The Integration And Control Of Animal Acylglycerol Metabolism

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The major function of TAG in animals is as a source of fatty acids to be used as metabolic fuel. A full description of the overall fuel economy of the body requires an understanding of the metabolism (storage, transport, synthesis, oxidation) of the fatty acids as well as of the integration of fat and carbohydrate metabolism.

3.6.1 Fuel economy: the interconversion of different types of fuels is hormonally regulated to maintain blood glucose concentration within the normal range and ensure storage of excess dietary energy in triacylglycerols

The maintenance of fuel reserves within fairly narrow limits is referred to as fuel homeostasis. Glycogen is stored within muscle and liver cells (and in small amounts elsewhere, e.g. in the brain). Because glycogen is stored in a hydrated form (Section 3.3.1) the amount that can be stored in a cell is limited, and also it would be disadvantageous for the body to carry around excessive amounts of this fuel source. In adult humans the liver glycogen store is typically about 100 g, and it is probably no coincidence that this is approximately the amount of glucose needed by the brain in 24 h. Glycogen stores are for immediate, 'emergency' use in maintaining carbohydrate supply for tissues that require it, such as the brain, and their total amount is held within fairly narrow limits. Protein is potentially a major energy source (the adult human body has about 12.5 kg of protein). However, there is no specific storage form of protein, and so all body protein plays some role (enzymes, structural, etc.). Therefore, it is not generally used as a fuel store, and indeed its degradation during starvation is specifically spared. Again, therefore, its amount appears to be controlled within fairly narrow limits. In contrast, humans and other mammals appear to have an almost infinite capacity for storage of TAG, achieved both by expansion of existing adipocytes (so that a mature, fat-filled human adipocyte may be 0.1mm in diameter) and by an increase in the number of adipocytes.

The various fuels can be interconverted to some extent. Excess carbohydrate and protein can be converted into fat, and amino acids from protein can be converted into carbohydrate (as happens during starvation as a means of supplying new glucose). However, fatty acids cannot be converted into carbohydrate (Section 3.5.3; Fig. 3.9) and do not appear to be significant precursors of amino acids.

The constituent glycerol from triacylglycerols that have been hydrolysed to release fatty acids may, however, be an important precursor of glucose during starvation.

When the energy in the diet exceeds immediate requirements, excess carbohydrate is preferentially used to replenish glycogen stores. Excess protein tends to be oxidized after satisfying the tissues' needs for protein synthesis. Any remaining excess of either fuel, or of fat, then tends to be converted into triacylglycerol for storage in adipose tissue. Adipose tissue TAG is the ultimate repository for excess dietary energy.

The control of these interconversions is mediated by the amount of energy in the diet, the nature of the dietary constituents and the concentrations of the relevant hormones in the blood. A dominant role is played by insulin. Its concentration in the blood helps to co-ordinate the flow of fuel either into storage or from the stores into various tissues as required. High concentrations of insulin characterize the fed state when ample fuel is available from the diet; low levels signal the starved state when the animal's own reserves need to be called upon. However, it has recently become clear that dietary fatty acids themselves may play an important role in regulating the expression of various genes involved in these processes (Section 5.3).

3.6.2 The control of acylglycerol biosynthesis is important, not only for fuel economy, but for membrane formation, requiring close integration of storage and structural lipid metabolism

An important concept of metabolism is that of turnover, which envisages the continual renewal, involving biosynthesis and breakdown, of body constituents. The rates of turnover may differ widely between different biological molecules in different tissues. The rates of forward and backward reactions may also differ. Thus, when there is net synthesis of the tissue component, the rate of synthesis is faster than the rate of breakdown, but both may be proceeding simultaneously. When metabolic control is exercised, by whatever means, it may affect the rate of synthesis, the rate of degradation, or both. Turnover allows a fine degree of control of metabolic pathways. Control may be exercised on the synthesis or degradation of enzymes catalysing the metabolic reactions at the level of gene transcription or translation, or on the allosteric control of enzymes by small molecules or cofactors.

