J.K. DrackleyDepartment of Animal Sciences, University of Illinois,
Urbana, Illinois, USA
Introduction
Traditionally, the study of lipid absorptionand metabolism in farm animals hascentred on the role of dietary lipids inprovision of dietary energy and inprocesses of fat deposition in meat, milkand eggs. Interest in these areas remainsintense because of the importance of fatdeposition in determining the efficiencyand, hence, profitability, of meat and milkproduction. The content and compositionof fat in animal products has becomeincreasingly important to consumer per-ceptions of the healthfulness of meat andmilk. Although a normal part of animalgrowth or milk synthesis, fat synthesisdecreases the efficiency of conversion offeed nutrients into lean meat or low-fatmilk products that consumers desire.Furthermore, metabolism of lipids in theliver is an integral component of animalproduction and is a key factor in develop-ment of metabolic disorders such as ketosisand fatty liver. Consequently, this chapterwill focus primarily on aspects of diges-tion, absorption and transport of dietarylipids, lipid synthesis, lipid mobilizationand lipid metabolism in the liver.
In addition to the primary and well-studied roles of lipids as energy substrates,however, many new roles of lipids asbiological mediators and second messengersin processes of signal transduction havebeen discovered in recent years. Anoverview of these functions will also bepresented.
Digestion, Absorption and Transportof Dietary Lipids
Digestion and absorption of lipids in non-ruminants
Lipids consumed by non-ruminant animalsare predominantly triacylglycerols (tri-glycerides), with the exception of herbi-vorous animals such as horses and rabbitsthat may consume considerable amounts ofgalactolipids from vegetative material.Proteolytic activity in the stomach helps torelease lipids from feed matrices, and theacid conditions and churning activitycaused by gastric motility serve to dispersethe lipids into a coarse emulsion. Lipaseactivity is present in the stomach, whichmay arise from enzymes synthesized and
secreted by the salivary glands (salivarylipase) as well as by the fundic region ofthe stomach (Gargouri et al., 1989).Salivary lipase possesses significanthydrolytic activity at pH 3.5, near that ofthe stomach. Gastric lipase is present athigher activities in neonatal animals andhas higher hydrolytic activity toward milktriacylglycerols than does pancreatic lipase(Jensen et al., 1997). Gastric lipase attacksprimarily short- and medium-chain fattyacid linkages on the sn-3 position oftriacylglycerol, such as those prevalent inmilk of ruminants and swine.
Bile is essential for further lipid diges-tion and absorption in the small intestine(Brindley, 1984). The primary componentsof bile necessary for lipid digestion are thebile salts and phospholipids. Bile salts,which are responsible for emulsification oflipid droplets, are synthesized fromcholesterol in hepatocytes of the liver. Thebile salts are conjugated by the liver withthe amino acids taurine or glycine, whichincreases the water solubility anddecreases the cellular toxicity of the bilesalts. Pigs conjugate bile salts to bothtaurine and glycine, whereas poultry onlyproduce taurine conjugates (Freeman,1984). The structure of the bile salts is suchthat it provides a flat molecule, with oneside being polar and hydrophilic and theother non-polar and hydrophobic. Thus,the bile salts lie at the water–lipid interfaceand do not penetrate deeply into eithersurface. Bile salts in bile are present incylindrical structures termed bile micelles(Brindley, 1984). The presence of bileimparts detergent-like effects on dietarylipids, causing lipid droplets to be sub-divided into smaller and smaller droplets.
Pancreatic secretions into the smallintestine are also critical for lipid digestionand absorption. Dispersion of lipid by bilesalts enables attachment of the pancreaticpolypeptide colipase, which attractspancreatic lipase and enables it to interactat the surface of the lipid droplet (Brindley,1984). Although the pancreatic lipase itselfhas no specific requirement for bile salts,the increased surface area created by theaction of bile salts greatly increases the rate
of pancreatic lipase-catalysed triacyl-glycerol hydrolysis. Pancreatic lipasespecifically attacks the sn-1 and sn-3 link-ages of triacylglycerols, resulting in forma-tion of 2-monoacylglycerols and free fattyacids. Phospholipases, particularly of theA1 and A2 types, are secreted in pancreaticjuice and convert the phospholipid lecithin(phosphatidylcholine) to lysolecithin(lysophosphatidylcholine).
Absorption of lipids is dependent onthe formation of mixed micelles and oncontinued movement of lipids from oildroplets in the intestinal lumen into themixed micelles. In the presence of bilesalts, the fatty acids and monoacylglycerolsproduced by pancreatic lipase action spon-taneously aggregate into mixed micelles.Lysolecithin produced from biliary anddietary phospholipids plays a key role information and stabilization of micelles. Inparticular, lysolecithin is highly efficient atsolubilizing highly non-polar lipids suchas stearic acid (18:0) into mixed micelles(Brindley, 1984). Formation of mixedmicelles is necessary to move the non-polar lipids across the unstirred waterlayer present at the surface of the intestinalmicrovillus membranes; this unstirredwater layer is thought to be the mainbarrier to lipid absorption (Brindley, 1984).Fatty acids and monoacylglycerols canenter the intestinal cells by simple diffu-sion into the lipid membrane, although thepresence of transmembrane carrier proteinshas been postulated (Glatz et al., 1997).
Absorption of fatty lipids into intestinalepithelial cells is an energy-independentprocess that is facilitated by maintenanceof a concentration gradient into the cells.Several putative fatty acid translocaseproteins have been identified in tissues,but their role and mechanism of actionhave not been resolved (Glatz et al., 1997).After fatty acids are absorbed into cells,they become bound to low-molecularweight (12–15 kDa) binding proteins (Glatzet al., 1997). These binding proteins aid infatty acid absorption, prevent accumula-tion of potentially toxic free fatty acids andmay direct fatty acids to the appropriateintracellular sites for metabolism.
98 J.K. Drackley
Most absorption of fatty acids andmonoacylglycerols takes place in thejejunum in mammals. In fowl, some fattyacid absorption has been demonstrated inboth the duodenum and the ileum. Theextensive degree of antiperistaltic or refluxactivity in the avian intestinal tract maycontribute to this more diffuse location oflipid absorption (Freeman, 1984). Bile saltsare not absorbed until they reach theterminal ileum, but instead cycle back tothe intestinal lumen to participate infurther micelle formation. The bile salts areabsorbed efficiently in the ileum by anactive transport process and are returned tothe liver (enterohepatic circulation) to bereincorporated into bile. In both pigs andfowl, this active recycling means that thequantity of bile salts that must be synthe-sized by the liver is quite low (Freeman,1984). Small quantities of bile salts are notreabsorbed but enter the large intestine,where they are converted into productsknown as ‘secondary bile salts’ byanaerobic gut bacteria. Loss of this quantityof bile salts in the faeces is the only routefor cholesterol excretion from the body.
Fatty acids are activated for furthermetabolism within intestinal epithelialcells by esterification to coenzyme A(CoA), a process that consumes two high-energy phosphate bonds from ATP. In non-ruminants, the acyl-CoA molecules largelyare re-esterified to triacylglycerols by themonoacylglycerol pathway, in which acyl-CoA molecules are added sequentially to 2-monoacylglycerols absorbed from theintestinal lumen. A smaller quantity oftriacylglycerol is formed by the a-glycerol-phosphate pathway. Absorbed lysolecithinis re-acylated in intestinal cells to formlecithin. Cholesterol is actively synthesizedfrom acetyl-CoA in intestinal cells of mostfarm animal species. Some of thecholesterol is esterified with a long-chainfatty acyl-CoA by acyl-CoA–cholesterolacyltransferase (ACAT) to form cholesterolesters.
Delivery of triacylglycerol from theintestine to other organs of the bodyrequires that these highly non-polar lipidsare packaged into a form that is stable in
aqueous environments. To do so, the non-polar lipids (triacylglycerol, cholesterolesters, fat-soluble vitamins) are surroundedby amphipathic compounds such as freecholesterol, phospholipids and specificproteins called apoproteins (Hussain et al.,1996). The major apoproteins synthesizedby intestinal cells of most species are apo-B48, apo-AI and apo-AIV. The resultingparticles, called chylomicrons, are quitelarge in mammalian species (50–500 nm)and contain by weight 85–95% triacyl-glycerol, 4–9% phospholipids, ~1% freecholesterol, ~0.5% esterified cholesteroland ~0.6% protein (Brindley, 1984). Thesize, but not number, of the chylomicronsincreases in proportion to larger dietaryintakes of lipid. Chylomicrons are secretedfrom the intestinal cells and enter thelacteals of the lymphatic system, whichthen drains into the venous blood at thethoracic duct. Fatty acids of less than 14carbons are not actively esterified byintestinal enzymes, and instead areabsorbed directly into the portal vein asfree fatty acids.
In fowl, the intestinally synthesizedlipoprotein particles are classified as verylow-density lipoproteins (VLDLs), and aremuch lower in triacylglycerol content thanmammalian chylomicrons or even VLDLsfrom pigs or humans (Freeman, 1984). Thelymphatic system is poorly developed infowl, and consequently the VLDL areabsorbed directly into the portal vein.
Fatty acid digestibility is high in non-ruminants, with values often >80% in pigsand poultry and >90% in pre-ruminantcalves (Doreau and Chilliard, 1997).Intestinal fatty acid digestibility decreaseswith increasing chain length and increaseswith increasing unsaturation. Absorptionof saturated fatty acids is greater when theyare in the sn-2 position of triacylglycerols,because they are absorbed as the 2-mono-acylglycerol after pancreatic lipase action.Fatty acid digestibility increases somewhatwith age in both pigs and poultry; fatdigestibility in young chicks in particularis quite poor because of the limited pro-duction of bile salts (Doreau and Chilliard,1997).
