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.
mitochondrial matrix. Consequently, oxidation of these fatty acids is not con- trolled by CPT-I.
The activity of CPT-I is inhibited by interaction with malonyl-CoA, the product of the first committed step of lipogenesis catalysed by acetyl-CoA carboxylase.
Insulin stimulates the activity of acetyl- CoA carboxylase. Conditions of negative energy balance as signalled by lower ratios of insulin to glucagon thus result in decreased concentrations of malonyl-CoA and increased rates of fatty acid oxidation.
Furthermore, in rats, the sensitivity of CPT-I to malonyl-CoA is decreased during times of low insulin or insulin resistance, which decreases the ability of the low concentra- tions of malonyl-CoA to inhibit acyl- carnitine formation and thereby further increases the rate of fatty acid oxidation (Zammit, 1996).
Classical studies (reviewed by McGarry et al., 1989) that delineated the control of CPT-I by malonyl-CoA in rats described this mechanism as a means of preventing simul- taneous oxidation and synthesis of fatty acids within the liver cell, a potential futile cycle. However, in cattle, sheep and swine, rates of lipogenesis are very low in liver, which obviates the need for such a control mechanism. Nevertheless, production of malonyl-CoA by acetyl-CoA carboxylase does occur in bovine, ovine and swine liver (Brindle et al., 1985), probably as a control mechanism for oxidation rather than as a quantitatively important site of fatty acid synthesis. Likewise, skeletal muscle and heart muscle are also non-lipogenic tissues that use fatty acids as energy sources. Both heart and skeletal muscle of rats contain a high activity of acetyl-CoA carboxylase of the -isoform (Kim, 1997). Physiological situations that lead to low insulin to glucagon ratios and decreased activity of acetyl-CoA carboxylase in these tissues result in increased rates of fatty acid oxida- tion. Whether the acetyl-CoA carboxylase present in the liver of ruminants and swine is similar to the -isoform of rats has not been determined.
Intramitochondrial oxidation of fatty acyl-CoA occurs through the -oxidation
pathway, resulting in formation of acetyl- CoA. During this process, electrons are transferred to FAD and NAD+ to form the reduced forms of these coenzymes, which in turn can donate electrons to the electron transport chain to drive ATP synthesis. The acetyl-CoA can be oxidized completely to carbon dioxide in the tricarboxylic acid (TCA) cycle. Alternately, acetyl-CoA can be diverted to formation of ketone bodies.
Ketogenesis is enhanced in times of increased fatty acid mobilization and uptake by the liver, when low ratios of insulin to glucagon cause activation of CPT-I that allows extensive uptake of fatty acids into mitochondria (Zammit, 1990).
Conversion of acetyl-CoA to ketone bodies rather than complete oxidation in the TCA cycle results in formation of less ATP per mole of fatty acid oxidized. For example, complete oxidation of palmitate in the TCA cycle, followed by oxidative phosphoryla- tion in the electron transport chain, yields 129 ATP per molecule of palmitate. In contrast, -oxidation of palmitate with acetyl-CoA converted to ketone bodies generates only 27 ATP per molecule of palmitate. Because the production of ATP must match its utilization for energy- requiring reactions in the liver, ketogenesis allows the liver to metabolize about five times more fatty acid for the same ATP yield. Conversion of fatty acids into water- soluble fuels may be an important strategy to allow the animal to cope with extensive mobilization of fatty acids during energy deficit.
In addition to control at the levels of fatty acid supply and CPT-I, ketogenesis is controlled by the activity of the key regula- tory enzyme, 3-hydroxy-3-methylglutaryl- CoA (HMG-CoA) synthase. This enzyme is controlled both through increased tran- scription and translation during prolonged energy deficit and by inactivation through succinylation (Emery et al., 1992).
Increased flux of metabolites such as pyruvate, propionic acid or glucogenic amino acids into the TCA cycle, resulting from greater feed intake and improved energy balance, results in increased pool size of succinyl-CoA, an intermediate of
the TCA cycle. The succinyl-CoA is used to add a succinyl group to a regulatory sub- unit of HMG-CoA synthase, which inactivates the enzyme.