In animals, net TAG synthesis occurs when energy supply exceeds immediate requirements. Most diets contain both fat and carbohydrates. When there is an excess of energy as carbohydrates, the body switches mainly to a pattern of carbohydrate oxidation so that these are used as the preferential metabolic fuel. When there is a considerable excess of carbohydrates for some time, then the tissues convert them into fatty acids that are esterified into acylglycerols. Conversely, when there is a preponderance of fat in the diet, fat synthesis from carbohydrate is depressed in the tissues and fat oxidation and fat storage will tend to predominate. This involves conversion of the products of fat digestion into lipoproteins (Section 5.2), which circulate in the bloodstream. When the lipoproteins reach the tissues, fatty acids are released from the acylglycerols at the endothelial lining of the capillaries, a process catalysed by the enzyme lipoprotein lipase (Section 3.5.2). The fatty acids are taken up into the cells (Fig. 3.10). Once inside, they are either oxidized or esterified into acylglycerols. In discussing the control of acylgly-cerol synthesis, we shall be discussing the ester-ification of fatty acids synthesized de novo or released from circulating lipoproteins. The control of fatty acid synthesis itself is discussed in Section 2.2.8.

Although acylglycerols may be synthesized in many animal tissues, the most important are: the small intestine, which resynthesizes TAG from components absorbed after digestion of dietary fats; the liver, which is concerned mainly with synthesis from carbohydrates and redistribution; adipose tissue, which is concerned with longer term storage of fat; and the mammary gland, which synthesizes milk fat during lactation. There is also turnover of a relatively smaller pool of TAG within muscle cells.

Metabolisme Lipid

Fig. 3.10 Central role of acyl-CoA in hepatic lipid metabolism. Acyl-CoA is formed either from esterification (sometimes called activation) of a fatty acid with CoA, or from the pathway of de novo lipogenesis (DNL). It may be utilized for P-oxidation in the mitochondria (catabolism) or for glycerolipid biosynthesis. The relative rates of these pathways are determined by the nutritional state, mediated largely by variations in plasma insulin concentration. When insulin concentrations are high (well-fed state) (shown as Ins +), generation of malonyl-CoA through the activity of acetyl-CoA carboxylase (1) inhibits entry of acyl-CoA into the mitochondrion by inhibition of carnitine palmitoyl transferase-1 (CPTi). At the same time, the pathway of glycerolipid synthesis is stimulated by insulin. Other enzymes/pathways shown are: (2) acyl-CoA synthase (also known as acid:CoA ligase); (3) acyl-CoA:glycerol phosphate 1-O-acyl transferase (GPAT); (4) acyl-CoA:1-acylglycerol phosphate 2-O-acyl transferase (LPAT); (5) phosphatidate phosphohydrolase (PAP); (6) diacylglycerol acyltransferase (DGAT); (7) pathway of phospholipid biosynthesis via CDP-DAG (see Figs 7.1 and 7.3) leading to phosphatidylinositol (Ptdlns), phosphatidylglycerol (PtdGly) and others; (8) 1,2-diacylglycerol:choline phosphotransferase, leading to phosphatidylcholine (PtdCho) and other phospholipids (see Figs 7.1 and 7.2). Glucose metabolism is also shown in outline; glucose (via glycolysis) leads to the provision of glycerol 3-phosphate, and via pyruvate dehydrogenase in the mitochondrion to acetyl-CoA. For acetyl-CoA produced by this route to be a substrate for de novo lipogenesis, it must be exported to the cytoplasm using the citrate shuttle.

Fig. 3.10 Central role of acyl-CoA in hepatic lipid metabolism. Acyl-CoA is formed either from esterification (sometimes called activation) of a fatty acid with CoA, or from the pathway of de novo lipogenesis (DNL). It may be utilized for P-oxidation in the mitochondria (catabolism) or for glycerolipid biosynthesis. The relative rates of these pathways are determined by the nutritional state, mediated largely by variations in plasma insulin concentration. When insulin concentrations are high (well-fed state) (shown as Ins +), generation of malonyl-CoA through the activity of acetyl-CoA carboxylase (1) inhibits entry of acyl-CoA into the mitochondrion by inhibition of carnitine palmitoyl transferase-1 (CPTi). At the same time, the pathway of glycerolipid synthesis is stimulated by insulin. Other enzymes/pathways shown are: (2) acyl-CoA synthase (also known as acid:CoA ligase); (3) acyl-CoA:glycerol phosphate 1-O-acyl transferase (GPAT); (4) acyl-CoA:1-acylglycerol phosphate 2-O-acyl transferase (LPAT); (5) phosphatidate phosphohydrolase (PAP); (6) diacylglycerol acyltransferase (DGAT); (7) pathway of phospholipid biosynthesis via CDP-DAG (see Figs 7.1 and 7.3) leading to phosphatidylinositol (Ptdlns), phosphatidylglycerol (PtdGly) and others; (8) 1,2-diacylglycerol:choline phosphotransferase, leading to phosphatidylcholine (PtdCho) and other phospholipids (see Figs 7.1 and 7.2). Glucose metabolism is also shown in outline; glucose (via glycolysis) leads to the provision of glycerol 3-phosphate, and via pyruvate dehydrogenase in the mitochondrion to acetyl-CoA. For acetyl-CoA produced by this route to be a substrate for de novo lipogenesis, it must be exported to the cytoplasm using the citrate shuttle.

