Lipid metabolism in the mammary gland or ruminant animals

Prog. Lipid Res. VoL 17. pp. 347 to 395. © PergamonPress Ltd 1979. Printedin Great Britain

0163-7827 79,0501-0347105.00 0

LIPID METABOLISM IN THE MAMMARY GLAND OF RUMINANT ANIMALS J. H. MOORE and W. W. CHRISTIE Department of Biochemistry. The Hannah Research Institute. Ayr, Scotland KA6 5HL CONTENTS

I. EARLIER CONCEPTS OF THE ORIGIN OF MILK FAT II. THE CONTRIBUTION MADE BY BLOOD LIPIDS TO MILK FAT SYNTHESIS A. Blood lipid composition B. Identity of the blood lipid fraction taken up by the mammary gland C. Hydrolysis of triglycerides during uptake by the mammary gland and the role of iipoprotein lipase III. FATTY ACID SYNTHESISIN THE MAMMARY GLAND A. Introduction

B. Source of carbon for fatty acid synthesis C. Activation of substrates D. The non-utilization of glucose carbon for fatty acid synthesis E. Source of NADPH for fatty acid synthesis F. Acetyl CoA carboxylase G. Fatty acid synthetase H. Synthesis of short- and medium-chain fatty acids I. Desaturation J. Chain elongation IV. ESTERIFICAT1ONOF FATTY ACIDS IN MAMMARY TISSUE

A. Triglyceride biosynthesis B. Phospholipid biosynthesis V. VI. VII. VIII.

CHOLESTEROL AND CHOLESTERYL ESTER METABOLISM1N MAMMARY GLAND THE SECRET,ON OF MILK FAT RELATED REVIEW ARTICLES REFERENCES

I. E A R L I E R

CONCEPTS

OF THE ORIGIN

OF

MILK

347 354 354 355 361 365 365 366 368 369 370 373 374 375 378 379 379 379 385 386 387 389 389

FAT

According to Maynard e t al., 273 the earliest investigations into the nature of the precursors of ruminant milk fat were reported by Foa it4 in 1912 who found that, when the mammary gland of a sheep was perfused with a suspension of olive oil in Ringer's solution, the gland secreted fat with an iodine value less than that of olive oil. The gland also secreted volatile fatty acids, although these were absent from the circulating fluid. Foa tt4 also noted that in both sheep and goats the iodine value of the fat in the arterial blood was greater than that of the fat secreted in the milk and concluded that blood triglycerides were the precursors of milk fat but the mammary gland selectively secreted fat with an iodine value less than that of the blood triglycerides. From the analyses of blood from the jugular and subcutaneous mammary veins, then considered to represent respectively the blood flowing to and from the mammary gland, Meigs e t at. 274 showed that there was a decrease in the phospholipid content and an increase in the inorganic phosphorus content of the blood as it traversed the mammary gland of the lactating cow. Meigs e t al. 274 considered that the results obtained by Foa t ~4 were unreliable and proposed .that it was the blood phospholipids that were utilized for milk fat synthesis in the mammary gland and that part of the excess phosphorus was returned in inorganic form to the blood draining the gland. Consistent with this proposition was the finding by Doulkin and Helman 96 of a positive correlation between blood lecithin concentration and milk fat production in lactating cows. Maynard e t a/. 272 had previously drawn attention to the close correlation that existed between the concentrations of blood lipids and the amount of milk fat produced by cows during the lactating cycle. Maynard 27~ also pointed out that the levels of blood lipids were invariably greater in lactating than in non-lactating cows. However, the 347

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J.H. Moore and W. W. Christie

conclusions of Meigs e t a / . 274 were, in turn, criticized by Blackwood 48 who was able to show that the concentration of phospholipids in the mammary venous blood was significantly lower than that in the jugular blood of both lactating and non-lactating cows. Thus, the differences in blood composition reported by Meigs et al. 274 could not necessarily be ascribed to the utilization of blood phospholipids for milk fat synthesis in the mammary gland. Blackwood 4s emphasized that for milk secretion studies of this type it was necessary that analytical values for mammary venous blood should be compared with those for arterial blood; in effect, no differences were found in the phospholipid concentrations of blood from the radial artery and mammary vein of lactating cows. Results in complete agreement with those of Blackwood 48 were published simultaneously by Lintzell T M and somewhat later by others; 139"253'273 thus, there appeared to be no uptake of blood phospholipids by the ruminant mammary gland. Consistent with these findings were those of Aylward et al. 13 who analyzed the blood and milk lipids of lactating cows after each animal had been given an oral dose of iodinated triglycerides. Blood lipids were separated into phospholipids and nonphospholipids by precipitation with acetone and magnesium chloride, and although about 70 and 30~o of the total blood fatty acids were found in the phospholipid and nonphospholipid fractions, respectively, about 90~o of the total lipid-bound iodine in the blood was found in the nonphospholipid fraction. Considerable amounts of iodinated fatty acids were secreted into the milk and these were directly related to the amounts of iodinated fatty acids in the nonphospholipid fraction in the blood. Aylward et al. 13 concluded that the blood phospholipids could not be the precursors of milk fat and that this function could be attributed to the blood triglycerides or cholesteryl esters, or to both of these blood lipids. Although Lintzell T M working with lactating goats. and G r a h a m e t a l . 139 and Maynard et al. 273 working with lactating cows found that arterial blood contained significantly higher concentrations of total lipid than did mammary venous blood, this difference was not reflected in the concentrations of free and esterified cholesterol in the blood flowing to and from the mammary gland. The mean values summarized in Table ! are those of Maynard e t a / . , 273 who also established that there was no difference in the total lipid content of arterial and mammary venous blood in non-lactating cows. Lintzell, T M Graham e t a / . 139 and Maynard e t a / . 273 interpreted their results as indicating that the only class of blood lipids thai( could contribute to milk fat synthesis in the ruminant mammary gland was the "neutral fat" fraction (the difference between the concentration of total lipids and the sum of the concentrations of phospholipids, free cholesterol and cholesteryl esters). Although it seemed likely that the neutral fat fraction could be accounted for mainly by blood triglycerides, Maynard et al. 2~3 recognized that final proof of the uptake of blood triglycerides would have to await the development of a direct method for the determination of the concentrations of triglycerides in the arterial and venous blood samples. However, ruminant milk fat characteristically contains appreciable proportions of fatty acids with 4-12 carbon atoms; under normal circumstances, these fatty acids are virtually absent from the neutral lipid fraction in the blood. Thus, although the weight of evidence indicated that the blood neutral lipid fraction could donate fatty acids for milk fat synthesis in the mammary gland, it was clear that blood lipids were not the only source of milk fatty acids or that some of the fatty acids taken up from the neutral lipid TABLE 1. Concentration of Lipids in the Blood of Lactating Cows (mg/100ml plasma): Mean Values given by Maynard et al. 2~3

Total lipids Phospholipids Free cholesterol Esterified cholesterol

Arterial blood

Mammaryvein blood

441 242 41 105

426 243 41 106

Lipid metabolism in the mammary gland of ruminant animals

349

fraction in the blood were modified in the mammary gland before being secreted as milk fat. This latter view was supported by Hilditch and his colleagues (see Hilditch ~6°'161) who developed an ingenious theory of the origin of the C4-C12 fatty acids in ruminant milk fat. This theory is outlined briefly as follows. From his classical studies on the chemical constitution of natural fats, Hilditch t6°'16~ concluded that, in spite of the striking differences in the fatty acid compositions of triglycerides isolated from a variety of plant and animal sources, the constituent fatty acids appeared to be assembled into triglyceride molecules by a common biosynthetic process that favored the elaboration of "mixed" rather than simple triglycerides. Thus, any individual triglyceride molecule, whether it be of plant or animal origin, tended towards maximum heterogeneity in its fatty acid composition. Consequently, appreciable proportions of triglyceride molecules containing three saturated fatty acids would be expected to occur only in those fats in which saturated fatty acids constituted more than about 60 moles % of the total fatty acids. This relationship between the saturated fatty acid and saturated glyceride contents of a variety of plant and animal fats is shown in Fig. 1 in which are plotted some of the results obtained by Hilditch and co-workers (see Hilditch~e~). The saturated glyceride contents were determined by oxidizing the triglyceride sample in acetone with potassium permanganate; the unchanged saturated glycerides were then separated from the azelao-glycerides derived from the triglycerides containing unsaturated fatty acid residues. ~62 Although the compositions of virtually all of the plant oils and most of the animal fats (e.g. rat and rabbit depot fat) examined at that time were found to conform with the rule of "even distribution", notable exceptions to this rule were apparent in the compositions of the milk and depot fats of ruminant animals. The results plotted in Fig. 1 for ruminant milk and depot fats are also taken from the publications of Hilditch and co-workers (see Hilditch16~). It was noted that the divergencies from the rule of "even distribution" were associated in ruminant depot fats with decreased proportions of oleic acid that were counterbalanced by increased proportions of stearic acid and in ruminant milk fat, with decreased proportions of oleic acid that were counterbalanced by increased proportions of the saturated acids of lower molecular weight. To explain these divergencies, Hilditch proposed that ruminant depot and milk triglycerides were synthesized according to the rule of "even distribution", but that a proportion of the resulting oleo-glycerides were then subjected to further transformation. In ruminant depot fat, this transformation of preformed oleoglycerides involved the hydrogenation of certain of the oleoyl residues to stearoyl residues while the acyl ester bond remained intact. 2°'21'~66 In milk fat, it was proposed 90i

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~ 60--

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._eme_e~e~e.o--o~o.~

g t.)

I I0

I 20

I 30

° I 40

[ 50

I 60

I 70

I 80

I 90

Content of saturated acids In total fatty acids, molar percontogo

FIG. 1. The relationship between the saturated fatty acid and saturated triglyceride contents of natural fats (see Ref. 161) H fats from non-ruminants, O----O ruminant milk fat.

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J.H. Moore and W. W. Christie

that the oleoyl residues in certain of the preformed oleo-glycerides were subjected to a process of combined oxidation and reduction that began at the terminal methyl group and resulted in the eventual shortening of the carbon chain. Again, there was no disruption of the acyl ester bond during this process. 165 During the period between 1930 and 1938, results appeared in the literature that seemed to lend some support to this concept of the origin of the short-chain fatty acids in ruminant milk fat. For example, Verkade and van der Lee 393 produced evidence that fatty acids could be degraded in the tissues of man by a process that involved the initial oxidation of the terminal methyl group. Two important nutrition experiments established an inverse relationship between the concentration of oleic acid and the concentration of the short-chain fatty acids in cows' milk fat. Hilditch and Thompson 16v found that the milk fat produced by cows given a dietary supplement of cod-liver oil contained appreciable proportions of the highly unsaturated C2o and C22 acids derived from the oil, decreased proportions of the short-chain fatty acids and increased proportions of oleic acid. These findings were explained by postulating, perhaps not unreasonably at that time, that in the mammary gland, the highly unsaturated C20 and C22 fatty acids preferentially combined with and thereby inhibited the enzyme system responsible for the degradation of oleic acid to the short-chain acids. Smith and Dastur 365 in 1938 withheld food from lactating cows for a period of several days and observed pronounced decreases in the molar proportions of the short-chain fatty acids in the milk fat. These decreases were almost entirely compensated for by an increase in the molar proportion of oleic acid. Although a number of alternative explanations of these findings were discussed, Smith and Dastur 3~5 did consider the possibility that inanition would result in a general reduction in metabolic activity in the mammary gland and hence, a reduction in the degradation of the oleic acid in oleo-glycerides to short-chain fatty acids. Perhaps the most striking evidence in favor of the view that the short-chain fatty acids were derived from the degradation of oleic acid was obtained by Hilditch and Longenecker 1~3 who established that the triglycerides of cows' milk fat contained, in addition to oleic acid, small proportions of the Ag~t° monoenoic acids shown in Table 2. It did not seem unreasonable to suppose that these C16, C14, C12 and Clo A 9:1° monoenoic acids would be intermediates in the degradation of oleic to short-chain saturated fatty acids in the mammary gland. The findings of Lintzell, 231 Graham et al "139 and Maynard et al. 273 were confirmed and extended by Shaw and Petersen, 35s'359 who also attempted to establish a quantitative relationship between the amount of neutral fat taken up from the blood and the amount of milk fat secreted by the mammary gland. In experiments with lactating cows, venous blood was taken from the subcutaneous abdominal vein, and arterial blood from the prepudic or internal iliac arteries at various times during the interval between milking. Arteriovenous differences in the concentration of neutral fat showed that immediately after milking only small amounts of blood lipids were taken up by the mammary gland. However, the rate of uptake of blood lipids increased gradually to a maximum value at the 4th hour after milking, and then remained relatively constant during the subsequent 7 hr period. Shaw and Petersen 359 calculated that the quantity of blood fat utilized by the mammary gland was sufficient to account for all of the milk fat secreted, and concluded, therefore, that the short-chain fatty acids in milk TABLE2. A9:10-MonoenoicFatty Acids in Cows' Milk Fat (Hilditch and Longenecker1"3) Molar ",, in cows' milk fat A9:10 A9:10 A9:10 A9:10

Oleic acid Hexadecenoicacid Tetradecenoicacid Dodecenoicacid Decenoicacid

CH3(CH2)-~"CH:CH(CH 2)~,' COOH

CH3(CH2)5."CH--CH(CH ~)7"COOH CH3(CH2)3"CH=CH(CH2)~"COOH CH3. CH2 •CHm.CH(CH2)7•COOH CH,~CH(CH2)7 "COOH

24.8 3.7 1.7 0.9 0.4

Lipid metabolism in the mammary gland of ruminant animals

351

fat must be derived from the breakdown of oleo-glycerides in the mammary gland as proposed by Hilditch and co-workers, t64'167 However, towards the end of the 1930's, evidence began to appear which indicated that an oxygen-rich substrate such as carbohydrate was being utilized by the lactating mammary gland for the synthesis of an oxygen-poor product such as fat. Using the arteriovenous difference technique in experiments with goats, Graham e t al.13s obtained values greater than 1.0 for the respiratory quotient of the lactating mammary gland in vivo. Confirmation of these results was obtained from similar experiments conducted with goats by Reineke e t al. 3'*2 who also reported that the respiratory quotient of lactating goat mammary gland in vivo was reduced to values less than 1.0 when the animals were fasted. In view of the fact that Smith and Dastur 365 had shown that the withholding of food from lactating cows resulted in decreases in the proportions of the short-chain fatty acids in the milk fat. Reineke e t al. 342 reasoned that it was these short-chain fatty acids that were synthesized from carbohydrate in the mammary gland. Arteriovenous difference studies with cows showed that the respiratory quotients of the lactating and non-lactating mammary glands were 1.20 and 0.76, respectively. 322 In a similar but more comprehensive study, Shaw 356 in 1946 found respiratory quotients of 1.27 and 0.61 for the mammary glands of lactating and non-lactating, non-pregnant cows, respectively. The respiratory quotient of the lactating mammary gland decreased to 0.88 after the cows had been fasted for 40 hr. From experiments in which lactating sheep were fed synthetic fatty acids containing odd numbers of carbon atoms, Appel e t al. 7 obtained evidence that the short-chain fatty acids of ruminant milk fat were not derived from the degradation of oleo-glycerides as had been proposed by Hilditch and co-workers. ~64'j 67 When these odd-numbered fatty acids were given to the sheep. no fatty acids with an odd number of carbon atoms and with less than 11 carbon atoms were detected in the milk fat. This finding of Appel et al. 7 suggested that the degradation of long-chain fatty acids to fatty acids containing less than 11 carbon atoms did not occur to any appreciable extent in the ruminant mammary gland. Since it had been well established that as much as 20% of the glucose in bovine arterial blood was removed in passing through the mammary gland, 139 it seemed possible that at least a proportion of milk fatty acids, perhaps the short-chain fatty acids, could be synthesized from glucose in the mammary gland. However, Mann and Shaw 261 could obtain no support for this hypothesis from experiments in which intravenous or intraabomasal infusions of glucose were administered to lactating cows or goats from which conventional food had been withheld for periods up to 70 hr. Even the highest rates of glucose infusion failed to prevent or diminish the decrease in the proportion of short-chain fatty acids in the milk fat. Studies on the origin of acetoacetic and fl-hydroxybutyric acids in mammalian tissues had shown that these ketone bodies were produced in the liver from the oxidative catabolism of fatty acids. Further oxidation of acetoacetic and fl-hydroxybutyric acids occurred only after they had been transported from the liver to various extrahepatic tissues. It occurred to Shaw and Knodt 357 that the lactating mammary gland could utilize ketone bodies either for the production of energy or for the synthesis of milk fatty acids. Arteriovenous difference studies with cows showed that fl-hydroxybutyric acid was utilized in substantial quantities by the lactating mammary gland, but not by the non-lactating gland. 357 The amount of fl-hydroxybutyric acid taken up by the lactating mammary gland was considerably greater in cows with ketonaemia than in normal cows. 355 In none of the conditions investigated was there any evidence for the uptake of acetoacetic acid by the mammary gland of the COW. 355"357 Shaw and Knodt 357 pointed out that the synthesis of fatty acids from fl-hydroxybutyric acid in the mammary gland would involve a high respiratory quotient, and would, therefore, be consistent with the findings of Graham e t al. 138 and Reineke e t a/. 342 In addition, Shaw and Knodt 357 calculated that the amount of fl-hydroxybutyric acid taken up by the lactating mammary gland would be sufficient to account for the short-chain fatty acids secreted in cows' milk fat. However, since the increased mammary uptake J.P.L.R. I 7'4~--("