In ruminants, CPT-I also is highly sensitive to inhibition by methylmalonyl- CoA (Brindle et al., 1985), which is an intermediate in the conversion of propionate to succinyl-CoA in the process of gluconeogenesis. This may constitute an additional adaptation of ruminants to link the supply of energy-yielding compounds from the diet with the need for hepatic fatty acid oxidation. Furthermore, the ability to distinguish between glucogenic molecules originating primarily through ruminal fermentation of dietary carbo- hydrates (propionate) and those originating from catabolism of endogenous amino acids (e.g. pyruvate) has been proposed as a unique adaptation of regulation of fatty acid oxidation in ruminants during energy deficit situations (Zammit, 1990).
Accelerated ketogenesis in response to low blood glucose from insufficient dietary energy intake may occur in both lactating cows and pregnant ewes. The increased ketogenesis is probably a factor of increased mobilization of free fatty acids from adipose tissue, increased uptake of fatty acids by the liver, increased activity of CPT-I, decreased sensitivity of CPT-I to malonyl-CoA and increased activity of HMG-CoA synthase.
Ketogenesis has been shown to occur at lower rates in swine than in many other species (Odle et al., 1995; Adams et al., 1997). This may be due to limitations both in the ability of swine to form acylcarnitines for transport into the mitochondria (Odle et al., 1995) and in the activity of HMG-CoA synthase (Adams et al., 1997). Limitations are particularly pronounced in neonates, with some developmental increase in oxida- tive capacity observed with advancing age in pigs (Adams et al., 1997; Yu et al., 1997).
Peroxisomal metabolism
An alternate pathway for -oxidation in liver is found in peroxisomes, which are
subcellular organelles present in most tissues (Singh, 1997). The peroxisomal pathway for -oxidation functions similarly to the mitochondrial pathway, with notable exceptions. First, the first dehydrogenase step of mitochondrial - oxidation is replaced with an oxidase step (acyl-CoA oxidase) in the peroxisome, resulting in formation of hydrogen peroxide rather than reduced NAD+. Second, peroxisomes do not contain an electron transport chain. As a result of these factors, peroxisomal -oxidation results in capture of less energy as ATP than does mitochondrial -oxidation. Another unique aspect of the peroxisomal pathway is that two enzymic activities of the -oxidation pathway (enoyl-CoA hydratase and 3-hydroxyacyl-CoA dehydrogenase) are performed by a single multifunctional protein, called the bifunctional protein.
Peroxisomal -oxidation is active with very long-chain fatty acids (20 carbons or more) that are relatively poor substrates for mitochondrial -oxidation. Because peroxisomal -oxidation enzymes are induced in rats by situations leading to increased supply of fatty acids in the liver, such as high dietary fat, starvation and diabetes, the peroxisomal -oxidation path- way has been discussed as an ‘overflow’
pathway to help cope with increased flux of fatty acids (Singh, 1997).
Recent investigations in dairy cows (Grum et al., 1994), sheep (Hansen et al., 1995) and pigs (Yu et al., 1997) have shown that the livers of these farm animals possess relatively high peroxisomal - oxidation activity. In neonatal pigs, peroxisomal -oxidation increases rapidly after birth in response to milk intake and may be an adaptive mechanism to aid in oxidation of long-chain fatty acids from milk in the face of relatively low capacity for mitochondrial fatty acid oxidation (Yu et al., 1997). In dairy cows, peroxisomal -oxidation is not induced by dietary fat during lactation or by starvation (Grum et al., 1994), but increases in response to dietary fat and nutrient restriction during the periparturient period. Subsequent to the increased peroxisomal -oxidation
prepartum, less triacylglycerol accumulates in the liver at the time of calving (Grum et al., 1996). This may be an adaptation to allow increased metabolism of fatty acids during their extensive mobilization.
During the last decade, research has identified specific nuclear receptors that are activated by fatty acids and chemicals that cause peroxisomal proliferation in rodents. These receptors, called peroxisome proliferator-activated receptors (PPARs), in turn bind to specific peroxisome prolifera- tor response elements (PPREs) located in the regulatory region of a number of genes whose products are associated with lipid metabolism (Schoonjans et al., 1996).
These include long-chain acyl-CoA synthetase; the peroxisomal enzymes acyl- CoA oxidase and bifunctional protein; the mitochondrial enzymes CPT-I, medium- chain acyl-CoA dehydrogenase and HMG- CoA synthase; and the microsomal cytochrome P450 enzymes CYP4A1 and CYP4A6, which catalyse -oxidation.