In the enterocytes of the small intestine, the rate of influx of dietary fatty acids appears to be the main factor controlling the rate of TAG synthesis via the 2-monoacylglycerol pathway. The glycerol 3-phosphate pathway seems to supply a basal rate of TAG synthesis between meals in the enterocyte. In other tissues, in which the glycerol 3-phosphate pathway is predominant, TAG synthesis is more tightly regulated by the prevailing nutritional state.

The nutritional regulation of acylglycerol synth esis has been mostly studied in liver and adipose tissue. In neither tissue, however, is our understanding complete, mainly because of the difficulty of isolating the relevant enzymes, which are insoluble and membrane-bound. Attempts to purify the enzyme diacylglycerol acyltransferase, for example, have been described as 'masochistic enzymol-ogy'. This has changed as modern methods of cloning by homology searching (screening cDNA libraries for related sequences) have been applied.

The cellular concentrations of the substrates for the acyltransferases that catalyse acylglycerol esterification, acyl-CoA and glycerol 3-phosphate, are influenced by nutritional status. Since glycerol 3-phosphate is produced from dihydroxyacetone phosphate, an intermediate in the pathways of glycolysis and gluconeogenesis, the main factors influencing the amounts of glycerol 3-phosphate available for acylglycerol synthesis are those that regulate the levels and activities of the enzymes of glucose metabolism. Starvation reduces the intra-cellular concentration of glycerol 3-phosphate severely, whereas carbohydrate feeding increases it. Intracellular concentrations of acyl-CoA increase during starvation.

In adipose tissue there is evidence that the supply of glycerol 3-phosphate may regulate TAG synthesis. Insulin stimulates glucose uptake by adipo-cytes and, by implication, glycerol 3-phosphate production. Certainly TAG synthesis in adipose tissue increases in the period following a meal, as is necessary to accommodate the influx of fatty acids from lipoprotein lipase (itself stimulated by insulin) acting on circulating TAG in the capillaries. This might equally, though, reflect stimulation of the enzymes of TAG synthesis by insulin and other factors. One such factor is the 76-amino acid peptide known as acylation stimulating protein (ASP). The production of ASP from adipocytes will be described in more detail in Section 5.3.4.

In the liver, however, there is little evidence that intracellular concentrations of glycerol 3-phosphate or acyl-CoA are important in regulating acylgly-cerol synthesis. Instead, the rate of acylglycerol synthesis in the liver appears to reflect the relative activities of the competing pathways for fatty acid utilization, acylglycerol synthesis and P-oxidation (Fig. 3.10). Entry of fatty acids into the mitochondrion for oxidation is mediated by carnitine pal-mitoyl transferase 1 (CPT1; Section 2.3.1), and this enzyme is powerfully suppressed by an increase in the cytosolic concentration of malonyl-CoA, the first committed intermediate in fatty acid synthesis (Section 2.2.3). The formation of malonyl-CoA is stimulated under well-fed conditions (when insulin levels are high; Section 2.2.8) and so fatty acid oxidation will be suppressed and acylglycerol synth esis favoured under these conditions. The opposite will be true in starvation or energy deficit. An important question is, whether there is also coordinated regulation of the enzymes of acylglycerol synthesis. The answer appears to be yes, but pinpointing the locus of control has proved difficult.

