Lipid metabolism in liver and selected tissues and in the whole body of ruminant animals

Pro~. Lipid Res. Vol. 18, pp. 117 164

0163-7827/80/0201-0117/$05.00/0

© Pergamon Press Ltd 1980. Printed in Great Britain

LIPID METABOLISM IN LIVER AND SELECTED TISSUES AND IN THE WHOLE BODY OF RUMINANT ANIMALS A. W. BELL School of Agriculture, La Trobe University, Bundoora, Victoria 3083, Australia

CONTENTS I. INTRODUCTION II. LIPID METABOLISM IN SPECIFIC TISSUES

A. Liver 1. Fatty acid synthesis de novo 2. Fatty acid uptake 3. Fatty acid metabolism 4. Triglyceridemetabolism 5. Sterol formation and metabolism 6. Regulation of hepatic lipid metabolism B. Skeletal muscle l. Uptake of fatty acids and ketone bodies 2. Metabolism of lipid substrates C. Heart D. Brain E. Kidney IlL

ADAPTATION AND REGULATION OF LIPID METABOLISM IN THE WHOLE ANIMAL

A. The fed animal 1. General aspects 2. Effectsof dietary manipulation B. Fasting and undernutrition C. Growth and development D. Pregnancy E. Lactation F. Environmental temperature 1. Cold exposure 2. Heat exposure G. Metabolic disorders 1. Bovine ketosis and ovine pregnancy toxemia 2. Diabetes 3. Hypocalcemia and hypomagnesemia 4. Low milk-fat syndrome IV. REFERENCES

I. INTRODUCTION This review has two distinct parts. In the first, the detailed examination of lipid metabolism in specific tissues of ruminant animals, discussed in earlier reviews in this series, 3°4'479 is extended to liver and several other organs and tissues. In the ruminant, these organs have less influence on the synthesis and disposal of lipids than do adipose tissue or the lactating m a m m a r y gland, and this is reflected by the relatively small literature specifically concerned with lipid metabolism in other than adipose and m a m mary tissues. Nevertheless, lipids are important in the metabolism of all of the tissues which are discussed here (and m a n y which are not). Liver, skeletal and cardiac muscle, brain and kidney were chosen partly because of their quantitative significance, particularly in fatty acid catabolism, but also because in ruminants, they have a number of interesting metabolic characteristics which distinguish them from the equivalent nonruminant tissues. J.P.L.R. 18/3 --4

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The second part of the review has a rather different aim and that is to examine the way in which the intermediary metabolism of lipids in the whole organism is regulated under a number of different nutritional, physiological and pathological conditions commonly encountered by the ruminant animal. In particular, the intention has been to integrate, at a more physiological level, much of the information contained in this and the earlier reviews 3°4'479 on cellular metabolism in individual tissues. Of necessity, the number of nutritional, physiological and other situations which are considered is limited, Nevertheless, it is hoped that their range and variety are sufficient to illustrate both the complexity and distinctiveness of lipid metabolism at the whole-animal level of organization in ruminants. 11 LIPID METABOLISM IN SPECIFIC TISSUES A. Liver 1. Fatty Acid Synthesis de novo

In ruminants, as in other mammals, the liver makes extensive use of fatty acids tbr catabolism or incorporation into glycerides and complex lipids, Unlike many nonruminant species, the ruminant derives little of these fatty acids from synthesis de no~'o within the liver itself, a9 Like many of the special features of ruminant metabolism, this would appear to be an adaptation to the pattern of energy substrates supplied by the ruminant mode of digestion. 3~5 The synthesis of fatty acids in liver, as in other lipogenic tissues, requires an extramitochondrial supply of acetyl CoA. In non-ruminants, this is largely derived from carbohydrate substrates via oxidative decarboxylation of pyruvate within the mitochondrion. Although there have been several hypotheses to explain the translocation of acety[ CoA from mitochondria to the cytoplasmic site of fatty acid synthesis, it is now generally accepted that it occurs via the citrate-cleavage pathway. 241'32v This pathway involves intramitochondrial incorporation of the acetyl group into citrate, followed by translocation into the cytoplasm and ATP-dependent cleavage to form acetyl CoA and oxaloacetate. Indirect evidence had for a long time suggested that little glucose was used for lipogenesis in the liver (or other tissues) of the ruminant (for a review of the older literature, see Lindsay258). This was first substantiated by Hanson and Ballard] 7~ who attributed it to very low activities of the hepatic enzymes ATP citrate lyase (EC 4.1.3.81 and NADP malate dehydrogenase (EC 1.1.1.40) in sheep and cows. Hardwick ~v9 had previously shown that ATP citrate lyase activity was almost absent from goat liver, and the ruminant liver's virtual lack of an active citrate-cleavage pathway has since been confirmed in a number of laboratories.~ 96,203.508 These observations on the inability of the ruminant to use glucose for hepatic fatty acid synthesis de novo are consistent with other features of its carbohydrate metabolism. In the normally-fed ruminant, very little dietary carbohydrate appears as glucose in the portal circulation and hepatic gluconeogenesis accounts for almost all of the animal's glucose supply. 68'25°'259 It is, therefore, not surprising that the ruminant liver was shown to be an invariable net exporter of glucose in V/I)O28''~°'220'457 and to lack the high-Kin hepatic glucokinase (EC 2.7.1.2) which appears to control concentration-dependent glucose uptake in non-ruminant animals. 41 It is well known that the activities of ATP citrate lyase and NADP malate dehydrogenase in rat liver are highly sensitive to carbohydrate intake (e.g. AUman et al,5), The possibility that glucose supply is the major limiting factor for activity of the citrate cleavage pathway and lipogenesis in the ruminant liver was tested by Ballard et al. 37 They found that, in young sheep fed a high carbohydrate diet, or infused abomasally or intravenously with glucose for several weeks, hepatic lipogenesis from glucose and acetate was increased. This was most striking for fatty acid synthesis from acetate in the intravenously-infused group. As shown in Table I,

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TABLE1. Hepatic Activitiesof ATP Citrate Lyase and NADP Malate Dehydrogenasein YoungSheep and Rats (/~molsubstrate convertedor product formed min/g tissue at 37°C; mean values from ref. 37). For details of animals and treatments refer to text and ref. 37 Species

Treatment

Sheep

Controls Carbohydrate-fed Abomasal glucose infusion Intravenous glucose infusion

Rats

ATP citrate lyase

NADP-malate dehydrogenase

0.062 0.149 0.056

0.18 0.49 0.32

0.150

1.09

1.64

6.20

activities of both key enzymes in the citrate cleavage system were increased from very low values, but these changes were variable and even the highest values were very much lower than those for the rat. These adaptive changes showed that glucose supply was at least partly responsible for the low rate of fatty acid synthesis de novo in ruminant liver. However, it should be noted that the elevated rates of lipogenesis were still low by comparison with normal rates in the non-ruminant, especially those obtained by treatments other than direct intravenous infusion of glucose. Also, previous studies of preruminant calves 3a'a4 and yearling steers 5°a suggested that hepatic citrate cleavage activity was reasonably resistant to dietary manipulation; nor were there large changes during the transition of the unweaned calf to mature ruminant. 199 In fed ruminants, acetate, produced by carbohydrate fermentation in the gut and absorbed into the portal circulation, is a major source of energy for extrahepatic tissues. 9 However, it has been clearly demonstrated that little of this acetate is taken up by the liver in vitro 252"2a°'295 or in vivo. 2a'7#'110"193"456 In addition, the capacity of the ruminant liver for lipogenesis from acetate was shown to be low relative to that of adipose tissue, although acetate was considerably more important than glucose. "r°'196'2°3'2°4 The mode of control of acetate uptake by tissues is not fully understood, although the activation of acetate to acetyl CoA is thought to be an important regulatory step.l°9 This reaction is catalyzed by acetyl CoA synthetase (EC 6.2.1.1), of which low to moderate activities have been detected in liver of sheep and cattle. 1°9'177'228'361'42s Quraishi and C o o k 361 reported that this enzyme was localized predominantly in the mitochondria of the bovine liver which, with the absence of an active citrate-cleavage pathway, would largely exclude activated acetate from extramitochondrial lipogenesis. However, it is uncertain whether this explanation for low rates of hepatic lipogenesis from acetate is tenable for sheep liver, which was shown apparently to have most acetyl CoA synthetase in the cytoplasm. 22s'42s Whatever the intracellular location of this enzyme, its total activity was invariably much less than that of acetyl CoA hydrolase (EC 3.1.2.1) in ruminant liver. 22s'361,42s The relative activities of these two antagonistic enzymes are almost certainly more important in the regulation of cellular acetate utilization than the activity of either alone. Yet another possible explanation for the low rate of acetate utilization by ruminant liver was provided by observations that hepatic mitochondrial acetyl CoA synthetase has a greater specificity for the activation of C3-C 7 fatty acids, and only a very low capacity for activation of acetate. 1°9 In support of this work, utilization of acetate by sheep liver in vitro was clearly inhibited by the presence of either propionate or butyratef152'42~ The synthesis of long-chain fatty acids requires not only a supply of extramitochondrial acetyl CoA, but also large amounts of NADPH. In liver and other tissues of non-ruminants, the decarboxylation of malate via the citrate-cleavage pathway is a major supplier of this NADPH. 241"327 The source of reducing equivalents for the relatively small amount of fatty acid synthesis which does occur in the ruminant liver is not known, but it has been presumed that sufficient glucose was available for metabolism via the

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hexose monophosphate pathway) 9 the other main source of N A D P H in non-ruminants. Certainly, substantial hepatic activities of key enzymes for this pathway in sheep and cows have been reported. ~'~4'17~'366 Yet another source of N A D P H could be the NADPisocitrate dehydrogenase pathway which was recently shown to be significant in ruminant mammary tissue, 5~ and may also operate in adipose tissue. 5°'~79 Its importance in ruminant liver remains to be examined. Ballard et a l . ) ~ in their excellent review, attempted to reconcile the high rate of gluconeogenesis with the low rate of lipogenesis in the ruminant liver in terms of a competition for cytoplasmic oxaloacetate. This intermediate, which is produced by several pathways, is removed by the obligatory pathways of both gluconeogenesis {via conversion to phosphoenolpyruvate) and lipogenesis (via conversion to malate and thence to pyruvateL Ballard et ~tl.39 showed that the relative activities of the enzymes involved in these pathways greatly favor gluconeogenesis in the liver of the adult ruminant. Also, incorporation of label from [2-~C]glutamate into fatty acids, which can occur only via labeling of oxaloacetate, was negligible in bovine liver. ~78

2.

F a t t y Acid

Uptake

The liver is a most important site for the removal of free fatty acids (FFA) from circulating blood plasma. 438 In the only experiments on ruminants in which the hepatic uptake and total body flux of FFA were measured simultaneously, Bergman et al. 69 showed that the liver in conscious fed sheep took up about 25~',] of all FFA entering the blood-stream There is considerable evidence, mostly obtained from use of the isolated perfused rat liver, that hepatic uptake of FFA is not regulated by metabolic events within the hepatocyte but is a function of the concentration of FFA in plasma, at least up to concentrations of 2-3 raM. ~ss The detailed observations of Soler-Argilaga et al. 432 suggested further that the limiting factor for fatty acid uptake is "the kinetic rate of equilibrium between the associated and dissociated form of FFA and albumin", i.e. a combination of the FFA :albumin molar ratio and blood flow. Certainly, in the intact conscious sheep, the fractional extraction of FFA by the liver remained constant at about 10Yoover a wide range of physiological states and arterial FFA concentrations, z2°'4ss'4s6 However, there was some evidence of saturation of the rate of uptake when arterial concentrations exceeded 2 mM during noradrenaline infusion. 45s A fractional uptake of 10Yo is somewhat less than most estimates for conscious intact non-ruminants (e.g. Basso and Have149). This may perhaps be attributable to the relatively high fraction of cardiac output received by the ruminant liver 174 and, therefore, greater rate of presentation of FFA to the liver, The major individual FFA in ruminant blood plasma are palmitic, stearic and oleic acids. 94 Although the sheep liver in vivo extracted about 10~il of the total plasma FFA circulating through it, Thompson and colleagues 4ss'456 found considerable differences in the relationships between arterial levels of these individual FFA and their uptake by the liver. These are illustrated in Fig, 1, in which mean values for hepatic uptake of individual FFA are plotted against corresponding values for arterial concentration, obtained under a variety of physiological conditions, it is notable that for palmitic and oleic acids there is a similar linear relationship between uptake and arterial level, whereas uptake of stearic acid is low and variable, regardless of concentration. In qualitative terms, these differences are similar to those demonstrated for the perfused rat liver in which the rate of uptake of FFA was inversely related to the chain length of the saturated fatty acids and directly related to the degree of unsaturation (i.e. number of double bonds).'* 3~ The sheep liver also takes up small amounts of triglyceride fatty acids from plasma. By infusing labeled chylomicron triglyceride into conscious sheep and measuring its uptake by different organs, Bergman et al. 69 showed that the liver took up about 2% of the triglyceride fatty acids circulating through it. This represented about 10°/ of the total removal of triglyceride fatty acids from the blood and some 10'~ of this hepatic uptake

Lipid metabolismin liver and selected tissues in ruminants '7

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(p.) FIG. 1. The relation between hepatic uptake and arterial plasma concentration of free palmitic (0), stearic (,t) and oleic (I) acids in sheep. Individualvalues are means from refs 455 and 456. was released back into the circulation as FFA. All of these values were considerably less than those similarly obtained from conscious dogs in the same study. 69 3. Fatty Acid Metabolism

(a) Activation. In liver, as in all mammalian tissues, fatty acids must be converted to fatty acyl thioesters of CoA at the expense of ATP hydrolysis before they can be further metabolized. This "activation" is catalyzed by acyl-CoA synthetases (EC 6.2.1.1-3), which can be broadly classified as being specific for short-, medium- or long-chain fatty acids (see Groot et al. t69 for a recent and comprehensive review). In ruminants, the hepatic activity of the short- and medium-chain fatty acyl synthetases (EC 6.2.1.1. and EC 6.2.1.2) has, understandably, been studied in some detail. Although the proteins in question have yet to be isolated and identified, measurements of substrate specificity have strongly suggested the existence of at least two distinct enzymes for the acylation of acetate, propionate and butyrate, respectively, in the livers of sheep 1°9'228'425 and cattle, ts'3g4 Furthermore, the relative rates of activation of these short-chain acids (propionate > butyrate >> acetate) agreed very well with their observed rates of hepatic uptake in vivo. 28"74"110"456 It has also been shown that in bovine liver homogenates not only do the relative activities of acetyl, propionyl and butyryl CoA synthetases favor propionate, and to a lesser extent, butyrate metabolism, but that the presence of propionate or butyrate can inhibit acetate activation; propionate can also inhibit butyrate activation. 15 This, together with their low absolute activities, suggests that these enzymes have a considerable influence on the regulation of short-chain fatty acid metabolism in ruminant liver Is'~ 09 and largely explains earlier reports of metabolic interactions among short-chain fatty acids.in this tissue in vitro. 252,280.421 At least some butyryl CoA synthetase activity in bovine liver may be due to the presence of the mitoehondrial medium-chain acyl CoA synthetase (EC 6.2.1.2) isolated by Mahler et al. 268 This enzyme showed a broad substrate specificity (C4-C12), with a preference for octanoate, but its metabolic significance in the intact ruminant has yet to be determined. Long-chain acyl CoA synthetase (EC 6.2.1.3) activity does not appear to have been measured directly in ruminant liver. In non-ruminant liver, it is localized in the endoplasmic reticulum and on the outer mitochondrial membrane, and may be part of a multienzyme complex.169 Its hepatic activity has been shown to greatly exceed rates of fl-oxidation in mitochondria 348 and esterification with ~-glycerophosphate in microsomes, 26s the two main pathways for consumption of long-chain acyl CoA. I n the absence of evidence to the contrary, it must be assumed that these characteristics also apply to

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long-chain acyl CoA synthetase in ruminant liver and that in ruminants also, this enzyme is not rate-limiting for the hepatic uptake and subsequent metabolism of long-chain fatty acids. (b) Fatty acid desaturation and chain elongation. In ruminants, dietary fatty acids are largely absorbed from the small intestine as stearic acid because of extensive biohydrogenation in the rumen of oleic, linoleic and linolenic acids, which are the principal fatty acids in feed lipids. 18° However, all major lipid fractions in the ruminant liver contain a significant proportion of oleic acid 94 and it has been postulated that the ratio of stearic to oleic acid is related to fatty acid desaturase activity in ruminant tissues.l°6 In mammalian hepatocytes, as in cells from all aerobic organisms, long-chain saturated fatty acids are desaturated to their 9,10-cis-monounsaturated derivatives by an enzyme system localized in the microsomes. 136 This system requires electron donors (such as reduced pyridine nucleotides) and molecular oxygen as obligatory co-factors 79 and acts on stearyl CoA. In the most extensive study of desaturase activity in ruminant tissues to date, Wahle 481 found the desaturase activity in sheep liver microsomes to be considerably lower than that in similar preparations from rat and chicken liver. Similarly, Cook and Reiser 1°6 found low activities in sheep compared with rat liver, but the results of Payne and Masters 354 suggested similar levels in the two species. Microsomes from sheep liver also had much lower desaturase activity than those from sheep adipose depots, particularly those situated subcutaneously. 354'481 It has been suggested that membrane-bound desaturase activity may be related to cytosolic fatty acid synthetase activity, either via topographical proximity of the two systems or the presence of a transport system between themff 81 However, the evidence is not conclusive and this suggestion has been challenged in a recent review. 479 Certainly, synthetase and desaturase activities are both low in the sheep liver, and the question of whether or not fatty acids synthesized de novo are preferentially desaturated may not be important for this tissue. The fact that the ruminant liver obtains most of its long-chain fatty acids from plasma FFA, yet apparently discriminates against stearic acid 455'456 may have a much greater effect on hepatic fatty acid composition. The elongation of fatty acids in ruminant liver has received little attention but it seems probable that, as in other mammalian hepatocytes, fatty acids are elongated by two different pathways, as reviewed by Seubert and Podack. 4°9 These are located in the microsomes and mitochondria, respectively. The microsomal enzyme system catalyzes the elongation of fatty acyl CoA to longer chain acids in the presence of malonyl CoA and NADPH, while the mitochondrial system requires acetyl CoA and both NADH and NADPH. Both systems elongate unsaturated fatty acids more rapidly than their saturated homologs. The enzyme system in the outer and inner membranes of bovine liver mitochondria has recently been characterized, sl although its physiological significance is not yet known. (c) Esterification and complex lipid synthesis. The few published data on fatty acid esterification in ruminant liver suggest that the process differs little from that in nonruminants. It is now evident that the rat liver does not esterify activated fatty acids exclusively by the classical glycerol phosphate pathway, since glyceraldehyde-3-phosphate and dihydroxyacetone phosphate, as well as glycerol-3-phosphate, were shown to stimulate fatty acid esterification. 37° The existence of alternative pathway(s) in bovine liver was suggested by the recording of significant esterification of palmitic acid without the addition of glycerol-3-phosphate. 65 As in other species, the enzymes responsible for the acylation of glycerol-3-phosphate in bovine liver were found to be localized in the outer mitochondrial membrane and the endoplasmic reticulum/~ 3 It was also shown z~3 that mitochondria esterified glycerol-3phosphate more rapidly with palmitate than with linoleate, and formed relatively more monacylated products than did microsomes; microsomes showed no such preference for palmitate. The high rate of lysophosphatidic acid formation in mitochondria is consistent with the recent identification of a protein with relatively high iysophospholipase activity (EC 3.1.1.5) in beef liver mitochondria. 118,465