Lipid Metabolism 99
Digestion and absorption of lipids in ruminants
Ruminant and non-ruminant animals differwith respect to strategies for lipid diges-tion, primarily because of the nature of thedietary lipids and the microbial processeswithin the rumen (Moore and Christie,1984). In forage-fed ruminants, dietarylipids consist primarily of galactolipidsand other glycolipids that are rich inlinolenic acid (18:3). Cereal grains andother concentrate ingredients contributetriacylglycerols that are high in linoleicacid (18:2). Oilseeds and animal fatscontribute triacylglycerols. A variety ofcommercial fat products are available,including calcium soaps of long-chain fattyacids, saturated or protected triacyl-glycerols, or mostly saturated free fattyacids. Phospholipids are smaller com-ponents of both grains and forages.
Bacteria and protozoa in the rumenhydrolyse complex lipids (glycerides) into
their constituent long-chain fatty acids,sugars, organic bases (choline, ethanol-amine, serine) and glycerol. Thus, therumen is the primary site of complex lipidhydrolysis, rather than the small intestineas in non-ruminants and pre-ruminants.The glycerol and sugars are fermentedrapidly to volatile fatty acids (mainlyacetic, propionic and butyric).
Unsaturated fatty acids are hydro-genated extensively to saturated fatty acids(stearic, 18:0 and palmitic, 16:0) byruminal bacteria and protozoa. The processof hydrogenation, which only occurs onfree fatty acids, requires a mixed popula-tion of microbial species (Doreau andChilliard, 1997). The biohydrogenation oflinoleic acid occurs through the sequenceof reactions shown in Fig. 5.1. The firstisomerization reaction converts the cis-9,cis-12 linoleic acid to the cis-9, trans-11form, known as conjugated linoleic acid(CLA). Most of the CLA that is produced inthe rumen is hydrogenated to trans-11
100 J.K. Drackley
Fig. 5.1. General pathway for ruminal microbial hydrogenation of linoleic acid (cis-9, cis-12 18:2) throughconjugated linoleic acid (CLA; cis-9, trans-11 18:2) and trans-vaccenic acid (TVA; trans-11 18:1) to stearicacid (18:0).
octadecenoic acid (vaccenic acid), whichsubsequently is hydrogenated to stearicacid. However, small amounts of transisomers and CLA escape hydrogenationand are absorbed from the small intestine.These isomers are incorporated into milkand meat, which explains their relativelyhigh content in ruminant products.
Eight positional isomers of CLA arepossible, but the predominant product inthe rumen is the cis-9, trans-11 isomer.Research has demonstrated that thiscompound has widespread effects in anumber of biological systems, includinginhibition of carcinogenesis, decreasedbody fat accumulation, modulation of theimmune system and prevention of athero-sclerotic lesions (Belury, 1995). Factorsthat increase the accumulation of CLA inthe rumen and its subsequent absorptioninto milk and meat currently are thesubject of intense research effort.
Rumen microorganisms also synthe-size fatty acids, most of which are incor-porated into cell membrane phospholipids.Bacteria synthesize odd-chain fatty acidscontaining 15–17 carbons, as well asbranched-chain fatty acids, which also arerelatively unique to ruminant fats (Doreauand Chilliard, 1997). As a consequence ofthe unique actions of the rumen microbes,;85% of the lipids entering the smallintestine of ruminants are free fatty acids,which are predominantly saturated andadsorbed to the surface of small feedparticles. At the prevailing pH in therumen, most of the fatty acids are presentas salts of sodium, potassium or calcium.The remaining 15% of lipids reaching theduodenum consist mostly of bacterialphospholipids.
Pancreatic juice and bile enter theduodenum through the common bile ductand are essential for lipid digestion andabsorption in the small intestine.Ruminants secrete more taurine-conjugatedbile salts than glycine-conjugated bile saltsbecause the former are more soluble at thelow pH found in the ruminant smallintestine (Moore and Christie, 1984). Bilesalts are essential to dissociate fatty acidsadsorbed to feed particles and enable
micelle formation. Phospholipase A2secreted in pancreatic juice becomes activein the upper jejunum where the pH is morefavourable and hydrolyses fatty acids fromthe sn-2 position of phospholipids. Themajor phospholipid in the intestine isphosphatidylcholine (lecithin), whichenters in bile, pancreatic juice and digestafrom the abomasum. The resultant productof phospholipase-catalysed hydrolysis islysophosphatidylcholine (lysolecithin),which is an excellent detergent for forma-tion of mixed micelles from the highlysaturated fatty acids in the ruminant smallintestine.
Bile salts and lysolecithin promotemicelle formation from free fatty acids. Themixed micelle associates with the brushborder of the intestinal epithelium andfacilitates transfer of the hydrophobic fattyacids across the unstirred water layer at thesurface of the brush border membranes.Fatty acids and lysolecithin then diffuseacross intestinal cell membranes into thecells. The bile salts are not absorbed in thejejunum, but continue to form micelles.Most bile salts are absorbed in the ileumand are returned to the liver to be reincor-porated into bile.
Within the small intestinal cells, fattyacids are re-esterified to glycerol-3-phosphate to form triacylglycerols. Theglycerol-3-phosphate is formed from bloodglucose via glycolysis. Pre-ruminants con-suming milk triacylglycerol function likenon-ruminants and absorb large amounts of2-monoacylglycerol that can be re-esterifiedto form triacylglycerol. Along with apo-lipoproteins (B48, AI and AIV), cholesteroland phospholipids, the triacylglycerols arepackaged into lipoprotein particles that aresecreted from the cells and enter thelacteals to be carried into the lymph andreach the peripheral circulation. Theseparticles are analogous to chylomicrons innon-ruminants but are classified morecorrectly as VLDLs because of their smallsize in functioning ruminants. This is afactor of the highly saturated nature of thetriacylglycerols, the low dietary fat contentof ruminant diets and the constant flow ofdigesta into the intestine as compared with
Lipid Metabolism 101
the episodic nature of digesta entry in meal-feeding non-ruminants. Recent evidencealso indicates that some triacylglycerol-rich lipoproteins may be secreted into theportal vein of calves and functioningruminants (Bauchart, 1993).
Fatty acid digestibility in ruminants isusually lower and more variable than thatin non-ruminants (Doreau and Chilliard,1997). Intestinal digestibility does not differappreciably between 16- and 18-carbonfatty acids (average of 79 and 77%, respec-tively), and is slightly greater forunsaturated than saturated fatty acids (77,85 and 83% for 18:0, 18:1 and 18:2, respec-tively; Doreau and Chilliard, 1997).
Lipid transport: lipoprotein metabolism
With the exception of free fatty acids,which circulate bound to serum albumin,lipids circulate as components of largelipoprotein particles. Lipoproteins generallyare classified according to their buoyantdensity, which is determined by the rela-tive proportions of lipids and proteins. Thelargest lipoproteins are the chylomicrons,followed by VLDLs. These are also the leastdense materials because they carry largelipid loads with relatively small proteincontents. High-density lipoproteins (HDLs)are the smallest particles and the mostdense, having higher amounts of proteinand less lipid. Low-density lipoproteins(LDLs) have densities between those ofHDLs and VLDLs.
Intestinally derived lipoproteins rich intriacylglycerols (chylomicrons, VLDL) func-tion to deliver dietary long-chain fatty acidsto peripheral tissues (Fig. 5.2). The liveralso secretes VLDLs as a way to packageendogenous triacylglycerols for transport inplasma. Following secretion from intestinalcells or liver, these triacylglycerol-richlipoproteins acquire apo-CII from circulat-ing HDLs (Hussain et al., 1996). Apo-CII isan activator of the enzyme lipoproteinlipase (LPL), which is responsible for clear-ance of plasma triacylglycerol (Braun andSeverson, 1992). LPL is present in mosttissues and is found in high activities in
adipose tissue, lactating mammary gland,heart and skeletal muscle. Synthesis of LPLoccurs in the parenchymal cells of thetissue; the LPL is secreted from the cellsand translocated to the interior surfaces ofcapillaries perfusing the tissue. There, thehighly glycosylated LPL is anchored to thevascular surface of the endothelial cells byinteractions with heparin sulphate proteo-glycans on the cell surface (Braun andSeverson, 1992).
As chylomicrons and VLDLs movethrough the capillary beds, they becometrapped by LPL through interactions of thecarbohydrate moieties of apo-B and LPL.Binding is facilitated by the presence ofapo-CII in the triacylglycerol-rich lipo-proteins. Triacylglycerol hydrolysis occursrapidly, with release of free fatty acids andmonoacylglycerols. The fatty acids candiffuse into the cells or exit the tissue inthe venous blood. Although LPL is aproduct of a single gene in all tissues, itstranscription is regulated differently indifferent tissues through the presence oftissue-specific cis-acting elements (Braunand Severson, 1992). For example, LPLactivity is higher in adipose tissue duringmid-gestation, and is high in mammarygland during lactation. In cows, adiposeLPL increases markedly during mid- to latelactation to restore energy reserves(McNamara, 1991). During fasting, theactivity of LPL decreases in adipose tissueand increases in the heart. Thus, LPL mayhelp to direct dietary fatty acids to appro-priate tissues depending on the nutritionalstate of the animal, which in turn issignalled by insulin and other hormones.
After triacylglycerol hydrolysis, thelipoprotein particles remaining are termedremnants (from chylomicrons) andintermediate-density lipoproteins (IDLs;from VLDLs). Continued triacylglycerolhydrolysis by LPL and, in some species,hepatic lipase, eventually decreases thesize of the particles so that they can beremoved by the liver. Remnants and IDLsare actively removed by the liver in mostspecies via interaction with the apo-B,Ereceptors (Hussain et al., 1996). Otherportions of IDLs are converted to LDLs,
102 J.K. Drackley
which are end-products of the intravascularmetabolism of VLDLs. The LDLs, which arerich in cholesterol esters and phospho-lipids, are taken up by receptors in theskeleton, intestine, liver, adrenals andcorpus luteum (Fig. 5.2).