The enzymes that may be involved in regulation of mammalian acylglycerol biosynthesis include the acyltransferases that link successive molecules of acyl-CoA to the glycerol backbone, and phos-phatidate phosphohydrolase (PAP) (Fig. 3.6). The first of these enzymes is GPAT (Section 3.4.1). It has been suggested that GPAT and CPT1 (see above), both expressed on the outer mitochondrial membrane, represent an important branch point in fatty acid metabolism, leading acyl-CoA into esterifica-tion or oxidation, respectively. GPAT expression is generally regulated in parallel with the rate of TAG synthesis in different nutritional states. This regulation is brought about by a number of factors including insulin, for which there is a specific response element in the promoter region of the mitochondrial GPAT gene. GPAT expression is regulated almost exactly in parallel with the expression of fatty acid synthase.

Nutritional and hormonal factors appear to influence the mitochondrial GPAT activity more than the microsomal. Thus, when fasted rats are given a diet low in fat and rich in carbohydrate, mitochondrial GPAT activity increases sixfold with little change in the microsomal activity. Similarly in perfused rat liver, inclusion of insulin in the perfusion fluid increases the mitochondrial GPAT activity four times more than the microsomal. During starvation, hepatic mitochondrial GPAT activity decreases, but the overall capacity for TAG biosynthesis in the liver remains unchanged provided that P-oxidation is inhibited. It seems that in starvation, the decrease in GPAT activity is due primarily to competition by CPT1 for acyl-CoA (Section 2.3.1.6).

The activity of the next enzyme in the pathway, LPAT, is increased 2.5-fold in liver post-natally and about 60-fold during the differentiation of 3T3-L1 adipocytes. Changes in mRNA for the enzyme in response to dietary changes have not been reported.

The activity of PAP, like that of GPAT, generally runs parallel to the potential for overall acylglycerol synthesis in that tissue, and it has been suggested that PAP is the major locus for regulation of TAG biosynthesis. However, it now appears more likely that control is 'shared' by a number of enzymes. PAP activity in liver is increased by high levels of dietary sucrose and fat, by ethanol and by conditions, such as starvation, that result in high concentrations of plasma non-esterified fatty acids. It is also increased in obese animals. It is decreased in diabetes and by administration of drugs that result in a reduction of circulating lipid concentrations. The factors that tend to increase the activity of PAP are also those that result in an increased supply of saturated and monounsaturated fatty acids to the liver, namely those fatty acids normally esterified in simple acylglycerols. If PAP activity is low, the substrate for the enzyme - phosphatidic acid, the central intermediate in lipid metabolism - does not accumulate, but becomes a substrate for the biosynthesis of acidic membrane phospholipids such as phosphatidylinositol (Sections 7.1.6 and 7.9). Phospholipid metabolism makes more extensive demands on a supply of unsaturated fatty acids than does simple acylglycerol metabolism and the activities of enzymes that divert phosphatidic acid into phospholipid rather than TAG metabolism tend to be elevated in conditions where unsaturated fatty acids predominate (Fig. 3.10).

PAP exists in the cytosol and in the endoplasmic reticulum. There is another isoform associated with the cell membrane, but this is thought to be involved more with signal transduction than TAG biosynthesis. The cytosolic enzyme is physiologically inactive, but translocates to the membranes of the endoplasmic reticulum on which phosphatidate is being synthesized. The translocation process seems to be regulated both by hormones and substrates. Cyclic-AMP (cAMP), for example, displaces the enzyme from membranes, whereas increasing concentrations of non-esterified fatty acids and their CoA esters promote its attachment. Insulin, which has the effect of decreasing intracellular concentrations of cAMP, ensures that the translocation is more effective at lower fatty acid concentrations. The mechanisms that cause these changes are not yet established but may involve the reversible phosphorylation of PAP. It seems, therefore, that the membrane-associated PAP is the physiologically active form of the enzyme and the cytosolic form represents a reservoir of potential activity. This phenomenon of translocation is seen with some other enzymes involved in lipid metabolism. Enzymes that exist in different locations in the cell and can regulate metabolism by moving from one location to another are called ambiquitous enzymes. Other examples in lipid metabolism are the CTP:phosphorylcholine cytidylyl transferase, which regulates the biosynthesis of phosphati-dylcholine (Section 7.1.5), and hormone-sensitive lipase in adipocytes (Section 3.6.3).