352

J.H. Moore and W. W. Christie

of fl-hydroxybutyric acid observed in lactating cows with ketonaemia was accompanied by a marked decrease in the proportion of short-chain fatty acids in the milk fat, Shaw 355 concluded that fl-hydroxybutyric acid was probably not the precursor of the short-chain fatty acids in cows' milk fat. In the 1940's, considerable advances were made in the understanding of digestion in the ruminant animal, a topic reviewed by Elsden and Phillipson. ~°7 It was shown that cellulose and other dietary carbohydrates underwent microbial fermentation in the rumen to yield considerable amounts of volatile acids, principally acetic, propionic and butyric acids, and that these volatile acids were absorbed into the bloodstream from the rumen, reticulum and omasum. During the same period, the work of Rittenberg and Bloch 343 established the importance of acetate as a precursor for the synthesis of fatty acids in mammalian tissues. Thus, it seemed possible that the short-chain fatty acids secreted in ruminant milk fat might be synthesized in the mammary gland from acetic acid taken up from the blood. In a series of experiments with cows and sheep, McClymont 254'255 observed that variations in the concentration of acetic acid in arterial blood were closely correlated with variations in the concentration of acetic acid in rumen fluid. The concentration of acetic acid in arterial blood attained maximum values, 8-14 mg/100 ml, 2-5 hr after feeding but declined to 2-6 mg/100ml 16 hr after feeding and to 1.5 mg/100ml 72 hr after feeding. Arteriovenous difference studies showed that the lactating mammary gland of the cow absorbed large amounts of acetic acid from the bloodstream, and that the amount taken up was closely correlated with the concentration of acetic acid in the arterial blood. Smaller amounts of acetic acid were taken up by the non-lactating mammary gland of the cow. When lactating cows were fasted, the resulting decreases in the content of short-chain fatty acids in the milk fat were closely associated with the decreases in the concentration of acetate in the arterial blood and with the decreases in the amount of acetic acid taken up by the mammary gland. However, from the calculated amounts of acetic acid taken up by the lactating mammary gland of the cow, McClymont 25s concluded that a large proportion of this acetic acid must be utilized for purposes other than the synthesis of short-chain fatty acids. Towards the end of the 1940's, major contributions to our knowledge of lipid metabolism in the ruminant mammary gland were made by Folley and his colleagues, who began a series of experiments with preparations of mammary tissues in vitro. When incubated with glucose as the sole substrate, slices from the mammary glands of lactating rats and rabbits respired actively and exhibited a respiratory quotient greater than 1.0. On the other hand, mammary slices from lactating sheep, goats and cows respired with only a relatively small oxygen uptake and exhibited a respiratory quotient of less than 1.0.~18 These results indicated that carbohydrate could be utilized for fat synthesis in non-ruminant mammary tissue but not to any significant extent in ruminant mammary tissue, a finding that appeared to be in conflict with the values obtained by earlier workers ~3a.322'342'356 for the respiratory quotient of the ruminant mammary gland in vivo. Realizing the importance of acetate as a substrate for fat synthesis, and the fact that a respiratory quotient greater than 1.0 would be recorded irrespective of whether a tissue was synthesizing fat from glucose (C6H1206) or acetate (C2H4021, Folley and French ~19 incubated mammary slices from lactating rats, rabbits, sheep and cows with acetate as the sole substrate. The slices from the ruminant mammary glands respired actively and exhibited respiratory quotients in excess of 1.0, but the slices from the non-ruminant mammary glands exhibed respiratory quotients less than 1.0. These results indicated that when acetate was the only available substrate, ruminant mammary tissue could synthesize fat but non-ruminant mammary tissue could not. Although the latter observations with rat and rabbit mammary tissues might be construed to be inconsistent with the results of Rittenberg and Bloch, 34~ Bloch and Kramer 5° had shown that glucose strongly stimulated the synthesis of fatty acids from acetate by rat liver slices. Balmain et al. 18 incubated mammary slices from lactating rats or sheep either with [14C]acetate alone or with I-~4C]acetate plus glucose. When [t4C]acetate was the sole substrate, the incorporation of 1~C into fatty acids by rat mammary slices was very small, but

Lipid metabolismin the mammary gland of ruminant animals

353

appreciable incorporation was observed with sheep mammary slices. However, the presence of glucose in the incubation medium resulted in a 100-fold increase in incorporation of 14C from acetate into fatty acids by rat mammary slices. On the other hand, glucose in the incubation medium resulted in only a 3-fold stimulation of the incorporation of 14C from acetate into fatty acids by sheep mammary slices. In an early application of a double isotope technique to this problem, Balmain et al. 19 incubated mammary slices from lactating rats or sheep with a mixture of [~aC]acetate and [14C]glucose. About 38 and 62% of the carbon in the fatty acids synthesized by rat mammary slices were derived from acetate and glucose, respectively. In contrast, about 97 and 3% of the carbon in the fatty acids synthesized by sheep mammary slices were derived from acetate and glucose, respectively. Thus, when presented with a choice of substrate, there was a marked preferential utilization of acetate carbon for fatty acid synthesis by sheep mammary tissue. Glucose was the favored substrate for fatty acid synthesis by rat mammary tissue but the differential utilization of these two substrates was much less pronounced than it was with sheep mammary tissue. Results indicating that a considerable proportion of ruminant milk fatty acids were synthesized in the mammary gland from acetate taken up from the blood were obtained from an experiment in which [l-14C]acetate was administered intravenously to a lactating goat from which blood and milk samples were taken at various time intervals thereafter, a25 The milk fatty acids were separated into (a) steam-volatile, water-soluble acids, (b) steam-volatile, water-insoluble acids (c) non-volatile, saturated acids, and (d) nonvolatile, unsaturated acids. The specific activities of all four fractions of milk fatty acids reached maximum values 3-4 hr after the injection of labeled acetate. At each time interval, the specific activities of the four fractions of milk fatty acids were considerably greater than the specific activity of the total plasma fatty acids, and during the first 12 hr after the administration of labeled acetate, the specific activities of the two steamvolatile fractions of milk fatty acids were considerably greater than those of the two non-volatile fractions. These results also indicated that the short-chain fatty acids in the milk fat were not derived from the degradation of long-chain fatty acids in the mammary gland. The milk fat samples from this experiment were fractionated further by Popjak et al. a26 who obtained values for the specific activities of oleic acid and of all the individual saturated fatty acids from C4 to C~s. In addition, the isolated butyric, hexanoic and octanoic acids were degraded chemically and the specific activities of the constituent carbon atoms determined. From these results, it was concluded that all of the milk fatty acids up to and including palmitic acid were synthesized in the mammary gland by a stepwise elongation process in which a shorter-chain acid was condensed with acetate or with a C2 compound derived from acetate. The very low specific activities of the stearic and oleic acids isolated from the milk fat indicated that these acids were not synthesized from acetate in the mammary gland. Stearic and oleic acids could have been synthesized in the mammary gland but only by some process that did not utilize acetate as a precursor, However, since it had already been established that the blood "neutral lipid" fraction can be taken up by the mammary gland, ~ag,2aL273 it seemed reasonable to assume that the C~a acids in ruminant milk fat were derived mainly from the blood. Determination of the specific activities of the individual carbon atoms in the isolated hexanoic acid revealed that only about 40% of the butyric acid in the milk fat could have been synthesized in the mammary gland by the condensation of two acetate molecules, and the remaining 60% appeared to be derived from a non-isotopic C4 compound. It seemed unlikely that this C4 compound was butyric acid itself, for although butyric acid is produced in, and absorbed from, the rumen, its concentration in the peripheral blood is particularly low. 2S4 On the other hand, ruminant blood contains appreciable concentrations of fl-hydroxybutyric acid, and this acid had previously been shown to be taken up by the mammary gland of the lactating cow. aS~ Thus, Popjak et at. 326 w e r e inclined to the view that the non-isotopic C4 compound was fl-hydroxybutyric acid that had been taken up from the blood of the lactating goat. With regard to the origin of fl-hydroxybutyric acid in the blood of ruminants, Masson

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J . H . Moore and W. W. Christie

and Phillipson 27° and Kiddie et al. 187 observed that the amount of butyric acid in the blood draining the rumen of sheep was considerably less than would have been expected from the amount of butyric acid absorbed from the rumen. This observation suggested that butyric acid was metabolized in the rumen wall. This was confirmed when Pennington 321 showed that although acetic, propionic and butyric acids were metabolized in vitro by preparations of the epithelium obtained from the rumen, reticulum and omasum of sheep, these tissues exerted a marked preference for the utilization of butyric acid, a large proportion of which was converted to ketone bodies. Thus, by the mid-1950's, the available evidence indicated that in ruminant animals the fatty acids secreted in milk triglycerides were derived from two metabolic processes. In the first of these, fatty acids with 18 carbon atoms were taken up by the mammary gland from a "neutral lipid" fraction, probably the triglyceride fraction, in the blood. In the second process, fatty acids with 4-16 carbon atoms were synthesized de not~o in the mammary gland from precursors such as acetic and perhaps fl-hydroxybutyrate that were taken up from the blood. In the last 20 years or so, considerable advances have been made in our understanding of these two processes which will now be discussed in some detail. 1I. T H E

CONTRIBUTION MILK

MADE

BY B L O O D

LIPIDS

TO

FAT SYNTHESIS

A. B l o o d L i p i d C o m p o s i t i o n

A full account of the blood lipids of ruminant animals is given by Christie 7s but attention should be drawn to certain salient features of ruminant blood lipid composition that are particularly pertinent to a detailed consideration of lipid precursors in the blood that are utilized for milk fat synthesis in the mammary gland. Plasma triglyceride levels in the lactating cow, about 10 mg/100 ml plasma or about 3 g/100 g total plasma lipid, 42"286'336 and in the lactating goat, about 20 mg/100ml plasma or about 5 g/100 g total plasma lipid, 6 are considerably smaller than in most non-ruminant animals. For example, the concentrations of triglycerides in the plasma and in the total plasma lipids of man are about 95 mg/100ml and 15g/100g, respectively; 1°~ corresponding values for the plasma triglycerides of the rabbit, a herbivorous but non-ruminant animal, are about 60mg/100ml and 20g/100g, respectively. 2a7 As in the plasma of most animals, the concentration of unesterified fatty acids in ruminant plasma is small, 3.5-5.8 mg/100 ml. 6"42"286 Thus, cholesteryl esters and phospholipids together account for about 95% of the total lipids in the plasma of ruminant animals. According to Raphael et al. 336 61% of the total serum lipids of the lactating cow circulate as HDL2 (d, 1.21-1.063), 31% as H D L l (d, 1.063-1.040), 7~o as LDL (d. 1.040-1.066) and 1% as VLDL (d, < 1.006); similar values have been reported by Wendiant and Davis 4°4 and for steers by Dryden et al. 9a This distribution is quite different from that observed for human serum in which about 23 and 77% of the total lipids are associated with H D L (d, > 1.063) and LDL (d, < 1.063), respectively. ~1'321 Although the results of certain groups of investigators cast doubt on the existence of chylomicrons in bovine s e r u m , 4 2 ' 1 3 ° ' 3 3 6 ' 3 7 5 others have claimed that chylomicrons are present but in very low concentrations, ag'ga'99"4°4 Thus, according to Wendlant and Davis, 4°4 only about 2°~o of the total serum lipids are associated with chylomicrons in the lactating cow. From the values given by Barry et al., 27 it may be calculated that about 3°,, of the total serum fatty acids are associated with chylomicrons in the blood of the lactating goat; low concentrations of chylomicrons in the serum of the lactating goat have also been reported by West et al. 4°5 The investigations of Raphael e t al. 336 have shown that the lipid associated with the VLDL (d, <1.006) in the serum of the lactating cow contains 57% triglyceride, 6"/,, cholesterol, 22% cholesteryl esters and 16~0 phospholipids. The lipid circulating as serum L D L (d, 1.006-1.040) was shown to contain 12% triglyceride, 14% cholesterol,

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46% cholesteryl esters and 28% phospholipids. Similar values have been reported by Stead and Welch. 375 There is some disagreement about the lipid composition of the HDL in bovine serum. According to most sources, z1°'141"336'375 cholesteryl esters and phospholipids together account for 91Y'/o or more of the total lipids associated with the HDL (d, 1.063-1.21) in bovine serum. Free cholesterol accounted for the remainder, but none of these investigators could detect more than trace amounts of triglycerides in this Iipoprotein fraction. On the other hand, Bickerstaffe39 found that about 14 and 20% of the total triglycerides in the serum of a lactating cow were associated with "high density" and "very high density" lipoprotein fractions, respectively. Unfortunately, the density characteristics of these two lipoprotein fractions were not defined in absolute terms so it is difficult to compare these findings with those of Raphael et ai. 336 and Stead and Welch. 37s Bickerstaffe et al.42 also stated that the HDL in the serum of lactating cows contained triglycerides, but no quantitative details were given. Consistent with the findings of Bickerstaffe et a/., 39'42 Jonas 179 reported that "triglycerides determined directly were roughly 20% of the total lipid" in bovine HDL (d, 1.063-1.125). It is also evident that the HDL (d, 1.063-1.210) isolated from bovine serum by Dryden et al. 9s'99 contained triglycerides but no quantitative results were given. Under normal conditions, 16:0, 16:1, 18:0 and 18:1 together account for about 90% of the total fatty acids in the triglyceride and unesterified fatty acid fractions in the plasma of ruminant animals; these two lipid fractions usually contain no more than about 5% of polyunsaturated fatty acids. Although the triglycerides in ruminant plasma contain only small concentrations of polyunsaturated fatty acids, there is evidence that these fatty acids are not evenly distributed between the triglycerides of the various lipoprotein fractions. Thus, the results of Dryden et al. 9a show that in bovine serum the polyunsaturated fatty acid contents of the LDL and chylomicron triglycerides were considerably smaller than those of the HDL triglycerides. Approximately 97% of the total polyunsaturated fatty acids in ruminant plasma circulates as cholesteryl esters and phospholipids. 98'2s4"286 This partition of plasma polyunsaturated fatty acids in favor of cholesteryl esters and phospholipids is also observed in non-ruminant animals 2s7 and in man 39S but the partition is not as marked as it is in the plasma of ruminant animals. The composition and characteristics of the protein moieties of the serum lipoproteins have been reviewed in detail by Eisenberg and Levy. I°6 Briefly, the total apoproteins of chylomicrons consist of about 25% apoprotein B (N-terminal glutamic acid), 15% apoprotein C-I (C-terminal serine, N-terminal threonine) and 15% apoprotein C-II (C-terminal glutamic acid, N-terminal threonine) and 45% apoprotein C-Ill (C-terminal alanine, N-terminal serine). Similarly, the total apoproteins of VLDL are comprised of about 50% apoprotein B, 10% apoprotein C-I, 10% apoprotein C-II and 30% apoprotein C-Ill. Apoprotein B accounts for more than 95% of the total apoproteins of LDL. Apoprotein A-I (C-terminal glutamine, N-terminal aspartic acid)and apoprotein A-II (C-terminal glutamine, N-terminal pyrrolidone carboxylic acid) account for 60% and 30%, respectively, of the total apoproteins of HDL which contain only small proportions of apoprotein B (2%), apoprotein C-I (~%), apoprotein C-II (2%) and apoprotein C-Ill

(4%). B. Identity of the Blood Lipid Fraction taken up by the Mammary Gland

During the period between 1950 and 1960, great advances were made in the techniques of lipid analysis; this period saw the emergence of gas-liquid chromatography, 175 gasliquid radiochromatography, a27 the separation of lipid classes by chromatography on columns 25"~l'xla'169 or thin-layers 26° of silicic acid, and the ultracentrifugal separation and analysis of serum lipoproteins, s5 In addition, methods became available for the direct determination of triglyceride concentrations.7L2sl These more precise techniques of lipid analysis were soon applied to blood samples obtained from arteriovenous difference studies in a number of different laboratories. A particularly comprehensive study