The HDLs are synthesized andsecreted by the liver and small intestine assmall discoidal particles consisting of aphospholipid bilayer containing only apo-Aand free cholesterol (Fielding and Fielding,1995). The particles become spherical ascholesterol esters are formed via thelecithin–cholesterol acyltransferase (LCAT)
reaction (Fig. 5.2). This enzyme, synthe-sized in liver and secreted into plasma,binds to the discoidal HDLs and catalysesthe transfer of a fatty acid (usually linoleicacid) from the sn-2 position of lecithin(phosphatidylcholine) to free cholesterol,forming cholesterol esters and lysolecithin.The non-polar cholesterol esters move intothe interior of the particle, causing it tobecome spherical and to enlarge as morecholesterol ester is formed. Lysolecithin istransferred to albumin in plasma. TheHDLs acquire excess surface components(phospholipids, apo-C, apo-E) from VLDLs
Lipid Metabolism 103
Fig. 5.2. Schematic diagram of lipoprotein metabolism in farm animals. Chylomicrons or very low-densitylipoproteins (VLDLs) secreted from the intestine or liver acquire apoprotein CII (apo-CII) from newly secreted(nascent) high-density lipoproteins (HDLs). Triacylglycerols in chylomicrons or VLDLs are hydrolysed bylipoprotein lipase (LPL) in peripheral tissues, which is activated by apo-CII and allows fatty acid uptake bytissues. The remaining particles (remnants or intermediate-density lipoproteins, IDLs) are cleared by the liveror undergo further triacylglycerol hydrolysis to produce low-density lipoproteins (LDLs). Excess surface com-ponents (phospholipids, PLs; apoproteins C and A; free cholesterol, chol) are transferred to HDLs. LDLs aredegraded in the liver or after receptor-mediated uptake in peripheral tissues. HDLs take up excess cholesterol(chol) from peripheral tissues and convert it to cholesterol esters by the action of lecithin cholesterol acyl-transferase (LCAT); lysolecithin is released into plasma, and cholesterol esters enter the core of HDLs. HDLscan deliver cholesterol to tissues or return it to the liver for conversion to bile salts. In some species,cholesterol esters may be transferred from HDLs to LDLs by cholesterol ester transfer protein.
or chylomicrons as these particles aremetabolized by LPL in peripheral tissues.
These metabolic functions of HDLsresult in these particles carrying out a cycleknown as reverse cholesterol transport, inwhich HDLs pick up excess free cholesterolfrom tissues and convert it to cholesterolester (Fielding and Fielding, 1995). TheHDL particles thus enlarge and grow lessdense as their content of cholesterol estersincreases. The HDLs then transfercholesterol esters to the liver for conver-sion into bile acids (the only route forexcretion of cholesterol from the body),and the now smaller HDLs can return torepeat the cycle (Fig. 5.2). Clearance ofHDL particles occurs in liver and bone.
The basic scheme of lipoproteinmetabolism as just discussed exhibitsmany species variations (Hollanders et al.,1986). Pigs have high concentrations ofLDLs, similarly to humans, and are oftenused as models of human lipoproteinmetabolism. In horses and ruminants,HDLs are the predominant lipoproteinsand serve to deliver cholesterol to steroido-genic tissues (liver, ovary, adrenals, testis)and to a variety of tissues for membranesynthesis. Many of the functions that LDLsplay in transport of cholesterol esters inhumans and rabbits are replaced by HDLsin ruminants and horses. In ruminants,there is considerable overlap in the densityrange of LDLs and HDLs, which makesseparation by traditional ultracentrifugalmethods difficult (Bauchart, 1993).
Lipid Synthesis and Mobilization
Lipid synthesis
LipogenesisLipogenesis (lipid synthesis) refers in thestrictest sense to synthesis of fatty acidsand not to the esterification of those fattyacids to glycerides. Adipose tissue is themain site of lipogenesis in non-lactatingcattle, sheep, goats, pigs, dogs and cats(Beitz and Nizzi, 1997). In poultry,similarly to humans, the liver is the majorsite of lipogenesis, while in rodents (rats
and mice) both liver and adipose tissue areimportant lipogenic sites. The mammarygland of lactating farm animals activelysynthesizes fatty acids.
De novo lipogenesis occurs in thecytosol and is a sequential cyclical processin which acetyl (2-carbon) units are addedsuccessively to a ‘primer’ or initial startingmolecule, usually acetyl-CoA but also 3-hydroxybutyrate in the lactating mam-mary gland of ruminants. The source of theacetyl units is acetyl-CoA, derived eitherfrom glucose through glycolysis in non-ruminants or pre-ruminants, or fromacetate via rumen fermentation of dietarycarbohydrates in ruminants. In functioningruminants, glucose is not used for fattyacid synthesis, which serves to spareglucose for other essential functions.
The nature of the mechanism thatminimizes lipogenic use of glucose byruminants remains unclear. Since thediscovery that activities of ATP-citratelyase and NADP-malate dehydrogenasewere lower in bovine adipose tissue thanin liver and adipose tissue of rats (Hansonand Ballard, 1967), it was believed thatthese enzymes limited lipogenic use ofglucose by ruminants. However, subse-quent research showed that lactate wasused by bovine adipose tissue (Whitehurstet al., 1978) and mammary gland (Forsberget al., 1985) at rates similar to those ofacetate. Lactate, after being converted topyruvate, is metabolized similarly topyruvate produced from glycolysis.Consequently, lactate also requires theenzymes ATP-citrate lyase and NADP-malate dehydrogenase in order to beconverted to fatty acids. The activity ofATP-citrate lyase in bovine adipose tissueand mammary gland, albeit lower than thatin rat tissues, is still sufficient to allow theobserved rates of lactate conversion to fattyacids (Forsberg et al., 1985). Moreover,ATP-citrate lyase activity is at least equalto that of acetyl-CoA carboxylase, the rate-limiting step in fatty acid synthesis (Beitzand Nizzi, 1997). Probably the most likelyexplanation for the low rate of incorpora-tion of glucose into fatty acids in ruminantadipose tissue and mammary gland is the
104 J.K. Drackley
limited flux of glucose carbon past thetriose phosphate stage in glycolysis(Forsberg et al., 1985), because of the highdemand for glycerol-3-phosphate fortriacylglycerol synthesis, and the activemetabolism of glucose in the pentosephosphate pathway to produce NADPH.
The rate-limiting step in fatty acidsynthesis is catalysed by the enzymeacetyl-CoA carboxylase (Hillgartner et al.,1995). This enzyme converts acetyl-CoA tomalonyl-CoA, which is the actual ‘donor’of acetyl units in the elongation process.Two forms of the enzyme, termed a and b,are found in animals (Kim, 1997). The a-form is the enzyme found in lipogenictissues that regulates the rate of fatty acidsynthesis. The b-form is found in non-lipogenic tissues and is associated withcontrol of mitochondrial fatty acid oxida-tion (discussed later). The a-form of theenzyme is subject to several levels of meta-bolic regulation from signals of nutrientstatus. Insulin, released when dietaryenergy is plentiful, activates the enzymeand so promotes fat storage. Increasedconcentrations of citrate and isocitrate,which also would signal increased sub-strate availability for storage as fat, activatethe reaction. In contrast, glucagon and thecatecholamines inhibit its activity viacyclic AMP (cAMP)-dependent phos-phorylation. In this way, fat synthesis isinhibited during times when mobilizationof energy stores is required. Increased con-centrations of fatty acyl-CoA in the cytosolinhibit the reaction, a form of negativefeedback. In addition to short-term changesin enzyme activity caused by thesehormones and metabolites, the abundanceof the enzyme protein is also regulated.Starvation decreases the amounts of boththe mRNA and the protein, while refeedingafter a fast causes a large increase intranscription and translation of mRNA foracetyl-CoA carboxylase (Hillgartner et al.,1995).
The fatty acid synthase enzymecomplex consists of two multifunctionalpolypeptide chains, each containing sevendistinct enzyme activities necessary toelongate a growing fatty acid (Smith, 1994).
The two polypeptide chains are arrangedhead-to-tail, resulting in two separate sitesfor synthesis of fatty acids; thus eachenzyme complex can assemble two fattyacids simultaneously. The activity of theenzyme complex is not limiting to theoverall rate of fatty acid synthesis. Theoverall reaction for synthesis of onemolecule of palmitic acid is:
In non-ruminants the hydrogen donor,NADPH, is generated through metabolismof glucose in the pentose phosphate path-way and in the malic enzyme reaction. Inruminants, cytosolic isocitrate dehydro-genase can generate over one-half of theNADPH needed through metabolism ofacetate (Beitz and Nizzi, 1997). Theremainder of the NADPH in ruminants isderived from glucose metabolism in thepentose phosphate pathway. The presenceof glucose enhances fatty acid synthesis inruminants, probably through enhancedproduction of NADPH. Regulation of fattyacid synthase is largely through intra-cellular concentrations of dietary or synthe-sized fatty acids, which decrease its activity(Smith, 1994). High-fat diets decrease theintracellular concentration of fatty acidsynthase, whereas refeeding after a fastincreases its concentration. High concen-trations of insulin increase the abundanceof fatty acid synthase, whereas growthhormone, glucagon and glucocorticoidsdecrease its abundance (Hillgartner et al.,1995).
Lipogenesis generally increases asanimals age, although changes are depot-specific and may be modulated by diet(Smith, 1995). Thus, lipogenesis in internaladipose depots such as perirenal fat ismore active earlier in the growth stage, andless active as the animal reaches physio-logical maturity. Somatotropin treatment ofpigs and cattle leads to decreased lipo-genesis, primarily by decreasing the sensi-tivity of adipose cells to the actions ofinsulin (Etherton and Bauman, 1998).