The diacylglycerol formed by the action of PAP can be used either for TAG synthesis (Fig. 3.6), or for glycerophospholipid synthesis (Fig. 7.1). The biosynthesis of phospholipids takes precedence over that of TAG when the rate of synthesis of diacylglycerol is relatively low. This ensures the maintenance of membrane turnover and bile secretion, which are more essential processes in physiological terms than the accumulation of TAG. The precise mechanisms for the preferred synthesis of phosphoglycerides are not certain, however. Among the factors involved is probably a relatively low Km of choline phospho-transferase for diacylglycerol. A factor that may eventually limit the biosynthesis of phosphati-dylcholine is a limitation in the supply of cho-line and it is well recognized that a major effect of choline deficiency is fatty liver indicating a diversion away from phospholipid biosynthesis into TAG biosynthesis.

The last enzyme in the biosynthesis of TAG, DAGAT, is highly expressed in tissues that have a high rate of TAG synthesis, including small intestine and adipocytes. However, the level of expression is relatively low in the liver, which is surprising and leaves open the possibility that yet another enzyme is still awaiting identification. As yet, little is known of its regulation, although it has been claimed to be an important locus of control of TAG synthesis by acylation stimulating protein (ASP; Section 5.3.4) in adipocytes. There is evidence for short-term regulation of its activity by reversible phosphorylation, but this has not yet been shown conclusively.

Further research into the hormonal and nutritional control of these processes may give insights into how to control the common diseases of lipid metabolism (Section 5.4).

3.6.3 Mobilization of fatty acids from the fat stores is regulated by hormonal balance, which in turn is responsive to nutritional and physiological states

In physiological states demanding the consumption of fuel reserves, the resulting low concentrations of insulin turn off the biosynthetic pathways and release the inhibition of hormone-sensitive lipase within adipocytes. The activity of hormone-sensitive lipase is regulated in the short term by a cascade mechanism illustrated in Fig. 3.11. In the longer term it is also regulated by control of transcription; its expression is up-regulated, for instance, during prolonged fasting.

Short-term regulation is brought about by reversible phosphorylation. The phosphorylated enzyme is active and the dephosphorylated form inactive. Changes in activity in vivo, or in intact cells, are much greater than can be achieved when the purified enzyme is phosphorylated in vitro and acts on a synthetic lipid emulsion. This has led to

Hsl Macrophages

Fig. 3.H Short-term regulation of the activity of hormone-sensitive lipase (HSL) in adipocytes. HSL is regulated in the short term by reversible phosphorylation brought about by the enzyme protein kinase A (also known as cyclic AMP-dependent protein kinase). Protein kinase A is activated by the binding of cyclic AMP (cAMP), generated by the action of adenylate cyclase on ATP. In turn, adenylate cyclase is regulated by membrane-associated heterotrimeric guanine-nucleotide binding proteins, known as G-proteins. These link cell-membrane hormone receptors with adenylate cyclase. There are stimulatory (Gs) and inhibitory (Gi) G-proteins that link with the appropriate receptors. Insulin suppresses the activity of HSL by causing its dephosphorylation. This reflects activation of a particular phosphodiesterase (PDE), which breaks down cAMP and therefore reduces the activity of protein kinase A. There are constitutively expressed protein phosphatases that return HSL to its inactivated state under these conditions.

HSL is active against the triacylglycerols (TAG) and diacylglycerols (DAG), but less so against monoacylglycerols (MAG). There is a specific, highly active MAG lipase in adipose tissue. The overall reaction is the liberation from stored TAG of three non-esterified fatty acids (NEFA) and a molecule of glycerol.

Fig. 3.H Short-term regulation of the activity of hormone-sensitive lipase (HSL) in adipocytes. HSL is regulated in the short term by reversible phosphorylation brought about by the enzyme protein kinase A (also known as cyclic AMP-dependent protein kinase). Protein kinase A is activated by the binding of cyclic AMP (cAMP), generated by the action of adenylate cyclase on ATP. In turn, adenylate cyclase is regulated by membrane-associated heterotrimeric guanine-nucleotide binding proteins, known as G-proteins. These link cell-membrane hormone receptors with adenylate cyclase. There are stimulatory (Gs) and inhibitory (Gi) G-proteins that link with the appropriate receptors. Insulin suppresses the activity of HSL by causing its dephosphorylation. This reflects activation of a particular phosphodiesterase (PDE), which breaks down cAMP and therefore reduces the activity of protein kinase A. There are constitutively expressed protein phosphatases that return HSL to its inactivated state under these conditions.