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The rate of fatty acid esterification in sheep liver slices in vitro reported by Payne and Masters T M was low, most of the substrate fatty acids being recovered in the FFA fraction. This may have been partly for methodological reasons, however, as in accompanying experiments on rats, hepatic glyceride synthesis was low relative to other published values. In contrast, bovine liver homogenates were found to esterify fatty acids more rapidly than adipose tissue homogenates (results presented on a tissue weight basis) and substrate fatty acids were incorporated into mono-, di- and triglycerides, and into phospholipids. 65 Of the fatty acids that were esterified, most were incorporated into phospholipids in sheep 3~4 and cows. 65 This was particularly notable for linoleic acid in the sheep, and for stearic and linoleic acids in the cow. Of the common long-chain fatty acids, only palmitic was found predominantly in di- or triglycerides. Incorporation into cholesteryl esters was also apparently limited in the sheep, a54 a finding which is consistent with recent evidence that in this species, virtually all cholesteryl esters are synthesized by the plasma lecithin-cholesterol acyltransferase (LCAT) system. 31a There appear to be no published values for the rate of cholesteryl ester synthesis in bovine liver. The plasma LCAT system was also shown to be active in cattle 321 but on the basis of the apparent exclusion of oleic acid from the plasma acyltransferase reaction, the authors concluded that at least some bovine plasma cholesteryl esters must have been of hepatic origin. The factors responsible for the relatively low rate of glyceride synthesis in ruminant liver, and for the diversion of fatty acids to phospholipids in preference to other complex lipids, are not clear. It has been suggested that in non-ruminants, the tissue concentration of glycerol-3-phosphate is the determining factor in the rate of glyceride biosynthesis. 463 This, in turn, will be affected by factors which regulate the levels and activity of the key enzymes of glycolysis, gluconeogenesis and lipogenesis. T M In the starved rat, when gluconeogenesis was active and lipogenesis depressed, hepatic levels of glycerol-3-phosphate were markedly depressed, 463 These conditions normally obtain in the liver of the fed ruminant, but the response of esterification rate to addition of glycerol-3-phosphate in sheep liver slices was reported to be variable and inconclusive. 354 Apart from its synthesis via the glycolytic and pentose phosphate pathways, glycerol-3-phosphate can be formed by direct phosphorylation of free glycerol by glycerol kinase (EC 2.7.1.30). Sheep liver was shown to take up considerable amounts of glycerol from circulating plasma 456 and glycerol kinase from bovine liver has been partially isolated and characterized 170 but the physiological significance of this reaction in ruminant liver remains to be defined. The control of glyceride synthesis in the mammalian liver has been recently reviewed elsewhere.~Sg'467 (d) Oxidation. Few papers have dealt specifically with the oxidation of long-chain fatty acids in ruminant liver, but there is little reason to believe that its basic mechanisms and mode of control differ from those of other mammalian tissues, as reviewed by Greville and Tubbs. 167 The uptake of FFA by the ruminant liver, can be considerable (see Section II. A.2) and it is generally accepted that the rate of fl-oxidation of fatty acids in tissues is largely controlled by their availability, i.e. plasma concentration. ~s~ The ruminant liver's capacity to oxidize long-chain fatty acids appears to be relatively low as Connelly et al. 1°5 detected only a negligible conversion of t4C-palmitate to ~4CO2 in the isolated perfused goat liver, although oxidation to ketone bodies and acetate was considerably greater. Koundakjian and Snosweli233 showed that in sheep liver mitochondria, palmitic and stearic acids were oxidized at a rate o.nly 30% of that in rat liver mitochondria. While it is almost certain that fatty acid oxidation is carnitine-dependent in intact tissues, there are conditions under which it can be largely unaffected by the addition of carnitine in rat liver mitochondria. ~67 However, under such conditions, oxidation was completely carnitine-dependent in sheep liver mitochondria. 233 It was suggested that the high activity of carnitine acetyltransferase (EC 2.3.1.7) relative to that of carnitine palmitoyltransferase (EC 2.3.1.21) in sheep liver mitochondria 4z7 might have enabled the former enzyme to compete more effectively for endogenous carnitine, thus severely limiting the oxidation of long-chain fatty acids in the absence of added carnitine. In support of this idea, Snoswell and Henderson 427 found only small amounts of long-chain fatty acyl

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carnitine esters relative to that of acetylcarnitine in sheep liver. Work by Shepherd et al.4~ 1 suggested that the conversion of palmitoyl CoA to palmitoylcarnitine (catalyzed by carnitine palmitoyltransferase) is the rate-limiting step in the oxidation of palmitate in rat liver mitochondria, although this theory has recently been challenged. 84 The carnitine palmitoyltransferase in beef liver mitochondria was recently isolated and characterized, 131'132 but little is known about its metabolic properties. The role of carnitine acetyltransferase and the metabolic importance of acetylcarnitine in the ruminant liver will be discussed in a later section. As discussed in Section I.A.1, the uptake and activation of acetate by ruminant liver is low in spite of the high acetate concentration in portal venous blood. Predictably, therefore, acetate was oxidized very slowly in sheep liver homogenates 296 and mitochondria. 233'421 Propionate and butyrate were more readily oxidized than acetate in liver slices in vitro from fed and fasted sheep, 252 in accordance with their relative rates of uptake by the intact liver TM and of mitochondrial activation. 1°9'421 However, most propionate taken up by the liver in vivo was converted to glucose 73"254 and small amounts only of butyrate and other short- to medium-chain fatty acids reached the liver.74'11 o.45~ (e) Ketogenesis and endogenous acetate production. A variable and sometimes major fraction of the long-chain fatty acids entering the fl-oxidation pathway in the liver are catabolized to form the ketone bodies acetoacetate and 3-hydroxybutyrate. Thus, Koundakjian and Snoswel1233 found that acetoacetate production accounted for 63°~ of oxygen uptake during palmitoylcarnitine oxidation by liver mitochondria from fed sheep. Surprisingly, this proportion was little different in liver slices from 4-day fasted ewes, some of which were pregnant and ketotic. 449 There is good evidence for a direct relationship between fatty acid uptake and the rate of hepatic ketogenesis in the perfused rat liver (e.g, Morris 3°7) and it has been proposed that, during periods of elevated fatty acid supply, more fatty acids go to ketogenesis and less enter the TCA cycle, thereby regulating total yield of energy from oxidation. 293 Such a theory implies that TCA cycle activity varies inversely with the rate of ketogenesis, perhaps via changes in the activity of citrate synthase (EC 4.1.3.7). Alternatively, hepatic ketogenesis from long-chain fatty acids has more recently been described as an "overflow" process, in which TCA cycle activity is not necessarily altered and ketogenesis results simply from a rate of acetyl CoA production in excess of the mitochondrial capacity for citrate synthesis. 266 That one of these mechanisms operates in the ruminant liver seems likely, as judged from the concurrent increases in the liver's uptake of fatty acids and output of ketones in vivo in fasted and/or pregnant sheep 22° and in fasted lactating cows. 27 However, before the regulation of hepatic ketogenesis in the ruminant is discussed further, some special features of ketone body metabolism in these animals should be considered. It is now well established that, in contrast to most non-ruminants, circulating levels of ketone bodies are high in fed sheep and cattle, and the ratio of 3-hydroxybutyrate to acetoacetate is usually greater than 10:l. 25'233 Experiments done in vitro 356 and in vivo 28'22°'489 have shown that much of this 3-hydroxybutyrate is produced in the rumen epithelium, almost certainly from the metabolism of ruminal butyrate; indeed, the results of Katz and Bergman 22° suggested that in fed sheep, splanchnic ketogenesis is virtually confined to this tissue. However, more recent work has indicated that in fed lactating and non-lactating cows, the ketogenic role of the liver relative to other tissues is by no means negligible. 2a'426 The main precursor of 3-hydroxybutyrate in the liver of fed animals is likely to be butyrate which has escaped metabolism in the rumen epithelium. In fasted animals, there is no doubt that the liver assumes the major role in ketogenesis. This has been clearly demonstrated in vivo for pregnant and non-pregnant ewes 22° and lactating cows 27 (Table 2). Under such conditions, ruminal production of butyrate virtually ceased, the liver's uptake of FFA increased greatly and the total rate of ketone production was considerably greater than that in the fed animal. In fasted sheep, the conversion of ~4C-FFA to ~4C-3-hydroxybutyrate was sufficient to account for the entire production of 3-hydroxybutyrate in the whole animal. 2~5"3'~1 Studies of enzyme activities and subcellular distribution have shown that acetoacetate

Lipid metabolismin liver and selectedtissues in ruminants

125

production in bovine liver almost certainly occurs in mitochondria via the 3-hydroxy-3methylglutaryl CoA (HMG CoA) pathway, 26'266 as in the rat. 498 Also, unlike rat liver, bovine liver had an active cytoplasmic H M G CoA pathway, which was stimulated during ketosis. 266 The enzymological basis for the considerably greater hepatic production of 3-hydroxybutyrate observed in vivo 2~'28'22° is much less clear. Several independent investigators have shown that the activity of 3-hydroxybutyrate dehydrogenase (EC 1.1.1.30), the key enzyme for the reduction of acetoacetate to 3-hydroxybutyrat¢ in all mammalian tissues studied previously (e.g. Wieland496), is absent from liver mitochondria and low in the cytosol in sheep and cattle. 233'314'4s6 It was also demonstrated that, contrary to an earlier postulation, 31'~ 3-hydroxybutyrate dehydrogenase activity was not induced by increased hepatic ketogenesis. 486 This, together with observations of a dramatic fall in the 3-hydroxybutyrate:acetoacetate concentration ratio in liver and blood of ketonemic animals, 25'233 suggests that this enzyme may be rate-limiting for 3-hydroxybutyrate synthesis in the ketogenic ruminant liver. Even so, there remains a marked disparity between values for enzyme activities measured in vitro and observed rates of hepatic production of 3-hydroxybutyrate in vivo. In the fed non-ketotic ruminant, the relatively low rate of hepatic ketogenesis is probably controlled mainly by the supply of butyrate in portal venous blood which, in turn, is a function of level of feeding and diet quality. In starved or spontaneously ketotic animals, much higher rates of ketone-body production must also be strongly influenced by the supply of ketogenic precursors (in this case, long-chain fatty acids) but the ultimate mode of regulation almost certainly involves other factors. Krebs 236 proposed that hepatic ketogenesis in the cow is regulated at the level of the TCA cycle by the availability of oxaloacetate and its precursors for combination with acetyl CoA derived from fatty acid oxidation. In support of this idea, Baird and co-workers 21'22 found a major decrease in hepatic concentrations of oxaloacetate and related intermediates in ketotic cows, and an increasing proportion of the hepatic uptake of fatty acids was apparently diverted to ketogenesis during the development of ketonemia in fasted pregnant ewes21 and lactating C O W S . 2 7 In contrast, Ballard et a/. 3s'4° found no change in the hepatic concentrations of oxaloacetate or malate, but a sharp decrease in citrate concentration in spontaneously ketotic cows. This was attributed to a possible inhibition of citrate synthase activity. Various other factors which affect the supply of acetyl CoA may be involved, including the rate of esterification of fatty acids, the mitochondrial redox state and the supply of coenzyme A. These should be considered in future studies of the control of ketogenesis in the ruminant liver, As discussed in an earlier section, in the fed ruminant, copious amounts of acetate of dietary (or "exogenous") origin are released from the rumen into the portal blood stream, but little is taken up by the liver. However, in lactating ewes 1tl and lactating and non-lactating COWS,28'426 a substantial net release of acetate from the liver in vivo has been consistently observed (Table 2). This phenomenon has been examined in some detail by Snoswell and his colleagues, who showed that the enzymic capacity of the liver to produce acetate from acetyl CoA in vitro could account for the observed rates of release in vivo in sheep 11~ and cows. '~26 It was suggested that the "endogenous" production of acetate in the liver may be an alternative to ketogenesis as a means of relieving an intramitochondrial build-up of acetyl CoA derived from fatty acid oxidation. ~ The balance between ketogenesis and acetate production may be determined by the availability of carnitine, with adequate supplies favoring acetate production and vice versa (A. M. Snoswell, personal communication), It was recently reported that increased hepatic ketogenesis in the fasted lactating cow is accompanied by a marked decrease in the liver's release of acetate 27 and in non-lactating sheep, fasting caused a fall in the ratio of free carnitine to acetylcarnitine in the liver, despite a large increase in the concentration of total acid-soluble carnitine. '~27 Early experiments on metabolism of acetate in ruminant liver in vivo suggested that substantial hepatic acetate production was confined to the lactating animal. 74'~ to.11 ~.456 Recently, however, acetate production by non-lactating cow liver in vivo and in vitro was J.P.L.R. 18/3

8

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A . W . Bell

TABLE 2. Hepatic Metabolism m vivo of Acetate and Ketone Bodies in Sheep and Cattle (mean values calculated from refs 27, 28, 74, 11 I, 220, 4261 Net hepatic productionS(/~mol/kg b. wta/4/min) Species Sheep Sheep Sheep

Cow Cow Cow

Nutritional or physiological status Fed Fasted (3 days) Fed, lactating (4 weeks) Fed, lactating (8 weeks) Fed, non-pregnant Fed, twin-pregnant Fasted 13 days), non-pregnant Fasted (3 days), twin-pregnant Lactating Non-lactating Lactating Non-lactating Fed, lactating Fasted (6 days), lactating

Acetate 0 2 40 6* -204 73 47 52 2

3-hydroxybutyrate

Acetoacetate

References 74 I 11

2 11 16 25 51 17 27 28 21 38

- 2 - 3 7 7 - 12 1 - 8 - 7 - 8 7

220

28 426 27

*1 animal only. tNegative values indicate net uptake.

reported, 426 and similar observations have been recorded in the starved non-lactating ewe, in which the net release of acetate was inversely related to arterial acetate concentration (A. M. Snoswell and D. B. Lindsay, personal communication), and in the growing steer (G. E. Thompson and A. W. Bell, unpublished observations). The metabolic significance of endogenous acetate production, its mode of control and its relation to ketogenesis in the ruminant F~ver are poorly understood and clearly require further investigation. 4. Triylyceride Metabolism

(a) Lipoprotein formation and release. In non-ruminant animals, the very low density lipoproteins (VLDL) synthesized and secreted by the liver are the major carrier by which triglycerides are transported in the plasma, though some is found as chylomicrons and some as VLDL of intestinal origin. ~34"~88 It has also been established that in both the isolated perfused rat liver 23° and intact human liver in vivo, ~a5 the major source of VLDL triglyceride is FFA taken up from circulating plasma. The sequence of the hepatic production of VLDL was recently summarized by Stein and Stein. 439 Briefly, lipoproteins were first observed in the transition zone between the rough and smooth endoplasmic reticulum, i.e. between sites of protein and glyceride (as well as phospholipid and sterol) synthesis, respectively. They were then transported to the Golgi apparatus, encased in membrane-bound secretory vacuoles, and released into the circulation by fusion of the secretory vacuole with the cell membrane. In ruminants, the concentration of VLDL in plasma is extremely low. 94 Nevertheless, the metabolic importance of VLDL as a primary source of lipid for extrahepatic tissues appears to have been established, particularly in the lactating animal. 342'36° It is not known whether the very low circulating level of this lipoprotein fraction is due to a low rate of hepatic synthesis and release or the exceptional avidity of extrahepatic tissues for VLDL triglyceride. In support of the latter suggestion, the rate of turnover of plasma VLDL in the lactating cow was rapid relative to that of other lipoproteins 159.346 as in other species, ~33 and plasma VLDL concentration was appreciably higher in non-lactating than in lactating COWS. 372'471 On the other hand, Bergman et al. 69 reported that despite considerable uptake of FFA by the sheep liver, little 14C taken up in labeled FFA appeared in circulating VLDL triglyceride fatty acids.