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was that reported by Annison et al. 6 Samples of arterial and mammary venous blood were taken from lactating goats given intravenous infusions of [U-14C]palmitic acid, [U-14C]oleic acid or [U-14C]stearic acid. During the experiment, the animals were maintained on a normal plane of nutrition. There were no significant uptakes by the mammary gland of free or esterified sterol, total phospholipids or any of the various plasma phospholipid fractions such as phosphatidylcholine, phosphatidylethanolamine, sphingomyelin or lysophosphatidylcholine; moreover, there were negligible changes in the fatty acid compositions of these plasma lipid fractions during passage through the mammary gland. Plasma triglycerides were taken up by the mammary gland in amounts calculated to be equivalent to 63-829/o of the output of milk triglycerides. The small arteriovenous differences in the concentrations of unesterified fatty acids and the fact that the composition of the arterial unesterified fatty acids was very similar to that of the venous unesterified fatty acids suggested the absence of significant uptakes of unesterified fatty acids by the mammary gland. However, during transit through the gland, there were marked decreases in the specific activities of the ~4C-labeled stearic, oleic and palmitic acids in the plasma unesterified fatty acid fraction. Thus, it appeared that there was an uptake of labeled unesterified fatty acids from the plasma but that this uptake was counterbalanced by an equivalent release of unlabeled unesterified fatty acids into the venous blood. When stearic acid was the only t4C-labeled unesterified fatty acid in the arterial blood, t4C appeared in both stearic and oleic acids in the venous unesterified fatty acid fraction. It seems improbable that the microsomal desaturase system in the lactating cell was responsible for this conversion of stearic to oleic acid. The fact that the unesterified fatty acids in the venous blood contained none of the shorter-chain fatty acids known to be synthesized in the lactating cell suggests that no equilibrium is established between arterial unesterified fatty acids and those fatty acids that result from metabolic processes in the lactating cell. A possibility worthy of further investigation is that stearic acid is converted to oleic acid by a desaturase system in the cells of the capillary endothelium. Results similar to those of Annison et al. 6 have been obtained from arteriovenous difference studies in vil~o or from experiments with isolated perfused mammary glands in a number of related studies,4.27.3a,39.,2,43.109.154,155.223.225.235.269.4.05 and have confirmed that triglycerides are the only plasma lipid class that can be taken up by the mammary glands of ruminant animals on an adequate plane of nutrition. Although Annison et al. 6 and other groups of investigators could find no arteriovenous differences in the concentration of plasma unesterified fatty acids across the ruminant mammary gland under normal dietary conditions, Barry et a/. 27 did discuss the possibility that, in an abnormal nutritional state such as fasting, the gland could well take up unesterified fatty acids from the blood. Fasting has already been shown to result in increased concentrations of unesterified fatty acids in the plasma of man for example, 93'137 and it was accepted that increased concentrations of plasma unesterified fatty acids generally indicated an increased mobilization of fatty acids from adipose tissue depots. 94'121 Moreover, arteriovenous difference studies on liver, kidney, heart and muscle preparations from various non-ruminant animals indicated that the uptake of unesterified fatty acids by these tissues was determined by the concentration of this plasma lipid fraction.~22 Increased concentrations of plasma unesterified fatty acids were observed also when food was withheld from ruminant animals.2 ~6.309.340.34.1 Eventually, Kronfeld 2~6 was able to demonstrate with lactating cows that significant arteriovenous differences in plasma unesterified fatty acid concentration did occur across the mammary gland but only when the concentration in arterial plasma exceeded 300p-equiv per liter. The work of Kronfeld216 was confirmed by Linzell, 232 who showed that when food was withheld from lactating goats for 24hr, there was a 4-fold increase in the concentration of unesterified fatty acids in the arterial plasma and that this increase was accompanied by a change from a negligible to a highly significant uptake of plasma unesterified fatty acids by the mammary gland. Whether or not there is a net uptake of plasma unesterified fatty acids by the ruminant mammary gland would thus appear to depend on the nutritional state of the animal. From time

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to time, there have been reports of the uptake of plasma cholesteryl esters by the ruminant mammary gland (e.g. Emery e t al.'°9). However, since the milk fat and plasma cholesteryl esters of the lactating cow, respectively, contain 1-3% and 80-90% polyunsaturated fatty acids, 377'378 it would seem highly unlikely that significant amounts of milk fatty acids can be derived from the plasma cholesteryl esters. The realization that lipoproteins function as vehicles for the transport of otherwise insoluble lipids in blood plasma led a number of research groups to investigate, by arteriovenous difference studies, the mammary uptake of triglycerides from different plasma lipoproteins. Barry e t ai. 27 utilized ultracentrifugal techniques to separate chylomicrons, low-density lipoproteins (d, 1.005-1.019) and lipoproteins (d, > 1.019) from the arterial and mammary venous plasma of lactating and non-lactating goats. Significant mammary uptakes of esterified fatty acids (determined by the method of Stern and Shapiro 382) associated with chylomicrons and low-density lipoproteins (d, 1.005-1.019) occurred in lactating but not in non-lactating goats; there was no mammary uptake of esterified fatty acids from lipoproteins (d, < 1.019). Microscopic examination under dark ground illumination revealed greater numbers of chylomicrons in arterial than in venous plasma of lactating animals, a difference not observed in non-lactating animals. Although the triglyceride contents of the lipoprotein fractions were not determined directly, each fraction was analyzed for phospholipid and cholesterol. Since no mammary uptake of phospholipid and cholesterol occurred, it was reasoned that the arteriovenous differences in esterified fatty acids were due to the mammary uptake of triglycerides from the chylomicrons and low-density lipoproteins (d, 1.005-1.019). From the results of experiments on the uptake of 3H-labeled chylomicrons by the isolated perfused mammary gland, Lascelles e t a / . 223 also concluded that the udder of the lactating goat removes considerable proportions of chylomicrons from the blood. West et al. 4°5 prepared doubly-labeled chylomicrons from the intestinal lymph of a goat given a duodenal infusion of [14C]glyceryl tripalmitate and [3H]palmitic acid. The washed chylomicrons were then infused into the mammary arteries of lactating goats. Arteriovenous differences in the concentrations of blood lipids indicated that there were considerable uptakes of triglycerides by the mammary gland from chyiomicrons and lowdensity fl-lipoproteins (d, 1.006-0.93); there were no arteriovenous differences in the concentrations of triglycerides in the high-density fl-lipoproteins (d, 1.063-1.006) or highdensity g-lipoproteins (d, 1~21-1.063). The fact that there were virtually no arteriovenous differences in the specific radioactivities of either the glycerol or fatty acid moieties of the triglycerides in the chylomicrons and low-density fl-lipoproteins (d, 1.006-0.93) indicated that the triglycerides taken up by the mammary gland were representative of the triglycerides present in these two plasma lipoprotein fractions. It also indicated that there was no release of triglycerides from the mammary gland into the venous plasma. In a comparison of the lipid compositions of jugular and mammary venous serum obtained from a lactating cow, Bishop et al. 46 found no significant differences in the concentrations of the lipids associated with the lipoproteins not precipitated with dextran sulphate (a-lipoproteins), and there we're no differences either in the concentrations of cholesteryl esters or phospholipids associated with the lipoproteins precipitated with dextran sulphate (fl-lipoproteins). However, the concentration of triglycerides circulating as dextran precipitable lipoproteins in jugular serum was about 4 times greater thafi the corresponding concentration in mammary venous serum. Bishop et al. 46 argued that no appreciable error would be introduced by calculating arteriovenous differences from results obtained for jugular and mammary venous serum, and concluded, therefore, that the triglycerides associated with dextran precipitable lipoproteins constituted the major serum lipid precursor of milk fat. From a similar arteriovenous difference study with lactating cows, Benson et al. as obtained results also indicating that the blood triglycerides taken up by the mammary gland were those associated principally with dextran sulphate precipitable lipoproteins. A more precise identification of the lipid component taken up from the blood by the mammary gland of the cow was attempted

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by Glascock and Welch, ~30 who utilized the ultracentrifugation and electrophoretic procedures outlined by Brumby and Welch 57 to separate and characterize the serum lipoproteins precipitated with dextran sulphate. Dextran sulphate precipitable lipoproteins in bovine serum were found to consist of very low-density lipoproteins, VLDL (d, < 1.019) and two low-density lipoprotein fractions LDLI (d, 1.019-1.039) and LDL2 (d, 1.039-1.050); lipoproteins with densities greater than 1.050 (ct-lipoproteins) were found not to be precipitated with dextran sulphate, s7 Glascock and Welch ~3° administered triglycerides containing [3H]palmitic acid into the rumen of a lactating cow and again calculated arteriovenous differences from the results of the analyses of jugular and mammary venous serum. Significant uptakes by the mammary gland of VLDL and LDL triglycerides from the blood were observed but the small uptake of LDL2 triglycerides was judged to be nonsignificant. Of the total triglycerides contained in the lipoproteins precipitated with dextran sulphate, 60~o, 25~o and 15% were associated with the VLDL. LDL1 and LDL2 fractions, respectively. Specific radioactivity measurements indicated that the triglycerides associated with the VLDL and LDL1 behaved as a single metabolic pool from which those associated with the LDL2 were excluded. These findings of Glascock and Welch 13° were in broad agreement with those obtained with lactating cows by Gooden and Lascelles ~3s in an experiment which involved the ultracentrifugal analysis n 4 of the serum lipoproteins in blood obtained from the carotid artery and subcutaneous abdominal vein. Esterified fatty acids were taken up by the mammary gland mainly from serum VLDL (d, < 1.005) but also from chylomicrons. There was no uptake of esterified fatty acids from serum LDL (d, 1.005-1.20) or from serum HDL (d, 1.20). Since Gooden and Lascelles ~3s were able to demonstrate that phospholipids and cholesteryl esters were not taken up by the mammary gland, they judged that the arteriovenous differences in esterified fatty acids were due to the mammary uptake of triglycerides from serum VLDL (d, < 1.005) and from chylomicrons. From an arteriovenous difference study with a lactating cow, Bickerstaffe 39 obtained somewhat different results and concluded, that. in addition to the mammary uptake of chylomicrons and low-density lipoprotein triglyceride, appreciable amounts of triglycerides were taken up by the mammary gland from high-density and very high-density lipoproteins. Of the total contribution from plasma lipoprotein triglyceride to milk triglyceride, Bickerstaffe 3~ calculated that 19°/0 was derived from the chylomicrons, 47'!o from low-density lipoproteins, 13j% from high-density lipoproteins and 21°~, from very high-density lipoproteins. Bickerstaffe et al. 42 also reported that the major proportion of the triglycerides taken up by the mammary gland was derived from low-density lipoproteins, but "in three out of four experiments with two cows, there was a significant mammary uptake of triglycerides from the high-density lipoproteins." Although these findings for the contribution made by high-density lipoproteins appear to be at variance with the findings of other groups, it is difficult to make valid comparisons since Bickerstaffe et al. 39"4z did not give absolute values for the densities of the "high-density'" and "very high-density" lipoprotein fractions. As an alternative to arteriovenous difference studies, Glascock et at. lz9 administered an oral dose of olive oil triglycerides labeled with [3H]stearic acid to a lactating cow and collected samples of blood and milk at various time intervals thereafter. In agreement with previous work, t28 there was an efficient transfer of radioisotope to milk fat and the specific activity of the total milk lipids reached maximum values at about 21 hr. However, the specific activity of the total blood lipids did not reach maximum values until about 35 hr after the administration of labeled fatty acids. Moreover, the specific activity of the total blood lipids was considerably less than that of the total milk lipids. Glascock et al ~29 deduced that the blood lipids of the experimental cow must have contained very small proportions of some component with a very high specific activity and that this component was taken up by the mammary gland for the synthesis of milk fat. Glascock and Wright 132 could obtain no information on the identity of this component from a similar experiment in which a lactating cow was given an oral dose of triglycerides containing [3H]stearic acid and in which the total lipids were

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extracted from the blood samples and fractionated on columns of silicic acid. The specific activities of the plasma phospholipids, cholesteryl esters, triglycerides and free fatty acids were all found to be considerably less than the specific activities of the milk lipids. Continuing the search for the blood lipid component that functions as a precursor in the synthesis of milk lipids, Glascock et a1.131 utilized the dextran sulphate precipitation technique to obtain ~t- and fl-lipoprotein fractions from the blood of lactating cows that had been given triglycerides containing [3H]stearic acid. The specific activity of the total lipids associated with the fl-lipoproteins was about the same a s that of the milk lipids but was much greater than that of the total lipids associated with the ~t-lipoproteins. Further fractionation showed that the specific activities of the individual lipid components of the fl-lipoproteins, with the exception of the triglycerides, were less than the specific activity of the milk fat. The maximum specific activity of the blood fl-lipoprotein triglycerides was 2.5-times greater than, and occurred somewhat earlier than the maximum specific activity observed for the milk fat. These findings were confirmed by Bishop et al. 46 in an experiment in which a lactating c o w was given an intrarumenal injection of triglycerides containing [3H]palmitic acid. The maximum specific activity of the triglycerides associated with the fl-lipoproteins in the blood was about 4-times greater than and was observed some hours before the maximum specific activity of the total milk lipids. Thus, although the weight of evidence from arteriovenous difference studies and product-precursor relationships indicate that the triglycerides taken up by the ruminant mammary gland are those that circulate in the bloodstream as chylomicrons, when present, and as low-density lipoproteins (d, < 1.05), there are a number of inconsistencies in the results that have appeared in the literature. For example, the results of Glascock and Wright ~32 seemed to exclude the possibility of milk fatty acids being derived from the total pool of blood triglycerides. Subsequent work by Glascock et al. T M indicated that "only a small proportion of the total circulating triglycerides is used for milk fat synthesis." This "small proportion" was identified as that present in the lipoprotein fraction (d, < 1.05) precipitable with dextran sulphate. The implication is, therefore, that there are substantial proportions of triglycerides in a serum lipid fraction that do not donate fatty acids to the mammary gland for milk fat synthesis and that these triglycerides circulate in the blood as lipoproteins (d, > 1.05) not precipitable with dextran sulphate. Yet the analyses reported by Brumby and Welch 5T and Stead and W e l c h 375 for the serum lipoproteins of the lactating cow clearly show that the lipoproteins (d, > 1.05) not precipitable with dextran sulphate contain negligible proportions of triglycerides. On the other hand, Bickerstaffe 39 has claimed that triglycerides are present in and are taken up by the mammary gland from all lipoprotein fractions in the blood of the lactating cow. Under normal dietary conditions, 16:0, 18:0 and 18:! are the principal fatty acids present in the blood triglycerides of ruminant animals and it follows, therefore, that these are the fatty acids that are normally taken up by the mammary gland. However, it is clear that certain dietary modifications that cause changes in the fatty acid composition of the blood triglycerides also "result in changes in the composition of the fatty acids that are taken up by the mammary gland. For example, the inclusion of 12:0 or 14:0 in the diet of the lactating cow increased the concentration of these two fatty acids in the plasma triglycerides and in the total fatty acids taken up by the mamm~try gland. 376 When lactating cows were given diets containing safflower oil particles coated with formaldehyde-treated casein, the 18:2 content of the plasma triglycerides was increased as was the amount of 18:2 assimilated by the mammary gland. 135 Nevertheless, there is evidence that the composition of the fatty acids taken up by the mammary gland is not determined solely by the composition of the fatty acids in the plasma triglycerides. In arteriovenous difference studies with lactating goats, Annison et al. 6 and West et al. 4°5 noted that the total triglycerides in the arterial plasma contained higher concentrations of 18:0 and lower concentrations of 18:1 than did the total triglycerides in the mammary venous plasma. One possible explanation was that the roam-

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mary gland preferentially removed those plasma triglycerides containing high concentrations of 18:0 and low concentrations of 18:1. From the results of experiments with cows given normal diets, Moore e t al. 286 concluded that the mammary gland selectively assimilated from the plasma a triglyceride fraction containing low concentrations of 18:2 and that there was in the plasma an 18:2-enriched triglyceride fraction that contributed little or no fatty acids to the mammary gland for milk fat synthesis. These observations 6'286"4°~ may be explained partly in terms of the differences in distribution of triglyceride fatty acids between the various serum lipoprotein fractions. The analyses of Dryden e t al. 9a and Stead and Welch 37~ have shown that, under normal dietary conditions, the VLDL and LDL triglycerides in bovine serum (taken up by the mammary gland) contain higher concentrations of 18:0 and lower concentrations of 18:2 than do the HDL triglycerides (not taken up by the mammary glandt. However, even within a particular lipoprotein fraction, it would appear that there is some selective assimilation of triglyceride fatty acids by the mammary gland. In the triglycerides of the dextran sulphate precipitable lipoproteins in the arterial blood of lactating cows given normal diets, Benson et al. 38 found higher concentrations of 18:0 and lower concentrations of 18:1 and 18:2 than in the corresponding triglyceride fraction in the mammary venous blood. In the jugular serum of lactating cows, Glascock and Welch 13o found higher concentrations of 18:0 in the VLDL (d, < 1.0191 and LDL~ (d, 1.019-1.039) triglycerides than in the triglycerides of the corresponding lipoprotein fractions in the mammary venous serum. There were no jugular-mammary venous differences in the fatty acid compositions of the LDL2 (d, 1.039-1.050) triglycerides, a lipoprotein triglyceride fraction not taken up by the mammary gland. In experiments with cows given diets containing sa~ower oil coated with formaldehyde-treated casein, Gooden and Lascelles ~35 found that the chylomicron and VLDL (d, <1.005) triglycerides in the arterial blood contained higher concentrations of 18:0 and 18:2, and lower concentrations of 16:0 and 18:1 than did the corresponding triglyceride fractions in the mammary venous blood. They indicated that when cows were given a dietary supplement of polyunsaturated oil protected against biohydrogenation in the rumen, there was a preferential assimilation by the mammary gland of those chylomicrons and VLDL triglycerides that contained high concentrations of 18:2. However, it should be noted that from a group of control cows given no protected polyunsaturated oil supplement. Gooden and Lascelles 135 obtained results similar to those of Glascock and Welch, 13° i.e. 18:0 was the only fatty acid that was selectively assimilated by the mammary gland from VLDL triglycerides in the blood. At present, it is difficult to explain the apparent selective uptake by the mammary gland of triglycerides from specific lipoprotein classes but it would seem logical to implicate the substrate specificity of mammary iipoprotein lipase, the enzyme involved in the uptake of triglycerides from the blood (see Section II.C). It is not known whether lipoprotein lipase exerts a higher specificity for certain molecular species of triglycerides. but there is evidence that the size of the iipoprotein particle plays an important part in determining the susceptibility of the constituent triglycerides to hydrolysis by lipoprotein lipase. Chylomicrons vary in diameter between 120 and l l00nm whereas VLDL vary in diameter between 21 and 76 n m. 155 Within each of these two iipoprotein classes, triglycerides associated with the larger particles are more susceptible to hydrolysis by lipoprotein lipase than are triglycerides associated with the smaller particles.~°o Dryden et al. 98 and Stead and Welch 375 determined the fatty acid compositions of the triglycerides in the various lipoprotein classes separated from bovine serum and is evident from their results that the mean 18:0 content of the triglycerides in each class decreased as the mean diameter of the particles in each class decreased. It is not unreasonable to suppose that the same relationship exists within a lipoprotein class, i.e. the 18:0 content of the triglycerides associated with the larger VLDL particles was greater than that of the triglycerides associated with the smaller VLDL particles. A relationship of this type could well explain the preferential uptake by the mammary gland of 18:0 from VLDL triglycerides. It should be emphasized that the results of Dryden e t al., os