Lipid Metabolism 105
Other acyl-CoA molecules such aspropionyl-CoA can be used as primers bythe fatty acid synthase complex. In thiscase, odd-carbon numbered fatty acids willbe produced, most commonly of 15 or 17carbon length. In addition, methylmalonyl-CoA can replace malonyl-CoA in theelongation reactions, resulting in branched-chain (methyl-branched) fatty acids. Inmost lipogenic tissues, these fatty acids areonly minor products, but in sebaceous(skin) glands of some species the produc-tion of methyl-branched fatty acids may besubstantial (Smith, 1994). In ruminants,higher concentrations of odd-chain andbranched-chain fatty acids are found inmilk and adipose tissue because of thegreater synthesis of these fatty acids byrumen bacteria.
In adipose tissue, the predominantproduct of the lipogenic pathway ispalmitic acid. In the mammary gland oflactating animals, however, large quantitiesof fatty acids <16 carbons in length aresynthesized. This is due to the action ofspecific chain-terminating mechanisms,which differ between ruminants and non-ruminants. In ruminants, the fatty acidsynthase complex allows the release ofshort- and medium-chain fatty acyl-CoAesters, which are incorporated rapidly intomilk fat. In non-ruminants, a specificenzyme, thioesterase II, is responsible forhydrolysing the thioester bond of the 8–14carbon acyl chain, thus releasing themedium-chain fatty acids (Smith, 1994).
Elongation and desaturationThe end-product of the de novo lipogenicpathway in animal tissues is usuallypalmitic acid, yet this fatty acid constitutesonly 20–30% of total fatty acids in adiposetissue lipids (Rule et al., 1995).Considerable amounts of stearic (18:0) andoleic (18:1) acids are present in adiposetissue lipids, and may arise either fromintestinally derived triacylglycerol-richlipoproteins or by conversion frompalmitic acid in adipose tissue. Elongationof palmitic acid (16:0) to stearic acid occursby the action of fatty acid elongase, foundin the microsomal fraction (endoplasmic
reticulum) of adipocytes. Malonyl-CoA isthe source of the additional two carbons.Fatty acid elongase is found in much largeractivities in bovine adipose tissue than inmammary gland, liver, muscle or intestinalmucosa (Smith, 1995).
The concentration of stearic acid intissue lipids is regulated by the presence ofstearoyl-CoA desaturase (D9 desaturase),which converts stearic acid to oleic acid.This microsomal enzyme is a mixedfunction oxidase that inserts a double bondnine carbons from the methyl end of thefatty acid. Considerable activity of stearoyl-CoA desaturase is found in mammarygland, muscle and duodenal muscosa, butlittle activity is found in bovine liver(Smith, 1995). The primary function of theenzyme seems to be to regulate lipidfluidity by preventing excessive accumula-tion of the very high-melting stearic acid.
Glycerolipid synthesisFew free (non-esterified) fatty acids arefound in the animal body; rather, most fattyacids are found esterified to glycerol asglycerolipids such as triacylglycerols andphospholipids. In adipose tissue and thelactating mammary gland, most fatty acidsare esterified to form triacylglycerols as anon-toxic form of energy storage or fortransfer to the young, respectively. In liverand other tissues, most fatty acids areesterified to form phospholipids as com-ponents of intracellular and plasma mem-branes. The liver actively synthesizestriacylglycerols when presented with highconcentrations of non-esterified fatty acidsfrom the blood.
The enzymes necessary for glycerolipidbiosynthesis are found in the microsomalfraction of cells. The general pathways ofesterification of fatty acids are shown inFig. 5.3. Acyl chains from acyl-CoA aretransferred consecutively to glycerol-3-phosphate produced via glycolysis.Production of diacylglycerol (diglyceride)from phosphatidate by phosphatidatephosphohydrolase and subsequent produc-tion of triacylglycerol from diacylglycerol bydiacylglycerol acyltransferase may beregulatory steps for triacylglycerol synthesis,
106 J.K. Drackley
but these enzymes have not been wellcharacterized in farm animals.Esterification of fatty acids in adiposetissue increases with increasing energyintake in meat animals (Rule, 1995) and islower in times of dietary energy deficit,such as during early lactation in dairycows (McNamara, 1991).
Fatty acid composition of milk, muscle and body fat
A variety of fatty acids are found incomplex lipids of animal tissues. Thesefatty acids range primarily from 14 to 20carbons in length, with varying degrees ofunsaturation. Characteristic profiles of fattyacids are found in individual tissues andamong species of animals. Sample profilesof 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 lipidsfrom non-ruminants such as pigs becauseof ruminal biohydrogenation of dietary
unsaturated fatty acids. Experimental post-ruminal infusions of unsaturated oils andfeeding formaldehyde-protected oils tosheep and cattle results in increasingunsaturation of adipose tissue lipids (Ruleet al., 1995). In pigs and chickens, increas-ing amounts of dietary fat will result inadipose tissue lipids reflecting the fattyacid composition of the dietary fat. Bodyfat generally becomes softer in thesespecies with supplementation of fats andoils, because the relative amounts of denovo synthesized palmitic acid decreaseand those of 18-carbon unsaturated fattyacids increase (Rule et al., 1995).
Bovine milk fat contains considerableamounts of fatty acids shorter than 14carbons that are synthesized within themammary gland (Table 5.2). The fatty acidcomposition of milk fat can be alteredmarkedly by supplementation of the dietwith fat (Palmquist et al., 1993). Dietarylong-chain fatty acids suppress de novosynthesis of short- and medium-chain fatty
Lipid Metabolism 107
Fig. 5.3. Major pathways of esterification of fatty acids to glycerolipids in farm animals. The key enzymesinvolved are: (1) glycerophosphate acyltransferase; (2) lysophosphatidate acyltransferase; (3) phosphatidatephosphohydrolase; (4) diacylglycerol acyltransferase; and (5) monoacylglycerol acyltransferase. Pi, inorganicphosphate. Adapted from Rule (1995).
acids in the mammary gland. Unprotectedfats lead to only slight increases in poly-unsaturated fatty acids in milk fat, but maylead to appreciable increases in oleic acidbecause of intestinal and mammarydesaturation of stearic acid produced byruminal biohydrogenation of dietaryunsaturated fatty acids (Table 5.2). Thebovine mammary gland readily incorporatesunsaturated fatty acids presented to it(LaCount et al., 1994). Producing milk withmore monounsaturated and polyunsaturatedfatty acids depends on the development ofpractical strategies to protect dietaryunsaturated fatty acids from hydrogenationby rumen microbes. Currently, formalde-hyde treatment of protein–fat mixtures is thebest methodology for rumen protection(Doreau and Chilliard, 1997), but regulatoryapproval may limit its application in manycountries.
Lipolysis
Mobilization of fatty acids from adiposetissue triacylglycerols (lipolysis) occursduring times of negative energy balance orin response to stresses. The reactionproceeds by the sequential release of fattyacids from the glycerol backbone. The fattyacids released increase the size of the intra-cellular free fatty acid pool and, in the
absence of stimuli to re-esterify those fattyacids, they diffuse from the cell into theblood. The free fatty acids are adsorbedquickly to binding domains on serumalbumin, and circulate to various tissues asa fatty acid–albumin complex. Physiologicalstates characterized by high rates of lipo-lysis, such as early lactation in dairy cowsand sows (McNamara, 1991), often are alsocharacterized by relatively lower concen-trations of albumin in the blood. Hence,the ratio of free fatty acids to albumin inblood increases, which favours greateruptake of the free fatty acids by tissues ofthe body because more fatty acids occupylower affinity binding sites on the albuminmolecule. Furthermore, the increased ratioof fatty acids to albumin increases the sizeof the tissue free fatty acid pool, which inturn increases re-esterification of fattyacids in adipose tissue and thus providesfeedback regulation on lipolysis (Metz andvan den Bergh, 1977).
The initial step in lipolysis is catalysedby hormone-sensitive triacylglycerol lipase.This enzyme is activated by binding ofhormones that stimulate formation of cAMPby adenyl cyclase. In mammals, the primary
108 J.K. Drackley
Table 5.1. Typical profiles of major fatty acidsfound in lipids from subcutaneous adipose tissue orlongissimus muscle from cattle, sheep and pigs (gkg21). (Adapted from Rule et al., 1995.)
Table 5.2. Fatty acid composition of milk fat fromcows fed a basal low-fat diet or the basal dietsupplemented with tallow. (Adapted from Palmquistet al., 1993.)
Diet (g kg21 of methyl esters)
Fatty acid Basal Basal + tallow
4:0 33 356:0 27 23a
8:0 18 13a
10:0 40 26a
12:0 46 29a
14:0 130 103a
14:1 15 1315:0 13 10a
16:0 299 28416:1 17 1817:0 6 8a
18:0 90 10418:1 172 233a
18:2 22 16a
18:3 6 9a
aDifferent from basal diet, P < 0.05.
agonists for this reaction are thecatecholamines, such as epinephrine andnorepinephrine. In poultry, glucagon is themajor lipolytic hormone. Binding of thesehormones to cell surface receptors causesactivation of adenyl cyclase, depending onthe balance between activation of thestimulatory guanine nucleotide-bindingprotein (Gs protein) and activation of theinhibitory Gi protein (Lafontan and Langin,1995). Receptor types vary among tissuesand species in the relative activation ofthese two G proteins. Agonists that activateb-adrenergic receptors cause activation ofGs. Activation of Gs activates adenyl cyclase,which increases the concentration of cAMPin the cell. In turn, cAMP activates proteinkinase A, which phosphorylates the regula-tory subunit of hormone-sensitive lipase.The activated hormone-sensitive lipasethen catalyses lipolysis of triacylglycerol.