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Factors involved in the regulation of VLDL secretion, and its relationship with ketogenesis in the isolated perfused rat liver have been recently reviewed by Heimberg et al. 188 They concluded firstly that the hepatic output of VLDL triglyceride is directly related to FFA uptake by the liver (and therefore to plasma concentration of FFA), and secondly, that the liver's capacity to secrete VLDL is less than its ability to take up and esterify fatty acids; when the uptake of fatty acids exceeds that necessary to sustain a maximal rate of secretion of VLDL, triglyceride accumulates in the liver and the rate of ketogenesis accelerates, both these responses being proportional to fatty acid uptake. In the absence of any supporting data, it can only be guessed that these conclusions might also apply to the mode of control of hepatic VLDL secretion in the normal fed ruminant. They are, however, consistent with several observations made during recent studies of the etiology of fatty infiltration of the liver in starved and/or ketotic cows. 87'374 Fatty infiltration of the liver, invariably associated with ketonemia, has been observed in cows with spontaneous lactation ketosis, 39j'395 in ewes with pregnancy toxemia 1.9'35° and in undernourished or starved COWS,87'227 sheep 147'289'35° and goats. 5°4 In fasted lactating cows, this condition was associated with elevated serum FFA and decreased serum low density lipoprotein leyels, s7 elevated uptake of FFA and decreased release of triglyceride by the liver, 3T4 and increased hepatic ketogenesis. 27 In the perfused rat liver, significant ketogenesis did not occur until triglyceride accumulation was observed, tab Similar data on the sequence of these events in the ruminant liver are not available, although a general relationship between plasma concentration of FFA, degree of ketonemia and liver triglyceride levels was reported for the starved sheep. 35° It has been suggested that a shortage of glycerol-3-phosphate precursors may account for the accumulation of lipid in the liver. 67"227 However, several studies showed that most of this lipid is in the form of triglyceride a7'35° and that the rate of incorporation of fatty acids into liver triglyceride may even increase. 5°4 If the rate of hepatic triglyceride synthesis is maintained, what then limits VLDL formation and release? Brumby et al. 8~ speculated that since triglyceride accumulation was accompanied by decreases in the percentages of phospholipid and cholesterol in liver, availability of one or more of these constituents may have limited lipoprotein synthesis. In contrast, it has been postulated that the amounts of phospholipid and cholesterol secreted in VLDL are dependent on triglyceride secretion, and are thus regulated by factors which affect the latter. 1as The alternative suggestion, that lipoprotein synthesis was limited by the amount of available apoprotein, seems more attractive. In the cows used by Brumby et al., s7 there was a marked decrease after starvation in the volume of rough endoplasmic reticulum in hepatocytes, which was strongly suggestive of decreased hepatic protein synthesis. 375 There is also some evidence that the symptoms of bovine ketosis and associated changes in serum lipoproteins can be alleviated by treatment with methionine, possibly via improved apoprotein synthesis and export of hepatic lipids. 279 Whether hepatic protein synthesis is an important factor in the regulation of VLDL secretion in well-fed healthy ruminants remains to be determined. There is an urgent need for research into the mode of synthesis and secretion of VLDL and their control in the ruminant liver. The availability of techniques for studying liver metabolism in vivo in large animals and a new awareness of the possible metabolic importance of VLDL as a vehicle for lipid transport, particularly to the lactating mammary gland, should contribute to fulfilling this need. (b) Lipolysis. Although the sheep liver was shown to be active in the removal of cholesterol from circulating chylomicrons, its uptake of triglyceride fatty acids was limited, and recirculation of the fatty acids so taken up was almost negligible. 69 It seems likely that the ruminant liver is similarly inactive in the hydrolysis of triglyceride from other triglyceride-rich lipoproteins, such as VLDL, as is the case in other species. 234'294 Studies with the isolated perfused goat liver showed significant hepatic incorporation of fatty acids from serum low density lipoproteins (LDL) (1.006 < density < 1.063) in the perfusate.l'~° However, the relevance of these observations to the intact animal is uncertain, particularly in view of the low triglyceride content of LDL in ruminant plasma. 94

128

A.W. Bell

5. Sterol Formation and Metabolism

Cholesterol, which is an important precursor of steroid hormones, bile acids, vitamin D and cholesteryl esters, can probably by synthesized from acetate in all mammalian tissues 416 according to the pathway elucidated by Bloch 7s and other workers. In ruminants and other herbivores, this endogenous synthesis assumes particular importance because no cholesterol is present in the normal diet. In most mammals studied to date, the primary site of endogenous cholesterol synthesis is the liver 4j6 but it was recently found that the rate of incorporation of acetate into cholesterol in ruminant liver was considerably less than that in adipose tissue 256 or, small intestine particularly the jejunum. 256"313"399 The significance of the latter observation is not known, but it is consistent with a report of similarly high rates of cholesterogenesis in the ileum of the guinea-pig'~62 which, like the ruminant, is a natural herbivore. It has been established that, in the non-ruminant, the rate-limiting step in the pathway of hepatic cholesterol synthesis is the conversion of 3-hydroxy-3-methylglutaryl CoA (HMG CoA) to mevalonic acid, catalyzed by the enzyme HMG CoA reductase (EC 1.1.1.34). This reaction is inhibited by cholesterol feeding, and to a lesser extent, fasting. 416 The effect of dietary factors on cholesterol synthesis in the ruminant has been largely ignored, However, it was recently found that feeding sheep with a lipid supplement which was "protected" from ruminal degradation caused a substantial increase in cholesterol synthesis in the small intestine in vivo 1°7"313 and in v i t r o . 313'399 At the same time, the rate of incorporation of 14C-acetate into hepatic cholesterol was markedly inhibited in vivo, although not in vitro. The elevation of intestinal cholesterogenesis was consistent with previous observations of increased plasma levels of free and esterified cholesterol in ruminants fed protected 75 and unprotected TM lipid supplements. It was suggested that this increased synthesis might occur in order to provide extra cholesterol for the transport of absorbed f a t . 1 0 7 ' 3 1 3 ' 3 9 9 It is notable that in ruminants, cholesteryl esters are an even more important vehicle for lipid transport than in most non-ruminants. 94"95 The fact that hepatic cholesterol synthesis decreased in 1.'it~o1°7'313 but not in vitr03~ 3.399 strongly suggests that the response in vivo was a direct consequence of the increased extrahepatic supply of endogenous cholesterol. Such a response is perhaps comparable with that of many non-ruminants to increased exogenous (dietary) cholesterol. Also, it has been reported that high blood levels of endogenous cholesterol can eventually be followed by inhibition of hepatic cholesterogenesis in non-ruminants. 392 Similarly, fasting apparently reduces hepatic cholesterol synthesis in ruminants, as indicated by the significant fall in liver concentration of cholesterol in starved cows. s7 The mechanisms by which hepatic cholesterol synthesis is controlled are not fully understood. An important factor could be the specific accumulation of cholesterol at the microsomal membrane, which is also the site of HMG CoA reductase synthesis. 4~° The secretion and enterohepatic recirculation of bile may also be involved. A high molecular weight lipoprotein was recently purified from bovine bile, which specifically inhibited the activity of HMG CoA reductase from rat liver in vitro. 2a7 In the only study of its kind on ruminants, Bergman et al. ~9 showed that in the sheep, as in the dog and other non-ruminants, the liver is a major site of removal of cholesterol from chylomicrons and presumably other plasma lipoproteins. 6. Regulation of Hepatic Lipid Metabolism

Lipid metabolism in the ruminant liver is characterized by a low rate of fatty acid synthesis de novo, with acetate as the principal carbon source for that which does occur, a consequent dependence on the uptake of exogenous long-chain fatty acids for synthesis of glycerides and complex lipids and for oxidation, and a relatively high rate of ketogenesis, even when the animal is in positive energy balance. For convenience's sake, the regulation of each of these aspects of lipid metabolism is separately discussed in the preceding sections but there are several important common factors. In particular, the scale and control of lipogenesis and ketogenesis must be considered in relation to each

Lipid metabolism in liver and selectedtissuesin ruminants

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other and against the background of the ruminant liver's constant and over-riding function in gluconeogenesis. That gluconeogenesis is a major and continuous function of the ruminant liver, regardless of nutritional or physiological state, is indicated by the high and relatively constant hepatic activities of all the key gluconeogenic enzymes in vitro 25'*°'1~5 and the invariable net export of glucose from the liver in vivo 2a'T°'22°'as7 under a variety of conditions. The metabolic consequences of this constant drain on carbon sources, reducing equivalents and energy has special implications with respect to the liver's capacity for l!pogenesis and ketogenesis. In particular, the availability of oxaloacetate may be an important common factor in the regulation of gluconeogenesis, lipogenesis and ketogenesis, since this intermediate is obligatory in the pathways of gluconeogenesis and lipogenesis, as well as being necessary for entry of acetyl CoA into the TCA cycle. Detailed discussion of the regulation of lipogenesis in the ruminant liver is probably irrelevant since, even in animals fed high-concentrate diets, the rate of hepatic fatty acid synthesis is very low. Both this and the liver's high rate of gluconoegenesis would appear to be adaptations to the chronic lack of exogenous (dietary) glucose in the portal blood stream. As pointed out by Ballard et al., 39 the very low hepatic activity of NADP malate dehydrogenase in the ruminant must place lipogenesis at a constant disadvantage in any competition with gluconeogenesis for oxaloacetate. Increased activity of this enzyme was induced by greatly increasing the exogenous supply of glucose to sheep, 37'259 but it is notable that lipogenesis from acetate was stimulated much more than that from glucose. This conforms with the suggestion of Lindsay 259 that hepatic fatty acid synthesis from acetate may be limited by the generation of NADPH via the pentose phosphate pathway. In the non-lactating non-pregnant ruminant with no exceptional demands on its glucose supply, hepatic ketogenesis in excess of that occurring from butyrate of dietary origin appears to be regulated by those factors which control the rate at which fatty acids are released from adipose tissue and taken up by the liver. Such factors include fasting or prolonged undernutrition, exercise and cold exposure, and in this regard the ruminant is little different from other animals. However, the extra demands imposed on the glucose supply by the fetus in late pregnancy or by the mammary gland in peak lactation can result in very great increases in hepatic ketogenesis. Krebs 23s'236 distinguishes the latter as "pathological", as opposed to "physiological", ketosis and has attributed its occurrence to an increased rate of gluconeogenesis and consequent aciate shortage of.oxaloacetate. The evidence for this theory was considered in an earlier section, and has also been recently reviewed by .Baird. 2° The metabolic disorders characterized by hypoglycemia and hyperketonemia in pregnant and lactating ruminants are discussed in Section III.G. A further illustration of the interrelationship between the different aspects of hepatic lipid metabolism is the possibility that ketogenesis is partly regulated by the liver's ability to esterify fatty acids and export triglyceride in lipoproteins. The quantitative significance of lipoprotein secretion and the factors which control it are potentially important aspects of lipid metabolism in the ruminant liver which need further study.

B. Skeletal Muscle 1. Uptake of Fatty Acids and Ketone Bodies

In ruminants, as in other mammals, skeletal muscle has a low resting metabolic rate relative to that of most other tissues and consequently, a lower rate of utilization of energy substrates on a weight basis. However, its large mass and capacity for greatly increasing its metabolism during contraction ensure that its demand for fatty acids and other energy substrates has a major effect on the lipid metabolism of the whole animal. This has been illustrated by recent studies of exercising 211 and shivering 61 muscle in sheep and cattle.

130

A.W. Bell

The uptake of circulating lipid substrates, including long-chain fatty acids, acetate and ketone bodies, by skeletal muscle in v i v o has been estimated in sheep and cattle by measurement of arteriovenous concentration differences and blood flow in hind-limb tissues. This approach has several advantages, including accessibility of a large mass of skeletal muscle with only minor surgical interference to the conscious intact animal. However, although the hind limb is predominantly skeletal muscle, it also contains small but metabolically significant amounts of adipose and other tissues. This has particular implications with respect to values for net exchange of plasma FFA, since these will simply reflect the net difference between the simultaneous uptake (by muscle and adipose tissue) and release (by adipose tissue) of FFA. Thus, both net uptakes and releases of FFA by the hind limb have been observed in sheep 2'°'211 and young steers, ~7.60 depending on the animal's physiological and nutritional state. Obviously, such measurements have only limited value. Differentiation of the metabolic roles of muscle and the small deposits of adipose tissue with which it is intimately associated remains a problem but it is possible to distinguish between the simultaneous uptake and release of FFA by these tissues. The masking of uptake by release of FFA in the human forearm was first demonstrated by Zierler and Rabinowitz, 5°9 who used minute doses of insulin to block FFA release. Subsequently, 14C-labeled FFA has been used as a tracer to directly measure FFA uptake by the exercising human forearm 173 and leg, 186 and this approach was recently adopted in our laboratory to make similar measurements in the hind leg of the young steer. 6: Some of the results are shown in Table 3. Although there was a net release of FFA from the leg in steers recently fed or fasted for 24 hr, the uptake of FFA was considerable, particularly in the fasted animals. This uptake by a single leg accounted for 13-14%,', of total entry rate of FFA in both fed and fasted steers. Thus, it seems likely that even the substantial wdues for net uptake of FFA by the sheep hind limb, reported by Jarrett e t al,, 2 ~ 1 significantly underestimated true uptake. Peripheral tissues, including skeletal muscle, also take up fatty acids from circulating triglycerides through the action of lipoprotein lipase (EC 3.1.1.34) within adjacent capillary endothelial cells. 4°3 Lipoprotein iipase activity has not been measured in ruminant muscle but small net uptakes of triglyceride fatty acids have been detected in the bovine hind limb, 56 and Bergman eta/., 6 9 while not distinguishing between individual organs and tissues, showed that the lower body (hind legs and rump) of the sheep took up about 30~o of the triglyceride fatty acids removed by the whole body. Payne and Masters 35k~ found significant amounts of radioactivity in skeletal muscle of 10-week-old lambs, 4 hr after injection of ~4C-iabeled triglyceride but much of this activity could have been taken up as FFA, following triglyceride hydrolysis elsewhere in the body. That skeletal muscle accounts for a considerable fraction of the total utilization of circulating acetate was first suggested by Reid, 376 and confirmed by Holdsworth e t al. ~9~ The latter workers killed a sheep 8 rain after injection of ~4C-acetate; about 33°% of the activity recovered in various organs and tissues was in skeletal muscle. Subsequently, acetate uptake by the hind limb has been measured in sheep 123"21°'211,228,263 and young steers, sT"Ss As illustrated in Fig. 2, a feature of these results was the dependence of uptake on arterial concentration of acetate over the complete range of values normally seen in ruminant b l o o d . ~7'58"22s It, therefore, seems reasonable to assume that utilization of TABLE{3. Metabolism in vivo of Plasma FFA in the Whole Body and Hind Limb of Young Steers (mean values + S.E.M. from ref. 62) Whole body

Treatment Fed Fasted (24hr)

Arterial concentration (/amol/l) 138 _+24 341 +_32

Entry rate (#mol/min) 138 + 23 273 + 19

Hind limb Uptake (/amol/minj

Release (tLmol/min)

19.5 _ 4.5 35.6 _+4.7

34.3 + 8.2 44.1 ± 7.4

Lipid metabolismin liver and selected tissues in ruminants

131

$0C

400 ,1[ ~

30G a:

~

20C

I0(

~oo

,oo

A.to,.t

,~o

,~o ,o'oo ,~'oo

I

1400

~c°t.t.] (,~M)

FIG. 2. The relation between arterial concentration and arteriovenous concentration difference across the hind limb of blood acetate in sheep (open symbol)and steers (closed symbols).Individual valuesare means from refs 57 (A), 58 (O) and 228 (O). The regressionline is described by the equation Y = 0.343X + 31.9 where Y= arteriovenousdifferenceand X = arterial concentration; r = 0.893 (P < 0.001). acetate by muscle and other peripheral tissues is controlled mainly by those factors which govern the rate of entry of acetate into the peripheral circulation. The decreased uptake of acetate by the hind limb in alloxan-diabetic sheep has been attributed to the restricted entry of glucose into muscle. 2t°'22s This possibility is discussed in more detail in Section III.G.2. The capacity of mammalian skeletal muscle to take up and utilize ketone bodies has been well known for some time, 5°° but only recently did the elegant experiments of Ruderman and his colleagues asa'39° show that, if present in sufficient quantities in blood, ketones are the preferred fuel for resting muscle in the isolated perfused rat hindquarter. The uptake of 3-hydroxybutyrate and acetoacetate by the hind limb has been measured in conscious sheep 12a'2t°'2t1 and steers 56 under a variety of conditions, and in anesthetized sheep infused intravenously with acetoacetate, z63 As might be expected, uptake in fed animals, although variable, was generally much higher than that in fed non-ruminants and in all animals, it was directly related to arterial concentration over a very wide range of values. Such concentration-dependent uptake, even at low circulating concentrations, is consistent with the conclusion of Ruderman and Goodman 388 that uptake of ketones by muscle is not .limited by diffusion into the cell, but rather by the kinetic characteristics of 3-oxoacid-CoA transferase (EC 2.8.3.5). As in the perfused rat hindquarter, t6t'389 diabetes diminished the uptake of ketone bodies by the sheep hind limb, despite increases in blood concentration. 2to

2. Metabolism of Lipid Substrates In skeletal muscle, the ultimate metabolic fate of FFA, acetate and ketones must be mainly oxidation. In the case of acetate and ketones, this is almost certainly a straightforward process. However, the picture may not be so simple for FFA in view of increasing, if indirect, evidence that skeletal muscle preferentially catabolizes its endogenous triglyceride fatty acids, rather than immediately oxidizing FFA taken up from plasma.l~4 This idea is based on observations of a consistent discrepancy between the directly measured oxidation of plasma FFA and the total oxidation of lipid as calculated from RQ values, as well as a pronounced delay between the uptake of ~4C-FFA by muscle and the appearance of 14CO2 in venous blood, despite apparently rapid mixing of labeled with unlabeled CO2 within the tissue. Certainly, substantial incorporation of injected ~4C-FFA into muscle glycerides was demonstrated in growing lambs, 353 and in a recent study of FFA oxidation in the bovine hind limb, 14CO 2 production accounted for only 5-30~ of the simultaneous uptake of :4C-FFA.62 However, further analysis of the latter results provided only qualified support for the hypothesis outlined above.