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Stead and Welch, a75 Glascock and Welch, ~a° and Benson et al. as were obtained from animals given normal diets from which only very small amounts of 18:2 would be absorbed. In contrast, the unique dietary supplements given to cows by Gooden and Lasceiles 135 would result in the absorption of large amounts of 18:2 from the small intestine, and the incorporation of this 18:2 into chylomicrons and VLDL triglycerides in the mucosal cells. Under these conditions, freshly absorbed 18:2 would enter the blood circulation, via the thoracic lymph duct, as triglycerides associated with the larger chylomicron and VLDL particles. 1°6 The susceptibilities of these particles to attack by lipoprotein lipase might account for the preferential assimilation by the mammary gland of 18:2 from chylomicron and VLDL triglycerides observed by Gooden and Lascelles ~35 in cows given diets containing polyunsaturated oil protected against rumenal hydrogenation. It is also possible that arteriovenous differences in the fatty acid composition of lipoprotein triglycerides might result from the release into the venous blood of triglycerides synthesized in mammary tissue ° or from an exchange between triglyceride fatty acids and unesterified fatty acids during the passage of blood through the mammary gland, t3° C. Hydrolysis of Triglycerides during Uptake by the Mammary Gland and the Role of Lipoprotein Lipase

The evidence reviewed by Robinson and French 3*s indicated that the uptake by extrahepatic tissues of triglyceride fatty acids from chylomicrons and VLDL in the bloodstream involved the hydrolysis of the triglycerides by lipoprotein lipase, an enzyme that had been studied principally in adipose tissues. Quigley et al. T M and Korn 215 noted that bovine milk contained a lipase with properties very similar to those of lipoprotein lipase. To gain further insight into the physiological function of this milk enzyme, McBride and Korn 2.3 studied lipoprotein lipase activity in the mammary tissue of guinea pigs as related to mammary gland development and function. Lipoprotein lipase activity was virtually absent from mammary tissue during most of the period of pregnancy but 24 hr before parturition, enzyme activity began to increase and attained maximum values 2 hr after parturition; thereafter, enzyme activity remained relatively constant throughout the entire period of lactation.. Lipoprotein lipase activity was hardly detectable in mammary tissue after the cessation of lactation. Similar findings with guinea pig mammary tissue were reported simultaneously by Robinson, a44 who also confirmed from inhibition and activation studies that the lipolytic activity was specifically due to lipoprotein lipase. In subsequent experiments with guinea-pig mammary tissue in various stages of physiological actiyity, McBride and Korn 2'*6 showed that the extent of chylomicron uptake by the gland correlated well with the lipoprotein lipase activity of the tissue. Moreover, when lactating guinea pigs were given intravenous injections of chylomicrons in which the triglycerides were doubly labeled with t4C in the glycerol and 3H in the fatty acid moieties, respectively, the 3H/t*C ratio in the lipids of the mammary tissue was much greater than the corresponding ratios in the injected chylomicrons, the serum lipids or the liver lipids. T M These experiments with guinea pigs provided strong evidence that appreciable hydrolysis of chylomicron triglycerides occurs during uptake by the mammary gland and that this hydrolysis is catalyzed by the lipoprofein lipase in mammary tissue. Some evidence that a similar mechanism was involved in the uptake of blood triglycerides by the ruminant mammary gland was first obtained by Barry et al. 27 from mammary arteriovenou.s difference studies with lactating goats. The uptake of triglycerides and low-density lipoproteins was found to be associated with a release of lipoprotein lipase from the mammary tissue into the bloodstream; enzyme activity in the mammary venous plasma was about 4--times greater than that in the arterial plasma. These results received support from those of Lascelles et ai. 223 who reported that the uptake of chylomicrons by the isolated perfused goat mammary gland resulted in increased concentrations of

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lipoprotein iipase and unesterified fatty acids in the perfusate after circulation through the gland. West et al. 4°5 infused chylomicrons containing triglycerides doubly labeled with [14C]glycerol and [3H]palmitic acid into the mammary arteries of lactating goats and were able to detect t~C-labeled free glycerol and 3H-labeled unesterified fatty acids in the mammary venous plasma. The radioactivities from both glycerol and fatty acid were incorporated into milk triglycerides and, from the differences between the t4C/3H ratio in the plasma chylomicron triglycerides and that in the milk triglycerides, it was concluded that at least 50,eJ,]of the chyiomicron triglycerides taken up by the mammary gland was hydrolyzed at some stage before incorporation into milk fat. The results of similar experiments in which an emulsion of [3H]glycerol tripaimitate and glycerol tri-[14C]palmitate was infused into the jugular veins of lactating goats indicated that at least 80~,~, of the infused tripalmitate was hydrolyzed during uptake and subsequent secretion as milk fat by the mammary gland. The time-courses of the incorporation of [3HI in the glycerol and [t4C] in the fatty acid moieties of the milk fat were virtually identical, irrespective of whether the goats were given an intravenous infusion of free [3H]glycerol plus [14C]palmitic acid or an emulsion of [3H]glyceryl tri-[t4C]palmitate. These findings provided further support for the view that plasma triglycerides are largely or completely hydrolyzed during uptake by the mammary gland. Although lipoprotein lipase, the enzyme responsible for this hydrolysis, has been studied in homogenates of bovine mammary tissue, ~° most investigators have found cows" milk to be a particularly convenient starting material for the isolation and purification of the enzyme. For example, Egelrud and Olivecrona ~°4 and Kinnunen et al. ~ss employed affinity chromatography on Sepharose containing covalently-linked heparin to obtain from bovine skim milk a lipoprotein lipase preparation that appeared to be about 80~o pure as judged by polyacrylamide gel electrophoresis. The protomeric molecular weight of this enzyme was estimated, by sodium dodecyl sulphate-polyacrylamide gel electrophoresis, to be 62,000 and its hydrolytic activity was inhibited by protamine sulphate and by 1.0M sodium chloride. In assay systems containing emulsified long chain triacylglycerols as substrates, the activity of the purified lipoprotein lipase was increased 20-fold by the addition of apolipoprotein C - I I . 177'3°3'3°4 In addition, there have been reports that lipoprotein lipase activity is stimulated by the presence of phospholipids in the assay system. 56'~°6'~77 Lipoprotein lipase purified from cows' milk has been shown to exert a pronounced s.pecificity for the acyi ester bond in position 1 of triacylglycerols which are hydrolyzed by the enzyme via the following reaction sequence: triacylglycerol --~ 2,3-diacylglycerol ~ 2-monoacylglycerol ---, glycerol. 288"29°'3°6"3°7 According to Nilsson-Ehle e t al. 296 the rate of production of glycerol was smaller than the rates of production of di- and monoacylglycerols and 2-monoacylglycerol accumulated during the reaction. Nilsson-Ehle et al. 296 also concluded that the isomerization of 2-monoacylglycerol to 1- and 3-monoacylglycerol occurred before cleavage of the last remaining acyl ester bond and the production of glycerol. Morley and Kuksis 289 could find no evidence for fatty acid specificity in the hydrolysis of triacylglycerols by milk lipoprotein lipase. Much of the detailed mechanism of the uptake of blood triglycerides by the mammary gland and the part played by lipoprotein lipase in this process has been elucidated by investigations with non-ruminant species. These investigations will be described briefly since the limited evidence that is available indicates that similar mechanisms obtain in the ruminant mammary gland. With regard to the site of lipoprotein lipase activity, Schoefl and French 352 used light and electron microscopy together with histochemical techniques to examine mammary tissue from rats, mice and guinea pigs that had been given maize oil by stomach tube or intravenous injections of an artificial fat emulsion or chyle collected from the thoracic ducts of rats given diets containing maize oil. High proportions of the chylomicrons in the blood permeating the mammary tissues were observed to be adhering to the lumenal surface of the capillary endothelium. Often, these chyiomicrons appeared to be partially embedded in the endothelial cells

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and to be fused with the outer aspect of the endothelial plasma membrane. After the mammary tissues had been incubated for 2.5-3 hr, impressive histochemical evidence was obtained for the extensive hydrolysis of fatty acyl ester bonds in those chylomicrons adhering to the capillary endothelium. Under electron microscopy, these partially degraded particles adhering to the lumenal surface appeared as "ghosts". A particularly important observation was that during incubation of the mammary tissues, no lipid hydrolysis occurred in the few chylomicron particles that did not adhere to the capillary endothelium and had remained freely suspended in the lumen of the blood vessels. An earlier finding that lipoprotein lipase usually cannot be detected in blood but can be rapidly released into the circulation following the injection of a small dose of heparin had already indicated that the enzyme is normally bound at the endothelial surface of the blood vessel wall. a45 Experiments in v i t r o had also shown that lipoprotein lipase is effectively adsorbed onto chylomicrons, a46 Thus, Schoefl and French 352 considered that this adsorption process might be responsible for the close adhesion of the chylomicrons to the capillary endothelium observed in vivo. They also concluded that their results were consistent with the view that chylomicrons were hydrolyzed by lipoprotein lipase at the lumenal surface of the endothelium and that this hydrolysis was prerequisite to the uptake of chylomicron lipid by the mammary cells. The findings of Schoefl and French aS: for rat mammary tissue have been confirmed and extended to rat adipose tissue by Blanchette-MacKie and Scow49 and by Scow and co-workers, 353'354 who proposed that glycerides traversed the endothelial cells within a membrane bound system. In addition to the hydrolysis of triglycerides in chylomicrons attached to or partially enveloped by capillary endothelial cells, Scow et al. 353'354 obtained cytochemical evidence for the hydrolysis of glycerides in the vacuoles and microvesicles of the capillary endothelium and in the subendothelial space. It seems probable that lipoprotein lipase is synthesized as an inactive proenzyme in the alveolar cells of the mammary gland and that activation occurs only after transport to the lumenal surface of the capillary endothelium where the proenzyme encounters the apoprotein C-II contained in the lipoprotein substrate. 3°3 In further studies on the part played by lipoprotein lipase in the uptake of triglycerides by tissues, Mendelson and S c o w 276 and Scow et al. 354 perfused the mammary glands of lactating rats with doubly labeled chylomicrons obtained from chyle collected from the thoracic ducts of rats given [3H]glyceryl trioleate and maize oil containing ['4C]palmitic acid. Arteriovenous difference data showed that chylomicron triglycerides were taken up by the mammary gland and it was calculated that, of the triglyceride removed from the bloodstream, 60~o was retained in the tissue and 40~o was released into the bloodstream as unesterified fatty acids and glycerol. During the first 2 min of the chylomicron infusion, the ratio of [14C]unesterified fatty acids to I-3H]glycerol in the venous blood was far greater than the ratio of ['4C] to [3H-i in the infused chylomicrons. This suggested that monoglycerides or diglycerides were formed during the initial stages of the process of triglyceride uptake by the mammary gland. However, neither monoglycerides nor diglycerides could be detected in the venous blood. The 14C/3H ratio in the triglycerides isolated from the perfused rat mammary tissue was about the same as that in the infused chylomicrons. This finding contrasts with those obtained from experiments in which chylomicrons containing doubly labeled triglycerides were injected into lactating guinea-pigs 246.247 or lactating goats; 4°5 the ~4C/3H ratios in the milk Iriglycerides secreted by goats and in the triglycerides present in the mammary tissues of guinea pigs were greater than the corresponding ratios in the injected chylomicrons. Mendelson and S c o w 276 considered that these differences might be due to a greater utilization of free glycerol or partial glycerides for triglyceride synthesis in rat mammary tissue. Although Scow et al. a54 found low levels of lipoprotein lipase activity in the venous blood collected from the perfused rat mammary glands, the infusion of heparin caused an immediate release of enzyme from the mammary tissue which resulted in a 100-fold increase in lipoprotein lipase activity in the venous blood. They calculated that the activity released during the first 3 rain of heparin infusion was equivalent to

364

J.H. Moore and W. W. Christie

about 5~ of the total enzyme activity in the gland and concluded that the enzyme released must have been present originally in the capillary endothelial cells of the mammary tissue. When the perfusion of rat man]mary gland with doubly labeled chylomicrons was stopped, labeled triglycerides disappeared from the venous blood almost immediately but the release of [14C]unesterified fatty acids and particularly [3H]glycerol into the venous blood continued for some minutes afterwards. Mendelson and S c o w 276 considered that this finding supported the view that hydrolysis of chylomicrons did not occur in the bloodstream. When the perfused mammary glands were unsuckled for 18 hr, the uptake of triglyceride from the blood, the release of unesterified fatty acids and glycerol to the blood, the retention of fatty acids and glycerol by the tissue, and the lipoprotein lipase activity in the tissue were all considerably less than the corresponding values for suckled mammary glands. From the biochemical and cytochemical evidence available, it is possible to put forward the following mechanism for the uptake of chylomicron and VLDL triglycerides by mammary tissue; this mechanism is similar to that proposed by Scow e t a / . T M Chylomicrons and VLDL, which provide the substrate (triglyceride) and the cofactors (apolipoprotein and phospholipid) necessary for optimum lipoprotein lipase activity, become attached to and partially enveloped by the capillary endothelial cells. It is not known whether a specific receptor other than lipoprotein lipase itself is involved in the attachment of the chylomicrons to the endothelial surface. Hydrolysis of triglycerides by lipoprotein lipase occurs only in those chylomicrons attached to the capillary endothelium, and is initiated possibly by the cleavage of the acyl ester bond in position sn-1. A proportion of the resulting diglycerides may be hydrolyzed further at the lumenal surface to produce monoglycerides; these monoglycerides and the remaining diglycerides are taken up into microvesicles and are transported across the endothelial cells. Hydrolysis of the second acyl ester bond of the diglycerides continues as the microvesicles cross the endothelium and the products of hydrolysis, i.e. fatty acids and monoglycerides, are released into the subendothelial space where a proportion of the monoglycerides are hydrolyzed with the production of free glycerol. Fatty acids, monoglycerides and some free glycerol enter the mammary alveolar cell and are utilized for the synthesis of milk triglycerides. A proportion of the free glycerol, the magnitude of which may depend on the glycerokinase activity in the mammary tissue, is released into the bloodstream. Fatty acids produced from the hydrolysis of chylomicron triglycerides at the endothelial surface are released into the bloodstream and appear to equilibrate with the plasma unesterified fatty acids. However, under normal dietary conditions, a fraction of the plasma unesterified fatty acids, equal in amount to that released into the bloodstream from the hydrolysis of chylomicron triglycerides, is taken up by the mammary tissue and utilized for milk fat synthesis. This mechanism was evolved mainly from the results of experiments with mammary tissues of non-ruminant animals but it is broadly consistent with the more limited information that is available for ruminant mammary tissues. For example, although large proportions of low-density lipoprotein triglycerides have been shown to undergo complete hydrolysis during uptake by the mammary gland of the lactating goat, 4°5 there is evidence that smaller proportions are hydrolyzed only to 2-monoglycerides which are then utilized directly for the synthesis of milk triglycerides in the alveolar cells. 92 An explanation is also provided for the observation of West et al. 4°5 and others that. when 14C-labeled unesterified fatty acids and 3H-labeled glycerol were infused into the mammary artery of the lactating goat, there were significant transfers of 14C and 3H into milk fat, decreases in the specific radioactivities of blood glycerol and unesterified fatty acids during passage through the gland but no arteriovenous differences in the blood concentrations of unesterified fatty acids and glycerol. Another relevant observation of West et al. 4°5 was that, when an emulsign of [3H]-glyceryl trioleate plus glyceryl tri-[14C]palmitate was infused into the jugular vein of a lactating goat, [3H]glyccrol and [~4C]palmitate rapidly appeared in the mammary lymph. When the infusion of the emulsion was stopped, the concentration of [14C]palmitate in the mammary lymph

Lipid metabolism in the mammary gland of ruminant animals

365

decreased rapidly but relatively high concentrations of [3H]glycerol continued to appear in the mammary lymph for some considerable period thereafter. The relationship between lipoprotein lipase activity in mammary and adipose tissues during pregnancy and lactation in the rat has been studied by Hamosh e t al. 14s In adipose tissue, the high levels of iipoprotein lipase activity observed during the greater part of pregnancy decreased markedly during the last 3 days before parturition and remained at low levels throughout lactation. On the other hand, enzyme activity in mammary tissue was small during the first 20 days of pregnancy but increased abruptly during the last few days of pregnancy and then decreased sharply at parturition. This was followed by a substantial increase in lipoprotein lipase activity during the first 2 days post partum after which time, enzyme activity remained at high levels throughout the remainder of the lactation period. The result of a more limited study with cows 36°'361 indicate that similar relationships exist between lipoprotein lipase activities in ruminant mammary and adipose tissues during pregnancy and lactation. The prevention of suckling resulted in a decrease in lipoprotein lipase activity in the mammary tissue of lactating guinea pigs 243 and rats t4s but it increased the activity of the enzyme in the adipose tissue of lactating rats. 14s Lipoprotein lipase activity in the mammary tissue of lactating rats was also reduced when the mammary ducts were ligated and the gland became engorged with milk; 148 this finding could well explain the marked decrease in enzyme activity observed in rat mammary tissue on the day of parturition. Since suckling is known to cause an immediate release of prolactin and oxytocin into the bloodstream, 82,s3 Scow e t al. 354 and Zinder e t al. 416 investigated the effects of these hormones on the liproprotein lipase activity of rat mammary tissue. Hypophysectomy in lactating rats decreased lipoprotein lipase activity in mammary tissue to levels of activity observed in non-lactating mammary tissue; conversely, it increased the enzyme activity in adipose tissue to levels normally observed in the adipose tissue of non-lactating rats. A complete reversal of these changes was brought about when prolactin and oxytocin were administered to hypophysectomized lactating rats; the administration of oxytocin alone to hypophysectomized lactating rats had no effect on lipoprotein lipase activity in mammary and adipose tissues. In groups of lactating rats in various physiological states, Zinder e t al. 4 ~ noted that there was an impressive inverse correlation between the lipoprotein lipase activities in mammary and adipose tissues. An increase in the lipoprotein lipase activity of mammary tissue was noted by Falconer and Fiddler 112 when prolactin was injected intraductally to pseudopregnant rabbits. They also demonstrated a very short half-life for lipoprotein lipase in the mammary tissue of pseudopregnant rabbits given intraductal injections of cycloheximide or actinomycin D. From experiments with explants of bovine mammary and adipose tissues, Emery t°a reported that the addition of prolactin to the incubation medium increased the lipoprotein lipase activity of mammary tissue but the hormone appeared to have little or no effect on the enzyme activity of adipose tissue.