Inhibition of lipolysis depends on agreater activation of Gi proteins, whichinhibit adenyl cyclase and increase theactivity of phosphodiesterase, the enzymethat degrades cAMP. Agonists that bind toa-adrenergic receptors activate Gi and thussuppress lipolysis (Lafontan and Langin,1995). Factors such as insulin, adenosineand the E series of prostaglandins areassociated with decreased activity ofhormone-sensitive lipolysis. Treatment ofanimals with somatotropin results in anindirect stimulation of lipolysis by increas-ing the sensitivity of adipose tissue to theeffects of the catecholamines. Somatotropincauses this increased sensitivity bydiminishing the ability of the Gi proteins toinhibit adenyl cyclase. Thus, suppressionof the inhibitory controls of lipolysisallows higher rates of lipolysis to occurduring treatment with somatotropin.
Another factor controlling the relativedegree of lipolysis is the degree to whichfatty acids are re-esterified to form triacyl-glycerols before they can diffuse out of thecells. Insulin stimulates uptake of glucoseand glycolysis, increasing the supply ofglycerol-3-phosphate available for esterifica-tion. Insulin also stimulates the activity ofthe esterification pathway. Control of lipo-lysis is interwoven tightly with regulation
of lipogenesis, so that the overall functionof adipose tissue to accrete or releaseenergy stores is coordinated according tothe physiological needs of the animal.
Metabolism of Lipids in the Liver
Oxidation and ketogenesis
The liver takes up free fatty acids fromblood in proportion to their concentration.Within the hepatocytes (liver cells), long-chain fatty acids of 14 carbons or more areactivated by acyl-CoA synthetases found inthe microsomes and outer mitochondrialmembrane. Acyl-CoA may either enter themitochondria for oxidation or be esterifiedwithin the endoplasmic reticulum (micro-somes). Under conditions of increased fattyacid uptake, the liver often produces largeamounts of the ketone bodies, acetoacetateand b-hydroxybutyrate, in the processknown as ketogenesis. The two mainfactors regulating the degree to which fattyacids are oxidized by the liver are thesupply of fatty acids to the liver via lipo-lysis and the partitioning within hepato-cytes between mitochondrial oxidation andmicrosomal esterification.
No acyl-CoA synthetase enzymes thatcan activate fatty acids with 14 carbons ormore are present within the mitochondrialmatrix (McGarry et al., 1989). Therefore,entry of these long-chain fatty acids intothe mitochondria is regulated effectively bythe activity of the enzyme carnitinepalmitoyltransferase I (CPT-I). This enzymeis an integral membrane protein of the outermitochondrial membrane, and catalysesthe formation of fatty acyl-carnitine fromfatty acyl-CoA and free L-carnitine. Theacyl-carnitine molecules are then trans-ported across the mitochondrial membraneby a specific carrier protein, and are recon-verted to acyl-CoA within the mitochondrialmatrix by the action of CPT-II, a peripheralprotein of the inner mitochondrial mem-brane. Short- and medium-chain fatty acids(12 carbons or less) pass through the mito-chondrial membrane and are activated byacyl-CoA synthetases found within the
Lipid Metabolism 109
mitochondrial matrix. Consequently,oxidation of these fatty acids is not con-trolled by CPT-I.
The activity of CPT-I is inhibited byinteraction with malonyl-CoA, the productof the first committed step of lipogenesiscatalysed by acetyl-CoA carboxylase.Insulin stimulates the activity of acetyl-CoA carboxylase. Conditions of negativeenergy balance as signalled by lower ratiosof insulin to glucagon thus result indecreased concentrations of malonyl-CoAand increased rates of fatty acid oxidation.Furthermore, in rats, the sensitivity of CPT-Ito malonyl-CoA is decreased during timesof low insulin or insulin resistance, whichdecreases the ability of the low concentra-tions of malonyl-CoA to inhibit acyl-carnitine formation and thereby furtherincreases the rate of fatty acid oxidation(Zammit, 1996).
Classical studies (reviewed by McGarryet al., 1989) that delineated the control ofCPT-I by malonyl-CoA in rats described thismechanism as a means of preventing simul-taneous oxidation and synthesis of fattyacids within the liver cell, a potential futilecycle. However, in cattle, sheep and swine,rates of lipogenesis are very low in liver,which obviates the need for such a controlmechanism. Nevertheless, production ofmalonyl-CoA by acetyl-CoA carboxylasedoes occur in bovine, ovine and swine liver(Brindle et al., 1985), probably as a controlmechanism for oxidation rather than as aquantitatively important site of fatty acidsynthesis. Likewise, skeletal muscle andheart muscle are also non-lipogenic tissuesthat use fatty acids as energy sources. Bothheart and skeletal muscle of rats contain ahigh activity of acetyl-CoA carboxylase ofthe b-isoform (Kim, 1997). Physiologicalsituations that lead to low insulin toglucagon ratios and decreased activity ofacetyl-CoA carboxylase in these tissuesresult in increased rates of fatty acid oxida-tion. Whether the acetyl-CoA carboxylasepresent in the liver of ruminants and swineis similar to the b-isoform of rats has notbeen determined.
Intramitochondrial oxidation of fattyacyl-CoA occurs through the b-oxidation
pathway, resulting in formation of acetyl-CoA. During this process, electrons aretransferred to FAD and NAD+ to form thereduced forms of these coenzymes, whichin turn can donate electrons to the electrontransport chain to drive ATP synthesis. Theacetyl-CoA can be oxidized completely tocarbon dioxide in the tricarboxylic acid(TCA) cycle. Alternately, acetyl-CoA can bediverted to formation of ketone bodies.Ketogenesis is enhanced in times ofincreased fatty acid mobilization anduptake by the liver, when low ratios ofinsulin to glucagon cause activation ofCPT-I that allows extensive uptake of fattyacids into mitochondria (Zammit, 1990).Conversion of acetyl-CoA to ketone bodiesrather than complete oxidation in the TCAcycle results in formation of less ATP permole of fatty acid oxidized. For example,complete oxidation of palmitate in the TCAcycle, followed by oxidative phosphoryla-tion in the electron transport chain, yields129 ATP per molecule of palmitate. Incontrast, b-oxidation of palmitate withacetyl-CoA converted to ketone bodiesgenerates only 27 ATP per molecule ofpalmitate. Because the production of ATPmust match its utilization for energy-requiring reactions in the liver, ketogenesisallows the liver to metabolize about fivetimes more fatty acid for the same ATPyield. Conversion of fatty acids into water-soluble fuels may be an important strategyto allow the animal to cope with extensivemobilization of fatty acids during energydeficit.
In addition to control at the levels offatty acid supply and CPT-I, ketogenesis iscontrolled by the activity of the key regula-tory enzyme, 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) synthase. This enzyme iscontrolled both through increased tran-scription and translation during prolongedenergy deficit and by inactivation throughsuccinylation (Emery et al., 1992).Increased flux of metabolites such aspyruvate, propionic acid or glucogenicamino acids into the TCA cycle, resultingfrom greater feed intake and improvedenergy balance, results in increased poolsize of succinyl-CoA, an intermediate of
110 J.K. Drackley
the TCA cycle. The succinyl-CoA is used toadd a succinyl group to a regulatory sub-unit of HMG-CoA synthase, whichinactivates the enzyme.
In ruminants, CPT-I also is highlysensitive to inhibition by methylmalonyl-CoA (Brindle et al., 1985), which is anintermediate in the conversion ofpropionate to succinyl-CoA in the processof gluconeogenesis. This may constitute anadditional adaptation of ruminants to linkthe supply of energy-yielding compoundsfrom the diet with the need for hepaticfatty acid oxidation. Furthermore, theability to distinguish between glucogenicmolecules originating primarily throughruminal fermentation of dietary carbo-hydrates (propionate) and those originatingfrom catabolism of endogenous aminoacids (e.g. pyruvate) has been proposed asa unique adaptation of regulation of fattyacid oxidation in ruminants during energydeficit situations (Zammit, 1990).
Accelerated ketogenesis in response tolow blood glucose from insufficient dietaryenergy intake may occur in both lactatingcows and pregnant ewes. The increasedketogenesis is probably a factor ofincreased mobilization of free fatty acidsfrom adipose tissue, increased uptake offatty acids by the liver, increased activity ofCPT-I, decreased sensitivity of CPT-I tomalonyl-CoA and increased activity ofHMG-CoA synthase.
Ketogenesis has been shown to occur atlower rates in swine than in many otherspecies (Odle et al., 1995; Adams et al.,1997). This may be due to limitations bothin the ability of swine to form acylcarnitinesfor transport into the mitochondria (Odle etal., 1995) and in the activity of HMG-CoAsynthase (Adams et al., 1997). Limitationsare particularly pronounced in neonates,with some developmental increase in oxida-tive capacity observed with advancing agein pigs (Adams et al., 1997; Yu et al., 1997).
Peroxisomal metabolism
An alternate pathway for b-oxidation inliver is found in peroxisomes, which are
subcellular organelles present in mosttissues (Singh, 1997). The peroxisomalpathway for b-oxidation functionssimilarly to the mitochondrial pathway,with notable exceptions. First, the firstdehydrogenase step of mitochondrial b-oxidation is replaced with an oxidase step(acyl-CoA oxidase) in the peroxisome,resulting in formation of hydrogenperoxide rather than reduced NAD+.Second, peroxisomes do not contain anelectron transport chain. As a result of thesefactors, peroxisomal b-oxidation results incapture of less energy as ATP than doesmitochondrial b-oxidation. Another uniqueaspect of the peroxisomal pathway is thattwo enzymic activities of the b-oxidationpathway (enoyl-CoA hydratase and 3-hydroxyacyl-CoA dehydrogenase) areperformed by a single multifunctionalprotein, called the bifunctional protein.
Peroxisomal b-oxidation is active withvery long-chain fatty acids (20 carbons ormore) that are relatively poor substrates formitochondrial b-oxidation. Becauseperoxisomal b-oxidation enzymes areinduced in rats by situations leading toincreased supply of fatty acids in the liver,such as high dietary fat, starvation anddiabetes, the peroxisomal b-oxidation path-way has been discussed as an ‘overflow’pathway to help cope with increased fluxof fatty acids (Singh, 1997).