132

A.W. Bell

Endogenous acetate production may be an alternative to complete oxidation of FFA to CO2 in ruminant muscle. Moderate activities of acetyl CoA hydrolase (EC 3.1.2.1) were found in sheep leg muscles 228 and the production of acetate in the sheep hind limb has been directly demonstrated in vivo. 3s8 Although the activity of acetyl CoA synthetase, which is essential for the activation and further metabolism of acetate, is low in ruminant muscle, 228'361 acetate is efficiently metabolized by this tissue. Labeled acetate taken up by sheep muscle in r,ivo ~93 and in v i t r o 295 was rapidly converted to CO2 or TCA cycle intermediates and over a period of several hours, the ~4C-acetate taken up by the hind limb in conscious sheep was almost completely converted to 14CO2 (A. Domanski, personal communication). Values for uptake of acetate by muscle and other ruminant hind-limb tissues in /)//)O 57"58"123"210'211"228'z63 may therefore be reasonable approximations of oxidation rates, especially during muscular activity. 5v'58"21~ It has been assumed that all ketone bodies taken up by ruminant skeletal muscle are oxidized but this has not yet been verified by experiments in vivo. Domanski et al. 123 found only a low rate of conversion of ~4C-3-hydroxybutyrate to ~4CO2 in the hind limb of fed sheep, while Lindsay and Setchell, 263 who used a similar preparation in anesthetized sheep infused with aceotacetate, were unable to detect a net efflux of ~4CO2 from muscle, even after infusion of ~4C-3-hydroxybutyrate for several hours. TABLE 4. Free Carnitine and Carnitine Ester Concentrations in Skeletal Muscle of Normal and Alloxandiabetic Sheep and Rats (mean values from refs 297, 355, 428 and 430) Concentrations (nmol/g wet wt)

Species

Muscle

Sheep

Biceps femoris

Rat Rat

Soleus Gastrocnemius

State Normal Diabetic Normal Normal Diabetic

Acetylcarnitine

Free carnitine

Total acidsoluble carnitine

1820 12,000 < 50

9860 3240 442

12,900 17,100 627 1830 837

.....

References 428 430 355 297

Skeletal muscle of sheep, 428'43° goats 429 and cattle 148 was found to contain unusually high concentrations of acetylcarnitine and free carnitine, relative both to other ruminant tissues and to skeletal muscle of non-ruminants (Table 4). Snoswell and Koundakjian 428 also found a reciprocal relationship between the concentration of total acid-soluble carnitine and that of acid-soluble CoA. This relationship was evident over a range of values obtained from lambs (5-16 days) and adult sheep, with lamb muscle having lower carnitine and higher CoA levels, and also between different values for individual muscles in the same animal. Carnitine was confined to the cytosol whereas almost all CoA was found to be mitochondrial. On the basis of these observations, Snoswell and Koundakjian 42s suggested that the role of muscle carnitine and carnitine acetyltransferase (EC 2.3.1.7) is to provide a buffer system, which would remove mitochondrial acetyl groups during periods of increased fatty acid oxidation for "storage" as acetylcarnitine in the cytosol. They calculated that in a 50kg sheep about 6g of acetyl groups could be "stored" in this way, which may reflect the extensive use of acetate and other fatty acids but limited availability of CoA in ruminant muscle. Little or no carnitine was exported from skeletal muscle to extramuscular tissues in normal or alloxan-diabetic sheep. 43° C. H e a r t

Heart, like active skeletal muscle, has an avid demand for a variety of energy substrates, and numerous studies on non-ruminants have highlighted the importance of lipids as sources of energy for maintaining the very high rate of myocardial oxidative metabolism (for recent reviews, see Gilbertson, 1~8 Neely et al., 312 Spitzer434). The rela-

Lipid metabolism in liver and selected tissues in ruminants

133

tive importance of the different fuels available to the ruminant heart has been studied much less intensively but the limited available evidence suggests that this organ may differ little from that of most non-ruminants, particularly in its avidity for ketone bodies. Plasma FFA have been deemed a preferred fuel for oxidation in non-ruminant cardiac muscle, 312 but their role in the myocardial metabolism of the ruminant is not yet clear. On a weight basis, the heart of the 20-week-old lamb took up considerable amounts of FFA and triglyceride fatty acids from circulating plasma, 353 but the metabolic fate of these fatty acids other than incorporation into glycerides and phospholipids was not examined. The ox heart was also found to contain substantial amounts of triglyceride and phospholipidf129 The importance of adequate phospholipid synthesis, particularly for structural integrity of cell membrane systems in a tissue such as heart, seems obvious but that of extensive triglyceride formation is less clear, It may be that endogenous triglycerides in heart fulfil the same role as that recently postulated for skeletal muscle triglycerides,114 and that little or no fatty acids taken up from plasma are oxidized before incorporation into triglycerides. Certainly, triglycerides in the isolated, perfused rat heart were extensively catabolized when FFA were absent from the perfusate, T M and Spitzer 434 has suggested the existence of at least two pools of long-chain fatty acids in the myocardium. Although the oxidation of long-chain fatty acids by ruminant heart has not yet been measured in vivo, bovine heart mitochondria were shown to be wellequipped in vitro for carnitine-dependent oxidation of long-chain acyl CoA, 86'231 via a carnitine:acylcarnitine exchange system. 36s The uptake of acetate per weight of tissue by the sheep heart in vivo 193 and in vitro 29s was high relative to that of other ovine tissues, which is consistent with the relatively high activity of acetyl CoA synthetase found in ruminant heart, 1°9'22s'361 In sheep heart, this activity was apparently almost exclusively located in the mitochondria, 22s'42s where it presumably functions to generate acetyl CoA for oxidation in the TCA cycle. In contrast, in bovine heart, much of the enzyme was found in the cytosol, 36~ although the possibility of mitochondrial leakage during sample preparation cannot be ruled out. It is notable that in sheep heart mitochondria, acetyl CoA synthetase appeared to be highly specific for C2 and Ca fatty acids t°9 which, in view of the very low levels of propionate in arterial blood, suggests that acetate suffers virtually no competition from other shortchain fatty acids. The importance of acetate as a fuel for heart in the normal fed ruminant is difficult to estimate. In anesthetized sheep fasted for 24 hr myocardial uptake of acetate accounted for only 9Yo of oxygen consumption by the heart, even assuming complete oxidation. 263 However, these animals were also infused with acetoacetate and it has been shown that sheep heart slices in vitro oxidize ketone bodies in preference to acetate and other short-chain acids. 253 In spite of its relatively low acetyl CoA hydrolase activity, sheep heart showed a considerable capacity for acetate production in vitro. 22a In view of the heart's inability to divert acetyl CoA to acetoacetate, it seems likely that, like skeletal muscle, it can relieve "acetyl pressure" by releasing free acetate from the mitochondrion. Alternatively, excess mitochondrial acetyl groups may be transferred into the cytosol under the action of carnitine acetyltransferase for storage as acetylcarnitine. 42s When present in arterial blood in sufficient concentrations, ketone bodies are almost certainly the preferred fuel of ruminant heart. 253 Moderate activity of 3-hydroxybutyrate dehydrogenase (EC 1.1.1.30), the key enzyme for 3-hydroxybutyrate utilization, was found in sheep heart mitochondria. 4s6 AS in most tissues from other species, but unlike many non-muscular tissues in the ruminant, the enzyme was tightly bound to the mitochondrial membrane; this and other properties of the enzyme isolated from beef heart were described by Sekuzu et al. 4°7 Lindsay and Setchell z6a have measured the uptake and oxidation in vivo of 3-hydroxybutyrate by the heart of anesthetized sheep infused with acetoacetate. The conversion of ~4C-3-hydroxybutyrate to ~4CO2 accounted for almost 80% of total CO2 production, which strongly supports the previously-mentioned contention that ketones are oxidized by sheep heart in preference to other substrates, such as acetate and glucose. 25a The contribution of ketone bodies to oxidative metaboJ.P.L.R. 1 8 3

4"

134

A.W. Bell

lism in the heart of conscious fed animals must be considerably less, but it still seems likely that the heart disposes of a relatively large fraction of the ketones (mainly 3-hydroxybutyrate) normally entering the peripheral circulation. D. Brain

The mammalian brain has a considerable requirement for long-chain fatty acids, principally for phospholipid synthesis, and it is now known that little of these fatty acids is derived from synthesis de novo. This, of course, particularly applies to the essential fatty acids, linoleic and linolenic acids. Brain phospholipids have a low content of these acids but an unusually high concentration of some of their longer-chain polyunsaturated derivatives. 9~ The latter may be synthesized via desaturation and chain elongation within the brain or be taken up by the brain after synthesis in extraneural tissues. Factors affecting the uptake and incorporation of fatty acids into brain lipids have been recently reviewed. ~20,4 ~5,503 Dynamic aspects of long-chain fatty acid metabolism by the ruminant brain have not been studied. However, the remarkably uniform fatty acid composition of brain phospholipids in a wide variety of mammals, including domestic cattle and several species of wild ruminants, ~2 suggests that the ruminant brain resembles that of other species in its requirement for and subsequent metabolism of FFA. This is interesting because the ruminant has a poor supply of linoleic and linolenic acids, yet these are the only source of certain polyunsaturated fatty acids for which the brain has a specific need. 4~ ~ It may be that the apparent ability of other ruminant tissues to conserve and efficiently use essential fatty acids 243 has evolved partly to allow the brain better access to these acids and their derivatives which, via their incorporation into phospholipids, seem to be so important for the structure and function of cerebral membranes. The oxidation of FFA by ruminant brain has not been measured, hut it is unlikely to be significant in view of" the lack of importance of short-chain fatty acids and ketone bodies as oxidizable substrates (see belowl, The uptake of acetate by ruminant brain was very low in vivo, 193'2~3"282'338 and in vitro, 295 which was consistent with the low activity of acetyl CoA synthetase observed in this tissue. ~09, z z 8,361 Although bovine brain mitochondria 53 and cerebral cortex slices 367 have shown some capacity to oxidize acetate in vitro, the uptake and oxidation of acetate accounted for no more than 2-3% of CO z production by the brain in vivo in conscious fed sheep. 263 In apparent contradiction of this and other studies, Kammula and Fong 2t9 reported relatively large uptakes (although very low rates of oxidation) of acetate by the brain of the conscious sheep. However, it has been suggested that in this study, the cerebral venous (dorsal sagittal sinus) catheter tip may have been sampling other than cerebral venous blood. 263 Certainly, the non-cerebral tissues of the ruminant head (mainly muscle) have been found to take up significant amounts of acetate. 9'3.6 The discovery that ketone bodies can be an important fuel for the human brain during prolonged starvation 337 led to numerous studies on other species. These mainly confirmed that ketones can be oxidized by the brain under certain conditions, particularly in neonatal animals (e.g. Hawkins et al., 18v Spitzer and Weng'~35). However, in keeping with its poor enzymatic capacity for utilization of acetoacetate 499 and 3-hydroxybutyrate,486 the sheep brain oxidized only limited amounts of these substrates in vitro, 253 and the recent detailed experiments of Lindsay and Setchell z63 appear to have confirmed earlier evidence zlS"282,33a that, even in highly ketonemic animals, the brain of sheep (and presumably other ruminants) relies almost exclusively on glucose for oxidation. As with acetate (see above), there is some evidence contrary to this that during ketonemia (acetoacetate infusion), ketones may make a significant contribution to ovine cerebral metabolism. 2~8 However, these results came from the same laboratory as that which found a relatively high cerebral uptake of acetatefl ~9 and the same explanation offered for the discrepancy between this and other studies z63 may apply. In addition to the brain's lack of adequate activities of the enzymes necessary for cerebral ketone utilization, ketones

Lipid metabolismin liver and selectedtissuesin ruminants

135

penetrated into the cerebrospinal fluid more slowly in sheep than in other species. 263 Lindsay and Setchel1263 proposed that, from a teleological viewpoint, it has been unnecessary for the sheep brain to evolve mechanisms for utilization of ketone bodies, largely because its metabolism can be supported by a much smaller fraction of the whole-body supply of glucose than in other species, particularly man. E. Kidney

Kidney, like heart, is a metabolically active tissue with a considerable demand for oxidizable energy substrates. Studies in vitro on material from several non-ruminant species have shown that renal cortical and medullary tissues have different metabolic characteristics. The more vascular cortex has a much higher rate of aerobic metabolism, which is supported by the oxidation of various substrates, among which FFA and ketones are prominent. The medulla, on the other hand, has a lower metabolic rate and relies mainly on glucose for its energy, via oxidation and glycolysis. These and many other aspects of renal substrate metabolism, including a section specifically on FFA metabolism, have been comprehensively reviewed elsewhere. 1°2 Unless otherwise specified, this section will deal only with lipid metabolism in the renal cortex of the ruminant, partly because cortical metabolism dominates that of the kidney as a whole, and partly because lipid metabolism is apparently much less significant in the medulla. The metabolism of FFA by the sheep kidney in vivo has been studied by measuring the incorporation of injected t4C-FFA into renal tissue lipids, 353 and also by measurement of A-V concentration differences and renal blood flow in the conscious animal. 221 On a weight basis, kidney was second only to liver in its uptake of FFA in lambs during rumen development (0--20 weeks) and most fatty acids were incorporated into phospholipids. 353 Contrary to results from similar experiments on non-ruminants, Kaufman and Bergman 221 found a consistent net output of FFA from the sheep kidney in vivo (Table 5). However, in this species, venous blood from the perirenal fat pad drains into the renal vein and a small positive A-V difference for FFA across the kidney could have been masked by a larger release of FFA from nearby adipose tissue. The renal cortex in the rat also contains readily metabolizable endogenous triglyceride,4s8 but it is not known whether fatty acids from this source can be released into the bloodstream. The quantitative importance of FFA as a fuel for ruminant kidney has not been assessed. However, they can be a significant source of energy in non-ruminants, particularly when their circulating levels are elevated, 102 and there is little evidence to suggest that the pattern of energy metabolism in the ruminant kidney differs greatly from that of other species. The sheep kidney has a large capacity for acetate metabolism, as indicated by its rate of uptake of labeled acetate in vivo 193 and in vitro, 295 and its very high acetyl CoA synthetase activity.22a'361 Most of the acetate taken up was oxidized, rather than used for non-oxidative purposes such as lipogenesis, t93'2°s which is consistent with the high rate of aerobic metabolism and gluconeogenic activity of the sheep kidney. 222'237 Thus, acetate is probably the major source of oxidizable substrate for kidney in the fed ruminant. 3-Hydroxybutyrate was readily oxidized by sheep kidney cortex slices 2sa and the sheep kidney was subsequently found to contain very high activities of the enzymes concerned with metabolism of both 3-hydroxybutyrate233'4a6 and acetoacetate. 499 However, evidence for the role of 3-hydroxybutyrate dehydrogenase, at least, should be treated with some caution. Unlike its intracellular distribution in the kidney of other species and in sheep muscle, this enzyme in ovine renal cortex was almost entirely confined to the cytosol, and there is some doubt about its stereospecificity for the naturally occurring D-isomer of 3-hydroxybutyrate.4a6'5°1 In accordance with the view that ketones are a preferred fuel for renal cortex, 4aa Kaufman and Bergman T M found that the kidneys in vivo took up about 20~o of all ketone bodies entering the bloodstream in both fed and fasted sheep. Results of this and a later study,222 which showed that 3-hydroxybutyrate and acetoacetate were taken up approximately according to their relative concentrations

136

A.W. Bell

TABLE 5. Renal Metabolism in vivo of FFA and Ketone Bodies in the Sheep (mean values from refs 221. 222) FFA

Nutritional or physiological status Fed Fasted (3 days) Fed, acidotic Fed, pregnant Fasted (3 days), pregnant

Total ketone bodies

Arterial plasma conc. (#mol/1)

Net renal production {pmol/min)

Arterial conc. (pmol/l)

Net renal* uptake !pmol/min~

Urinary excretion lpmol/min)

260 1040 250

48 82 60

470 710 380 576 1810

44 73 37 5,~ 63

2 1 1 3 6

*Approximately 80~o 3-hydroxybutyrate. 20°,/0 acetoacetate (see ref. 222).

in arterial blood (i.e. 80~ 3-hydroxybutyrate, 20~ acetoacetate), are summarized m Table 5. In contrast, similar work on starved humans showed an uptake of 3-hydroxybutyrate but a small net release of acetoacetate, a36 As in other species, ketone bodies were excreted in urine by the sheep kidney although renal clearance accounted for less than 10~ of the net renal uptake of ketones, even in pregnant and fasted animals 221'2z2 (Table 5). However, even the highest blood concentrations of total ketones barely exceeded 1.8 mM in these experiments, and earlier work had shown that the threshold for efficient reabsorption of ketone bodies in the renal tubules was a blood concentration of at least 2 mM, with some difference between the clearance rates of 3-hydroxybutyrate and acetoacetate.168,378

Ill.