HSL is active against the triacylglycerols (TAG) and diacylglycerols (DAG), but less so against monoacylglycerols (MAG). There is a specific, highly active MAG lipase in adipose tissue. The overall reaction is the liberation from stored TAG of three non-esterified fatty acids (NEFA) and a molecule of glycerol.

the realization that phosphorylation of hormonesensitive lipase is associated with translocation of the enzyme from a cytosolic location, to the surface of the intracellular lipid droplet. This may involve 'docking' with a protein, perilipin, that is associated with the lipid droplet surface, and is itself a substrate for phosphorylation under similar conditions to hormone-sensitive lipase.

Phosphorylation of hormone-sensitive lipase is mediated by the enzyme protein kinase A, or cAMP-dependent protein kinase. This in turn is activated by the binding of cAMP when cellular cAMP concentrations are elevated through increased activity of the enzyme adenylate cyclase. The activity of the cyclase is under the control of the catecholamines adrenaline (a true hormone, released from the adrenal medulla) and norad-renaline, a neurotransmitter released from sympathetic nerve terminals in adipose tissue. Binding of catecholamines to P-adrenergic receptors in the cell membrane activates adenylate cyclase through the intermediary G-proteins (proteins that bind GTP, and couple receptors to adenylate cyclase). The situation is complex because catecholamines can also bind to a-adrenergic receptors that act through inhibitory G-proteins to reduce adenylate cyclase activity, although the overall effect in vivo is usually towards activation. There is also a number of locally produced mediators, such as adenosine and prostaglandins (formed from dietary essential fatty acids; Section 2.4), that inhibit adenylate cyclase activity, again via specific cell-surface receptors and inhibitory G-proteins. This complex system undoubtedly exists to regulate fat mobilization extremely precisely.

Counter-regulation is achieved mainly by insulin, which acts via cell-surface insulin receptors, to activate a specific form of phosphodiesterase that catalyses the breakdown of cAMP. Under these conditions, protein phosphatases in the cell, which do not appear to be regulated but are present in high activity, dephosphorylate hormone-sensitive lipase, which is thus inactivated. Presumably hormone-sensitive lipase in vivo is continually cycling between phosphorylated and dephosphorylated states, with the balance determined by cellular cAMP concentrations. The phosphodiesterase can be inhibited by methylxanthines, such as caffeine, therefore leading to activation of hormone-sensitive lipase and fat mobilization. This is one reason why some athletes claim that drinking strong coffee before an endurance event improves their performance.

Hormone-sensitive lipase is most active against tri- and diacylglycerols, but relatively inactive against monoacylglycerols. There is a highly active monoacylglycerol lipase expressed in adipocytes that leads to the complete hydrolysis of stored TAG with release of three molecules of fatty acid and one of glycerol, which leave the cell. There is currently great interest in the question of whether the exit of fatty acids occurs by diffusion across the cell membrane or via a specific transport protein. A number of putative fatty acid transport proteins has now been identified, although it is not clear whether this step might be open to regulation. The non-esterified fatty acids (often referred to as free fatty acids, or FFA) are bound to plasma albumin in the circulation (Fig. 3.11). They may then be taken up by tissues such as muscle that utilize fatty acids as a major source of fuel, and by the liver where they may be used for oxidation or for the synthesis of new acylglycerols.

It has become clear recently that hormone-sensitive lipase is also responsible for TAG hydrolysis within skeletal muscle. The lipid droplets that are seen within muscle fibres on electron microscopy provide a local supply of fatty acids during intense exercise. The mechanisms for activation of muscle hormone-sensitive lipase may be similar to those within adipocytes, except that muscle contraction is also a stimulus to TAG hydrolysis. The mechanism by which contraction activates hormone-sensitive lipase is not yet known.

The co-ordination of TAG synthesis and breakdown in adipose tissue is illustrated in Fig. 3.12).

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