Recent investigations in dairy cows(Grum et al., 1994), sheep (Hansen et al.,1995) and pigs (Yu et al., 1997) haveshown that the livers of these farm animalspossess relatively high peroxisomal b-oxidation activity. In neonatal pigs,peroxisomal b-oxidation increases rapidlyafter birth in response to milk intake andmay be an adaptive mechanism to aid inoxidation of long-chain fatty acids frommilk in the face of relatively low capacityfor mitochondrial fatty acid oxidation (Yuet al., 1997). In dairy cows, peroxisomal b-oxidation is not induced by dietary fatduring lactation or by starvation (Grum etal., 1994), but increases in response todietary fat and nutrient restriction duringthe periparturient period. Subsequent tothe increased peroxisomal b-oxidation
Lipid Metabolism 111
prepartum, less triacylglycerol accumulatesin the liver at the time of calving (Grum etal., 1996). This may be an adaptation toallow increased metabolism of fatty acidsduring their extensive mobilization.
During the last decade, research hasidentified specific nuclear receptors thatare activated by fatty acids and chemicalsthat cause peroxisomal proliferation inrodents. These receptors, called peroxisomeproliferator-activated receptors (PPARs), inturn bind to specific peroxisome prolifera-tor response elements (PPREs) located inthe regulatory region of a number of geneswhose products are associated with lipidmetabolism (Schoonjans et al., 1996).These include long-chain acyl-CoAsynthetase; the peroxisomal enzymes acyl-CoA oxidase and bifunctional protein; themitochondrial enzymes CPT-I, medium-chain acyl-CoA dehydrogenase and HMG-CoA synthase; and the microsomalcytochrome P450 enzymes CYP4A1 andCYP4A6, which catalyse v-oxidation.Furthermore, the liver fatty acid-bindingprotein gene contains a PPRE. In rats, thegluconeogenic enzymes phosphoenol-pyruvate carboxykinase and malic enzymealso contain PPREs in their regulatoryregions. Although limited research hasbeen conducted with farm animals to date,it is attractive to speculate that the PPARsrepresent a molecular mechanism thatwould function to coordinate the activityof the metabolic machinery necessary forfatty acid metabolism with the supply offatty acids to tissues.
Esterification and export
Esterification is believed to ‘compete’ withoxidation for acyl-CoA in the liver of farmanimals. The pathways for esterification ofacyl-CoA to glycerolipids in liver aresimilar to those discussed earlier foradipose tissue. In rodents, the activities ofphosphatidate phosphohydrolase anddiacylglycerol acyltransferase appear to beincreased in times of high insulin; little isknown about regulation of these enzymesin farm animals. In dairy cows, hepatic
capacity for esterification of fatty acids isincreased around calving (Grum et al.,1996), which may contribute to the propen-sity of dairy cows to develop fatty liversaround the time of calving. The enzymesglycerophosphate acyltransferase, diacyl-glycerol acyltransferase and phosphatidatephosphohydrolase (Fig. 5.3) are potentialregulatory sites for accumulation of triacyl-glycerol in the liver, but data supportingtheir role are inconclusive.
The general mechanisms for synthesisand secretion of VLDLs from liver are wellknown (Bauchart, 1993). Apoprotein B isthe key component whose rate of synthesisin the rough endoplasmic reticulum isbelieved to control the overall rate of VLDLproduction. Lipid components that aresynthesized in the smooth endoplasmicreticulum are added to apoprotein B as itmoves to the junction of the two compart-ments. After being carried to the Golgiapparatus in transport vesicles, the apopro-teins are glycosylated. Secretory vesiclesbud off the Golgi membrane and migrate tothe sinusoidal membrane of the hepato-cyte. The vesicles fuse with the membraneand release the VLDLs into blood in thespace of Disse.
Ruminants and swine do not exporttriacylglycerol from the liver as VLDLs asefficiently as do poultry or laboratoryrodents. In particular, ruminants have avery low rate of VLDL export comparedwith rats, despite similar rates of esterifica-tion of fatty acids to triacylglycerols(Kleppe et al., 1988). Where the limitationin VLDL synthesis or secretion resides isunknown (Bauchart, 1993). Based on avail-able evidence, it appears that the rate ofsynthesis or assembly of VLDLs is morelikely to be limiting than is the secretoryprocess per se. Possible limitations includea low rate of synthesis or a high rate ofdegradation of apoprotein B, or deficientsynthesis of phosphatidylcholine orcholesterol.
The rate of export of triacylglycerolfrom the liver corresponds in general to therelative rate of de novo fatty acid synthesisamong species, with species such as cattleand pigs that do not synthesize fatty acids
112 J.K. Drackley
in the liver also having the lowest rates oftriacylglycerol export (Pullen et al., 1990).On the other hand, poultry and fishactively synthesize fatty acids in the liverand secrete VLDLs at very high rates. Ratesof VLDL export are intermediate for speciesthat have lipogenesis in both liver andadipose tissue, such as rats and rabbits. Inrats, the origin of the fatty acids incor-porated into triacylglycerol can affect therate of VLDL export. Dietary conditionsthat promote lipogenesis in liver alsostimulate VLDL output. In contrast, highfat diets or conditions that promotemobilization of fatty acids from adiposetissue decrease the rate of VLDL synthesisbut promote formation of a separate pool ofstorage triacylglycerol (Wiggins andGibbons, 1996). Because the latter condi-tion (uptake by the liver of fatty acidsmobilized from adipose tissue) is similar tothat usually encountered in ruminants,similar factors may govern the rate of VLDLsynthesis in ruminants (Bauchart, 1993).
Consequently, conditions in ruminantsthat promote extensive body fat mobiliza-tion usually result in accumulation oftriacylglycerol within the liver, potentiallyresulting in fatty liver. Problems with fattyliver in dairy cows are more likely in over-fattened cows, possibly as a result of highinsulin and its effects on fatty acidesterification in the liver, and increasedinsulin resistance in peripheral tissuessuch as adipose tissue. The mechanism ofclearance of accumulated triacylglycerolhas not been determined definitively. Nohormone-sensitive lipase is present in theliver of farm animals. In rats, the storedlipid droplets do not contribute appreciablyto synthesis of VLDLs (Wiggins andGibbons, 1996). Rather, it appears that thelipid droplet must be degraded by lysosomalacid lipases to free fatty acids, which thencan be metabolized by the liver (Cadórniga-Valiño et al., 1997).
Metabolism of Essential Fatty Acids
Animals can synthesize fatty acids withdouble bonds no closer than nine carbons
from the methyl end of the fatty acyl chain.For example, stearic acid (18:0) can bedesaturated to oleic acid (18:1) bydesaturase enzymes in liver, adipose tissue,intestinal mucosa and mammary gland.The convention for nomenclature of theposition of double bonds within the fattyacyl chain refers to the carbon numberstarting from the methyl carbon end of thefatty acid, with the methyl carbon referredto as the ‘v-carbon’. Thus, oleic acid isreferred to in shorthand notation as 18:1 v-9, because the double bond occurs at theninth carbon from the methyl end.Alternate nomenclature refers to this as the‘n-9’ or ‘D9’ position. Likewise, the enzymeactivity responsible for conversion ofstearic to oleic acid is usually referred to asD9-desaturase.
Polyunsaturated fatty acids withdouble bonds nearer to the end of the chainare required for normal formation of cellmembranes and synthesis of other keyregulatory molecules such as prosta-glandins (Sardesai, 1992). These fatty acidsfall into two groups, the v-6 series and v-3series. Because animal tissues are unable tosynthesize fatty acids with double bonds inthe v-6 or v-3 positions, such fatty acidsmust be supplied in the diet. In mostspecies, the parent compounds of thesefamilies, linoleic acid (18:2 v-6) andlinolenic acid (18:3 v-3), respectively, arethe only fatty acids that are required fromdietary sources. Consequently, these arereferred to as dietary essential fatty acids.These fatty acids can be elongated anddesaturated to produce longer chain fattyacids that are more highly unsaturated. Forexample, linoleic acid can be converted toarachidonic acid (20:4 v-6) beginning withD6-desaturation, followed by elongationand D5-desaturation (Fig. 5.4). However,because the position of the final doublebond in the chain is always fixed from themethyl end, linoleic acid cannot be con-verted to eicosapentaenoic acid (20:5 v-3)or docosahexaenoic acid (22:6 v-3). Catsand some other carnivores have very limitedactivities of the D6-desaturase enzyme, andthus require dietary arachidonic acid aswell as linoleic and linolenic acid.
Lipid Metabolism 113
The enzymes for elongation anddesaturation are found in the microsomalfraction of cells. The distribution ofenzyme activity varies among organs ofanimals, with the greatest activity in theliver and the adrenal glands and onlylimited activity in tissues such as the heart,kidneys and brain (Bézard et al., 1994).
One function of the essentialunsaturated fatty acids is in maintainingthe appropriate fluidity of cell membranes(Sardesai, 1992). Because unsaturated fattyacids have lower melting points thansaturated fatty acids, their presence inmembranes makes the membranes morefluid. Changes in the fluidity of cellmembranes can affect the degree to whichintegral membrane proteins such asreceptors are assembled into and diffuselaterally in the membrane. Such changescan also affect the activity of membrane-associated enzymes, change the expressionand function of receptors and alter thetransport of molecules across themembrane.
While the biochemical basis of therequirement for linoleic acid and its
metabolites in animal metabolism has beenrelatively well understood for some time,functions of the v-3 family of fatty acidshave been harder to delineate (Sardesai,1992). Docosahexaenoic acid is a com-ponent of lipids in the grey matter of thecerebral cortex of the brain and of thephotoreceptor membranes of the rod outersegment of the retina. As a result, depriva-tion of linolenic acid in pregnant femalerats leads to impairments in cognitive func-tion, learning and vision in the rat pups.Thus, the presence of adequate linolenicacid during fetal and neonatal develop-ment is critical.