ADAPTATION

AND

REGULATION

THE

WHOLE

OF

LIPID

METABOLISM

IN

ANIMAL

A. The Fed Animal 1. General Aspects

In the fed ruminant, metabolism is dominated by the extensive microbial fermentation of dietary carbohydrate and other organic constituents to short-chain fatty acids in the reticulo-rumen and, to a lesser extent, the cecum; ~8°'3~ 5 these account for at least 50°:~ of the intake of digestible energyfl 2't66'4°4 As described earlier in this review, of the three acids produced in significant amounts (acetic, propionic and butyric), acetic acid predominates; it is also metabolized least by ruminal epithelium and, after passage in hepatic portal blood, the liver. Therefore, large amounts of circulating acetate are available for post-hepatic metabolism in the fed ruminant. Much of this is oxidized in peripheral tissues; a'9 that which is surplus to oxidative requirements becomes by far the most important source of acetyl CoA for the synthesis de novo of long-chain fatty acids, as demonstrated by Hanson and Ballard 177,178 and subsequently by numerous other investigators) 96'2°a'2°4"5°a The role of acetate in lipogenesis in the fed ruminant assumes added importance, relative to the role of glucose in the non-ruminant, because the dietary intake and gastrointestinal absorption of preformed lipids is usually considerably lower in the normally-fed ruminant than in the non-ruminant, a~5 The other major short-chain fatty acids, propionate and butyrate, are also involved in lipogenesis after feeding through utilization of their metabolites, glucose and 3-hydroxybutyrate, respectively. Almost all propionate which reaches the liver is metabolized by way of the TCA cycle following its comiersion to succinate via methylmalonate:23 Some of this is oxidized to CO2, but much of the oxaloacetate so formed is used for glucose synthesis, which usually accounts for 40-60~o of all glucose produced by the fed animalfl ~°'259 Although this glucose is only a minor source of carbon for fatty acid syn-

Lipid metabolism in liver and selectedtissues in ruminants

137

thesis, at least partly because of the ruminant's lack of a functional citrate cleavage pathway, 177't96,2°3"5°s it is nonetheless very important in lipogenesis as a source of NADPH via metabolism in the pentose phosphate cycle5°5 and of glycerol-3-phosphate for esterification of long-chain fatty acids. Apart from its importance as a precursor of glucose, the little propionate which does escape hepatic metabolism was found to be involved in the synthesis of long-chain fatty acids with an odd number of carbon atoms, 4a i and more recently, of abnormal saturated branched-chain fatty acids in adipose tissue triglycerides, t2a't 55 This subject has been recently reviewed in greater detail by Garton. 1s4 Butyrate is extensively metabolized in tureen epithelium and the liver to 3-hydroxybutyrate via conversion to butyryl CoA, 3-hydroxy-3-methylglutaryl CoA and acetoacetate. 2s'22°'356'4a9 In addition to acetate, this 3-hydroxybutyrate contributes to fatty acid synthesis, especially in the lactating mammary gland. 3°4 Therefore, from the point of view of the supply of both carbon and reducing equivalents, the post-feeding increase in availability of short-chain fatty acids is crucial for lipogenesis in ruminant tissues, and lipogenesis is likely to be directly affected by any factors which alter the rate of ruminal production of short-chain fatty acids. The most important of these is level of feeding and the role of this and other factors in controlling short-chain fatty acid production in the ruminant have been reviewed by Leng T M and by Church. 9a Another less direct link between the availability of short-chain fatty acids and lipid metabolism in the fed animal may occur via the putative relationship between the rate of appearance of these acids in blood and the post-feeding increase in insulin secretion. 46'a5 Whatever its stimulus, insulin is almost certainly the major humoral influence on lipid metabolism in the fed ruminant as in the non-ruminant. Insulin has been shown to enhance lipogenesis from both glucose and acetate 35'43'226,47a's°s and also to inhibit catecholamine-stimulated lipolysis 299'3°°'5°6 in ruminant adipose tissue in vitro. Although these responses suggested that adipose tissue may be less sensitive to insulin in ruminants than in non-ruminants, insulin administration in vivo produced substantial decreases in plasma concentrations of F F A 7'50'66'191'239"272'461''.93 and glycerol, 66 and in the net output of these substrates from adipose tissue in the sheep. 226 It is also notable that the invariable decrease in plasma FFA level which followed feeding in the sheep was closely associated with increased plasma insulin. 45 The work of Bauman and his colleagues suggested that in the short term, at least, post-feeding changes in lipogenesis in ruminant adipose 2°5 and mammary tissues 29s may be regulated by the allosteric enzyme acetyl CoA carboxylase (EC 6.4.1.2), which catalyzes the conversion of acetyl CoA to malonyl CoA. This enzyme is activated by citrate, isocitrate and possibly, long-chain fatty acylcarnitine, while long-chain fatty acids and their activated derivatives have the opposite effect. 267 It has been tentatively suggested that insulin may be involved in the regulation of fatty acid synthesis at this level, t57,267 but the role of this and other factors in the mediation of the effects of feeding on the rate-limiting enzyme(s) in lipogenesis remain to be eluciated. In ruminants as in other animals, lipid synthesis occurs in most tissues of the body but in non-lactating ruminants, more than 90% of lipogenesis occurs in adipose tissue.196,204,290,354,4.79 The quantitative significance of mammary lipogenesis in the lactating animal is discussed in detail elsewhere in this series of reviews. 3°4

2. Effects of Dietary Manipulation Advances in our knowledge of the processes of digestion and metabolism in ruminants have led to intensive studies into possible means of dietary manipulation of metabolism. It is now clear that the metabolism of lipids, as of other nutrients, can to some extent be controlled by modification of the diet. Two major avenues by which this can occur will be considered here. Firstly, it has been known for some time that both chemical and physical characteristics of the ruminant diet affect not only the relative proportions of

138

A.W. Bell

short-chain fatty acids produced in the reticulo-rumen but also the energetic efficiency with which these acids are used for lipogenesis and other metabolic processes. Secondly, there is now much evidence that the degree to which ingested lipids are modified during passage through the forestomachs can be reduced by dietary means; quite spectacular results have been obtained by "protecting" dietary unsaturated fatty acids against ruminal hydrogenation by encapsulating them in formaldehyde-treated protein. There have been many studies of the effects of diet on the molar percentages of individual short-chain fatty acids in the rumen; these have been recently reviewed by Church. 9a Despite considerable variability between results from different laboratories, it is clear that relatively more acetic acid is produced from high-roughage diets, particularly those containing a high proportion of mature hay or mature pasture. Conversely, highconcentrate (usually cereal grain) rations tend to increase the proportion of propionic at the expense of acetic acid. In addition, rumen fermentation may be influenced by the physical form of the diet as grinding and pelleting of roughages and crushing or heat treatment of grain also tended to increase the molar percentage of propionic acid in the rumen. 45o The consequences for lipid metabolism of such diet-induced changes in the pattern of rumen fermentation have been most intensively studied in relation to their effects on milk triglyceride synthesis in the lactating cow. It is well-established that the feeding of highgrain low-fiber diets to dairy cows causes a depression in milk-fat content, and that this "low milk-fat syndrome" is associated with elevated levels of rumen propionate and usually a relative decrease in acetate. The metabolic etiology of the low milk-fat condition in dairy cows is discussed in more detail in Section III.G. Feeding of starch-rich diets, with associated increases in ruminal propionate production and blood glucose entry rate, apparently does not depress lipogenesis in adipose tissue of non-lactating ruminants. Rather, there was increased synthesis of odd-numbered and branched-chain fatty acids 127,155 and a strong relationship between blood glucose entry rate and body weight gain 225 and, presumably, rate of fattening. Despite their extensive biohydrogenation in the reticulo-rumen, dietary fatty acids can, to a limited extent, influence the fatty acid composition of plasma lipids. However, the unsaturated fatty acids which escape ruminal modification appear to be largely confined to the less metabolically active phospholipid and cholesteryl ester fractions. 3°6'43~ The dietary influence on lipid composition of body tissues, including adipose tissue and the mammary gland, is usually therefore minimal (see Christie95). However, when the diet contained a significant amount of lipid which was protected from ruminal lipolysis and hydrogenation by coating with a layer of formaldehyde-treated protein, 4°°'4°~ the dietary influence on lipid composition approached that seen in non-ruminants. The metabolic consequences of feeding dietary lipids protected in this way have been reviewed by Scott and Cook 399 and Christie. 95 Initial studies suggested that the unusually large amounts of dietary lipid reaching the small intestine had relatively little effect on the efficiency of digestion and absorption, ~92 although it has since been shown that the digestibility of protected fat may be reduced when fed in large amounts to growing lambs.~4~ Dietary unsaturated fatty acids protected from ruminal hydrogenation were absorbed and incorporated into lymph triglycerides and as such were apparently transported to the bloodstream in the normal manner. 399 Although Scott and Cook 39~ indicated that lymphatic triglyceride was transported primarily in chylomicrons, it has recently been suggested that chylomicrons as they exist in lymph and plasma of nonruminants are present in negligible amounts in ruminants and that dietary triglyceride is transported primarily in VLDL. 342'36° Certainly, in sheep fed formaldehyde-treated sunflower seed supplements, there were substantial increases in the proportion of linoleic acid in triglycerides carried in serum VLDL, as well as in the phospholipids and cholesteryl est~ers carried in this and the denser serum lipoproteins. 326'39s There was also evidence of increased cholesterogenesis, particularly in small intestine, which may largely account for the elevated levels of plasma cholesterol' observed in ruminants fed protected fat. 75.122

Lipid metabolismin liver and selectedtissuesin ruminants

139

It is quite clear that a greatly increased supply of circulating polyunsaturated fatty acids results in a concomitant change in the fatty-acid composition of tissue lipids, most notably in those which are important commercial products, i.e. skeletal muscle, adipose tissue and milk. These effects have been reviewed elsewhere. 95 There is less certainty, however, about the possible metabolic side effects of supplementing ruminants with large amounts of both dietary fat and polyunsaturated fatty acids. In the preruminant lamb, administration of safflower oil inhibited lipogenesis in perirenal adipose tissue474'4~6 and a similar effect was observed in the subcutaneous, but not perirenal adipose tissue of older lambs, a99 These observations contrast with results from fully-ruminating, growing lambs in which feeding of protected tallow or maize oil, but not protected sunflower seed oil plus soyabean oil resulted in reduced lipogenesis in both perirenal and subcutaneous tissues. 4~5 The latter results were supported by a study which indicated that both saturated (palmitic) and polyunsaturated (linoleic) acids inhibit the activity of one or more lipogenic enzymes in ovine adipose tissue in vitro but that polyunsaturated acids are less effective.'~77 More work is required to clarify this area but at present it does seem that in the short term, at least, the advantages of increasing the availability of preformed longchain fatty acids (especially polyenoic acids) may outweigh any disadvantages resulting from an associated depression of fatty-acid synthesis de novo. Studies of the long-term effects of feeding protected lipids rich in polyunsaturated fatty acids should be a future research priority. Another consideration is the possible effect on tissue peroxidation of a sudden increase in the polyunsaturated fatty acid status of animals adapted to low tissue levels of these acids. Certainly, the feeding of protected linoleic-rich oils to lactating ruminants was associated with a decreased oxidative stability of the milk. ~29'41a'4~4 Less is known about effects in other tissues but as recently suggested by Palmquist and his colleagues, a'~s the feeding of such supplements to ruminants may necessitate a reassessment of the dietary requirements for vitamin E (ct-tocopherol) and/or selenium, particularly in young animals. B. Fastinff and Undernutrition

In ruminants fed less than an ad libitum ration once or twice daily, there are pronounced diurnal variations in the rate of ruminal production, 165,166'aa4 in the rate of appearance in hepatic portal blood 45~ and in circulating levels of short-chain fatty acids. 45'as7,4ss Depending. on the level of feeding, the physical and chemical nature of the diet and its rate of ingestion, blood levels of acetate usually peak at between l and 2 mM within several hours of completion of a meal and then decline gradually to around 0.5 mM just before the next meal. During these diurnal changes, an inverse relationship between the circulating levels of acetate and FFA has been found. 45'56 Plasma FFA concentration was usually less than 0.2 mM after feeding but then increased with the decline in blood acetate to around 0.4--0.5 mu within 24 hr of the previous meal. Shortterm changes in FFA flux (entry rate) have not been measured over the same post-feeding period. However, changes in plasma glycerol paralleled those in FFA 56 and there seems little reason to doubt that there is an increase in the net release of fatty acids from adipose tissue following an increase in the rate of lipolysis and, possibly, a decrease in the rate of esterification of adipose tissue triglycerides. Such changes would be consistent with the decline in plasma insulin seen in ruminants after the post-feeding period. 44,45.93,276 When the period of fasting was prolonged beyond 24 hr, blood acetate concentration declined to a minimal level of about 0.2-0.3 raM, while plasma FFA levels increased to concentrations which, in the non-pregnant non-lactating animal, were usually maximal at 1.5-2.0 mM within 4 days after feeding. 7'3s2 At this time, the alimentary supply of acetate and other short-chain fatty acids was negligible and virtually all blood acetate appeared to be of endogenous origin; 74 it is, therefore, not surprising that acetate levels in the starved ruminant were very similar to those normally seen in non-ruminant animals. 22a

140

A.W. Bell

The flux of the major individual plasma FFA, palmitic, stearic and oteic acids, has been shown to reflect their plasma concentrations in sheep 11.24.5,262.491 and cattle 62'2°6 fasted for 1-3 days. Thus, Lindsay 26° estimated that, in sheep starved for several days, the total FFA entry rate of about 600 mmol/day would be ample to account for all of the animal's energy requirement, given that the fasting heat production of a 50 kg sheep is about 4500 k J/day and the energy equivalent of the major FFA in ovine plasma is about 10,900 k J/mole. Measurements of arteriovenous concentration differences for FFA in vivo across the fat tail of the Syrian sheep provided direct evidence that adipose tissue is indeed the source of the increased supply of FFA in the fasted ruminant. 1 This study also showed that the disproportionately greater increase in circulating oleic acid relative to other FFA often observed in the starved sheep 1'2°7'262 is related to the greater release of this fatty acid from adipose tissue, a finding consistent with the fact that triglycerides in peripheral adipose depots of the ruminant are richer in oleic acid than are plasma FFA.96,124A 26,183,244 Only a fraction of each of these fatty acids entering the bloodstream was completely oxidized to CO2,11'245'262 such that the estimated maximal contribution of total plasma FFA to respiratory CO2 in the starved sheep was no more than 400/o.2~° This is apparently at odds with indirect estimations, based on measurements of respiratory gas exchange, that fat catabolism accounted for about 80~o of energy expenditure in the fasted sheep. 162 The discrepancy is probably attributable to the incomplete oxidation of long-chain fatty acids to ketones and acetate in the liver and possibly other tissues. It has been directly demonstrated that as the concentration of plasma FFA increases during fasting, so does the hepatic uptake of FFA in sheep 22°'4s6 and cows. 27 The simultaneous increase in hepatic output of ketones accounted for 50-90~o of the increased u p t a k e of fatty acids, 27'22° which is consistent with the rate of transfer of ~4C from 14C-FFA to ketone bodies in the whole animal. 25s'341 The possible mode of control of hepatic ketogenesis has been discussed earlier in this review. Thus, circulating levels of 3-hydroxybutyrate and acetoacetate were elevated in non-pregnant non-lactating sheep and cattle fasted for several days, but the level of ketonemia was far less severe than that found in human diabetic ketoacidosis or the ketosis of pregnant or lactating ruminants (see Section III.G) and can be viewed as a "'physiological ketosis", as defined by Krebs, 2s5 which contributes to the metabolic homeostasis of the animal. From measurements of the rate of oxidation of ketones during short-term fasting, 1~ L i n d s a f "° has estimated that ketones may contribute up to 20°/~, of CO 2 production in the sheep starved for several days. The role of endogenous acetate production from the incomplete fl-oxidation of FFA in the starved animal is less clear. Palmquist T M demonstrated a marked increase in the rate of transfer of 14C from 14C-palmitate to acetate in the whole animal in fasted sheep and cows, and there is little doubt that the relative importance of endogenous synthesis as a source of acetate is considerably increased during starvation. 74 On the other hand, there is no evidence that the absolute rate of production of endogenous acetate in individual organs is increased. In non-pregnant non-lactating sheep, hepatic production was unchanged 74 and synthesis in leg muscle decreased significantly 358 after several days without food while in the lactating cow, hepatic release of acetate decreased during fasting. 27 The regulation of endogenous acetate production and its relation to hepatic ketogenesis in the starved ruminant is poorly understood and is clearly in need of further study. In the extensively-grazed ruminant, chronic undernutrition is more common than outright starvation, but its consequences for lipid metabolism have been less well studied. Indirect measurements of the utilization of body fat in sheep at different levels of submaintenance feeding suggested a direct relationship between fat catabolism and the severity of undernutrition. 162 This was supported by observations of a negative relationship between digestible energy intake and plasma FFA levels in underfed beef heifers. 104 As shown in Fig. 3, the relation between these two parameters was curvilinear over a range of sub- and supra-maintenance levels of feeding and it is notable that the asymptote of the curve coincided with maintenance energy intake. There are no reported values

Lipid metabolismin liver and selected tissues in ruminants

~E

~

i

a,

141

400

30C

20(

= x'~..~ I0(

21o Digestible energy

41o intake

~o (MJ.d -1)

FIG. 3. The relation betweendigestibleenergyintake and mean plasma FFA concentration over several weeksin beefheifers.Valuesare for individualanimalsand the symbolsrepresent different rations, as shown in ref. 194. for rates of entry and oxidation of FFA in partially starved ruminants; until these become available, it must be assumed that lipid catabolism is related to plasma FFA concentration at different levels of undernutrition in much the same way that it is in the completely fasted animal at different times after feeding. C. Growth and Development

Lipid metabolism in the fetal and neonatal ruminant has been recently and thoroughly reviewed elsewhere, a16 as have special features of the metabolic development of adipose tissue. 479 This material will not be duplicated in detail here, but it is worth reiterating several important features of lipid metabolism in the developing ruminant which have particular relevance to metabolic regulation and adaptation in the whole animal: (a) Unlike their counterparts in the adult, lipogenic tissues in the fetal and unweaned neonatal ruminant have a relatively active citrate cleavage pathway and make greater use of glucose as a source of carbon for lipogenesis. 43'177.178.a i i These characteristics disappear after weaning along with the direct supply of carbohydrate from the gut, which is additional evidence that it is the mode of digestion and associated pattern of substrate supply which is ultimately responsible for the very low activities of ATP-citrate lyase and NADP-malate dehydrogenase in the tissues of adult ruminants. (b) Unlike the placentae of some non-ruminant species, such as the rabbit, ~3°'~37 guinea-pig ~9° and rat, 2°2 the ovine (and presumably, the bovine) placenta is relatively impermeable to maternal circulating lipids including FFA. 13a'466 Together with the comparatively modest rates of synthesis.de novo of fatty acids in fetal tissues, this must contribute to the paucity of body fat in the new-born lamb 3 and calf. ~72 It also highlights the need for the neonatal ruminant to suckle fairly quickly after birth before its meager stores of energy are depleted. 3 (c) The ruminant placenta is particularly impermeable to linoleic acid, which can only be supplied to the fetus from the maternal circulation. 246'a23 Thus, lambs and calves are born with tissue levels of essential fatty acids which would produce severe clinical deficiency symptoms in other animals. 3°5 As discussed elsewhere, a~6 the degree to which the neonatal ruminant has adapted to this situation and its implications for normal postnatal growth and production remain to be established, particularly in animals subject to additional stresses in the critical few days after birth. (d) Lipogenesis in tissues of non-lactating growing ruminants is largely confined to adipose tissue. 37'40"177'193'196'204'295'354" AS indicated by Bauman and Davis, sl this

