerols, hormone-sensitive lipase will rapidly hydrolyse diacylglycerols, monoacylglycerols, retinol esters and cholesterol esters. In fact, diacylglycerols are hydrolysed ten times as rapidly as triacylglycerols. Within the triacylglycerol molecule, hormone-sensitive lipase preferentially hydrolyses ester bonds in the sn-1 and sn-3 positions, leaving free acids and 2-monoacylglycerols as the main end products.

Triacylglycerol hydrolysis in adipose tissue

The monoacylglycerol lipase is believed to be the rate-limiting enzyme in monoacylglycerol hydrolysis, i.e. the final step in triacylglycerol catabolism releasing free glycerol and fatty acids, and is found in the cytoplasm, the plasma membrane, and in lipid droplets. It is specific for monoacylglycerols and has no activity against di- or triacylglycerols. As it is the enzyme mainly responsible for deactivation of the endocannabinoid 2-arachidonoylglycerol and is highly active in malignant cancers, it is attracting pharmaceutical interest.

Free fatty acids released by the combined action of these enzymes are exported into the plasma for transport to other tissues in the form of albumin complexes, while the glycerol released is transported to the liver for metabolism by either glycolysis or gluconeogenesis. Eventually, the whole organelle can disappear, including the proteins, when they undergo a process of autophagy ('lipophagy'), i.e. the delivery of the organelles to lytic compartments for degradation. This process is especially important during starvation, and while it is mechanistically distinct from lipolysis, there is cross-talk between the two.

Other functions of lipid droplets: Not only does the adipocyte provide a store of energy but it manages the flow of energy through the formation of the hormone leptin, which stimulates secretion of various factors that communicate with other tissues, including cytokines, adiponectin and resistin. The synthesis of leptin is tightly controlled by adipocytes and its main function is believed to be the provision of information on the state of fat stores to other tissues. Lipid droplets may play a role in this process, since perilipin is required for the sensing function. As caveolae, which contain the proteins caveolins (and presumably sphingolipids) and are particularly abundant in adipocytes, modulate the flux of fatty acids across the plasma membrane and are involved in signal transduction and membrane trafficking pathways, it is evident that they have a major role in this aspect of lipid metabolism. In addition, insulin is the main hormone that affects metabolism and its receptor at the plasma membrane is located in caveolae. Release of proinflammatory cytokines can stimulate lipolysis and cause insulin resistance, in turn leading to dysfunction of adipose tissue and systemic disruption of metabolism. Thus, adipose tissue metabolism has profound effects on whole-body metabolism, and defects in these processes can have severe implications for the pathogenesis of diabetes and obesity in humans.

Lipid droplets accumulate within many cell types other than adipocytes, including leukocytes, epithelial cells and hepatocytes, especially during infectious, neoplastic and other inflammatory conditions. These are associated with a variety of enzymes, including protein kinases, which are involved in many different aspects of lipid metabolism, including cell signalling, membrane trafficking and control of the production of inflammatory mediators such as eicosanoids. For example, the triacylglycerols in cytoplasmic lipid droplets of human mast cells have a high content of arachidonic acid, which can be released by adipose triacylglycerol lipase as a substrate for eicosanoid production. Similarly, triacylglycerols in lipid droplets of the skin are a highly specific source of the linoleic acid that is required for the formation of the O-acylceramides, which are essential for epidermal barrier function. Lipid droplets in brain are vital for proper neuronal function. In the nucleus, they can sequester transcription factors and chromatin components and generate the lipid ligands for certain nuclear receptors.

In addition to their role in lipid biochemistry, lipid droplets participate in protein degradation and glycosylation. Their metabolism can be manipulated by pathogenic viruses with unfortunate consequences for the host, but they also serve as reservoirs for proteins that fight intracellular pathogens. In consequence, such lipid droplets and their enzyme systems may be markers for disease states and are also considered to be targets for pharmaceutical intervention.


2.4.  Brown Adipose Tissue

Most adipose tissue depots ('white fat') serve primarily as storage and endocrine organs that provide a reservoir of nutrients for release when the food supply is low. However, a second specialized form of adipose tissue, brown fat, is highly vascularized and rich in mitochondria and the iron-containing pigments that transport oxygen and give the tissue its colour and name. In humans, these depots tend to be located in subcutaneous areas around the neck, and elsewhere near the heart, kidney, pancreas and liver. Brown fat is able to oxidize fat so rapidly that heat is generated (“non-shivering thermogenesis”), and it is especially important in young animals and in those recovering from hibernation. In brief, during cold exposure, release of noradrenaline and stimulation of β-adrenergic receptors in the nervous system initiate a catabolic program that commences with rapid breakdown of cellular triacylglycerol stores and transient activation of a co-activator of peroxisome proliferator-activated receptor gamma (PPARγ), which results in the efficient utilization of fatty acids to producing heat. The key molecule is believed to be the uncoupling protein-1 (UPC1), which uncouples electron transport in the respiratory chain from ATP production with a highly exothermic release of chemical energy.

