Lipid synthesis Lipogenesis
Lipogenesis (lipid synthesis) refers in the strictest sense to synthesis of fatty acids and not to the esterification of those fatty acids to glycerides. Adipose tissue is the main site of lipogenesis in non-lactating cattle, sheep, goats, pigs, dogs and cats (Beitz and Nizzi, 1997). In poultry, similarly to humans, the liver is the major site of lipogenesis, while in rodents (rats
and mice) both liver and adipose tissue are important lipogenic sites. The mammary gland of lactating farm animals actively synthesizes fatty acids.
De novo lipogenesis occurs in the cytosol and is a sequential cyclical process in which acetyl (2-carbon) units are added successively to a ‘primer’ or initial starting molecule, usually acetyl-CoA but also 3-hydroxybutyrate in the lactating mam- mary gland of ruminants. The source of the acetyl units is acetyl-CoA, derived either from glucose through glycolysis in non- ruminants or pre-ruminants, or from acetate via rumen fermentation of dietary carbohydrates in ruminants. In functioning ruminants, glucose is not used for fatty acid synthesis, which serves to spare glucose for other essential functions.
The nature of the mechanism that minimizes lipogenic use of glucose by ruminants remains unclear. Since the discovery that activities of ATP-citrate lyase and NADP-malate dehydrogenase were lower in bovine adipose tissue than in liver and adipose tissue of rats (Hanson and Ballard, 1967), it was believed that these enzymes limited lipogenic use of glucose by ruminants. However, subse- quent research showed that lactate was used by bovine adipose tissue (Whitehurst et al., 1978) and mammary gland (Forsberg et al., 1985) at rates similar to those of acetate. Lactate, after being converted to pyruvate, is metabolized similarly to pyruvate produced from glycolysis.
Consequently, lactate also requires the enzymes ATP-citrate lyase and NADP- malate dehydrogenase in order to be converted to fatty acids. The activity of ATP-citrate lyase in bovine adipose tissue and mammary gland, albeit lower than that in rat tissues, is still sufficient to allow the observed rates of lactate conversion to fatty acids (Forsberg et al., 1985). Moreover, ATP-citrate lyase activity is at least equal to that of acetyl-CoA carboxylase, the rate- limiting step in fatty acid synthesis (Beitz and Nizzi, 1997). Probably the most likely explanation for the low rate of incorpora- tion of glucose into fatty acids in ruminant adipose tissue and mammary gland is the
limited flux of glucose carbon past the triose phosphate stage in glycolysis (Forsberg et al., 1985), because of the high demand for glycerol-3-phosphate for triacylglycerol synthesis, and the active metabolism of glucose in the pentose phosphate pathway to produce NADPH.
The rate-limiting step in fatty acid synthesis is catalysed by the enzyme acetyl-CoA carboxylase (Hillgartner et al., 1995). This enzyme converts acetyl-CoA to malonyl-CoA, which is the actual ‘donor’
of acetyl units in the elongation process.
Two forms of the enzyme, termed and , are found in animals (Kim, 1997). The - form is the enzyme found in lipogenic tissues that regulates the rate of fatty acid synthesis. The -form is found in non- lipogenic tissues and is associated with control of mitochondrial fatty acid oxida- tion (discussed later). The -form of the enzyme is subject to several levels of meta- bolic regulation from signals of nutrient status. Insulin, released when dietary energy is plentiful, activates the enzyme and so promotes fat storage. Increased concentrations of citrate and isocitrate, which also would signal increased sub- strate availability for storage as fat, activate the reaction. In contrast, glucagon and the catecholamines inhibit its activity via cyclic AMP (cAMP)-dependent phos- phorylation. In this way, fat synthesis is inhibited during times when mobilization of energy stores is required. Increased con- centrations of fatty acyl-CoA in the cytosol inhibit the reaction, a form of negative feedback. In addition to short-term changes in enzyme activity caused by these hormones and metabolites, the abundance of the enzyme protein is also regulated.
Starvation decreases the amounts of both the mRNA and the protein, while refeeding after a fast causes a large increase in transcription and translation of mRNA for acetyl-CoA carboxylase (Hillgartner et al., 1995).
The fatty acid synthase enzyme complex consists of two multifunctional polypeptide chains, each containing seven distinct enzyme activities necessary to elongate a growing fatty acid (Smith, 1994).