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effectively means that gluconeogenesis and lipogenesis are carried out in separate tissues, eliminating competition for carbon, reducing equivalents and energy. It also has implications for the pattern of physiological growth and for the nature of the commercial meat product. Studies of changes in body composition in growing sheep 4°5'*°6 and cattle ~72 have indicated that there are several distinct phases in the relationship between rates of fat deposition and body growth, each of which can be described by a linear equation. The physiological bases of these different rates of fat accretion appear to be related to both hyperplasia and hypertrophy in adipocytes during the phases of juvenile growth, and to hypertrophy only in the subsequent adult fattening phase. 184 This aspect of adipose tissue growth and its relationship with lipogenesis is discussed in detail elsewhere. 479 The remainder of this section is concerned with the changes in lipid metabolism which occur during and after weaning of previously milk-fed animals. Weaning commonly causes a pronounced decrease in the rate of deposition, or even a net loss, of body fat in young ruminants. 3°2'4°5'4°6 This has been attributed mainly to the decrease in energy intake which is associated with weaning 76"t42 and is particularly evident in rapidly-weaned animals which have not established a fully functional rumen. 4s5 Fatty acid synthesis is almost certainly limited by the sudden decrease in availability of the hitherto major lipogenic precursor, glucose, as evidenced by the sudden decrease in plasma glucose levels in lambs weaned at 3.5-9.5 weeks after birth. ~42 Presumably, supplies of acetate, the major lipogenic precursor in the adult ruminant, would also be limited before the complete development of ruminant digestion. Increased lipolysis of adipose tissue triglycerides was indicated by marked increases in plasma FFA concentrations in lambs weaned 3.5 and 5.5 but not 7.5 or 9.5 weeks after birth, with the elevation being larger and more persistent in the early-weaned group. ~42 Not surprisingly, there was a close negative correlation between plasma FFA concentration and intake of dietary energy over the 30-day period after weaning at 3.5 and 5.5 weeks. This study suggested that while early weaning may confer a significant nutritional stress upon the young ruminant with profound effects on lipid metabolism, later weaning is not inevitably associated with catabolism of body fat. Similarly, Searle 4°4 found that gradual adjustment of lambs to a high energy solid diet before weaning resulted in rates of fat deposition after weaning equal to those of milk-fed lambs of the same age. Weaning hastens the process of forestomach development which is already in progress, particularly in young ruminants with previous access to solid food as well as milk. 485 This transitional period is associated with the gradual loss of the distinctive features of lipid metabolism in the young animal 3t6 and the acquisition of those of the adult ruminant, 180,479 as summarized earlier in this section. There appears to have been only a single detailed attempt to define these changes in terms of tissue metabolism in 1)ivo 353 a s opposed to the much more common measurement of whole body composition. In this study, ~4C-labeled fatty acids were injected as FFA or triglyceride into lambs and both transport kinetics and rate of incorporation into tissue lipids were measured. Lambs aged 1, 10 and 20 weeks were used, being regarded as functionally monogastric, transitional and fully ruminant, respectively. The most notable changes were a progressive age-dependent fall in the rate of turnover of plasma FFA (especially for oleic and iinoleic acids) and a vastly increased total incorporation of fatty acids into individual tissues only after rumen development was complete (20 weeks); most of the FFA was apparently oxidized in younger lambs. In particular, the rate of uptake of FFA and triglyceride by adipose tissue was still low at 10 weeks. D. Preonancy

As noted in a recent review, 36 in several ruminant and non-ruminant species, a biphasic change in whole-body lipid metabolism has been observed during pregnancy: early pregnancy is characterized by lipid deposition while lipid mobilization predominates in late pregnancy. In early pregnancy, when fetal energy demands are negligible, it

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seems likely that the rate of lipogenesis in adipose tissue is largely a function of feed intake and the associated rate of insulin secretion. The stimulus for increased feed intake and thus increased fat deposition is not clear, although enhanced progesterone secretion has been implicated, possibly through its suppression of estrogen secretion.19 There is limited evidence that estrogens have a lipolytic effect in ruminants, perhaps via stimulation of growth hormone secretion. 273 Any changes in the balance between lipogenesis and lipolysis in adipose tissue during late pregnancy may also be effected by changes in voluntary feed intake and energy balance. In well-fed pregnant ewes and cows, this balance, as indicated by plasma FFA concentration, was little different from that in non-pregnant animals fed at or above maintenance. 12s'163,22°'3al However, the likelihood of the animal falling into negative energy balance, with a consequent increase in fatty acid mobilization, is considerably greater during pregnancy. This is particularly so for the twin-pregnant ewe, which not only must support greater fetal demands for energy but is more likely to reduce its voluntary feed intake during late pregnancy, as° Thus, in the study of Reid and Hinks, T M plasma FFA levels were considerably higher in twin-bearing than in single-bearing ewes when both groups were fed ad libitum, and when results for all animals were pooled, there was a signficant correlation between mean levels of plasma FFA over the last month of pregnancy and total fetal weight at term. Leat and Ford 2'*s showed that FFA levels are a reasonable index of rates of entry and oxidation of FFA in pregnant as in non-pregnant sheep. This work also raised the possibility that FFA metabolism was enhanced by pregnancy per se, but interpretation of the evidence is difficult because different ~4C-labeled fatty acids were used as tracers in pregnant and non-pregnant animals and the nutritional status of the sheep was poorly defined. The pregnant uterus and its contents have considerable demands for both acetate and glucose, the major sources of acetyl units and reducing equivalents respectively for lipogenesis. It is now well established that the fetus and uteroplacental unit account for most if not all of the extra glucose synthesized by the pregnant ruminant, t°4'4°a but the effect of pregnancy on acetate production and utilization is less clear. Rates of entry and oxidation of blood acetate in fed late-pregnant ewes were comparable with those in similarly nourished non-pregnant animals. 26t Fetal uptake did not constitute a significant drain on maternal supplies of acetate in ewes 92'asg'a9a or cows, 1°4 but utilization by the metabolically active uterine and placental tissues was appreciable. ~°4 The supply of acetate to reproductive tissues is presumably maintained at the expense of other maternal tissues when feed intake.and ruminal acetate production is reduced. It is possible that not only fatty acid synthesis de novo but also uptake and re-esterification of preformed plasma fatty acids by adipose tissue is reduced during late pregnancy. The fraction of FFA entering the bloodstream which was oxidized to CO2 was higher in pregnant ewes than in non-pregnant sheep. 24s'491 A further fraction apparently entered the fl-oxidation pathway and was incompletely oxidized to ketone bodies, even in well-fed pregnant animals, as indicatedby rates of hepatic production, 22° entry into the bloodstream 249 and blood levels of ketones, 16a'22°'23s'2'*9 all of which were elevated above normal values for non-pregnant sheep. It must, however, be emphasized that the level of ketonemia in these animals was mild compared with that seen in nutritionallystressed pregnant ewes (see Section III.G). During the few days before parturition, a marked increase in plasma FFA concentration was observed in ewes ~6a'a24'aa~ and cows, a°°'a°9'a~° and this effect usually extended into the first few weeks of lactation, a°°'aa~ Accompanying changes in the fatty acid composition of plasma FFA suggested that mobilization of subcutaneous depot fat was primarily responsible for the increase a°9,a~°'a2'~ and subsequent experiments have confirmed that in subcutaneous adipose tissue from parturient and postparturient cows, there is both increased lipolysis and greatly reduced re-esterification of fatty acids in vitro, aoo

The stimulus for these metabolic changes in the intact animal hasnot been established, but it appears to involve the complex interaction of a number of factors. Even in animals

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which maintained an adequate level of voluntary food intake throughout late pregnancy, there was an almost invariable decrease in appetite during the few days before parturition, 3a° which was presumably responsible for depressed rates of insulin secretion. The stress of parturition itself must augment the effects of a negative energy balance on fat mobilization, presumably via increased sympathetic nervous activity and release of noradrenaline at nerve endings in adipose tissue. Although it has been suggested that lipolysis in ruminant adipose tissue is relatively insensitive to catecholamines, 51 Metz and van den Bergh 3°° found 10-fold increases in rates of lipolysis when adipose tissue from pregnant and lactating cows was incubated with noradrenaline. In addition, the stimulatory effect of noradrenaline was increased after parturition. Increased secretion of a number of hormones directly involved in the process of parturition, such as estrogens and prostaglandin F2~ (itself a product of the placental metabolism of polyunsaturated long-chain fatty acids, 257j may also affect lipid metabolism before and during parturition. E. Lactation

As mentioned in the previous section, the elevated levels of plasma FFA observed in parturient ewes and cows extended into the first few weeks of lactation, 3°°'381 despite a marked increase in voluntary food intake 9°'171'38° and the consequent increase in the supply of ruminal acetate and other short-chain fatty acids. •°2 During early lactation when the increased energy demands of the mammary gland and non-mammary tissues such as the liver exceed the increase in dietary intake, the deficit is apparently filled by increased release of long-chain fatty acids from adipose tissue. Thus, plasma FFA levels were considerably higher during the first 3 months of lactation in high-yielding than in low-yielding cows fed identical rations. 182 The relationship between adipose tissue lipolysis and energy balance in the ruminant during early to mid-lactation is now well established. 36'4'79 Of greater interest is the physiological basis for the considerable genetic variation in the way in which dietary energy is partitioned between milk production and synthesis of body tissues, both between individual animals of the same breed and between breeds. In a recent study of genetic differences in the endocrine control of energy metabolism in the lactating cow, Hart and his colleagues ~82 showed that the higher plasma FFA concentrations observed in high-yielding dairy cows compared with low-yielding beef cows were associated with higher levels of growth hormone and lower levels of insulin in plasma (Fig. 4). These persistent and significant differences were not apparent when the cows were not lactating. The role of growth hormone as a lipolytic agent in ruminant animals seems to be less well established than the antilipolytic role of insulin. A positive correlation between plasma levels of FFA and growth hormone has been demonstrated in experiments on lactatinglS2.as5 and non-lactating ruminants 45'191 and growth hormone levels are com~ monly elevated during early and peak lactation when the milking animal's energy deficit is likely to be greatestfl 3a'4a° Also, administration of exogenous growth hormone increased plasma FFA levels in cattle 497 and sheep. 47 However, increased lipolysis is not always associated with increased plasma growth hormone in ruminants, 285'46° and the rapid lipolytic activity of the bovine hormone in vitro has been attributed to non-somatotrophic peptide impurities. 248 The roles of insulin in the regulation of lipid metabolism in the lactating and non-lactating ruminant have been recently reviewed. 36'5° Plasma insulin levels were low in high-yielding cows 182 and further depressed in underfed lactating animals. 198.39-7 This is consistent with observations of decreased lipoprotein lipase activity,412 decreased reesterification 3°° and increased lipolysis3°°'5°5 in vitro, in adipocytes from cows in early lactation, and of decreased plasma FFA levels after intravenous injection of insulin into milking animals. 2~9 On the other hand, it does not explain the apparent increase in lipogenic capacity of adipose tissue after the onset of lactation. 32 Although insulin and, to a lesser extent, growth hormone may be prime factors in mediating the mobilization of body fat during early to mid-lactation, the question of how

Lipid metabolismin liver and selected tissues in ruminants

iL Lt

~E W

~

145

2

~7

I

II $ ~11

,50 ao

Average

80

t20

180

Dry

stage of lactation (days)

FIG.4. The averageconcentrationsof insulin, growth hormone and FFA in plasma of high-yielding (open bars) and low-yielding(solid bars) cows at various stages of lactation and during the dry period. (Valuesfrom ref. 182). the secretion of these hormones is controlled in the lactating animal remains unresolved. In non-lactating sheep, there was a clear relation between the amount of dietary organic matter digested in the gastrointestinal tract and plasma levels of insulin and growth hormone, 48 and all of the non-neural factors so far implicated in the stimulation of insulin secretion are related to feed intake and digestion. 46'8s However, the marked differences in plasma insulin and growth hormone between high- and low-yielding cows, shown in Fig. 4, were maintained despite virtually identical intakes of digestible organic matter. 182 It would appear that the secretion of these hormones is somehow sensitive to overall energy balance as well as to level of feed intake. The moderate mobilization of adipose tissue fatty acids considerably augments the amount of energy readily available to the lactating ruminant. However, as discussed elsewhere, 3°4 the net uptake of plasma FFA by the lactating mammary gland was small unless circulating levels were substantially greater than those normally seen in the fed animal. 14 Instead, much of the increased supply of long-chain fatty acids appears to be converted to other blood metabolites for which the lactating gland has an avid demand viz. acetate, 3-hydroxybutyrate and triglyceride. As discussed earlier in this review, the hepatic production of "endogenous" acetate was considerably greater in lactating ewes t 11 than in non-lactating sheep, 74'456 although this difference was not as great in dairy cows. .26 Non-hepatic tissues have also been observed to produce acetate in the lactating animal, including the mammary gland, l°J2,t4 skeletal muscle 35s and head tissues, 9 but in skeletal muscle at least, acetate production was significantly lower in lactating than in non-lactating sheep. 3s8 Hepatic ketogenesis from long-chain fatty acids was also discussed earlier in Section II.A.3. The effect of lactation on FFA uptake by the liver has yet to be reported but a moderate increase might be expected to result from the previously discussed increase in plasma FFA concentration. Certainly, the net hepatic release of 3-hydroxybutyrate was greater in well-fed lactating cows than in non-lactating animals, 28 a finding consistent with previous reports of modest elevations in blood ketone levels in fed lactating ruminants. a63,asl Blood 3-hydroxybutyrate concentration was significantly greater in highyielding cows than low-yielding aniinals during lactation but not during the dry period, t 82 It is not known whether the increased supply of plasma FFA during early lactation contributes to an enhanced rate of triglyceride synthesis in the liver. Although the rate of

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hepatic release of triglyceride (presumably incorporated in VLDL) has recently been measured in vivo in lactating cows, 374 similar data for non-lactating animals are not available. However, if VLDL synthesis and secretion is stimulated by increased hepatic uptake of FFA in the lactating animal, any increase must fall short of the demands of the mammary gland since plasma concentrations of VLDL were shown to be significantly lower in lactating than in dry COWS, TM .372,4-71 presumably because of the rapid clearance of VLDL triglyceride by the u d d e r . 13'42'160"242'492 Limited studies of the kinetics of lipoprotein metabolism in the whole animal have shown that the small pool of plasma VLDL is turned over very quickly in the lactating cow 159,346 but once again, no comparison with the non-lactating animal was made. Also, it was not possible to distinguish between exogenous (dietary) and endogenous (hepatic) contributions to the supply of plasma VLDL. Factors affecting hepatic lipoprotein formation and release in lactating and non-lactating ruminants are discussed in Section II.A.4. The way in which the balance between hepatic synthesis of acetate, ketone bodies and triglyceride is controlled in the lactating ruminant is poorly understood, although it seems likely that the rate of entry of fatty acids into the oxidation pathway is partly regulated by the liver's ability to esterify FFA taken up from plasma, and to synthesize lipoproteins for export, as in non-ruminant animals. 185 Why acetate is apparently synthesized as a significant alternative to ketones as a product of incomplete oxidation of long-chain fatty acids is not yet clear, although the advantages of increased hepatic acetogenesis to the heavily-lactating ruminant seem obvious, in view of the much greater importance of acetate relative to 3-hydroxybutyrate as a source of C2 units for mammary synthesis of short- to medium-chain fatty acids. 344 After mid-lactation, there is an increasing diversion of dietary nutrients away from the mammary gland to non-mammary tissues. Most notable are the increases in body weight, and presumably in synthesis of adipose tissue triglyceride which occur at the expense of milk fat production and which are accompanied by a decreasing net efflux of fatty acids from adipose tissue, as indicated by plasma FFA concentration.182 The kinetics of whole-body metabolism of plasma FFA have been studied in cows near the end of lactation, using 3H-labeled palmitic acid as a tracer 2°6 but unfortunately, data on early-lactating and dry animals are not available for comparison. Changes in the partition of dietary energy during the course of lactation must be mediated hormonally, but progressive changes throughout lactation in plasma concentrations of several hormones, including insulin and growth hormone, were not as great as differences between highand low-yielding cows at any stage. 182 This, of course, does not exclude the possibilities that the relative sensitivity of the major target organs, mammary gland and adiposc tissue, to these hormones is somehow modified, or that the concentrations of individual hormones are less important than their levels relative to each other. Bovine ketosis and the low milk-fat syndrome are two disorders of lipid metabolism which are peculiar to the lactating ruminant. These are discussed in Section III.G.