III. F A T T Y A C I D S Y N T H E S I S

IN T H E M A M M A R Y

GLAND

A. Introduction

With the elucidation of the mechanism of //-oxidation of fatty acids to acetate units, 1'*°'242 it was a s s u m e d that the synthesis of fatty acids in a variety of animal tissues, including mammary tissue, occurred by a reversal of this fl-oxidation pathway. Thus, Hele e t al. 157 concluded that the fatty acid synthesis in cell-free preparations of lactating rabbit mammary tissue occurred by a stepwise condensation of C2 units through the reversal of fl-oxidation. However, 1958 saw the publication of the first of a series of papers by Wakil and his colleagues 55.127.396-398 who showed that the

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J . H . Moore and W. W. Christie

principal mechanism of fatty acid synthesis in many animal tissues involved two basic reactions. 1. CH3COSCoA + HCO3 + ATP--* HOOC. CH2" COSCoA + ADP + Pi. 2. CH3COSCoA + 7HOOC'CH2.COSCoA + 14NADPH ---~CH3"CH2(CH2"CH2)o'CHz'COOH + 7CO 2 + 14NADP + 8CoA + 6H20. The first reaction concerns the carboxylation of acetyl CoA to malonyl CoA and is catalyzed by acetyl CoA carboxylase. 399 The second reaction is catalyzed by a group of enzymes, known collectively as the fatty acid synthetase. Ganguly t 24 established that these two reactions were mainly responsible for the de novo synthesis of fatty acids in bovine mammary tissue. A partially-purified fatty acid synthetase, prepared by Becker and Kumar 3s from the supernatant fraction of lactating goat mammary tissue, showed an absolute requirement for maionyi CoA and NADPH in catalyzing the incorporation of ~4C from [l-tgC]acetate into 4:0, 6:0, 8:0 and 10:0 plus longer-chain fatty acids. Experiments with this enzyme preparation and [1-~4C]acetyl C0A plus rnalonyi CoA or acetyl CoA plus [2-t4C]malonyl CoA showed that carbon atoms 1 and 2 of 4:0 were derived from malonyl CoA and carbon atoms 3 and 4 from acetyl CoA; 6:0 and 8:0 were synthesized by successive additions of C2 units derived from malonyl C o A . 35

B. Source of Carbon for Fatty Acid Synthesis

Mainly from the results of radioactive experiments in vivo, there is now ample evidence that in the lactating ruminant animal acetate and fl-hydroxybutyrate taken up by the mammary gland from the blood 221 are the two important and, under most conditions. perhaps the only sources of carbon for fatty acid synthesis de novo. Thus, when lactating goats were given intravenous infusions of [1-14C]acetate ~ or when isolated mammary glands of lactating goats were perfused with D(-)-fl-hydroxy[l-~4C]butyrate,235 t4C was actively incorporated into the milk fatty acids from 4:0 to 16:0. The specific radioactivities of the fatty acids from 4:0 to 14:0 were similar but the specific radioactivity of 16:0 was considerably less than that of the other labeled fatty acids (Table 3). No t4c was detected in the Ct8 acids of the milk fat and it was concluded that part of the 16:0 and :all of the Cts fatty acids in the milk fat were derived from the blood lipids. Similar findings were reported for lactating goats by Walker et al. 4°° and for lactating cows by others. 2'45"2z6'3°5 The earlier views of Popjak et al. 326 that fatty acids were synthesized in the mammary gland of the goat by the stepwise condensation of C2 units derived from acetate were fully supported by Ahrens and Luick, 2 who determined the intramolecular distribution of ~4C in the milk fatty acids secreted by cows given intravenous injections of [l-t4C] or [2-t4C]ace TABLE 3. The Compositions and Specific Radioactivities of the Milk Fatty Acids Secreted b~, [a} Lactating Goats given Intravenous Infusions of [I-14C]Acetate 6 and ib) The Isolated Goat M a m m a r y Gland Perfused With Dl-)fl-Hydroxy-[1-14C]butyrate T M

Fatty acid 4:0 6:0 8:0 10:0 12:0 14:0 16:0 18:0 18:1 18:2

.

(at [l-t4C]-acetate infusion Composition Specific [g/100 g) radioactivity 1.3 2.8 8.3 12.9 3.6 10.2 24.5 9.8 23.3 1.5

13.3 13.8 15.3 13.9 16.8 12.6 4.3 --

(b) D{-- !fl-hydroxy-[1-~4C]butyrate Composition Specific Ig/100 gl radioactivity 1.7 2.2 2.7 9.6 4.3 7.7 28.3 7.9 26.6 3.0

61.6 51.7 65.6 55.8 64.8 51.4 35.2

Lipid metabolism in the mammary gland of ruminant animals

367

tate. After the cows had been given an injection of [l-l*C]acetate, the odd-numbered carbon atoms in the milk fatty acids were more highly labeled than were the evennumbered carbon atoms. Conversely, when [2-14C]acetate was administered, the evennumbered carbon atoms in the milk fatty acids were more highly labeled than were the odd-numbered carbon atoms. Another aspect of the conclusions of Popjak et al. 3 ' 6 received support from the work of Ahrens and Luick, 2 Lawrence and Hawke 226 and Walker et ai., 4 ° ° who determined the distribution of ~4C in the individual milk fatty acids secreted by cows or goats that had been given [t4C]acetate by intravenous injection or intramammary infusion; the results also provided indirect evidence that a C4 compound was utilized intact for fatty acid synthesis in the ruminant mammary gland. From the findings of experiments in which lactating cows were given intravenous injections of [1-14C], [2-14C] or [3-~4C]butyrate, 2 it appeared that this C 4 compound could be butyrate. However, realizing that under normal physiological conditions the intact C4 unit was more likely to be derived from fl-hydroxybutyrate than from butyrate, Luick and Kameoka 24° administered DL-fl-hydroxy-[3-~4C]butyrate by intravenous injection to lactating cows and found that carbon atom 3 of milk butyrate and carbon atom 5 of milk hexanoate were more highly labeled than were any of the other constituent carbon atoms in these two fatty acids. Luick and Kameoka 24° concluded, therefore, that fl-hydroxybutyrate provided the C4 skeleton that was utilized intact for fatty acid synthesis in the ruminant mammary gland, as had been tentatively suggested by Popjak et a/. 326 A quantitative assessment of the contribution made by the intact Ca unit to the synthesis of milk fatty acids was made by Bines and Brown, 45 who gave lactating cows intravenous injections of single tracer doses of Il-t4C]acetate of [1-t4C]butyrate and determined the specific radioactivities of the individual fatty acids in the milk fat (Table 4). From the results obtained from the cows given an injection of [1-~4C]acetate, it was calculated that about 60% of the milk butyrate was derived from an intact C,, unit and that the remainder originated from the condensation of two C2 units derived from acetate. When the cows were given an injection of [l-~4C]butyrate, it was rapidly converted into fl-hydroxybutyrate but not into acetate; l0 min after the injection, the specific activity of the blood fl-hydroxybutyrate was almost as great as that of the blood butyrate in spite of the fact that the concentration of fl-hydroxybutyrate in the blood was more than 3-times greater than the concentration of butyrate. After the injection of [1-~4C] butyrate, the relative specific radioactivities of the individual milk fatty acids decreased with increasing chain length (Table 4) and corresponded very closely to theoretical values calculated by assuming that the methyl-terminal C,, group of the fatty acids originated from a labeled 4-carbon precursor and that the additional carbons were added from an unlabeled pool of C4 units. Palmquist e t al. 3°5 administered [1-~4C]ace tate or D(-)-fl-hydroxy-[l,3-~4C]butyrate to lactating cows by intramammary infusion and determined the relative specific radioactivities of the individual fatty acids in the milk fat The results indicated that about 50~ of the butyrate in milk fat originated TABLE 4. Relative Specific Radioactivities (Butyrate = 100) of the Fatty Acids Secreted in the Milk Fat of Cows Given Intravenous Injections of [l-~4C]Acetate or [1-14C]Butyrate45

J.P.LR. i 7/4--1)

Fatty acid

Relative specific activity after injection of [1-14C]acetate

Relative specific activity after injection of [l-l'*C]butyrate

4:0 6:0 8:0 10:0 12:0 14:0 16:0 18:0

100 170 184 183 160 141 60 2

100 81 62 54 40 27 9 l

368

J.H. Moore and W. W. Christie

from condensation of two C2 units derived from acetate and about 50~ from the intact Ca skeleton of fl-hydroxybutyrate. The butyrate originating equally from these two sources provided a Ca unit which condensed sequentially with one or more C 2 units derived from acetate to form milk fatty acids from 6"0 to 16:0. All of the milk fatty acids from 6:0 to 12:0, most of the 14:0 and about 60~ of the 16:0 were synthesized by this process in the mammary gland; the remainder of the 14:0 and 16:0, and all of the C~ s fatty acids were derived from the constituent fatty acids of the plasma lipids. Thus, in about 50~o of the milk fatty acids that were synthesized de novo in the mammary gland, the four carbon atoms at the methyl-terminal end of the molecules originated from an intact Ca unit derived directly from fl-hydroxybutyrate, and the remaining carbon atoms originated from C2 units derived from acetate. In the other 50~o of the fatty acids that were synthesized de novo in the mammary gland, all of the carbon atoms originated entirely from C2 units derived from acetate. Palmquist et al. 3°5 calculated that the fl-hydroxybutyrate taken up from the blood by the mammary gland of the cow contributed only about 8~o of the carbon present in the total milk fatty acids. Although a subject of past controversy,2 the results of Bines and Brown as and Palmquist et al. 3°s seemed to establish that onJ.y a very small proportion of fl-hydroxybutyrate was cleaved to C2 units before being incorporated into milk fatty acids. The fact that little or no glucose carbon is utilized for fatty acid synthesis in the ruminant mammary gland is discussed in Section III.D of this article.

C. Activation o f Substrates

In the mammary tissue of lactating ruminants, acetate is taken up from the blood and enters into the alveolar cells but before this acetate can be utilized for fatty acid synthesis, it must be converted into acetyl CoA in the cytosol. The enzyme that catalyzes this conversion, acetyl CoA synthetase, is localized predominantly in the cytosolic fraction of lactating cow, sheep and goat mammary tissue, a 1,147.332.370 The substrate specificity of a purified acetyl CoA synthetase from lactating bovine mammary tissue was studied a°'3a3 and appreciable enzyme activity was found only with acetate, propionate and acrylate. Unfortunately, the activity with fl-hydroxybutyrate was not determined, but there was no activity with butyrate. The enzyme in bovine mammary tissue would thus appear to be similar to the acetyl CoA synt,hetase studied in bovine heart by Beinert et al., 37 Hele 157 and Campagnari and Webster. 65 During the period between the 30th day before parturition and the 40th day after parturition, there was a 40-fold increase in the activity of acetyl CoA synthetase in cow mammary gland 275 but, by the end of lactation, enzyme activity had decreased to almost undetectable levels. 263'332 Acetyl CoA synthetase is also present in the mitochondria of lactating cow and goat mammary tissue a°'a 1 but, in this intracellular location, the enzyme is concerned presumably with the activation of acetate that is oxidatively catabolized. According to Annison and Linzell, 5 about one half of the acetate taken up from the blood by the mammary gland of the goat is utilized for fatty acid synthesis and the other half is oxidatively catabolized. The mechanism of fl-hydroxybutyrate activation in ruminant mammary tissues appears to have received little attention. The specific butyryl CoA synthetase that occurs in other ruminant tissues a'a°'4°2 shows insignificant activity with fl-hydroxybutyrate4°z and it is unlikely that the synthesis of fl-hydroxybutyryl CoA could be catalyzed by the acetyl CoA synthetase in ruminant mammary tissue. An enzyme that could possibly be involved in the activation of fl-hydroxybutyrate in ruminant mammary tissues is the medium-chain acyl CoA synthetase first isolated from bovine liver by Mahler e t a / . 257 This enzyme is widely distributed in mammalian tissues 1a2 but it does not appear to have been studied in ruminant, mammary tissues. Although the mediumchain acyl CoA synthetase in bovine liver shows maximum activity with hexanoate, heptanoate and octanoate, it shows appreciable activity also with fl-hydroxybutyrate.257

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D. The Non-utilization o f Glucose Carbon f o r F a t t y Acid Synthesis

The earlier investigations of Balmain et al. Is indicating that there was a negligible contribution from glucose carbon for fatty acid synthesis in sheep mammary tissue have been fully confirmed by Bauman et al. 29 in experiments with cow mammary slices. Acetate carbon was readily incorporated into fatty acids by cow mammary slices but not by rat or sow mammary slices; on the other hand, glucose carbon was readily incorporated into fatty acids by rat and sow mammary slices. The marked preference for acetate carbon over glucose carbon for fatty acid synthesis has been observed also in other ruminant tissues by Ballard et a l ) 7 The utilization of glucose carbon for fatty acid synthesis in the tissues of non-ruminant animals involves the oxidative catabolism of glucose to pyruvate in the cytosol by the Embden-Meyerhof or hexosemonophosphate pathways; pyruvate subsequently enters the mitochondria and is oxidized to acetyl CoA by the multienzyme pyruvate dehydrogenase complex. This acetyl CoA must then be transported from within the mitochondria to the major site of fatty acid synthesis in the cytosol. However, the permeability of the mitochondrial membrane to CoA and its derivatives is very small and the rate of diffusion of acetyl CoA from within the mitochondria to the cytosol is insufficient to account for the observed rates of the synthesis of fatty acids from glucose. 12a'2as The mitochondrial membrane is relatively permeable to acetate but it is extremely unlikely that the acetate utilized for fatty acid synthesis in the cytosol is derived from direct diffusion across the mitochondrial membrane. The activity of acetyl CoA hydrolyase within the mitochondria is too small to support the observed rates of fatty acid synthesis from glucose. 23s The absence of carnitine acetyl transferase from the cytosol would seem to preclude the involvement of carnitine in the transport of acetyl CoA out of the mitochondria.3e'sa'5. 4"29s There is now good reason to believe that the citrate cleavage pathway provides the most important metabolic route whereby mitochondrial acetyl CoA is made available for fatty acid synthesis in the cytosol. 23s'373'374 In this pathway, acetyl CoA and oxaloacetate condense to form citrate in the mitochondria in which the enzyme responsible for this condensation, citrate synthetase, is found exclusively. Citrate is translocated across the mitochondrial membrane 2°9 and is cleaved in the cytosol to form acetyl CoA and oxaloacetate; ATP-citrate lyase, the enzyme that catalyzes this cleavage exists exclusively in the cytosol. The importance of the citrate cleavage pathway in the synthesis of fatty acids from pyruvate in the tissues of non-ruminant animals was demonstrated by Lowenstein et al. 239 and Watson and Lowenstein 4°1 in experiments with (-)-hydroxycitrate, an inhibitor of ATP-citrate lyase. With a cell-free system of rat liver mitochondria plus particle-free cytoplasm, it was found that the synthesis of fatty acids from [~4C]alanine, a process involving the production of intramitochondrial acetyl CoA, was effectively inhibited by (-)-hydroxycitrate. Under the same conditions, (-)-hydroxycitrate had no effect on the synthesis of fatty acids from acetate, a process involving the production of acetyl CoA in the cytosol. These findings are consistent with the observation that ATP-citrate lyase activity in rat mammary tissue increases after the onset of lactation but declines rapidly on weaning. 16'1s°.22°,372 The first indication that the differences between ruminant and non-ruminant mammary tissues in the utilization of glucose for fatty acid synthesis could be related to species differences in the activity of the citrate cleavage pathway emerged from experiments conducted by Hardwick et al. ~s2.~s3 Jn which isolated mammary glands of lactating goats were perfused with [U-J4C]glucose or [2-14C]acetate. In agreement with others, Hardwick et al. ls3 found that goat milk fatty acids were synthesized from [14C]acetate but not from [l'*C]glucose. However, [14C]acetate and [14C]glucose contributed equally to the synthesis of milk citrate; that glucose carbon contributed to the synthesis of citrate via acetyl CoA was confirmed for goat mammary tissue by Hardwick) 52 Since acetyl CoA was utilized for the synthesis of both citrate and fatty acids, Hardwick et a l ) s3 reasoned that, in goat mammary tissue, glucose and acetate did not contribute to the same pool of acetyl CoA. They also obtained evidence that