The two polypeptide chains are arranged head-to-tail, resulting in two separate sites for synthesis of fatty acids; thus each enzyme complex can assemble two fatty acids simultaneously. The activity of the enzyme complex is not limiting to the overall rate of fatty acid synthesis. The overall reaction for synthesis of one molecule of palmitic acid is:
Acetyl-CoA + 7 malonyl-CoA + 14 NADPH + 14 H+→palmitic acid + 7 CO2+ 8 CoA + 14 NADP+
+ 6 H2O (5.1)
In non-ruminants the hydrogen donor, NADPH, is generated through metabolism of glucose in the pentose phosphate path- way and in the malic enzyme reaction. In ruminants, cytosolic isocitrate dehydro- genase can generate over one-half of the NADPH needed through metabolism of acetate (Beitz and Nizzi, 1997). The remainder of the NADPH in ruminants is derived from glucose metabolism in the pentose phosphate pathway. The presence of glucose enhances fatty acid synthesis in ruminants, probably through enhanced production of NADPH. Regulation of fatty acid synthase is largely through intra- cellular concentrations of dietary or synthe- sized fatty acids, which decrease its activity (Smith, 1994). High-fat diets decrease the intracellular concentration of fatty acid synthase, whereas refeeding after a fast increases its concentration. High concen- trations of insulin increase the abundance of fatty acid synthase, whereas growth hormone, glucagon and glucocorticoids decrease its abundance (Hillgartner et al., 1995).
Lipogenesis generally increases as animals age, although changes are depot- specific and may be modulated by diet (Smith, 1995). Thus, lipogenesis in internal adipose depots such as perirenal fat is more active earlier in the growth stage, and less active as the animal reaches physio- logical maturity. Somatotropin treatment of pigs and cattle leads to decreased lipo- genesis, primarily by decreasing the sensi- tivity of adipose cells to the actions of insulin (Etherton and Bauman, 1998).
Other acyl-CoA molecules such as propionyl-CoA can be used as primers by the fatty acid synthase complex. In this case, odd-carbon numbered fatty acids will be produced, most commonly of 15 or 17 carbon length. In addition, methylmalonyl- CoA can replace malonyl-CoA in the elongation reactions, resulting in branched- chain (methyl-branched) fatty acids. In most lipogenic tissues, these fatty acids are only minor products, but in sebaceous (skin) glands of some species the produc- tion of methyl-branched fatty acids may be substantial (Smith, 1994). In ruminants, higher concentrations of odd-chain and branched-chain fatty acids are found in milk and adipose tissue because of the greater synthesis of these fatty acids by rumen bacteria.
In adipose tissue, the predominant product of the lipogenic pathway is palmitic acid. In the mammary gland of lactating animals, however, large quantities of fatty acids <16 carbons in length are synthesized. This is due to the action of specific chain-terminating mechanisms, which differ between ruminants and non- ruminants. In ruminants, the fatty acid synthase complex allows the release of short- and medium-chain fatty acyl-CoA esters, which are incorporated rapidly into milk fat. In non-ruminants, a specific enzyme, thioesterase II, is responsible for hydrolysing the thioester bond of the 8–14 carbon acyl chain, thus releasing the medium-chain fatty acids (Smith, 1994).
Elongation and desaturation
The end-product of the de novo lipogenic pathway in animal tissues is usually palmitic acid, yet this fatty acid constitutes only 20–30% of total fatty acids in adipose tissue lipids (Rule et al., 1995).
Considerable amounts of stearic (18:0) and oleic (18:1) acids are present in adipose tissue lipids, and may arise either from intestinally derived triacylglycerol-rich lipoproteins or by conversion from palmitic acid in adipose tissue. Elongation of palmitic acid (16:0) to stearic acid occurs by the action of fatty acid elongase, found in the microsomal fraction (endoplasmic
reticulum) of adipocytes. Malonyl-CoA is the source of the additional two carbons.
Fatty acid elongase is found in much larger activities in bovine adipose tissue than in mammary gland, liver, muscle or intestinal mucosa (Smith, 1995).
The concentration of stearic acid in tissue lipids is regulated by the presence of stearoyl-CoA desaturase (9 desaturase), which converts stearic acid to oleic acid.
This microsomal enzyme is a mixed function oxidase that inserts a double bond nine carbons from the methyl end of the fatty acid. Considerable activity of stearoyl- CoA desaturase is found in mammary gland, muscle and duodenal muscosa, but little activity is found in bovine liver (Smith, 1995). The primary function of the enzyme seems to be to regulate lipid fluidity by preventing excessive accumula- tion of the very high-melting stearic acid.