F. Environmental Temperature 1. Cold Exposure

During cold exposure, ruminants, like other eutherian mammals, must increase their rate of metabolic heat production in order to maintain normal body temperature. Until some years ago, it was widely believed that, in the rat and other non-ruminant laboratory animals, lipids were the preferred if not the exclusive fuel for the metabolic response to cold, even in fed animals. 339'34° Similarly, the calorimetric studies of Blaxter and his colleagues on cold-exposed sheep ~64 a n d c a t t l e 77 suggested that, over a range of cold environmental temperatures, the increase in metabolic heat production was entirely supported by catabolism of body fat. With the advent of new techniques for studying the oxidative metabolism of individual substrates in the whole animal, it became apparent

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that non-lipid substrates, and glucose in particular, can make a significant contribution to cold thermogenesis in ruminant 61'2 s6 as in non-ruminant animals.119,a01 Nevertheless, there is little doubt that lipids, mainly in the form of plasma FFA, are extremely important as a readily available and easily oxidizable source of energy during both acute and chronic cold exposure. Plasma concentrations of FFA quickly increased after the onset of cold stress in sheepS2.176.4.17.456 and cattle 57'6°'333'.54 and levels remained elevated during more prolonged exposure to cold, IS'57'121'333'5°7 The accompanying decrease in respiratory quotient 57'6°'456 suggested increased catabolism of body fat, presumably mobilized as FFA, and it has since been demonstrated directly that the cold-induced increase in plasma FFA levels in young cattle is indeed associated with an increased rate of entry into the blood-stream, as well as increased uptake and oxidation of these metabolites by peripheral tissues. 62 The latter observations are consistent with previous reports of increased turnover and oxidation of plasma FFA in several non-ruminant species during cold exposure. 292'a°t'352 Other plasma lipids are probably unimportant as sources of energy for the cold-exposed ruminant, although evidence on this point is far from conclusive. Thompson and Clough 454 found no changes in the plasma concentrations of cholesteryl esters, phospholipids or triglycerides during cold exposure of adult steers and new-born calves. On the other hand, significant decreases have been observed in plasma triglyceride concentrations in the young steer, that were most notable in animals fasted for 20 hr, 56 and in serum cholesterol concentrations in sheep 2°s and cattle. 269 Cold exposure caused a marked decrease in the plasma triglyceride level in the rat, which was confined to that carried in VLDL. a64 This was explained by subsequent observations of marked increases in the activity of lipoprotein lipase in several tissues, including brown fat, heart, lung, skeletal muscle and post-heparin plasma. 5.'365'3s6 Lipoprotein lipase activity in tissues from cold-exposed ruminants has not been measured, but only small and variable amounts of triglyceride fatty acids were extracted from plasma by peripheral tissues (principally skeletal muscle) of the young steer during cold expsoure. 56 In common with most smaller neonatal mammals, ruminants are born with brown adipose tissue and a well-developed capacity for non-shivering thermogenesis. The metabolism of brown adipose tissue and its role in the metabolic response to cold of the new-born animal is discussed in detail elsewhere. 316 Young lambs and calves lose their brown adipose tissue within weeks of birth and the available evidence suggests that in older animals, virtually all of the cold-induced increase in heat production occurs in shivering muscle. 63 For this reason, the metabolism of shivering muscle in vivo in conscious steers has been studied in some detail, by the simultaneous measurement of blood flow and arteriovenous difference in concentrations of various energy substrates in the hind limb, which is predominantly skeletal muscle but also contains adipose tissue and other non-muscular tissues. The greatly increased rate of oxidative metabolism in shivering muscle was supported by substantial increases in the uptake of blood acetate 58 and p l a s m a FFA 6°'62 as well as of non-lipid substrates. 61 Some of these results are shown in Fig. 5. Values for uptake of acetate and FFA are expressed in terms of the contributions each would make, if completely oxidized, to aerobic metabolism in leg tissues under thermoneutral and cold conditions. As might be expected, the potential importance of acetate declined with time after feeding and the reverse was true for FFA. As also shown in Fig. 5, the oxidation of FFA taken up by the hind limb was far from complete. 62 The metabolic fate of the fraction not immediately converted to CO2 is not yet known, but one possibility is that the production of endogenous acetate by skeletal muscle35s is increased by shivering. If so, values for net uptake of blood acetate would underestimate true uptake and the importance of this metabolite as a fuel for shivering muscle. Metabolism of both short- and long-chain fatty acids in tissues other than muscle is also considerably affected by cold exposure. Surprisingly, it was found that an acute cold stress (several hours) caused an increase in the rates of visceral release and hepatic uptake of acetate, propionate and butyrate in fed, but not fasted, sheep. 456 This observation was repeated in a subsequent experiment which also showed that the increased rate of

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Thermoneutral

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Thermoneutral

, tasted

3

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4

Cold,

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, ted

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2 1

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x o

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FIG. 5. Percent of oxygen uptake by the hind limb that can be accounted for by uptake of acetate and FFA, assuming complete oxidation, in fed and fasted steers exposed to thermoneutral and cold environments. Hatched areas represent percent of oxygen uptake accounted for by direct measurements of FFA oxidation to CO 2. (Similar data are not available for acetate.) Values are means calculated from data in refs 58 and 62. appearance of short-chain fatty acids in portal blood was not due to any effect of environmental temperature on rate of eating or level of feeding. T M The authors speculated on a number of possible explanations for this phenomenon, including the shortterm effect of cold on rectal, and presumably rumen, temperature 48v and gut motility, 495 which could temporarily increase rates of ruminal production and absorption of shortchain acids. Alternatively, the increased supply of long-chain fatty acids might lead to increased metabolism of these in substitution for short-chain acids in rumen epithelium. Cold exposure caused an increase in the hepatic uptake of plasma free palmitate and oleate proportional to the increase in the circulating levels of these acids, but a proportionately much smaller increase in the uptake of stearate. 456 The metabolic fate of the long-chain fatty acids within the liver was not examined but most were presumably oxidized. There is no direct evidence that lipolysis of adipose tissue triglycerides is the source of the increased supply of plasma FFA in cold-exposed ruminants, but there are several reasons for believing that this is so. Plasma concentration of glycerol is commonly used as an index of adipose tissue lipolysis in vivo and in sheep ]8'456 and cattle 5v'6° exposed to cold, the increase in plasma FFA concentration was invariably associated with a similar increase in the plasma glycerol level. At the same time, the increase in plasma oleic acid concentration was greater than that in the levels of other individual FFA, 4s6 a finding consistent with the relatively high oleate content of peripheral fat depots in ruminants. 96'1z6 Also, there was a significant increase in blood flow to adipose tissue in cold-exposed sheep ~v5 and cattle, 59 which could assist FFA release (although not lipolysis) by increasing the rate of presentation of albumin binding sites for FFA. 4°2 There is good evidence that, during acute cold stress, the mobilization of long-chain fatty acids from adipose tissue is controlled primarily by the sympathetic nervous system in ruminants as in other species. In severely stressed new-born lambs 4 and more moderately stressed new-born calves and adult steers, 454 the normal increase in plasma FFA concentration was abolished by application of a number of different adrenergic blocking agents, and the intravenous infusion of noradrenaline into animals exposed to a thermoneutral environment mimicked the lipolytic effect of cold e x p o s u r e . 4'1°°'325'452 This effect of increased sympathetic nervous activity was apparently mediated by release of

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noradrenaline at sympathetic nerve ends and not by increased secretion of adrenal medullary catecholamines, because the cold-induced increase in plasma FFA concentration was not affected by denervation of the adrenal medullae4s4 or by complete adrenalectomy (G. Alexander and A. W. Bell, unpublished observations). Several series of experiments by Thompson and his colleagues a9'I°1'115.453 have shown that the sympathetic stimulation of fat mobilization is probably initiated in the hypothalamus, and that the various stimulatory and inhibitory effects of several putative hypothalamic neurotransmitters follow a pattern similar to their effects on a number of other important physiological responses to cold. The regulation of FFA metabolism during more prolonged cold exposure of ruminants has not been specifically studied. There is evidence that sympathetic nervous activity remains elevated during chronic cold exposure of sheep 97 but the primary factor is likely to be the overall energy balance of the animal, as mediated by the net effect of changes in secretion of a number of hormones which influence the balance between lipogenesis and lipid mobilization and catabolism.

2. Heat Exposure Exposure of ruminants to hot environments has a number of direct and indirect effects on the metabolism of short-chain fatty acids of digestive origin and of other plasma lipids. A number of workers have observed a reduced concentration of shortchain fatty acids in bovine rumen fluid, 156'3°3'332 and this was attributed to the marked decrease in voluntary feed intake that invariably accompanies heat exposure in cattle, l°3'291'a°a'332'4a° However, when intake was kept constant by placing the feed refused by heat-stressed cattle in the rumen via a rumen fistula, the difference between heat-treated and control animals was still apparent. 224 The possibilities that rumen fatty acid levels were depressed by the diluting effect of increased water intake 224 or by increased rumen temperature 156 have been discounted. There is limited evidence that the rate of production and absorption into the bloodstream of rumen short-chain fatty acids may be enhanced by heat exposure. 291 O'Kellya2a observed marked seasonal changes in the composition of plasma lipids of grazing cattle in a tropical environment. In particular, plasma total and free cholesterol, phospholipid, and to a lesser extent, trigylceride levels were lower during the summer months, and subsequent work has shown that this was largely an effect of heat exposure, and not of the poorer nutrition usually experienced at this time of year. a22,329,3a° Similar changes were also found in steers after, but not during, short-term (6 hr) exposure to heat; these changes were maintained for several days afterwards in a relatively cool environment, a19 As well as decreasing the plasma levels of cholesteryl esters and phospholipids, heat exposure significantly reduced the percentage of linoleic acid in the cholesteryl ester fraction and in lecithin, and increased the ratio of free to total cholesterol. 322 The authors attributedthese observations to a severe reduction in the activity of the enzyme lecithin-cholesterol acyltransferase (LCAT). Such a mechanism may also explain the failure of heat-stressed lambs to show the normal increase in concentrations of linoleic acid in the cholesteryl ester and phospholipid fractions of plasma during the first week after birth, a2° since there is normally a rapid postnatal increase in plasma LCAT activity in the new-born lamb. 317 The essential fatty acid status of the new-born ruminant is poor, at least by non-ruminant standards (for a detailed consideration of this point see Noblea16); under practical husbandry conditions, heat stress may aggravate this situation. In cattle, as in other species, sebaceous gland secretions pro.vide an intact lipid film on the skin surface. Sebum output in young steers was increased under natural conditions during the summer months 4~a and also by prolonged exposure to an artificially hot environment.419 In the latter study, sebum fatty acid composition '~/as also altered, particularly by a greater than average increase in the secretion of linoleic acid, mainly in the triglyceride fraction. It was suggested that this may be involved both in increased

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skin moisture loss and in protecting the animal against bacterial action in warm environments, G. Metabolic Disorders The domestic ruminant is peculiarly susceptible to several metabolic disorders which intimately involve lipids. Such disorders are most evident during late pregnancy and early lactation when the metabolic demands of the fetus and uteroplacental tissues or lactating mammary gland can be so great as to severely disrupt the function of maternal tissues and even cause death. They are a consequence partly of the ruminant mode of digestion and nutrient supply, and partly of intense genetic selection for characteristics such as milk production and number and size of offspring. For the latter reason, some of the disorders discussed in this section, which include bovine ketosis, ovine pregnancy toxemia and the various manifestations of bovine and ovine hypocalcemia and hypomagnesemia, are often called "production diseases". Although diabetes is not a naturally occurring disease of any consequence in ruminant animals, the use of the drug alloxan or surgical pancreatectomy to induce diabetes has provided invaluable information on ruminant metabolism. Therefore, diabetes in sheep is discussed in addition to the common naturally occurring disorders. Although not a metabolic disorder in the pathological sense, the low milk-fat syndrome in dairy cows is also considered. 1. Bovine Ketosis and Ovine Pregnancy Toxemia Although bovine ketosis and ovine pregnancy toxemia are obviously different diseases, not least because they occur in different species and under different physiological conditions, they have a common metabolic etiology and will, therefore, be considered together here. There are a number of excellent reviews which cover the general subject of ruminant ketosis, as well as specific pathological features of the different syndromes in s o m e detail. 20'23'24'67"240'379'396 Bovine ketosis typically occurs in high-yielding dairy cows during the first 2 months of lactation. Pregnancy toxemia is most commonly seen in multiparous ewes during late pregnancy. Both conditions are associated with hypoglycemia, hyperketonemia and progressive loss of appetite, with development of hypoglycemia rather than hyperketonemia being regarded as the crucial initial metabolic change. 24° Also, both syndromes can to some extent be simulated under experimental conditions by food deprivation of heavily lactating cows or twin-pregnant ewes, although the severity of the naturally occurring clinical syndrome is rarely achieved. The spontaneously ketotic cow usually recovers following the onset of hypophagia and a marked decrease in milk yield. However, the prognosis for the ketotic ewe is often poor, even after abortion or normal parturition, unless the disease is treated during its early stages. As mentioned above, the primary event common to the etiology of both bovine lactation ketosis and ovine pregnancy toxemia appears to be hypoglycemia, which almost certainly occurs because gluconeogenesis cannot keep pace with the demand of the lactating mammary gland or pregnant uterus for glucose. Decreased plasma insulin 397 may be a major factor in the mobilization of FFA in incipiently ketotic COWS 2 2 " 3 6 2 ' 3 6 3 and sheep, 381'382 which leads to increased hepatic uptake of FFA and ketogenesis, 27'22° although the role of this and other hormonal changes in the induction of ruminant ketosis is far from unequivocal. 479 As discussed in Section II.A, the primary intracellular factor responsible for diversion of acetyl CoA to production of ketones in liver has been the subject of some controversy. The author inclines to the view of Baird 2° that a decrease in the supply of hepatic oxaloacetate and its precursors is crucial. Another major feature of hepatic metabolism during lactation ketosis and pregnancy toxemia is fatty infiltration of the liver. 149'35°'391'395 This was recently shown to be a consequence not only of increased uptake of FFA but also a markedly decreased release of triglyceride by the liver in fasted lactating cows. 374 In the starved pregnant ewe, the degree of fatty infiltration was directly related to the level of ketonemia. 3s°

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The kinetic studies of Leng 2.9 suggested that the hyperketonemia of ewes with pregnancy toxemia was a result of increased ketogenesis rather than of inhibition of ketone utilization by extrahepatic tissues. However, it has recently been shown that even mild fasting ketosis in non-pregnant ewes is associated with a decreased enzymatic capacity to catabolize ketone bodies in renal cortex and cardiac muscle. 473 Both these tissues are important users of blood ketone bodies in the sheep (see Sections II.C and II.E). In mild ketosis, renal clearance of ketone bodies accounted for less than 10~ of the net renal uptake of ketones in sheep 22~'222 but when blood levels exceeded a threshold of about 2 raM, there was a dramatic increase in the urinary excretion of ketones. 37s The advanced stages of ketoacidosis in pregnant ewes were associated with quite pronounced disturbances of renal function, including decreased renal plasma flow and glomerular filtration rate ~7,223,3,$9 which, by decreasing urinary excretion of ketones, must have further exacerbated the hyperketonemia and acidosis. Much of the above evidence regarding the metabolic etiology of spontaneous lactation ketosis and pregnancy toxemia has been gained from experiments in which food was withdrawn from heavily-lactating cows or late-pregnant multiparous ewes. While this approach certainly mimics many of the metabolic features of the naturally-occurring diseases, it should be borne in mind that it is usually difficult to induce the pathological symptoms experimentally. Reid 379 has suggested that the main difference between the experimental condition and the several natural syndromes of pregnancy toxemia is the development of severe ketoacidosis and central nervous depression in the latter. Thus, fasted pregnant or lactating ruminants may retain control of metabolic homeostasis whereas this is lost during development of spontaneous ketosis. The crucial factors involved in this breakdown of homeostasis remain to be elucidated. 2. Diabetes

Surgical pancreatectomy21a and the diabetogenic drug alloxan 2° s,210,2 t 2,277,3s3 have been used to study the metabolic effects of insulin deficiency in sheep and both these treatments produced a severe diabetes with hyperglycemia, elevated plasma FFA levels and ketoacidosis. However, the use of alloxan has been preferred because pancreatectomy was also associated with serious impairment of protein digestion and metabolism, probably caused by the restriction of pancreatic exocrine secretions. 2°9 The diabetic response to alloxan in the sheep was severe relative to that in non-ruminant animals as judged by survival, rate. of weight loss, degree of inappetance and severity of ketoacidosis. 383 It is interesting that the rapid onset of the symptoms of diabetes, which in untreated sheep resulted in death, occurred only after the concentration of blood ketones exceeded about 3 raM,3s3 a level considered to be critical in the appearance of the clinical signs of pregnancy toxemia in e w e s . 377 It is also notable that at about this concentration of total ketones (3-hydroxybutyrate plus acetoacetate), a plateau was reached in the relationship between concentration and entry rate for 3-hydroxybutyrate249 and acetoacetate, 71 i.e. further increases in concentration were associated with an unchanged rate of entry into the bloodstream. In addition to the hyperglycemia and hyperketonemia normally seen in the diabetic non-ruminant animal, alloxan diabetes in the sheep was characterized by unusually high levels of blood acetate as long as feed intake was maintained. 2~°'2~2'a77 Thus, Reid et a/. as3 observed a good correlation between blood VFA (mostly acetate) concentration and feed intake during the previous 24 hr in diabetic ewes; VFA levels were 3-5 mM in animals eating more than half their daily ration, but even after complete loss of appetite, they remained considerably higher than levels normally seen in the non-diabetic fasted sheep. This suggested that while most of the excess acetate must have been of alimentary origin when the sheep were still eating, the contribution of endogenous acetate was far from negligible. It was also surmised that the large increase in blood acetate was due to its impaired utilization in peripheral tissues rather than to increased production. This idea has since been substantiated by experiments which indicated that the rate of re-