370

J.H. Moore and W. W. Christie

pyruvate could not be utilized for fatty acid synthesis by the isolated goat mammary gland and concluded that pyruvate must be decarboxylated to acetyl CoA at a site where it was unavailable for the synthesis of milk fatty acids. Meanwhile, Spencer and Lowenstein37~ had reported that fatty acids were synthesized from [~4C]citrate by the supernatant fraction of rat mammary gland homogenates and that the initial step in this process, the cleavage of citrate into oxaloacetate and acetyi CoA was catalyzed by high levels of ATP-citrate lyase. The unavailability of acetyl CoA derived from pyruvate for fatty acid synthesis in goat mammary gland was then explained by Hardwick 152 who found that the high-speed supernatant fraction of this tissue contained virtually no ATP-citrate lyase activity. Although high activities of ATP-citrate lyase have been observed in mammary and other tissues of all non-ruminant animals investigated, the negligible activity of this key enzyme of the citrate cleavage pathway has now been confirmed for cow and sheep mammary tissue as well as for other tissues of ruminant animals.29.~51 The lack of citrate cleavage activity and the resulting inability to utilize glucose carbon for fatty acid synthesis in ruminant tissues is presumably an evolutionary response to the development of a digestive system in which little glucose is absorbed from the intestine due to the extensive conversion of dietary carbohydrate to acetate, butyrate and propionate in the rumen. The glucose in ruminant tissues must, therefore, be derived almost entirely from gluconeogenesis and it is perhaps not surprising that this glucose should be reserved for more specific metabolic functions such as in the synthesis of lactose in the mammary gland. On the other hand, ruminant tissues receive an abundant supply of acetate and fl-hydroxybutyrate which thus have become the principal sources of carbon for fatty acid synthesis. In this respect, it is of interest that Gumaa et al. 14~ found that the activity of acetate thiokinase in sheep mammary tissue was considerably greater than that in rat mammary tissue. E. Source of N A D P H for Fatty Acid Synthesis

A readily available supply of NADPH is necessary for active fatty acid synthesis, and since the mitochondrial membrane is relative impermeable to nucleotides, 2:7'329"35° it is essential that this NADPH is generated in the cytosol. In rat mammary tissue, there is evidence that the oxidation of glucose via the pentose phosphate pathway (Fig. 2) provides the major proportion of the NADPH required for fatty acid synthesis. 14~'1a4 With the onset of lactation in the rat, pronounced increases have been found in the activities of glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase in the particle-free supernatant fraction of mammary tissue homogenates.134 After weaning, marked decreases were observed in the activities of these two enzymes of the pentose phosphate pathway that are responsible for the generation of NADPH. 16`la'~'i45"146,a39 Lowenstein237 and Abraham et al. 1 demonstrated the transfer of 3H from [1-3H]glucose to NADP-[3H] and the subsequent incorporation of the 3H into the fatty acids synthesized by rat mammary tissue. An additional source of NADPH for fatty acid synthesis in rat mammary tissue would appear to be the malate transhydrogenation cycle in which oxaloacetate, produced from the cleavage of citrate, is converted in the cytosol to malate by the action of NAD-malate dehydrogenase. The resulting malate is then converted to pyruvate in the cytosol by the action of NADP-malate dehydrogenase. The cytosol of rat mammary tissue contains an active pyruvate carboxylase which catalyzes the conversion of pyruvate to oxaloacetate. 145'146 The overall result of this sequence of reactions which occurs entirely in the cytosol of rat mammary tissue is: NADH + NADP + A T P ~ N A D + NADPH + ADP + Pi In the particle-free supernatant fraction of rat mammary tissue homogenates, the activity of NADP-malate dehydrogenase increases immediately after parturition and decreases after weaning, t45' 146 It is clear that ruminant mammary tissue differs profoundly from rat mammary tissue in the origin of the NADPH that is utilized for fatty acid synthesis. This difference

Lipid metabolism in the mammary gland of ruminant animals Glucose

371

FATTY ACIDS

1

/ Glucose - 6 - phospt~o~e f PENTOSE ~f I PHOSPHATE . ~ ~ I PATHWAY e - phospho~,orlot e

\

..,../,-

J

NADP " ~ " ~ %

NADPH-J

/"

Ribulose - ,5- phosphote Triose phosphate

~ " ~ N A O H --"'~ioloocetote Pyruvdte"~.NADPH ~ "

~

Cttrote

= Acetyl CoA

Mitochondria

FIG. 2. Pathways for the generation of NADPH for fatty acid synthesis in the rat rfiammary gland.

is evident from the earlier work of Balmain et al. ~s now that a modern interpretation can be placed upon their findings. In experiments with rat mammary tissue slices, they observed that when [l~C]acetate was the sole substrate, there was very little incorporation of 14C into fatty acids but when unlabeled glucose was added to the incubation medium, there was a 100-fold increase in the amount of acetate carbon utilized for fatty acid synthesis. These findings were confirmed in more recent years by Bauman et a/., 29"a2 who showed also that the stimulatory effect of glucose on the incorporation of z~C from acetate into fatty acids by rat mammary slices occurred even in the presence of glycerol in the incubation medium The stimulatory effect of glucose thus appears to be due to its oxidation via the pentose phosphate pathway with the concomitant production of NADPH. In contrast to their findings with rat mammary tissue, Balmain et al. zs observed that sheep mammary tissue slices incorporated large amounts of ~4C into fatty acids when I-l~C]acetate was the sole substrate and that the addition of glucose to the incubation medium resulted in only a 3-fold increase in the utilization of acetate carbon for fatty acid synthesis. Similar results were obtained by Bauman et al. in experiments with COW29'32 andsheep a4 mammary tissue slices. Moreover, Bauman et ai. 3z showed that, when glycerol was present in the incubation medium, the addition of glucose resulted in only a 40?/o increase in the incorporation of acetate carbon into fatty acids by cow mammary tissue slices. Since the oxidation by the pentos6 phosphate pathway of the small amounts of endogenous glucose or glycogen contained in ruminant mammary tissue slices ~5 could not provide sufficient NADPH to support fatty acid synthesis, it seemed clear that in ruminant mammary tissue, but not in rat mammary tissue, there must exist a mechanism for the oxidation of acetate that resulted in the production of considerable amounts of NADPH in the cytosol. This mechanism was sought by Bauman and co-workers in a series of comparative investigations into the activities of NADP-coupled enzyme systems in the cytosol of ruminant and nonruminant mammary tissues. With regard to the enzymes of the pentose phosphate pathway, Bauman et al. z ° found that although the activities of 6-phosphogluconate dehydro-

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J . H . Moore and W. W. Christie

genase were similar in rat and cow mammary cytosol, glucose-6-phosphate dehydrogenase activity in rat mammary cytosol was about 6 times greater than that in cow mammary cytosol. In rat mammary cytosol, the activity of NADP malate dehydrogenase was about 200 times greater than the very low activity of this enzyme observed in cow mammary cytosol. On the other hand, NADP-isocitrate dehydrogenase activity in cow mammary cytosol was about 20 times greater than that found in rat mammary cytosol. According to Bauman e t al. 34 and Gumaa e t al. 147 the activities of NADPmalate dehydrogenase, glucose-6°phosphate dehydrogenase, 6-phosphogluconate dehydrogenase and NADP-isocitrate dehydrogenase in the cytosol of sheep mammary tissue were similar to the corresponding activities in the cytosol in cow mammary tissue. The low levels of NADP-malate dehydrogenase activity were consistent with the low levels of ATP-citrate lyase activity found in cow and sheep mammary cytoso129'34'147 and it was clear that in these tissues, the malate transhydrogenation cycle could not generate significant amounts of N A D P H for fatty acid synthesis. If glucose was available for oxidation by the pentose phosphate pathway, NADPH could be generated in cow and sheep mammary cytosol by the actions of glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase. However, the finding of extremely high levels of NADP-isocitrate dehydrogenase activity in cow and sheep mammary cytosol enabled Bauman e t al. 29 to propose the following mechanism whereby acetate could be metabolized to furnish N A D P H at the site of fatty acid synthesis (Fig. 3). Acetate passes into the mitochondria where it condenses with oxaloacetate to form citrate, part of which diffuses across the mitochondrial membrane and is converted to isocitrate in the cytosoi. The citrate remaining in the mitochondria is also converted to isocitrate, part of which diffuses into the cytosol. The highly active NADP-isocitrate dehydrogenase in cow and sheep mammary cytosol catalyzes the conversion of isocitrate to ct-ketoglutarate with Glucose

FATTY ACIDS

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Lipid metabolismin the mammarygland of ruminant animals

373

the production of NADPH at the site of fatty acid synthesis. The resulting ~t-ketoglutarate diffuses from the cytosol into the mitochondria where it is oxidized in the tricarboxylic acid cycle to furnish oxaloacetate which is utilized to condense with more acetate, thus completing what Bauman et al. 29 have termed the isocitrate cycle. This metabolic cycle was extended and modified by Gumaa et al., t47 who found that the activity of mitochondrial glutamate dehydrogenase in sheep mammary tissue was about 25 times greater than that in rat mammary tissue. They 147 proposed that after the oxidative decarboxylation of isocitrate in sheep mammary cytosol and the influx of the resulting ~-ketoglutarate into the mitochondria, only 50% of this ~-ketoglutarate was oxidized by the tricarboxylic acid cycle to produce oxaloacetate, NADH and ATP. The remaining 50% of the ~-ketoglutarate was converted to isocitrate and citrate by a reversal of the tricarboxylic acid cycle driven by glutamate dehydrogenase acting as an intramitochondrial transhydrogenase coupling malate dehydrogenase with isocitrate dehydrogenase. This sequence of metabolic reactions in ruminant mammary tissue would permit the sustained synthesis of fatty acids from acetate as the sole substrate. However, this situation is unlikely to obtain in the ruminant mammary gland in vivo and Bauman et al. 32"a4 have estimated that about one-half of the NADPH required for fatty acid synthesis in cow and sheep mammary glands is derived from the operation of the isocitrate cycle and about one-half from the operation of the pentose phosphate pathway; these estimates are consistent with those made by Gumaa et al. 14T for sheep mammary gland. As discussed by Bauman and Davis, 3°'3~ fatty acid synthesis in goat mammary gland in vivo would appear to involve a greater emphasis on the production of NADPH from the activity of the pentose phosphate pathway. Shirley et al. at~ and Mellenberger et al. 2~5 found that the initiation of lactation in the cow was associated with substantial increases in the cytosolic activities of NADP-isocitrate dehydrogenase, glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase in mammary tissue obtained by biopsy. F. A c e t y l C o A C a r b o x y l a s e

Acetyl CoA carboxylase is a biotin-dependent carboxylating enzyme, and its important role in fatty acid biosynthesis is illustrated by the fact that avidin effectively inhibits the synthesis of fatty acids from acetate by the particle-free supernatant fraction of lactating bovine mammary tissue. ~59,252 Although acetyl CoA carboxyIase is usually present in the cytosol of most mammalian tissues, there is some evidence that in rabbit mammary tissue in vivo, the enzyme might be loosely associated with microsomal particies. 1°1'369 Acetyl CoA carboxylase, which has been purified from the mammary tissue of lactating rats 2s° and rabbits, 262 exists in a catalytically inactive protomeric form and in an active polymeric form. The conversion of the protomeric form of the enzyme into the active polymeric form is promoted by tricarboxylic acids, notably citrate. 3°'aI Hansen et al. ~5° have shown that the acetyl CoA carboxylase of rabbit mammary tissue is inhibited by malonyl CoA. Long-chain fatty acids and their CoA derivatives also inhibit acetyl CoA carboxylase 279.3°°,a°~ and it has been postulated that the decreased rates of fatty acid synthesis observed in mammary tissue after weaning are due to the inhibition of acetyl CoA carboxylase by free fatty acids which accumulate in the mammary gland under these conditions. 228,279 In experiments with lactating cows, Moor~ and Steele28s observed that an increased mammary uptake of blood fatty acids resulted in a decrease in the de novo synthesis in the mammary gland of fatty acids from acetate and fl-hydroxybutyrate. This observation was thought tO be due to the inhibition of acetyl CoA carboxylase in the mammary tissue by the increased amounts of long-chain fatty acids that were taken up by the mammary gland from the blood. However, many enzymes, some of which are not even concerned with fatty acid biosynthesis, are inhibited also by long-chain fatty acids and their CoA esters and it is the view of many that this inhibition is due to the detergent properties of these long-chain fatty acids and their derivatives and is not likely to be of physiological significance.95,aoa.a7a, agl On

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J.H. Moore and W, W. Christie

the other hand, the work of Goodridge ~36 indicates that the inhibition of acetyl CoA carboxylase by palmitoyi CoA is reversible and competitive with citrate and, therefore. may play an important part in the regulation of fatty acid synthesis in vivo. From experiments with the supernatant fraction of bovine mammary tissue homogenates, Ganguly 124 observed that the activity of acetyl CoA carboxylase was smaller than the activities of other enzymes concerned with fatty acid synthesis, and concluded that acetyl CoA carboxylase catalyzed the rate-limiting step in fatty acid synthesis. This conclusion was supported by the results of Mellenberger et a/., 275 who reported that there was a marked positive correlation between acetyl CoA carboxylase activity and lipogenic capacity in bovine mammary tissue obtained at various stages in the lactation cycle. There were much poorer correlations between lipogenic capacity and the activities of other enzymes (e.g. acetyl CoA synthetase, fatty acid synthetase and NADP isocitrate dehydrogenase) involved in fatty acid synthesis in bovine mammary tissue. ~4.275 G. F a t t y Acid S y n t h e t a s e

Fatty acid synthetase occurs in the cytosol of animal cells 55'~71 where it exists as a tight complex of seven enzyme subunits together with a distinct structural component which contains a 4'-phosphopantetheine group.~S2 The seven enzyme subunits are acetyl transacylase, malonyl transacylase, fl-ketothioester synthetase, fl-ketothioester reductase, fl-hydroxyacylthioester dehydrase, enoylthioester reductase and acylthioester hydrolase. 368 This muitienzyme complex has been purified from several mammalian tissues including the mammary tissues of lactating rats, 367 guinea pigs, 387 rabbits, 68 COWS 2 ° 2 " 2 1 0 ' 2 5 8 " 2 5 9 and goats. 295 The properties of the fatty acid synthetases isolated from mammary and other tissues of various animals have been compared by Kinsella eta/. 202 and Smith and Abraham 368 and found to be very similar. However, the molecular weight of the fatty acid synthetase isolated from rabbit mammary tissue (910,0001 is about twice that of the enzyme complexes isolated from the mammary and liver tissue of other animals (480,000); it seems possible that the synthetase isolated from rabbit mammary tissue was a dimer of the native enzyme complex. 3°'31 With regard to the mechanism of the chain-elongation process, Phillips et a/. 323 and Joshi et al. ts2 have shown that the incubation of purified fatty acid synthetase with acetyl CoA or malonyl CoA results in the transacylation of the acetyl and malonyl groups to covalent binding sites on the synthetase and in the formation of stable acylenzyme intermediates. Peptic hydrolysis of these intermediates yielded three groups of acetyl peptides and two groups of malonyl peptides. From an investigation of the nature of the covalent bonding of acetyl and malonyl groups in these peptides, Phillips et a/. 323 and Joshi et al. t a2 concluded that the fatty acid synthetase contained three separate types of binding sites: (a) The - - O H group of a hydroxyamino acid, probably serine, moiety which binds both acyl groups but has a greater affinity for malonyl than for acetyl groups. There is competitive inhibition between acetyl CoA and malonyl CoA for this binding site. Ib) The --SH group of a 4'-phosphopantetheine moiety which also binds both acyl groups but has a greater affinity for acetyl groups than for malonyl groups. Ic) The --SH group of a cysteine moiety which binds acetyl groups but not malonyl groups. According to Phillips et a/. 323 and Joshi et al., ts2 the first step in the chain elongation process is the binding of an acetyl group to the fatty acid synthetase as the site containing the hydroxyamino acid moiety; this site has been termed the loading site. The acetyl group is then transferred from the loading site to the binding site containing the 4'-phosphopantetheine moiety, and a malonyl group is bound to the now vacant loading site. In the following step, the acetyl group is transferred from the SH group of the 4'-phosphopantetheine moiety to the SH group of the cysteine moiety, and the