Arachidonic acid in cell membranephospholipids can be released in responseto various signals that activate phospho-lipase activity. Arachidonic acid can thenbecome a substrate for conversion to theeicosanoids of the so-called series 2 type(prostaglandin E2, thromboxane A2, 12-hydroxyeicosatetraenoic acid andleukotriene B4), which have strong pro-inflammatory and pro-aggregatory actions.In contrast, the series 3 products pro-duced from eicosapentaenoic acid
114 J.K. Drackley
ω-6 Series
LA 18:2 ω-6
ω-3 Series
GLA 18:3 ω-6
20:3 ω-6
AA 20:4 ω-6
22:4 ω-6
22:5 ω-6
∆-6-Desaturase
∆-5-Desaturase
Elongase
∆-4-Desaturase
Elongase
18:3 ω-3 ALA
18:4 ω-3
20:4 ω-3
20:5 ω-3 EPA
22:5 ω-3
22:6 ω-3 DHA
Fig. 5.4. Metabolism of essential fatty acids of the v-6 (linoleic) and v-3 (linolenic) series. LA, linoleic acid;GLA, g-linolenic acid; AA, arachidonic acid; ALA, a-linolenic acid; EPA, eicosapentaenoic acid; DHA,docosahexaenoic acid.
released from cell membrane phospholipids(prostaglandin E3, thromboxane A3 andleukotriene B5) have anti- or weaklyinflammatory effects and only weakaggregation-promoting effects. Becauselinoleic and linolenic acid compete forthe same pathways of elongation anddesaturation, an increased supply of onewill decrease the elongation–desaturationproducts of the other that are incorporatedinto membrane phospholipids (Sardesai,1992). In this way, the fatty acid composi-tion of cell membranes can be altered bythe type of dietary fat. Interest has grownin whether sources of v-3 fatty acids suchas fish oil could be used to conferadvantages to animals by increasingeicosapentaenoic acid and decreasingarachidonic acid in cell membranes,thereby decreasing the influence of theseries 2 eicosanoid products. Numerousquestions remain about the effectivenessof such approaches, and to date few con-clusive data are available.
Several fundamental questions remainabout the metabolism of essential fattyacids in farm animal species. One of theseis how these fatty acids are transferred inutero to the fetus, given the complexnature of the placenta in ruminants andpigs. A second issue is how ruminants areable to obtain sufficient essential fattyacids in the face of extensive rumenmicrobial hydrogenation of dietaryunsaturated fatty acids (Noble, 1984). Asmall, and evidently at least marginallysufficient, amount of the essential fattyacids escapes hydrogenation in the rumenand is absorbed. Nearly all of the linoleic(and presumably linolenic) acid thatreaches the small intestine is incorporatedinto phospholipids (through reacylation oflysophosphatidic acid) and cholesterolesters (via the ACAT reaction). Theselipids have a very slow turnover in thebody, so that the essential fatty acids areretained for their critical functions (Noble,1984). Very few essential fatty acidsnormally are incorporated into the triacyl-glycerol fraction, which has a very rapidturnover in the body. However, if pro-tected unsaturated lipids are fed or the
rumen is by-passed experimentally toallow absorption of large amounts ofunsaturated fatty acids, essential fattyacids can be incorporated into lymphtriacylglycerols, which will then be trans-ferred to tissues and milk (LaCount et al.,1994). An additional factor in conserva-tion of essential fatty acids by ruminants isthat they oxidize linoleic acid lessefficiently than other more abundant fattyacids such as oleic, and less efficientlythan do non-ruminants (Reid andHusbands, 1985).
Role of Lipids in Cell Signalling andSignal Transduction
Cells within and among tissues of animalsmust communicate with one another toensure coordinated growth, differentiation,metabolism and apoptosis (regulated celldeath). A variety of endocrine (hormonal),paracrine and autocrine factors communi-cate such information to the surface ofneighbouring or distant cells. During thelast two decades, research has exploded onthe ways in which receptor-borne messagesare translated into intracellular function.These processes, which are referred to assignal transduction mechanisms, haveprofound effects on both normal growthand carcinogenesis (Eyster, 1998). One ofthe most exciting current areas of researchin lipid metabolism relates to the role oflipids as signalling compounds.
Recent evidence has shown thatvarious polyunsaturated fatty acids canserve directly as second messengers ormodulators of enzymes. Polyunsaturatedfatty acids play a key role in regulatingexpression of genes for lipid-metabolizingor lipogenic enzymes in both lipogenicand non-lipogenic tissues (Sessler andNtambi, 1998). Other lipids, such asplatelet-activating factor and the eico-sanoids, have regulatory effects on theinflammatory response. Platelet-activatingfactor is a type of ether-linked lipid calleda plasmalogen.
The first discovery of the involvementof phospholipids was of the agonist-
Lipid Metabolism 115
induced hydrolysis of phosphatidylinositol4,5-bisphosphate by phospholipase C toproduce inositol-1,4,5-trisphosphate anddiacylglycerols. In turn, the diacylglycerolsand the calcium mobilized by inositoltrisphosphate activate protein kinase C,which has many diverse effects on cellulargrowth and metabolism. Many hormonesand growth factors activate isoforms ofphospholipase C, including epinephrine andnorepinephrine, serotonin, thromboxaneA2, histamine, cholecystokinin, epidermalgrowth factor, nerve growth factor andplatelet-derived growth factor (Exton,1997).
During the last decade, phosphatidyl-choline also has been found to participatein cell signalling mechanisms, throughagonists that stimulate phospholipase Dactivation. The products of this reactionare choline and phosphatidic acid, whichmay in itself serve as a signallingmechanism as well as being convertedrapidly to diacylglycerol by phospha-tidate phosphohydrolase. Phosphatidicacid also may be converted by a specificphospholipase A2 to form lysophospha-tidic acid, which is recognized as animportant intercellular messenger, parti-cularly in stimulating growth. As dis-cussed earlier, hydrolysis of arachidonicacid from phosphatidylcholine byphospholipase A2 leads to formation ofthe eicosanoids.
Recently, a new pathway of cell signal-ling has been discovered that worksthrough sphingomyelin (Exton, 1997).Activation of sphingomyelinase byagonists such as tumour necrosis factor-a,interferon-g and 1,25-dihydroxychole-calciferol causes hydrolysis of sphingo-myelin to produce ceramide andphosphocholine. Ceramide is a potentintracellular signalling factor that haswidespread effects on cellular growth,differentiation and viability. Furthermore,ceramide can be converted by removal ofits fatty acyl group to sphingosine, whichis an inhibitor of protein kinase C.Sphingosine in turn can be phosphorylatedto form sphingosine-1-phosphate, which
has different cell signalling properties.Progress in this exciting area is extremelyrapid (Eyster, 1998) and it is likely thatadditional information on these pathwaysin farm animals will be forthcoming.
Future Perspectives
Although much is known about the basicpathways of lipid metabolism and theirregulation in farm animals, the processes oflipid accretion and lipid secretion in milkwill continue to assume great importancein future research. This prominence isstimulated by the tremendous impact thatlipid synthesis has on the efficiency, orinefficiency, of meat and milk production.In turn, this directly affects profitability oflivestock enterprises. Lipid metabolismalso plays a key role in development ofmetabolic disorders such as fatty liver,ketosis and pregnancy toxaemia, whichcontinue to plague livestock producers.Supplemental fats are now standardcomponents of diets fed to high-producingdairy cows, and are common in both swineand beef diets. Continued efforts toenhance the digestibility and utilization ofdietary fatty acids will improve theenergetic efficiency of milk and meatproduction.
Increased understanding of lipidmetabolism should lead to practicalapproaches to enhance the productivityand health of farm animals. Variousbiotechnological approaches to manipulatethe process of growth or milk production,such as somatotropin, target key aspects oflipid synthesis. Development of transgeniclivestock may be able to exploit desirablepathways or overcome limitations in othersto alter lipid metabolism. Finally, unravel-ling the roles and metabolism of the lipid-derived cell signalling mechanisms willhave a huge impact on understanding thecellular processes of growth in farmanimals, as well as on the cellularmechanisms underlying homeostaticactions of circulating hormones and growthfactors.
116 J.K. Drackley
References
Adams, S.H., Alho, C.S., Asins, G., Hegardt, F. and Marrero, P.F. (1997) Gene expression ofmitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase in a poorly ketogenic mammal: effectof starvation during the neonatal period of the piglet. Biochemical Journal 324, 65–73.
Bauchart, D. (1993) Lipid absorption and transport in ruminants. Journal of Dairy Science 76,3864–3881.
Beitz, D.C. and Nizzi, C.P. (1997) Lipogenesis and lipolysis in bovine adipose tissue. In: Onodera, R.,Itabashi, H., Ushida, K., Yano, H. and Sasaki, Y. (eds) Rumen Microbes and Digestive Physiologyin Ruminants. Japan Science Society Press, Tokyo/S. Karger, Basel, pp. 133–143.
Belury, M.A. (1995) Conjugated dienoic linoleate: a polyunsaturated fatty acid with uniquechemoprotective properties. Nutrition Reviews 53, 83–89.
Bézard, J., Blond, J.P., Bernard, A. and Clouet, P. (1994) The metabolism and availability of essentialfatty acids in animal and human tissues. Reproduction, Nutrition, Development 34, 539–568.
Brindle, N.P.J., Zammit, V.A. and Pogson, C.I. (1985) Regulation of carnitine palmitoyltransferaseactivity by malonyl-CoA in mitochondria from sheep liver, a tissue with a low capacity for fattyacid synthesis. Biochemical Journal 232, 177–182.
Brindley, D.N. (1984) Digestion, absorption and transport of fats: general principles. In: Wiseman, J.(ed.) Fats in Animal Nutrition. Butterworths, London, pp. 85–103.