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moval of intravenously-injected acetate from the blood of diabetic sheep was greatly decreased.2~o Also, it was shown that alloxan treatment markedly depressed the uptake of blood acetate by hind-limb tissues in vivo. 2x°'22s This was reversed by insulin treatment, which also significantly increased the uptake of blood 3-hydroxybutyrate.21° Reduced whole-body utilization of acetate 99.~s~'22s and ketone bodies 29 has also been observed in diabetic non-ruminant animals. Recent evidence suggested that a substrate (acetoacetate)-inhibitory effect on 3-0xoacid CoA-transferase was at least partly responsible for impaired ketone utilization in diabetic rat muscle, ~43 but the mechanism by which the uptake of acetate by skeletal muscle and possibly other tissues is reduced in the diabetic animal has not been identified. The restriction of glucose entry into muscle and the consequent effect of reduced availability of intracellular glucose may, however, be more important than any putative direct effect of insulin deficiency on transport of short-chain fatty acids into ceils. 21° Certainly, the observations of a several-fold increase in muscle concentration of acetylcarnitine bul relatively little change in the ratio [acetyl CoA]/[free CoA] in diabetic sheep suggested a considerable diversion from the normal mode of acetyl CoA metabolism. 428'43° Muscle metabolism of acetate in diabetic non-ruminants has not been studied in any detail, but reduction of acetoacetate uptake in the diabetic rat was tentatively attributed to an associated decrease in free CoA content of muscle and a resulting increase in the acetoacetate concentration needed to maintain a given level of acetyl CoA. 38'~ Thus, the build-up in intramuscular "acetyl pressure" in the diabetic sheep may be largely buffered by the carnitine/acetylcarnitine system according to the mechanism proposed by Snoswell and Koundakjian,428 while the CoA system may be more important in the rat and possibly other non-ruminants. The relative importance of muscle carnitine in the sheep is supported by observations that in normal animals its concentration is about 20 times that in rat muscle. 428 Also, as shown earlier in Table 4, these high levels were unchanged during alloxan diabetes, 428"43° whereas the initially much smaller amount of total carnitine in rat muscle decreased significantly during diabetes. 297 Carnitine may also play an important role in other tissues of the alloxan-diabetic sheep. In particular, there were very large increases in the hepatic content of total acid-soluble carnitine and its individual components, free carnitine and acetylcarnitine. 42a'43° Once again, the limited capacity of the CoA buffering system to accommodate increased "acetyl pressure" in sheep tissues appears to have been compensated by the relative availability of carnitine. The precise stimulus for the marked accumulation of carnitine in the liver of the diabetic sheep is not known. However, the phenomenon has been associated with the spontaneous reduction in feed intake which occurs during the terminal stages of alloxan diabetes. 43° Data from this study also indicated that the extra carnitine is synthesized within the liver itself, and not taken up from the bloodstream after mobilization from the very substantial extrahepatic stores, particularly in skeletal muscle. 3. Hypocalcemia and Hypomagnesemia

Clinical hypocalcemia of ruminants occurs most commonly as parturient paresis (or milk fever) in newly-calved dairy cows; in sheep, the disease is most prevalent during late pregnancy a3.216.4.59 Hypomagnesemia, often complicated by hypocalcemia, is manifested as grass tetany (or grass staggers) in grazing cattle and sheep, most frequently in lactating cows. 2.7'459 In contrast to the central importance of fatty acid catabolism in the development of the ketotic syndromes discussed earlier in this section, lipid metabolism has not been considered a significant factor in the etiology of metabolic disorders associated with calcium and/or magnesium deficiency in ruminants. However, in recent years, limited evidence has been obtained for a possible relationship between adipose tissue lipolysis and the appearance of clinical hypocalcemia and hypomagnesemia. Spontaneous hypocalcemia in newly parturient COWS 1 9 5 ' 1 9 7 ' 2 7 1 ' 2 7 5 and pregnant ewes 271 was accompanied by fatty acid mobilization, as indicated by elevated plasma

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FFA levels and the development of fatty liver. As discussed in Section III.D, increased FFA levels are not uncommon in normocalcemic ruminants during the period encompassing late pregnancy and early lactation, particularly during and soon after parturition. However, levels were higher in paretic than in non-paretic cows immediately after calving, 197"275 probably because of the diabetic-like hormonal state which has been shown to accompany parturient paresis, s°'264 and remained so even 2 weeks afterwards. 197 Even more notable was the significant negative correlation between plasma concentrations of calcium and FFA, which was observed in each of three separate studies on spontaneously hypocalcemic cows. 197,271,275 Also, calcium therapy of hypocalcemic cows and ewes was followed not only by improved calcium status but also a decrease in FFA levels, whereas experimentally induced hypocalcemia had no significant effect on plasma FFA concentrations.271 At about the same time, highly significant correlations between increased plasma FFA levels and hypocalcemia were demonstrated in sheep intravenously injected with catecholamiries or subjected to mild surgical stress. 27°'3°8 This effect was apparently independent of a catecholamine-induced secretion of calcitonin, because it also occurred in thyroidectomized animals. 27. Also, nicotinic acid, which is not known to directly influence calcium metabolism, reduced both the increase in FFA concentration and the hypocalcemia which followed injection of noradrenaline. 27° More recently, it has been shown that plasma magnesium concentration is similarly depressed during catecholamine-stimulated fatty acid mobilization. 357'373 This effect was also abolished by nicotinic acid. 373 There is evidence that the association between increased FFA and decreased calcium and magnesium concentrations in plasma of animals treated with lipolytic agents may be due to increased sequestration of these minerals by adipose tissue during lipolysis. In particular, there was a marked increase in the calcium content of subcutaneous adipose tissue in vivo in sheep following adrenaline treatment, and this was highly correlated with the fall in plasma calcium concentration. 3°8 Similar observations were made during corticotrophin-induced lipolysis in rabbits 2 and in both species, the uptake of calcium by adipose tissue was calculated to be great enough to explain the hypocalcemia. Also, the addition of various lipolytic agents to adipose tissue in vitro increased its accumulation of calcium in sheep 3°s and of calcium 6'**°'.9° and magnesium 13s in the rat. Understandably, these studies led to the suggestion that redistribution of plasma calcium into adipose tissue might contribute to post-parturient hypocalcemia in dairy cows. However, the results of two independent experiments designed to investigate this possibility have proved negative; if anything, the calcium content of adipose tissue from cows showing frank parturient paresis was lower than that in newly-calved but non-paretic animals. 197'275 In explanation, it was suggested that the response to experimentally stimulated lipolysis may be a transitory phenomenon and therefore, by implication, unimportant in the etiology of clinical hypocalcemia, tg~ This idea could be checked by prolonged infusion of lipolytic drugs and seems worthy of future investigation. At the same time, the potential importance of any factors, however transient, which might assist in triggering the onset of spontaneous hypocalcemia or hypomagnesemia in ruminants should not be underestimated. 4. Low Milk-fat Syndrome

The low milk-fat syndrome most commonly occurs in dairy cows fed diets with a high ratio of readily-digestible carbohydrate to roughage, or normal rations supplemented with oils rich in polyunsaturated fatty acids. It is characterized by markedly depressed yields of milk fat, as its name implies, as well as changes in the fatty acid composition of milk triglycerides. The condition cann6t be regarded as a metabolic disease in the normal pathological sense. It is, however, a source of considerable economic loss in areas where the feeding of high-concentrate rations to dairy cows is widely practised. From a purely scientific point of view, the etiology of the syndrome is especially relevant to this review,

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TABLe: 6. Metabolic Changes Associated with the Low Milk-fat Syndrome in Dairy Cows Fed High-concentrate, Low-roughage Diets Site Rumen

Blood

Adipose tissue Mammary gland

Parameter Molar ratio acetate:propionate Acetate production Propionate production Vitamin BI2 synthesis Acetate concentration and entry rate Glucose concentration and entry rate Insulin concentration FFA concentration and entry rate Triglyceride concentration Triglyceride content of unsaturated fatty acids Lipoprotein lipase activity Fatty acid uptake Lipogenic enzyme activity Lipoprotein lipase activity Fatty acid uptake Acetate uptake

Change

References

j, No change T ~ +t i" *, l ,~ ~"

30, 284. 445,446, 447 116 52 4~3 10, 281,445.447 10, t39 139, 214, 484 I0, 343 1~1,278, 27q 10, 278, 279

l "~ T No change ~ ~

~4 343 ~1,335 16 10, 343 10. 200, 447, 470

firstly because it appears to involve several interrelated aspects of lipid metabolism in a number of organs and tissues, and secondly because all of the various metabolic changes which characterize it can be traced back to events in the rumen. The major metabolic characteristics of the condition are summarized in Table 6. While it is agreed that diet-induced changes in rumen function are ultimately responsible for the appearance of the low milk-fat syndrome, several quite different hypotheses have been advanced to explain how this occurs. The main features of these, with evidence for and against, are outlined here; for detailed discussion of all but the most recent literature, the excellent reviews of Van Soest 468 and Davis and Brown ~t7 are recommended. Less detailed but more recent reviews are also available. 95'35x'479 In 1955 Balch and his colleagues 3° first showed that the low milk-fat condition in cows fed high-grain low-fiber diets was accompanied by a marked decrease in the molar ratio of acetic to propionic acids in rumen fluid, and numerous studies since then have confirmed this finding (e.g. McCullough, 284 Storry and R o o k , 4'*5"446 and Storry and Sutton44V). This led to the theory, documented by Van Soest, 46a that the reduced secretion of milk fat was primarily due to a decreased supply of blood acetate for m a m m a r y fatty acid synthesis. Observations of an accompanying decrease in the concentration 281'445'44; and m a m m a r y uptake 2°°'44~'47° of blood acetate, in addition to the elucidation of a major role for acetate as a lipogenic precursor in the ruminant m a m m a r y gland (see M o o r e and Christie3°4), gave added support to this hypothesis. However, subsequent work has cast some doubt that reduced availability of ruminal acetate is a key factor in the low milk-fat syndrome. Although the evidence for a decrease in the molar proportion of acetate in the rumen is unequivocal, Davis and his colleagues showed that this was due to a marked increase in the ruminal production of propionate, 52 rather than a decrease in acetate production.116 Also, although there have been numerous studies purporting to show improved milk-fat yields following supplementation of high-grain, low-fiber diets with acetate, these have yielded variable results. '~7 Nevertheless, in seeming contradiction of the data on ruminal acetate production, ~16 Annison e t al. ~° clearly demonstrated not only a decreased arterial concentration but also a substantially lower rate of entry of blood acetate in cows fed a low-roughage diet. This was associated with a concomitant decrease in m a m m a r y uptake of blood acetate. The authors attributed the reduced supply of blood acetate to a decrease in its endogenous production in body tissues (see Section II.A and II.B), which was supported by their observation of decreased release of acetate from the udder. As indicated by Davis and Brown, ~17 reduced m a m m a r y uptake of acetate in

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cows secreting low-fat milk could be an effect rather than a cause of depressed mammary lipogenesis. However, the good relationship between arterial level and mammary uptake of acetate in the study of Annison e t al. 1° gives renewed support for the latter idea. Alternative theories on the etiology of the low milk-fat syndrome have dwelt on the possible effects of a high rate of production of ruminal propionate, which undoubtedly occurs when cows are fed high-grain low-roughage diets. 52 Van Soest and Allen 469 observed decreased arterial levels and mammary extraction of ketone bodies in cows on high-concentrate rations and attributed this to the antiketogenic property of propionate. However, this hypothesis was effectively discredited by Palmquist e t al., a44 who measured the effect of a high-grain diet on both the rate of entry of 3-hydroxybutyrate into the bloodstream and its incorporation into milk fatty acids. Despite a substantial depression of milk-fat yield, the supply of 3-hydroxybutyrate in blood was not significantly affected. In any case, the estimated contribution of this metabolite as a source of carbon units for mammary fatty acid synthesis was judged too small to have any real impact on the overall rate of secretion of milk triglyceride. The idea that the glucogenic property of propionate is ultimately responsible for the production of low-fat milk was first proposed by McClymont 28~'283 and received support when he and other workers were able to depress milk-fat yield by intravenous infusion of glucose. ~46'283'444 It was postulated that enhanced hepatic gluconeogenesis from propionate, and the consequent increase in supply of blood glucose, stimulates insulin secretion. This, in turn, supposedly suppresses the release of FFA from adipose tissue sufficiently to decrease hepatic synthesis of VLDL triglyceride, thereby lowering the availability of this important precursor for milk-fat synthesis. This theory is attractive because most of its major assumptions have received some degree of direct experimental support. Numerous workers have demonstrated increased levels of blood glucose in cows fed high-concentrate rations (e.g. Annison e t al., ~° Evans e t a l., ~39 Jorgensen e t al. 2~ 7), but more important is the fact that this was the result of a marked increase in glucose entry rate.~ 0,~39 Not surprisingly, plasma insulin concentration was also elevated under these conditions. '39'2t4'484 This may explain the findings that feeding of high-concentrate rations to cows increased the lipoprotein lipase activity of adipose tissue, 64 but had no significant effect on this enzyme in the mammary gland, 16 because similar responses were obtained by intravenous administration of glucose or insulin, a69 This and other evidence that the enzymatic capacity for lipogenesis in adipose tissue is increased in concentratefed cows at'335 was supported by data obtained in vivo on the relative rates of incorporation of labeled fatty acids in plasma into adipose and mammary triglycerides, a43 In the latter study, decreased rates of milk-fat production were clearly associated with lower plasma FFA concentrations, a finding which is consistent with the subsequent observation of a decreased rate of turnover of free palmitic acid in plasma under similar dietary conditions, t° There has been some question about the magnitude of the effect of highgrain, low-roughage diets on circulating levels and mammary uptake of plasma triglycerides. 1~7 However, the recent experiments of Annison e t al., ~° who measured mammary blood flow as well as A-V differences in plasma triglyceride concentration, showed substantial decreases in the mammary uptake of these important precursors of milk fat, due to both lower arterial levels and reduced extraction by the udder. Further evidence that the supply of plasma triglyceride can limit milk-fat production was provided by observations that intravenous administration of cottonseed oil 444 or feeding protected tallow 44~ caused a marked recovery in milk-fat production in animals with previously depressed yields. Despite the considerable evidence outlined above for the glucogenic theory of milk-fat depression, there are grounds for reservations, which have been highlighted by Davis and his colleagues. ~17.t 52 In particular, the" effects on milk-fat yield of the administration of exogenous propionate 146.152,444 o r glucose t't6.t52,a69,'g't4 to cows on normal diets, in amounts equal to or greater than those made available through normal digestion and metabolism, have generally been much smaller than those seen in the naturally-occurring

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condition. The results of McClymont and Vallance, 283 which gave initial support to the hypothesis, would appear to be an exception. However, it is notable that the levels of plasma glucose, achieved by intravenous infusion in their experiments, were far higher than those normally seen in animals fed high-grain low-roughage rations. 11~ These doubts have led Frobish and Davis ~53 to propose an alternate explanation for the low milk-fat condition. Like the glucogenic theory, this hypothesis involves the increased ruminal production of propionate, but suggests a quite different mechanism for its effect. The suggestion is that increased production of propionate is coupled with decreased production of vitamin B12 (cyanocobalamin) in the rumen, which leads to impaired intermediary metabolism of propionate in the liver and accumulation of methylmalonic acid in the bloodstream. Methylmalonyl CoA has been shown to reduce fatty acid synthesis in non-ruminant tissues in vitro. 91'15° Certainly, there is evidence that the ruminal synthesis of vitamin B~2, upon which the ruminant is entirely dependent for its supply, 464 is depressed in sheep 448 and dairy cows 483 fed high-grain diets. Also, there is little doubt that vitamin BI: dificiency leads to impaired hepatic metabolism of propionate, and accumulation of methylmalonic acid. 422'424"433 However, direct experimental evidence that such a chain of events may be involved in the etiology of the low milk-fat syndrome is at best equivocal ~53 and more work is required to support or refute this theory. Milk fat can also be depressed by supplementation of normal diets for dairy cows with oils rich in unsaturated fatty acids, or addition of the fatty acids themselves. 95"~1; The mechanism by which this occurs has not been established, but the condition has some interesting common features with that produced by high-concentrate low-roughage diets. In particular, both disorders are characterized by decreased percentages of palmitic and stearic acids and increased percentages of C18 unsaturated fatty acids, including those with t r a n s - d o u b l e bonds, in milk and plasma triglycerides. 1~7 Early investigations into the metabolic consequences of feeding oil supplements suggested that a further common factor might be a pronounced change in the pattern of short-chain fatty acid fermentation in the rumen. 41° Most subsequent work has shown only minor changes in rumen short-chain fatty acids compared with those seen when feeding high-grain low-fiber diets (e.g. Beitz and Davis, 55 Brumby et al., 88 Steele and Moore 436 and Varman et al.472). Nevertheless, events in the rumen apparently have some influence because when the rumen was bypassed by giving oil supplements per abomasum 11~ or feeding them in a protected form, 347'442 there was little or no change in milk-fat yield. Reduced biohydrogenation of unsaturated long-chain fatty acids in the rumen has been reported to occur when cows are fed highly concentrated rations 1°'278'279 and such an effect may be even more pronounced when relatively large amounts of polyunsaturated fatty acids are present in the diet. McCarthy and his colleagues 27s'279 suggested that increased unsaturation of fatty acids in plasma lipoproteins may adversely affect mammary uptake and/or metabolism of plasma triglyceride, and this idea has received experimental support from another laboratory. 88'441'443 In apparent contradiction of this hypothesis, marked increases in the content of polyunsaturated fatty acids in plasma triglycerides occurred without any depression of milk-fat yield when oil supplements were protected from ruminal influence. ~°~44z However, as argued by Storry et al., 442 any direct effect of polyunsaturated fatty acids on mammary uptake of plasma triglyceride fatty acids may have been masked by the substantial increase in plasma triglyceride concentration usually associated with the feeding of protected lipids. The etiological complexity of the low milk-fat syndrome is perhaps best illustrated by the variety of seemingly different theories concerned with its metabolic basis. In view of the amount of evidence for each of these proposed mechanisms, it is suggested that future studies should seek to emphasize the multifactorial nature of the syndrome, following the example of Annison et al. ~° It seems likely that such an approach will unearth more common factors than differences among the existing theories. For instance, the suppression of FFA mobilization, which has been held responsible for a decreased availability of plasma triglycerides, may also be at least partly responsible for a shortage of blood

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acetate, since the latter has been attributed to reduced endogenous synthesis of acetate, ~° rather than decreased ruminal production.l~6

(Received 13 July 1979) REFERENCES 1. 2. 3. 4. 5. 6. 7. 8.

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