Glycerolipid synthesis
Few free (non-esterified) fatty acids are found in the animal body; rather, most fatty acids are found esterified to glycerol as glycerolipids such as triacylglycerols and phospholipids. In adipose tissue and the lactating mammary gland, most fatty acids are esterified to form triacylglycerols as a non-toxic form of energy storage or for transfer to the young, respectively. In liver and other tissues, most fatty acids are esterified to form phospholipids as com- ponents of intracellular and plasma mem- branes. The liver actively synthesizes triacylglycerols when presented with high concentrations of non-esterified fatty acids from the blood.
The enzymes necessary for glycerolipid biosynthesis are found in the microsomal fraction of cells. The general pathways of esterification of fatty acids are shown in Fig. 5.3. Acyl chains from acyl-CoA are transferred consecutively to glycerol-3- phosphate produced via glycolysis.
Production of diacylglycerol (diglyceride) from phosphatidate by phosphatidate phosphohydrolase and subsequent produc- tion of triacylglycerol from diacylglycerol by diacylglycerol acyltransferase may be regulatory steps for triacylglycerol synthesis,
but these enzymes have not been well characterized in farm animals.
Esterification of fatty acids in adipose tissue increases with increasing energy intake in meat animals (Rule, 1995) and is lower in times of dietary energy deficit, such as during early lactation in dairy cows (McNamara, 1991).
Fatty acid composition of milk, muscle and body fat
A variety of fatty acids are found in complex lipids of animal tissues. These fatty acids range primarily from 14 to 20 carbons in length, with varying degrees of unsaturation. Characteristic profiles of fatty acids are found in individual tissues and among species of animals. Sample profiles of muscle and adipose tissue of beef cattle, sheep and pigs are shown in Table 5.1.
Adipose tissue lipids from ruminants gener- ally are more highly saturated than lipids from non-ruminants such as pigs because of ruminal biohydrogenation of dietary
unsaturated fatty acids. Experimental post- ruminal infusions of unsaturated oils and feeding formaldehyde-protected oils to sheep and cattle results in increasing unsaturation of adipose tissue lipids (Rule et al., 1995). In pigs and chickens, increas- ing amounts of dietary fat will result in adipose tissue lipids reflecting the fatty acid composition of the dietary fat. Body fat generally becomes softer in these species with supplementation of fats and oils, because the relative amounts of de novo synthesized palmitic acid decrease and those of 18-carbon unsaturated fatty acids increase (Rule et al., 1995).
Bovine milk fat contains considerable amounts of fatty acids shorter than 14 carbons that are synthesized within the mammary gland (Table 5.2). The fatty acid composition of milk fat can be altered markedly by supplementation of the diet with fat (Palmquist et al., 1993). Dietary long-chain fatty acids suppress de novo synthesis of short- and medium-chain fatty
Fig. 5.3. Major pathways of esterification of fatty acids to glycerolipids in farm animals. The key enzymes involved are: (1) glycerophosphate acyltransferase; (2) lysophosphatidate acyltransferase; (3) phosphatidate phosphohydrolase; (4) diacylglycerol acyltransferase; and (5) monoacylglycerol acyltransferase. Pi, inorganic phosphate. Adapted from Rule (1995).
Fatty acyl-CoA
GLYCEROL-3-PHOSPHATE
Fatty acyl-CoA
Fatty acyl-CoA
1-ACYLGLYCEROL-3-PHOSPHATE (LYSOPHOSPHATIDATE)
1,2-DIACYLGLYCEROL-3-PHOSPHATE (PHOSPHATIDATE)
Cardiolipin
Phosphatidylglycerol Phosphatidylinositol
Monoacylglycerol
Phosphatidylcholine Phosphatidylethanolamine
Fatty acyl-CoA
Phosphatidylcholine 1,2-DIACYLGLYCEROL
TRIACYLGLYCEROL (1)
(2)
(3)
(4)
(5) Pi
acids in the mammary gland. Unprotected fats lead to only slight increases in poly- unsaturated fatty acids in milk fat, but may lead to appreciable increases in oleic acid because of intestinal and mammary desaturation of stearic acid produced by ruminal biohydrogenation of dietary unsaturated fatty acids (Table 5.2). The bovine mammary gland readily incorporates unsaturated fatty acids presented to it (LaCount et al., 1994). Producing milk with more monounsaturated and polyunsaturated fatty acids depends on the development of practical strategies to protect dietary unsaturated fatty acids from hydrogenation by rumen microbes. Currently, formalde- hyde treatment of protein–fat mixtures is the best methodology for rumen protection (Doreau and Chilliard, 1997), but regulatory approval may limit its application in many countries.