In mice, brown adipose tissue is especially important metabolically and can consume about 50% of dietary lipids and glucose when the animals are exposed to cold. Similarly, in humans, the activity of brown adipose tissue is induced acutely by cold and is stimulated via the sympathetic nervous system, and the relevance of this tissue to human metabolism is now becoming apparent. For example, there are suggestions that brown adipose tissue can behave as an endocrine system to secrete endocrine factors ('batokines') that may be favourable towards cardiovascular risk. For obvious reasons, there are efforts to determine whether sustained activation of brown fat by pharmaceutical means could be beneficial towards a number of human disease states, including obesity, diabetes and cardiovascular disease.


2.5.  Other Functions of Triacylglycerol Depots

Subcutaneous depots act as a cushion around joints and serve as insulation against cold in many terrestrial animals, as is obvious in the pig, which is surrounded by a layer of fat, and it is especially true for marine mammals such as seals. Those adipocytes embedded in the skin differ from the general subcutaneous depots and support the growth of hair follicles and regenerating skin, and they may also have a defensive role both as a physical barrier and by responding metabolically to bacterial infection

In marine mammals and fish, the fat depots are less dense than water and so aid buoyancy with the result that less energy is expended in swimming. More surprisingly perhaps, triacylglycerols together with the structurally related glyceryl ether diesters and wax esters are the main components of the sonar lens used in echo-location by dolphins and some whales. The triacylglycerols are distinctive in that they contain two molecules of 3-methylbutyric (isovaleric) acid with one long-chain fatty acid. It appears that the relative concentrations of the various lipids in an organ in the head of the animals (termed the ‘melon’) vary in such a way that they are able to focus sound waves.


2.6.  Triacylglycerol Metabolism in Plants and Yeasts

Fruit and seed oils are major agricultural products with appreciable economic and nutritional value to humans. The mesocarp of fruits is a highly nutritious energy source that attracts animals that help to disperse the seeds, and in plants such as the oil palm and olives a high proportion of the flesh contains triacylglycerols. Similarly in seeds, triacylglycerols are the main storage lipid and can comprise as much as 60% of their weight. Fruit lipids are not intended for use by the plant per se and are stored in lipid droplets in large irregular structures that break down readily, but seed lipids are required for the development of the plant embryo so their metabolism is of particular importance.

In plants, fatty acids synthesised in the plastid compartment are stored in the embryo or endosperm tissues of seeds as triacylglycerols in lipid droplets with a coherent surface layer of proteins and lipids. In addition to the common range of fatty acids synthesised in plastids, some plant species produce novel fatty acids, including medium- and very-long-chain components and those with oxygenated and other functional moieties. Some very specific means of diverting these to seeds and triacylglycerol production must exist to prevent disruption of the plant membranes. Seed development occurs in three stages - rapid cell division with no accumulation of storage material, rapid deposition of triacylglycerols and other energy-rich metabolites, and finally desiccation. In comparison, relatively little triacylglycerol biosynthesis and metabolism occurs in tissues other than developing seeds (but see the note on plastoglobules below).

During the period of oil accumulation in seeds, there must be a mechanism to hydrolyse the newly formed ACP esters of fatty acids and export the unesterified fatty acids to the endoplasmic reticulum, where they are converted to the CoA esters and triacylglycerols are synthesised by the Kennedy and other pathways described above. In yeast and plants, diacylglycerol esterification is the only committed step in triacylglycerol production and this occurs by mechanisms that can be both dependent or independent of acyl-CoA esters. The acyl-CoA-dependent route is catalysed by diacylglycerol:acyl-CoA acyltransferases (DGATs) with acyl-CoA and diacylglycerols as substrates. In plants, two membrane-bound enzymes (DGAT1 and DGAT2) and a cytosolic enzyme (DGAT3) are known, while the acyl-CoA-independent reaction utilizes a phospholipid:diacylglycerol acyltransferase with phospholipids as acyl donor and diacylglycerol as acyl acceptor to produce triacylglycerols and lysophospholipids. DGAT2 is especially important in those plant species with unusual fatty acid compositions.

In addition, a substantial proportion in some species is synthesised by a flux through the membrane phospholipid phosphatidylcholine, produced by both the eukaryotic and prokaryotic pathways with differing positional distributions (see our web-page on galactosyldiacylglycerols), in which diacylglycerols are generated by the action of a phosphatidate phosphatase as an intermediate. As phosphatidylcholine undergoes extensive remodelling and its fatty acid components are subject to modification, for example by desaturation to form linoleic and linolenic acids, the compositions and especially the positional distributions of triacylglycerols produced in this way can be very different from those synthesised by the ‘classical’ pathways. Phosphatidylcholine may also function as a carrier for the trafficking of acyl groups between organelles and membrane subdomains. As triacylglycerol synthesis continues, oil droplets accumulate in the endoplasmic reticulum and are surrounded by a monolayer of phospholipids and proteins, which in Arabidopsis include oleosins, a caleosin, a steroleosin, a putative aquaporin and a glycosylphosphatidylinositol-anchored protein.