Lipid metabolism in the mammary gland of ruminant animals

375

malonyl group is transferred from the OH group of the serine moiety to the SH group of the 4'-phosphopantetheine moiety. The 4'-phosphopantetheine moiety participates in the condensation reaction between acyl and malonyl groups and the resulting acetoacetyl group is bound to the SH group of the 4'-phosphopantetheine moiety which also binds higher fl-keto homologs such as the/~-ketooctanoyl group. The acetoacetyl group bound to the 4'-phosphopantetheine moiety of the synthetase complex constitutes the substrate for the subsequent reduction, dehydration and hydrogenation steps. The resulting butyryl group is transferred from the 4'-phosphopantetheine moiety to the - - S H group of the cysteine moiety which also possesses high specificity for the binding of higher saturated acyi homologs. A second malonyl group is transferred from malonyl CoA to the OH group of the serine moiety and from thence to the SH group of the 4'-phosphopantetheine moiety where it condenses with the butyrate bound to the SH group of the cysteine moiety. This process is repeated with the stepwise elongation of the fatty acyl chain. When the fatty acid synthetase purified from mammalian tissues or avian liver is incubated with acetyl CoA, malonyl CoA and NADPH, the main product of the reaction is unesterified palmitic acid. The enzyme responsible for the termination of the chain elongation sequence is palmitoyl thioesterase which cleaves the palmitic acid from the 4'-phosphopantetheine binding site. Palmitoyl thioesterase is considered to be an integral part of the fatty acid synthetase complex and has little or no hydrolytic activity on the thioesters of lauric or myristic acids. Although the fatty acid synthetase isolated from the mammary tissues of lactating ruminant animals shows absolute specificity for NADPH and malonyl CoA, it has a somewhat broader specificity with regard to the "primers" that are used for fatty acid synthesis. The initial transacylase or "loading enzyme" will utilize acetyl CoA, butyryl CoA or hexanoyl CoA with a distinct preference for butyryl CoA. 2°2,295 This finding is consistent with results that have been obtained from experiments involving intact bovine mammary tissue 197,, 98 and with the fact that the ruminant mammary gland may take up from the blood appreciable quantities of fl-hydroxybutyrate which may be utilized for fatty acid synthesis after being reduced to butyrate by an enzyme system that is present in the supernatant fraction of mammary tissue. 229 However, degradation of the fatty acids synthesized from [3-14C]DL-fl-hydroxybutyrate by the mammary supernatant fraction of lactating goats 222 and COWS252'364" indicated that, although the intact C4. unit of fl-hydroxybutyrate could act as a "primer" for fatty acid synthesis, some cleavage did occur and the resulting C2 units were utilized for chain elongation. It is well known that ~-hydroxybutyrate can be oxidized to acetate in bovine mammary tissue. 47'217'235 In agreement with Kumar et at., 222 Smith and McCarthy, 364 and McCarthy and Smith, 252 Kinsella ~92 found that cultures of functional mammary cells obtained from lactating cows utilized tgc from [1,3-14C]D(--)-fl-hydroxybutyrate for fatty acid synthesis partly via acetate and partly via an intact C4 unit. On the other hand, cultures of de-differentiated bovine mammary cells utilized fl-hydroxybutyrate for fatty acid synthesis via the acetate pathway only. Kinsella t92 concluded that the quantitative significance of each pathway was probably influenced by the metabolic or physiological state of the secretory cells a t t h e time of the experiment.

H. Synthesis of Short- and Medium-chain Fatty Acids Ruminant milk fat characteristically contains appreciable proportions of short- and medium-chain fatty acids which are synthesized in the mammary gland 33.34 but not to any extent in other ruminant tissues. 124,3za However, studies on the compositions of the fattyacids synthesized from [~4C]acetate by slices 275 and homogenates 2°° of mammary tissue obtained by biopsy from cows at various times before and after parturition revealed that the ability to synthesize short- and medium-chain fatty acids depended on the physiological state of the mammary gland. On the 18th day before parturition, only trace amounts of fatty acids from 4:0 to 10:0 were synthesized by cow mammary tissue: the synthesized fatty acids consisted of 16:0 (60%), 14:0 (30%),

376

J.H. Moore and W. W. Christie

18:0 (5~/o) and 12:0 (4%). On the 7th day before parturition, the synthesis of shortand medium-chain fatty acids had begun to increase and the synthesis of 16:0 and 18:0 had begun to decrease. These changes in the pattern of fatty acids synthesized by cow mammary tissue continued until the 7th day after parturition when the fatty acids from 4:0 to 12:0 accounted for about 40% and 16:0 accounted for only about 30~o of the total fatty acids synthesized. From the 7th to the 40th day after parturition, there was little change in the composition of the fatty acids synthesized by cow mammary tissue. Between the 18th day before parturition and the 20th day after parturition, there was a 30-fold increase in the rate of fatty acid synthesis by homogenates of cow mammary tissue. 2°° A similar increase in the rate of fatty acid synthesis by cow mammary slices was noted by Mellenberger et al. 2v5 An analogous situation has been observed in the rabbit, the milk fatty acids of which characteristically contain large proportions of 8:0 and 10:0. These fatty acids of mediumchain length are synthesized in the mammary gland but not to any extent in other rabbit tissues. 68 Strong and Dils 386-388 found that the major fatty acids synthesized from [14C]acetate by explants of rabbit mammary tissue on the 14th day of pregnancy were 16:0 (43%), 14:0 (18%), 18:0 (12~) and 18:1 (14%). Between the 18th day of pregnancy and the 2nd day after parturition, the incorporation of [~4C]acetate into 8:0 and 10:0 increased from 5 to 48% and from 7 to 43%, respectively, whereas the synthesis of 14:0, 16:0, 18:0 and 18:1 by the mammary explants decreased to negligible levels. In this respect, it is of interest to note that Kinsella 192'197 has observed that freshly dispersed lactating bovine mammary cells incorporated ~4C from [2-~4C]acetate predominantly into fatty acids from 4:0 to 16:0 in proportions consistent with those found by Mellenberger et al. 275 and Kinsella 2°° for slices and homogenates of cow mammary tissue obtained 7 days after parturition. In contrast, de-differentiated bovine mammary cells synthesized only trace amounts of fatty acids from 4:0 to 12:0 but incorporated [~4C]acetate into 14:0, 16:0, 18:0 and 18:1 in proportions similar to those observed by Kinsella ~94 for homogenates of cow mammary tissue obtained on the 18th day before parturition. The results of Mellenberger e t a / . 275 and Kinsella2°° for cow mammary tissue and those of Strong and Dils 386-38a for rabbit mammary tissue suggested that there was some hormonal control of the mechanisms involved in the mammary synthesis of the specific patterns of fatty acids that are so characteristically observed in cow and rabbit milk fat. Evidence that this is so in the rabbit was obtained by Strong e t al., 389 who found that when mammary explants from pseudopre~nant rabbits were cultured with insulin and corticosterone, 16:0, 14:0, 18:0 and 18:1 were the major fatty acids synthesized. When prolactin was added to the culture medium, there was a 40-fold increase in the rate of fatty acid synthesis by the mammary explants and this increase was accounted for mainly by the synthesis of 8:0 and 10:0. It is not known whether prolactin has a similar role in the control of the mechanisms responsible for the synthesis of short- and medium-chain fatty acids in the bovine mammary gland. The precise mechanism of the chain-length termination process, which is presumably responsible for the characteristic presence of short- and medium-chain fatty acids in the milk of ruminant animals is incompletely understood. The composition of the fatty acids synthesized in the lactating bovine mammary gland in vivo is similar to that of the fatty acids synthesized in v i t r o from [14C]acetate by primary cultures of bovine mammary cells ~°6'2°7 or by slices 27s and homogenates 2°° of lactating bovine mammary tissue but is quite different from that of the fatty acids elaborated by the fatty acid synthetase purified from the supernatant fraction of lactating bovine mammary gland homogenates. 2°2'21° At optimum concentrations of acetate, malonyl CoA and NADPH, Knudsen 2~° and Kinsella et al. 2°2 observed that 16:0 was the major fatty acid produced by the purified fatty acid synthetase. An analogous situation exists for the mammary gland of the rabbit which secretes milk fat with characteristically high concentrations of 8:0 and 10:0. Thus, the compositions of the fatty acids synthesized by the intact rabbit mammary gland 6s or by rabbit mammary gland slices 3a6 were similar to the

Lipid metabolismin the mammarygland of ruminant animals

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composition of the fatty acids in rabbit milk, However, with optimum concentrations of acetate, malonyl CoA and NADPH in the incubation medium, 16:0 was the major fatty acid produced by the fatty acid synthetase purified from the supernatant fraction of lactating rabbit mammary tissue. 6s Moreover, with regard to the nature of the fatty acids synthesized, it is clear that there are marked similarities between the fatty acid synthetases that have been purified from cow, 2°2"21° rabbit, es rat a67 and guinea pig 3s7 mammary tissues and from the livers of rats 5s'69 and pigeons. ~7~ These similarities led Knudsen 2~° and Carey and Dils 6s to conclude that the fatty acid synthetases in all mammalian tissues have similar specificities for chain-length termination and are not responsible per se for the synthesis of the fixed patterns of fatty acids that are so characteristically produced by particular tissues. Knudsen 21° and Carey and Dils 6a suggested, therefore, that the chain length termination process is superimposed upon but is not an inherent component of the fatty acid synthetase in any particular tissue. Substrate concentration and pH are known to influence the chain length of fatty acids synthesized by the fatty acid synthetase in vitro but it is doubtful whether either of these two factors play any part in determining the chain-length of fatty acids synthesized in vivo. a6s In experiments with the fatty acid synthetases purified from lactating cow 2°2'2~° and rabbit 67's7 mammary tissues, it was found that a decrease in the malonyl CoA:acetyl CoA ratio in the reaction medium resulted in increases in the proportions of the shorter-chain fatty acids synthesized and a decrease in the proportion of 16:0. However, the possibility that changes in the relative concentrations of acetyl CoA and malonyl CoA might regulate the chain-length of the fatty acids synthesized in vivo seems unlikely for a number of reasons. Thus, the conditions (i.e. low malonyl CoA :acetyl CoA ratios in the reaction medium) that favor the synthesis of short- and medium-chain fatty acids support only very low rates of fatty acid synthesis, s7'~5~'366 To attain rates of fatty acid synthesis that are equivalent to those observed with slices of lactating mammary tissue, purified mammary fatty acid synthetase must be in a medium containing high malonyl CoA:acetyl CoA ratios. Under these conditions, 16:0 is virtually the only fatty acid synthesized,s7,2°2'21°'36s In any event, it would seem unlikely that a pool of malonyl CoA accumulates during fatty acid synthesis in the mammary gland in vivo. 15° Moreover, the fatty acid synthetases of liver 1't9 and yeast a9° also produce short- and medium-chain fatty acids when incubated with low concentrations of malonyl CoA and high concentrations of acetyl CoA, yet only mammary tissue produces shortand medium-chain fatty acids in vivo. 36s Smith and Abraham 36s also reported that an increase in pH of the incubation medium increased the proportion of medium-chain fatty acids synthesized by the fatty acid synthetase purified from rat mammary tissue. Although they dismissed the possibility that pH could exert some control over the chain-length termination process in vivo, they did consider that a change in pH might simulate the effect of some physiological factor that induces the fatty acid synthetase to assume some structural configuration that favors the synthesis of medium-chain fatty acids. Strong et al. 3ss presented evidence for the existence in the cytosol o f lactating rabbit mammary tissue of a high molecular" weight protein factor that could modify the specificity of the fatty acid synthetase for termination of the growing acyl chain. With optimum concentrations of acetyl CoA, malonyl CoA and NADPH in the incubation medium, the purified fatty acid synthetase produced mainly 4:0 and 16:0 but, in the presence of this high molecular weight factor, the proportion of 8:0 plus 10:0 synthesized increased from 1 to 24%. By titration with specific antibodies, Smith and Abraham 36s removed the fatty acid synthetase from the soluble supernatant fraction of lactating rat mammary tissue. The addition of this cytosolic fraction to an incubation medium containing purified fatty acid synthetase and optimum concentrations of acetyl CoA, malonyl CoA and NADPH resulted in a decrease in the chain-length of the fatty acids synthesized. The synthesis of 8:0 and 10:0 by lactating rabbit mammary tissue could be explained if the factor in the soluble supernatant fraction was an acyl-thioester hydrolase that could specifically catalyze the release of medium-chain fatty acids from the

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J.H. Moore and W. W. Christie

fatty acid synthetase complex. Support for this concept was obtained by Knudsen and Dils 214 and Knudsen e t a / . , 212 who found that the supernatant fraction of lactating rabbit mammary tissue contained an acyl-thioester hydrolase that was active towards medium as well as long-chain acyl CoA esters. An acyl-thioester hydrolase was subsequently purified by Knudsen et al. 2~2 and Dils et al. 89 from the cytosol of lactating rabbit mammary tissue. When this enzyme was incubated with purified fatty acid synthetase and rate-limiting concentrations of malonyl CoA, fatty acids of medium chain-length were produced. Findings that are entirely consistent with those of Knudsen et al. 212,2~ 3 were reported by Carey 66 also from experiments with lactating rabbit mammary tissue. It is not known whether the mammary tissues of lactating ruminant animals contain specific acyl-thioester hydrolases that could account for the characteristic patterns of fatty acids that are observed in ruminant milk fat. However, it is clear that differences do exist between lactating goat and rabbit mammary tissues with regard to the mechanism of the production of short- and medium-chain fatty acids. In the particle-free supernatant fraction of lactating goat mammary tissue, Grunnet and Knudsen ~44 were unable to demonstrate the presence of an acyl-thioester hydrolase that was specific for mediumchain fatty acids. On the other hand, goat mammary microsomes appeared to contain a factor that could induce the purified fatty acid synthetase of goat mammary tissue to synthesize medium-chain fatty acids. 144 Although fatty acid synthesis in the mammary tissue of ruminants occurs predominantly by the malonyl pathway, some 4:0 appears to be synthesized by a pathway that does not involve malonyl CoA. For example, Becker and Kumar 3s showed that 4:0 could be synthesized from acetyl CoA in the supernatant fraction of lactating goat mammary tissue by an enzyme system that appeared to be independent of malonyl CoA and NADPH, and it seemed possible that this enzyme synthesized 4:0 by a reversal of fl-oxidation. It has been reported also that 4:0 can be synthesized by an avidininsensitive enzyme system in rabbit mammary tissue. 222'294 I. Desaturation

In agreement with the results obtained from experiments in l)ivo, 6"225 Kinsella ~gt found that freshly dispersed lactating bovine mammary cells actively desaturated 18:0 to 18:1, whereas de-differentiated mammary cells possessed relatively little desaturase activity. Kinsella and Heald 2°4 have reported that 18:0 is actively desaturated to 18:1 by bovine mammary tissue obtained 2 days before parturition but not by mammary tissue obtained 7-14 days before parturition. Subcellular fractionation of homogenates of lactating goat 4° and COW 196 mammary tissues showed that desaturation of 18:0 to 18:1 occurred exclusively in the microsomes. Mammary microsomes from non-lactating cows possessed very low desaturase activity. ~96 Desaturation of 16:0 to 16:1 also occurred with mammary microsomes from lactating goats but the extent of the desaturation of 16:0 to 16:1 was only about 20~,,, of that of 18:0 to 18:1. There was no evidence for the introduction of more than one double bond per fatty acid molecule and 14:0 and 12:0 were not desaturated. 4° According to Kinsella et a/. 196 and McDonald and Kinsella, 256 the desaturase of bovine mammary microsomes requires NADH and CoA esters of 18:0 or 16i0. The fatty acid specificity of the desaturase in bovine mammary tissue would seem to be somewhat different from that in goat mammary tissue for, according to McDonald and Kinsella,2s6 bovine mammary microsomes desaturate 18:0 to 18:1 and 16:0 to 16:1 with equal facility. However, as they 256 point out, this finding with bovine mammary microsomes in vitro is not consistent with results obtained with the bovine mammary gland in vivo. They also reported that the desaturase of bovine mammary microsomes was inhibited by palmitoyl CoA, oleoyl CoA, 1,2-diglycerides, L-at-glycerophosphate and lysophosphatidyicholine; sonication of the microsomal preparation increased desaturase acti.vity. The desaturation of 18:0 to 18:1 in the mammary gland of the lactating goat 43 and COW 79 is inhibited by sterculic acid, a cyclopropene fatty acid which has been shown to inhibit the conversion of

Lipid metabolism in the mammary gland of ruminant animals

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18:0 to 18:1 in other animal tissues. 334 Although Gerson et al. 126 have suggested that 18:1 may be synthesized in the bovine mammary gland from sources other than 18:0, Bickerstaffe and Johnson 43 and Kinsella and McCarthy 2°6 could find no direct fvidence for such a pathway. The desaturase activity observed by McCarthy et al. 249 in freshly secreted cows' milk was presumed to be due to the presence of microsomes in the milk but it is now known to reside in cytoplasmic droplets 76 (see Section VI). J, Chain EIon#ation