Braun, J.E.A. and Severson, D.L. (1992) Regulation of the synthesis, processing, and translocation oflipoprotein lipase. Biochemical Journal 287, 337–347.
Cadórniga-Valiño, C., Grummer, R.R., Armentano, L.E., Donkin, S.S. and Bertics, S.J. (1997) Effects offatty acids and hormones on fatty acid metabolism and gluconeogenesis in bovine hepatocytes.Journal of Dairy Science 80, 646–656.
Doreau, M. and Chilliard, Y. (1997) Digestion and metabolism of dietary fat in farm animals. BritishJournal of Nutrition 78 (Suppl. 1), S15–S35.
Emery, R.S., Liesman, J.S. and Herdt, T.H. (1992) Metabolism of long chain fatty acids by ruminantliver. Journal of Nutrition 122, 832–837.
Etherton, T.D. and Bauman, D.E. (1998) Biology of somatotropin in growth and lactation of domesticanimals. Physiological Reviews 78, 745–761.
Exton, J.H. (1997) Cell signaling through guanine-nucleotide-binding regulatory proteins (G proteins)and phospholipases. European Journal of Biochemistry 243, 10–20.
Eyster, K.M. (1998) Introduction to signal transduction: a primer for untangling the web of intra-cellular messengers. Biochemical Pharmacology 55, 1927–1938.
Fielding, C.J. and Fielding, P.E. (1995) Molecular physiology of reverse cholesterol transport. Journalof Lipid Research 36, 211–228.
Forsberg, N.E., Baldwin, R.L. and Smith, N.E. (1985) Roles of lactate and its interactions with acetatein maintenance and biosynthesis in bovine mammary tissue. Journal of Dairy Science 68,2550–2556.
Freeman, C.P. (1984) The digestion, absorption and transport of fats – non-ruminants. In: Wiseman, J.(ed.) Fats in Animal Nutrition. Butterworths, London, pp. 105–122.
Gargouri, Y., Moreau, H. and Verger, R. (1989) Gastric lipases: biochemical and physiological studies.Biochimica et Biophysica Acta 1006, 255–271.
Glatz, J.F.C., van Nieuwenhoven, F.A., Luiken, J.J.F.P., Schaap, F.G. and van der Vusse, G.J. (1997)Role of membrane-associated and cytoplasmic fatty acid-binding proteins in cellular fatty acidmetabolism. Prostaglandins, Leukotrienes and Essential Fatty Acids 57, 373–378.
Grum, D.E., Hansen, L.R. and Drackley, J.K. (1994) Peroxisomal b-oxidation of fatty acids in bovineand rat liver. Comparative Biochemistry and Physiology 109B, 281–292.
Grum, D.E., Drackley, J.K., Younker, R.S., LaCount, D.W. and Veenhuizen, J.J. (1996) Nutrition duringthe dry period and hepatic lipid metabolism of periparturient dairy cows. Journal of DairyScience 79, 1850–1864.
Hansen, L.R., Drackley, J.K., Berger, L.L., Grum, D.E., Cremin, J.D., Jr, Lin, X. and Odle, J. (1995)Prenatal androgenization of lambs: II. Metabolism in adipose tissue and liver. Journal of AnimalScience 73, 1701–1712.
Hanson, R.W. and Ballard, F.J. (1967) The relative significance of acetate and glucose as precursorsfor lipid synthesis in liver and adipose tissue from ruminants. Biochemical Journal 105,529–536.
Lipid Metabolism 117
Hillgartner, F.B., Salati, L.M. and Goodridge, A.G. (1995) Physiological and molecular mechanismsinvolved in nutritional regulation of fatty acid synthesis. Physiological Reviews 75, 47–76.
Hollanders, B., Mougin, A., N’Diaye, F., Hentz, E., Aude, X. and Girard, A. (1986) Comparison of thelipoprotein profiles obtained from rat, bovine, horse, dog, rabbit and pig serum by a new two-step ultracentrifugal gradient procedure. Comparative Biochemistry and Physiology 84B, 83–89.
Hussain, M.M., Kancha, R.K., Zhou, Z., Luchoomun, J., Zu, H. and Bakillah, A. (1996) Chylomicronassembly and catabolism: role of apolipoproteins and receptors. Biochimica et Biophysica Acta1300, 151–170.
Jensen, M.S., Jensen, S.K. and Jakobsen, K. (1997) Development of digestive enzymes in pigs withemphasis on lipolytic activity in the stomach and pancreas. Journal of Animal Science 75,437–445.
Kim, K.-H. (1997) Regulation of mammalian acetyl-coenzyme a carboxylase. Annual Review ofNutrition 17, 77–99.
Kleppe, B.B., Aiello, R.J., Grummer, R.R. and Armentano, L.E. (1988) Triglyceride accumulation andvery low density lipoprotein secretion by rat and goat hepatocytes in vitro. Journal of DairyScience 71, 1813–1822.
LaCount, D.W., Drackley, J.K., Laesch, S.O. and Clark, J.H. (1994) Secretion of oleic acid in milk fat inresponse to abomasal infusions of canola or high-oleic sunflower fatty acids. Journal of DairyScience 77, 1372–1385.
Lafontan, M. and Langin, D. (1995) Cellular aspects of fuel mobilization and selection in whiteadipocytes. Proceedings of the Nutrition Society 54, 49–63.
McGarry, J.D., Woeltje, K.F., Kuwajima, M. and Foster, D.W. (1989) Regulation of ketogenesis and therenaissance of carnitine palmitoyltransferase. Diabetes/Metabolism Reviews 5, 271–284.
McNamara, J.P. (1991) Regulation of adipose tissue metabolism in support of lactation. Journal ofDairy Science 74, 706–719.
Metz, S.H.M. and van den Bergh, S.G. (1977) Regulation of fat mobilization in adipose tissue of dairycows in the period around parturition. Netherlands Journal of Agricultural Science 25, 198–211.
Moore, J.H. and Christie, W.W. (1984) Digestion, absorption and transport of fats in ruminantanimals. In: Wiseman, J. (ed.) Fats in Animal Nutrition. Butterworths, London, pp. 123–149.
Noble, R.C. (1984) Essential fatty acids in the ruminant. In: Wiseman, J. (ed.) Fats in AnimalNutrition. Butterworths, London, pp. 185–200.
Odle, J., Lin, X., van Kempen, T.A.T.G., Drackley, J.K. and Adams, S.H. (1995) Carnitine palmitoyl-transferase modulation of hepatic fatty acid metabolism and radio-HPLC evidence for lowketogenesis in neonatal pigs. Journal of Nutrition 125, 2541–2549.
Palmquist, D.L., Beaulieu, A.D. and Barbano, D.M. (1993) Feed and animal factors influencing milkfat composition. Journal of Dairy Science 76, 1753–1771.
Pullen, D.L., Liesman, J.S. and Emery, R.S. (1990) A species comparison of liver slice synthesis andsecretion of triacylglycerol from nonesterified fatty acids in media. Journal of Animal Science68, 1395–1399.
Reid, J.C.W. and Husbands, D.R. (1985) Oxidative metabolism of long-chain fatty acids inmitochondria from sheep and rat liver. Evidence that sheep conserve linoleate by limiting itsoxidation. Biochemical Journal 225, 233–237.
Rule, D.C. (1995) Adipose tissue glycerolipid biosynthesis. In: Smith, S.B. and Smith, D.R. (eds) TheBiology of Fat in Meat Animals. American Society of Animal Science, Champaign, Illinois,pp. 129–143.
Rule, D.C., Smith, S.B. and Romans, J.R. (1995) Fatty acid composition of muscle and adipose tissueof meat animals. In: Smith, S.B. and Smith, D.R. (eds) The Biology of Fat in Meat Animals.American Society of Animal Science, Champaign, Illinois, pp. 144–165.
Sardesai, V.M. (1992) Nutritional role of polyunsaturated fatty acids. Journal of NutritionalBiochemistry 3, 154–166.
Schoonjans, K., Staels, B. and Auwerx, J. (1996) The peroxisome proliferator activated receptors(PPARs) and their effects on lipid metabolism and adipocyte differentiation. Biochimica etBiophysica Acta 1302, 93–109.
Sessler, A.M. and Ntambi, J.M. (1998) Polyunsaturated fatty acid regulation of gene expression.Journal of Nutrition 128, 923–926.
Singh, I. (1997) Biochemistry of peroxisomes in health and disease. Molecular and CellularBiochemistry 167, 1–29.
118 J.K. Drackley
Smith, S. (1994) The animal fatty acid synthase: one gene, one polypeptide, seven enzymes. FASEBJournal 8, 1248–1259.
Smith, S.B. (1995) Substrate utilization in ruminant adipose tissue. In: Smith, S.B. and Smith, D.R.(eds) The Biology of Fat in Meat Animals. American Society of Animal Science, Champaign,Illinois, pp. 166–188.
Whitehurst, G.B., Beitz, D.C., Pothoven, M.A., Ellison, W.R. and Crump, M.H. (1978) Lactate as aprecursor of fatty acids in bovine adipose tissue. Journal of Nutrition 108, 1806–1811.
Wiggins, D. and Gibbons, G.F. (1996) Origin of hepatic very-low-density lipoprotein triacylglycerol:the contribution of cellular phospholipid. Biochemical Journal 320, 673–679.
Yu, X.X., Drackley, J.K. and Odle, J. (1997) Rates of mitochondrial and peroxisomal b-oxidation ofpalmitate change during postnatal development and food deprivation in liver, kidney, and heartof pigs. Journal of Nutrition 127, 1814–1821.
Zammit, V.A. (1990) Ketogenesis in the liver of ruminants – adaptations to a challenge. Journal ofAgricultural Science (Cambridge) 115, 155–162.
Zammit, V.A. (1996) The role of insulin in hepatic fatty acid partitioning: emerging concepts.Biochemical Journal 314, 1–14.