Furthermore, the liver fatty acid-binding protein gene contains a PPRE. In rats, the gluconeogenic enzymes phosphoenol- pyruvate carboxykinase and malic enzyme also contain PPREs in their regulatory regions. Although limited research has been conducted with farm animals to date, it is attractive to speculate that the PPARs represent a molecular mechanism that would function to coordinate the activity of the metabolic machinery necessary for fatty acid metabolism with the supply of fatty acids to tissues.
Esterification and export
Esterification is believed to ‘compete’ with oxidation for acyl-CoA in the liver of farm animals. The pathways for esterification of acyl-CoA to glycerolipids in liver are similar to those discussed earlier for adipose tissue. In rodents, the activities of phosphatidate phosphohydrolase and diacylglycerol acyltransferase appear to be increased in times of high insulin; little is known about regulation of these enzymes in farm animals. In dairy cows, hepatic
capacity for esterification of fatty acids is increased around calving (Grum et al., 1996), which may contribute to the propen- sity of dairy cows to develop fatty livers around the time of calving. The enzymes glycerophosphate acyltransferase, diacyl- glycerol acyltransferase and phosphatidate phosphohydrolase (Fig. 5.3) are potential regulatory sites for accumulation of triacyl- glycerol in the liver, but data supporting their role are inconclusive.
The general mechanisms for synthesis and secretion of VLDLs from liver are well known (Bauchart, 1993). Apoprotein B is the key component whose rate of synthesis in the rough endoplasmic reticulum is believed to control the overall rate of VLDL production. Lipid components that are synthesized in the smooth endoplasmic reticulum are added to apoprotein B as it moves to the junction of the two compart- ments. After being carried to the Golgi apparatus in transport vesicles, the apopro- teins are glycosylated. Secretory vesicles bud off the Golgi membrane and migrate to the sinusoidal membrane of the hepato- cyte. The vesicles fuse with the membrane and release the VLDLs into blood in the space of Disse.
Ruminants and swine do not export triacylglycerol from the liver as VLDLs as efficiently as do poultry or laboratory rodents. In particular, ruminants have a very low rate of VLDL export compared with rats, despite similar rates of esterifica- tion of fatty acids to triacylglycerols (Kleppe et al., 1988). Where the limitation in VLDL synthesis or secretion resides is unknown (Bauchart, 1993). Based on avail- able evidence, it appears that the rate of synthesis or assembly of VLDLs is more likely to be limiting than is the secretory process per se.Possible limitations include a low rate of synthesis or a high rate of degradation of apoprotein B, or deficient synthesis of phosphatidylcholine or cholesterol.
The rate of export of triacylglycerol from the liver corresponds in general to the relative rate of de novofatty acid synthesis among species, with species such as cattle and pigs that do not synthesize fatty acids
in the liver also having the lowest rates of triacylglycerol export (Pullen et al., 1990).
On the other hand, poultry and fish actively synthesize fatty acids in the liver and secrete VLDLs at very high rates. Rates of VLDL export are intermediate for species that have lipogenesis in both liver and adipose tissue, such as rats and rabbits. In rats, the origin of the fatty acids incor- porated into triacylglycerol can affect the rate of VLDL export. Dietary conditions that promote lipogenesis in liver also stimulate VLDL output. In contrast, high fat diets or conditions that promote mobilization of fatty acids from adipose tissue decrease the rate of VLDL synthesis but promote formation of a separate pool of storage triacylglycerol (Wiggins and Gibbons, 1996). Because the latter condi- tion (uptake by the liver of fatty acids mobilized from adipose tissue) is similar to that usually encountered in ruminants, similar factors may govern the rate of VLDL synthesis in ruminants (Bauchart, 1993).
Consequently, conditions in ruminants that promote extensive body fat mobiliza- tion usually result in accumulation of triacylglycerol within the liver, potentially resulting in fatty liver. Problems with fatty liver in dairy cows are more likely in over- fattened cows, possibly as a result of high insulin and its effects on fatty acid esterification in the liver, and increased insulin resistance in peripheral tissues such as adipose tissue. The mechanism of clearance of accumulated triacylglycerol has not been determined definitively. No hormone-sensitive lipase is present in the liver of farm animals. In rats, the stored lipid droplets do not contribute appreciably to synthesis of VLDLs (Wiggins and Gibbons, 1996). Rather, it appears that the lipid droplet must be degraded by lysosomal acid lipases to free fatty acids, which then can be metabolized by the liver (Cadórniga- Valiño et al., 1997).