Lipolysis
Mobilization of fatty acids from adipose tissue triacylglycerols (lipolysis) occurs during times of negative energy balance or in response to stresses. The reaction proceeds by the sequential release of fatty acids from the glycerol backbone. The fatty acids released increase the size of the intra- cellular free fatty acid pool and, in the
absence of stimuli to re-esterify those fatty acids, they diffuse from the cell into the blood. The free fatty acids are adsorbed quickly to binding domains on serum albumin, and circulate to various tissues as a fatty acid–albumin complex. Physiological states characterized by high rates of lipo- lysis, such as early lactation in dairy cows and sows (McNamara, 1991), often are also characterized by relatively lower concen- trations of albumin in the blood. Hence, the ratio of free fatty acids to albumin in blood increases, which favours greater uptake of the free fatty acids by tissues of the body because more fatty acids occupy lower affinity binding sites on the albumin molecule. Furthermore, the increased ratio of fatty acids to albumin increases the size of the tissue free fatty acid pool, which in turn increases re-esterification of fatty acids in adipose tissue and thus provides feedback regulation on lipolysis (Metz and van den Bergh, 1977).
The initial step in lipolysis is catalysed by hormone-sensitive triacylglycerol lipase.
This enzyme is activated by binding of hormones that stimulate formation of cAMP by adenyl cyclase. In mammals, the primary Table 5.1. Typical profiles of major fatty acids
found in lipids from subcutaneous adipose tissue or longissimus muscle from cattle, sheep and pigs (g kg1). (Adapted from Rule et al., 1995.)
Fatty acid Cattle Sheep Pigs
Adipose tissue
14:0 40 40 10
16:0 280 260 240
18:0 110 160 130
18:1 430 410 440
18:2 30 30 120
Muscle
14:0 40 30 10
16:0 270 250 250
18:0 130 110 110
18:1 380 460 490
18:2 80 60 70
Table 5.2. Fatty acid composition of milk fat from cows fed a basal low-fat diet or the basal diet supplemented with tallow. (Adapted from Palmquist et al., 1993.)
Diet (g kg1of methyl esters)
Fatty acid Basal Basal + tallow
4:0 33 35
6:0 27 23a
8:0 18 13a
10:0 40 26a
12:0 46 29a
14:0 130 103a
14:1 15 13
15:0 13 10a
16:0 299 284
16:1 17 18
17:0 6 8a
18:0 90 104
18:1 172 233a
18:2 22 16a
18:3 6 9a
aDifferent from basal diet, P< 0.05.
agonists for this reaction are the catecholamines, such as epinephrine and norepinephrine. In poultry, glucagon is the major lipolytic hormone. Binding of these hormones to cell surface receptors causes activation of adenyl cyclase, depending on the balance between activation of the stimulatory guanine nucleotide-binding protein (Gs protein) and activation of the inhibitory Giprotein (Lafontan and Langin, 1995). Receptor types vary among tissues and species in the relative activation of these two G proteins. Agonists that activate -adrenergic receptors cause activation of Gs. Activation of Gsactivates adenyl cyclase, which increases the concentration of cAMP in the cell. In turn, cAMP activates protein kinase A, which phosphorylates the regula- tory subunit of hormone-sensitive lipase.
The activated hormone-sensitive lipase then catalyses lipolysis of triacylglycerol.
Inhibition of lipolysis depends on a greater activation of Gi proteins, which inhibit adenyl cyclase and increase the activity of phosphodiesterase, the enzyme that degrades cAMP. Agonists that bind to -adrenergic receptors activate Giand thus suppress lipolysis (Lafontan and Langin, 1995). Factors such as insulin, adenosine and the E series of prostaglandins are associated with decreased activity of hormone-sensitive lipolysis. Treatment of animals with somatotropin results in an indirect stimulation of lipolysis by increas- ing the sensitivity of adipose tissue to the effects of the catecholamines. Somatotropin causes this increased sensitivity by diminishing the ability of the Giproteins to inhibit adenyl cyclase. Thus, suppression of the inhibitory controls of lipolysis allows higher rates of lipolysis to occur during treatment with somatotropin.
Another factor controlling the relative degree of lipolysis is the degree to which fatty acids are re-esterified to form triacyl- glycerols before they can diffuse out of the cells. Insulin stimulates uptake of glucose and glycolysis, increasing the supply of glycerol-3-phosphate available for esterifica- tion. Insulin also stimulates the activity of the esterification pathway. Control of lipo- lysis is interwoven tightly with regulation
of lipogenesis, so that the overall function of adipose tissue to accrete or release energy stores is coordinated according to the physiological needs of the animal.