Scottish thistle At the onset of germination, water is absorbed and lipases are activated. The process of lipolysis begins at the surface of oil bodies, where the oleosins, which are the most abundant structural proteins, are believed to serve to assist the docking of lipases. They also control the size and stability of lipid droplets in seeds. A number of lipases have been cloned from various plant species and are typical α/β-hydrolases, with a conserved catalytic triad of Ser, His, and Asp or Glu as in patatin (an especially abundant lipolytic protein in potatoes), which are able to hydrolyse triacylglycerols but not phospho- or galactolipids. The most important of these is believed to be the 'sugar-dependent lipase 1 (SDP1)', which is a patatin-like lipase similar to the mammalian adipose triacylglycerol lipase discussed above, and is located on the surface of the oil body. This is active mainly against triacylglycerols to generate diacylglycerols, but presumably works in conjunction with di- and monoacylglycerol lipases to generate free fatty acids and glycerol.

The lipid droplets in seeds exist in close proximity with glyoxysomes (broadly equivalent to peroxisomes). These are the membrane-bound organelles that contain most of the enzymes required to oxidize fatty acids derived from the triacylglycerols via acetyl-CoA to four-carbon compounds, such as succinate, which are then converted to soluble sugars to provide germinating seeds with energy to fuel the growth of the seedlings and to produce shoots and leaves. In addition, they supply structural elements before the seedlings develop the capacity to photosynthesise. How the products of lipolysis are transported to the glyoxysomes for further metabolism has still to be determined, but a specific ‘ABC’ transporter is required to import fatty acids into the glyoxysomes in Arabidopsis. The free acids are converted to their coenzyme A esters by two long-chain acyl-CoA synthetases located on the inner face of the peroxisome membrane before entry into the β-oxidation pathway. All of these processes are controlled by an intricate regulatory network, involving transcription factors that crosstalk with signalling events from the seed maturation phase through to embryo development. After about two days of the germination process, the glycoxysomes begin to break down, but β-oxidation can continue in peroxisomes in leaf tissue.


Lipid droplets - plastoglobules: Triacylglycerol-rich lipid droplets have been observed in most cell types in plants, and although their origin and function are poorly understood, they contain all the enzymes required for triacylglycerol metabolism together with phospholipases, lipoxygenases and other oxidative enzymes. They are believed to be involved in stress responses, but they may have other specialized roles, for example in anther development, where triacylglycerols serve as a source of fatty acids for membrane biosynthesis. Fatty acids derived from triacylglycerols in lipid droplets are believed to be subjected to peroxisomal β-oxidation to produce the ATP required for stomatal opening and no doubt many other purposes. During senescence, lipid droplets accumulate rapidly in A. thaliana leaves. Antifungal compounds such as 2-hydroxy-octadecanoic acid are then produced from α-linolenic acid in these organelles, and it has been suggested that they function as intracellular factories to produce stable metabolites via unstable intermediates by concentrating the enzymes and hydrophobic substrates in an efficient manner. Lipid droplets that have been termed 'plastoglobules' are present in the curved regions of thylakoid membranes of chloroplasts, where they are involved in a wide range of biological functions from biogenesis to senescence via the recruitment of specific proteins. Plastoglobules are also implicated in the biosynthesis and metabolism of vitamins E and K.

Microalgae and yeasts: Triacylglycerol metabolism in lipid droplets in microalgae is under intensive study because of the potential for biodiesel production. It seems that similar processes occur as in higher plants, but with a simpler genome encoding few redundant proteins. The size and triacylglycerol content of lipid droplets in yeasts change appreciably in different stages of growth and development. As most of the important biosynthetic and catabolic enzymes involved in triacylglycerol metabolism are conserved between yeasts and mammals, the former are proving to be useful models for the study of triacylglycerol homeostasis.


2.7.  Triacylglycerol Metabolism in Prokaryotes

Study of the biosynthesis of triacylglycerols in bacteria has been stimulated by an awareness of the role of this lipid class in the pathogenesis of Mycobacterium tuberculosis and the relationship with antibiotic biosynthesis by Streptomyces coelicolor. For example, triacylglycerols are believed to be an energy reserve for the long-term survival of M. tuberculosis during the persistence phase of infection as well as a means by which unesterified fatty acids are detoxified. Increasing numbers of bacterial species are now known to produce triacylglycerols, with the first three steps catalysed by GPAT, LPAT and PAP enzymes comparable to those in other organisms. However, it has become apparent that the DGAT can be a dual-function CoA-dependent acyltransferase known as wax ester synthase/diacylglycerol acyltransferase, which accepts a broad diversity of acyl-CoA substrates for esterification of diacylglycerols or long-chain fatty alcohols for the synthesis of triacylglycerols or wax esters, respectively, depending on which intermediates are present in the organisms. Bacteria that lack such an enzyme are unable to produce these non-polar lipids.


An earlier document in this section dealing with triacylglycerols (Triacylglycerols. Part 1) discusses their structure and compositions in animals, plants and other organisms.


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Lipid listings Credits/disclaimer Updated: June 23rd, 2017 Author: William W. Christie LipidWeb icon