In most animal tissues, 16:0 synthesized by the malonyl pathway in the cytosol may be converted to 18:0 by chain elongation systems that occur in the mitochondria or microsomes. 299"384'397 Since experiments in vivo have shown that acetate cannot be incorporated into C~s fatty acids by the lactating mammary glands of cows and goats, 6'45 it can only be assumed that the mitochondrial and microsomal chain-elongation systems are inactive or are inhibited in these tissues. This contrasts with other ruminant tissues, e.g. bovine liver 39s which possess active chain-elongation systems. With freshly dispersed lactating bovine mammary cells, Kinsella ~92 observed that ~4C from [2-~4C]acetate was incorporated predominantly into fatty acids from 4:0 to 16:0, whereas virtually no ~4C was incorporated to 18:0 or 18:1. On the other hand, de-differentiated mammary cells incorporated relatively small amounts of 14C into short- and medium-chain acids but considerable amounts into 16:0, 18:0 and 18:1. It seems possible, therefore, that the functionally specialized lactating bovine mammary cell, while having gained the ability to synthesize lactose, fl-casein, fl-lactoglobulin, short- and medium-chain fatty acids, 3"t°3'192 loses the ability to elongate 16:0 to 18:0. IV. ESTER1FICATION OF FATTY ACIDS IN MAMMARY TISSUE A. Triglyceride Biosynthesis

The composition of milk lipids has been described in some detail by Christie. 75 By far, the major components are triglyeerides which comprise 97-98% of the total with diglycerides amounting to no more than 0.5% and phospholipids 1.0%. Triglyceride biosynthesis in mammary tissue has been the subject of several recent reviews.30,s6,3 ~3,36s The fatty acid components of the triglycerides are either synthesized in the tissue (see Section III above) or they are derived from the lipids circulating in the plasma (see Section II above). Before they can be utilized for glycerolipid synthesis in general and triglyceride synthesis in particular, they must be activated by conversion to the acyl-CoA esters by acyl-CoA synthetases. The properties of such enzymes have been reviewed by Hiibscher. 173 That they are present in mammary gland is beyond doubt since innumerable studies have shown that CoA and ATP are obligatory cofactors for esterification or that acyl-CoA esters are rapidly esterified by mammary cell preparations but the nature and cellular location of the acyl-CoA synthetases have y e t to be determined. Bickerstaffe and Annison 4t suggested that the failure of a goat mammary microsomal system to esterify medium-chain fatty acids was due to a lack of the appropriate activating enzyme in the microsomes and a similar phenomenon has been noted in rabbit mammary gland preparations. 52 Also, although acyl-CoA synthetases are usually believed to be particulate-bound, limited stearoyl-CoA synthetase activity only could be detected in bovine mammary mierosome preparations.196 In mammary tissue homogenates of rabbits, the activity of palmitoyl-CoA synthetase was shown to change in parallel with certain other enzymes of importance in terms of lipid metabolism, i.e. fatty acid synthetase, glycerolphosphate acyltransferase and phosphatidate phosphatase, from the 16th day of pregnancy until 15 days post partum. 362 Three routes have been defined for triglyceride biosynthesis in animal tissues in

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J. H. Moore and W. W. Christie

CHz OH

I I

C~O CH~O 2 PO3H-

(5)

CHz 02C . R

I I CH20PO3 HC~O

I

(6)

CH2OH CHOH

I CH20PO 3 H-

CHzO2C R -

CHOH

I CHz OPO3 H-

CH202C .R ~ CHOzC R'

I

CH20PO3 H(3)

CH2 02 C IR

CH2 02 C. R

CH2 OH

CliO 2 C . R' ~

CliO 2 C . R ' -

Clio 2 C • R'

I CH202 C. R"

I CHeOH

CH2OH

I

FIG. 4. Pathways of triglyceride biosynthesis. Steps 1, 2, 3 and 4 constitute the sn-glycerol-3-phosphat¢ pathway, steps ? and 4 are the monoglyceride pathway and steps 5, 6, 2, 3 and 4 represent the dihydroxyacetone phosphate pathway.

general, including the mammary gland. These are the sn-glycerol-3-phosphate, the monoglyceride and the dihydroxyacetone phosphate pathways as outlined in Fig. 4. The quantitative significance of each of th~se will vary with the species and has yet to be defined for ruminant mammary gland. The sn-glycerol-3-phosphate pathway 4°3 is undoubtedly the principal route for triglyceride biosynthesis in ruminant mammary tissue. In this, sn-glycerol-3-phosphate produced by the action of the enzyme glycerol kinase on free glycerol or by glycolysis is acylated sequentially on positions sn-1 and sn-2 (although l-acyl-lysophosphatidic acid as such is rarely isolated as an intermediate) to form phosphatidic acid. The phosphate group is removed by the action of phosphatidate phosphatase and the resulting diglyceride is acylated by an appropriate acyltransferase to form the triglyceride. The existence of this pathway in mammary gland of several species, including that of ruminants, has been demonstrated in a number of studies in which sn-glycerol-3-phosphate, phosphatidic acid and 1,2-diglycerides have been shown to be substrates and probable intermediates for triglyceride biosynthesis and in which the cofactor requirements were shown to be similar to those in other t i s s u e S . 11A1'74'88'170'218"245"311"330 Recently, for example, Patton 31t was able to detect phosphatidic acid in mammary tissue in vivo for the first time, and within 10 min of injecting 32p-phosphoric acid into lactating rats, highly labeled phosphatidic acid was present in the mammary gland. The uptake of glycerol, released as a consequence of lipoprotein lipase activity from the plasma triglyceride fraction by the mammary gland, was discussed earlier (see Section II.C). Definitive quantitative measurements of. this uptake have yet to be made but Bauman and Davis 3° have calculated that 50-60Yo of the glyceride-glycerol in milk lipids from the cow is derived from the glycerol of the plasma triglycerides. They assumed that this was the only significant exogenous source of glycerol and that all

Lipid metabolism in the mammary gland of ruminant animals

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the exogenous fatty acids absorbed by the mammary gland were utilized for milk fat synthesis. This free glycerol is phosphorylated by the enzyme glycerolkinase (EC 2.7.1.30), the presence of which has been demonstrated in vitro in mammary tissue from a variety of species including that of the cow and goat. 16''.1'7°'73'189'195 ATP and Mg 2+ are required cofactors and the enzyme is located in the cytosolic fraction of the mammary epithelial cell. 41'244 In common with many mammary gland enzymes, its activity was found to increase dramatically after parturition. 16 sn-Glycerol-3-phosphate is also produced from glucose via triose phosphates during glycolysis. For example, Luick and Kleiber T M infused labeled-glucose into lactating cows and calculated that 70% of the glyceride-glycerol in the milk fat was derived from plasma glucose but their methods did not differentiate between glycerol synthesized in the mammary gland itself and that synthesized elsewhere in the animal (e.g. in the liver) and transported in plasma to the mammary gland. In comparable experiments in the isolated bovine mammary gland, it was estimated that 16% of the milk fat glycerol was synthesized in situ from glucose4°6 while a minimum value of 23% was obtained in experiments with a perfused goat udder with a further 2% from acetate as precursor. 15a Evidence for the existence of two pools of glycerol in mammary gland was obtained in an experiment in which the activity of lipoprotein lipase was suppressed by heparin administration, The pattern of labeling of the glycerol moiety in different triglyceride species in milk after glucose infusion was appreciably different from that in untreated animals. 24a Although free glycerol can apparently diffuse into the mammary cell, it is also possible that it is transported across the cell membrane as a monoacyl-glycerol (monoglyceride), as is discussed below. The positional distributions of fatty acids in the triglycerides of ruminant milk fat were also discussed by Christie. 7s Probably the most striking feature is the positioning of butyric and hexanoic acids exclusively on position sn-3 but it is also noteworthy that high proportions of palmitic acid are found in positions sn-I and sn-2. It is probable that these distributions reflect to a considerable extent the specificities of the acyitransferases required for esterification at each position. Electron microscopy in combination with autoradiography and histological procedures was used to demonstrate that the esterifying enzymes were located predominantly in the endoplasmic reticulum in mouse mammary gland 379-3al and other evidence suggested that this could also be true of the guinea pig 21s and ruminant. 41 Although it has also been suggested that mitochondria may also contain such enzymes, ~1'24S'33° doubts have been expressed 3° as to the purity of the cell fractions used in the experiments concerned. Palmitic and oleic acids were esterified at comparable rates by a particulate fraction from bovine mammary tissue in vitro but stearic acid was esterified more slowly while the rate of esterification of linoleic acid was found to be only one-tenth that of palmitic acid. 12 Indeed, linoleic acid was shown to inhibit esterification of other fatty acids. In similar experiments with goat mammary microsomal preparations in vitro, palmitic and oleic acids were found to be esterified at comparable rates at their optimum concentrations and twice as rapidly as stearic and linoleic acids. 4~ 12:0, 14:0, 18:1, 18:2 and 18:3 fatty acids inhibited esterification at higher concentrations and 8:0 and 10:0 fatty acids were not esterified to any significant extent at any concentration. Dispersed bovine mammary cells absorbed and esterified 16:0, 18:0, 18:1 and 14:0 fatty acids at comparable rates but that for linoleic acid was lower, t93 In contrast, in freshlysecreted goat milk in which the enzymes are present in endoplasmic reticulum contained within an intact plasma membrane, 76 all. the unsaturated fatty acids tested were found to be esterified at comparable rates in vitro and more rapidly than palmitic and other saturated fatty acids. 73 The results of these experiments cannot readily be rationalized at present; the differences in the observed esterification rates may reflect such factors as differences in activation rates, acyltransferase activities or the relative availability of acyl accepters in the cell while the integrity of the membranes in the subcellular preparations used may also be important. The specific activity of the palmitoyl-CoA-sn-glycerol-3-phosphate acyltransferase was found to increase 37-fold at parturition in the guinea pig 219 and similar if less pro-

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J.H. Moore and W. W. Christie

nounced effects have been observed in the rat 62 and in ruminants. 2°4'36~ Gross and Kinsella 143 and Kinsella and Gross 2°3 have studied the properties of the palmitoylCoA:sn-glycerol-3-phosphate acyltransferase in bovine mammary gland microsomes in t,itro. They observed that palmitoyl-CoA was, by far, the preferred substrate and was esterified at up to ten times the rates observed when myristoyl-, stearoyl- or oleoyl-CoA were used and suggested that the acylation of sn-glyceroyl-3-phosphate with palmitoylCoA was of particular significance in initiating triglyceride biosynthesis. In these experiments, the product was phosphatidic acid and no lysophosphatidic acid was detected. The findings a r e i n accord with the natural distribution of palmitic acid in milk triglycerides. Marshall and Knudsen 26. reported that these acyltransferases were not capable of esterifying the CoA derivatives of short-chain fatty acids such as butyric or hexanoic acids but they later 266 conceded that there might have been inadequacies in their experimental procedures. It, therefore, remains to be proved whether butyryl-CoA can be esterified to position sn-1 in vitro but this does not seem likely as it is not found naturally in this position. A goat mammary homogenate was found to use sn-glycerol-3phosphate in vitro as an acceptor for hexanoyl and octanoyl-CoA with diglycerides as the main product isolated rather than triglycerides which were formed from longerchain acyi-CoA derivatives. 33° It was also reported that butyryl-CoA was used similarly but at a very low rate. When l-palmityl-sn-glycerol-3-phosphate was the acyl acceptor with a bovine mammary microsomal preparation in vitro, a number of longer-chain acyl-CoA derivatives were esterified rapidly though palmitoyl-CoA was once more the preferred substrate. 2°t'2°3 Again, this finding is in keeping with the natural distribution of palmitic acid in milk fat and contrasts with the specificity of the corresponding enzyme in most other mammalian tissues which tend to show a preference for unsaturated fatty acids. 16s'3°2 As might be anticipated from the natural distributions of fady acids in "bovine milk fat, l-oleyl-sn-glycerol-3-phosphate was found to be a much poorer acceptor of acyl-CoA esters than was the I-paimityl derivative. 2°1'-'66 The chain-length specificity of the acyl-transferase for short-chain fatty acids was shown to be Ct6 > C1, > C12 > C~o > Ca while the relative proportions of each component found naturally in position sn-2 of the triglycerides naturally were in the same order. 266 Although the rate of the reaction varied with the nature of the fatty acids in position sn-1, the specificity for particular CoA-ester~ was not affected so that acylation of position sn-2 appeared to occur with non-correlative specificity. 363 No acyltransferase activity of this kind was found when butyryl- or hexanoyl-CoA were the acyl donors. The palmitoyl-CoA:monopalmityl-sn-glycerol-3-phosphate acyltransferase of rabbit mammary gland was shown by Caffrey and co-workers 59-64 to consist of two isoenzymic species. One of these was active with monomeric or dispersed substrate molecules and was inhibited by micelles while the second was active with micelles but not with monomers. Although it has yet to be determined whether such isoenzymes exist in bovine mammary tissue, the kinetic evidence was not inconsistent with this state. 2°1 In rat mammary tissue, 392 on the other hand, the acyltransferases would only accept acyl-CoA derivatives in monomeric form. The nature and cellular location of phosphatidate phosphatase, the enzyme that catalyzes the hydrolysis of the phosphate bond in phosphatidic acid yielding diglycerides in mammary gland, appear to be similar to that in most other tissues. It is generally considered to be the rate-limiting enzyme in triglyceride biosynthesis and exists in two forms, one particulate-bound and the other in the cytosolic fraction. 173 In goat mammary tissue, a microsomal system contained all the enzymes required for triglyceride biosynthesis in vitro but the yield of triglycerides was increased by addition of the cytosolic fraction. 4~ The presence of an MgZ+-dependent phosphatidate phosphatase has been demonstrated in the microsomal fraction of cow mammary tissue. 265 A 1,2-diacylglycerol acyltransferase in microsomes from cow mammary gland was shown to esterify [14C]labeled butyric, hexanoic and palmitic acids as the CoA esters

Lipid metabolism in the mammary gland of ruminant animals

383

to microsomal-bound or ethanol-solubilized 1,2-diglycerides in vitro. 265 Mg 2+ was not a cofactor. While this finding certainly provided an explanation for the occurrence of butyric acid uniquely in position sn-3 of the triglycerides, some aspects of the mechanism require further study. For example, butyrate and palmitate comprised over 40 and under 10%, respectively, of the fatty acids in this position in milk triglycerides (see Christie 75) so the mammary gland must esterify the former more rapidly than the latter in vivo. On the other hand, the enzyme only used butyroyl-CoA more effectively than palmitoylCoA in vitro when the CoA esters were present at high concentrations. The intracellular concentrations of the various substrates relative to each other may, therefore, play a key role in determining the composition of position sn-3. It has recently been suggested that a similar mechanism may be involved in the incorporation of short- and mediumchain fatty acids in position sn-3 of rat milk triglycerides. 23° In addition, it was shown that although some sn-l,2-diacylglycerols were better acceptors than others, this had no effect on the specificity for the acylation of position sn-3, i.e. the acylation of position sn-3 also occurred with non-correlative specificity. The probable physiological significance of the short-chain fatty acids in position sn-3 of ruminant milk triglycerides is that they give the milk fat a sufficiently low melting point to be readily secreted as liquid droplets. Jenness 176 has pointed out that the melting point of a triglyceride is lowered by increased proportions of short-chain or unsaturated fatty acids and by asymmetric positioning of fatty acids on the glycerol moiety. Polyunsaturated fatty acids are not readily available to the ruminant mammary gland so short-chain fatty acids must serve the purpose. Also, Kinsella 196 could not detect any oleic acid formed de novo from stearoyl-CoA in vitro in position sn-2 of bovine milk triglycerides and suggested that this might have been esterified exclusively to position sn-3. If this were so, there would exist a possibility that the desaturase activity was a regulatory step in triglyceride biosynthesis. Although the microsomal enzymes in freshly-secreted milk showed some preference for positioning the newlysynthesized oleic acid in position sn-3 in vitro, considerable amounts were also found in position sn-1. ~3 Knudsen 2~1 has provided some evidence that, in rabbit mammary gland, one factor involved in the control of the synthesis of short-chain fatty acids could be esterification at position sn-3. It should be noted that compartmentation of the various enzymatic reactions within the cell may impose some selectivity of esterification not apparent from studies of the specificities of the acyltransferases in homogenized tissue and that the presence of pathways of triglyceride biosynthesis other than the sn-glycerol-3-phosphate pathway may affect the overall structure of the triglycerides. Evidence has been obtained for the existence of the dihydroxyacetone phosphate pathway for triglyceride biosynthesis (shown schematically in Fig. 4) in microsomes from mammary gland of lactating mice. 33~ NADPH rather than NADH was required and di- and triglycerides were the products; in the absence of both NADPH and NADH, the product was acyldihydroxyacetone phosphate. It has yet to be determined whether such a pathway operates in ruminant mammary gland. In the monoglyceride pathway for triglyceride biosynthesis (see Fig. 4), 2-monoglycerides, assumed to be formed by hydrolysis of chylomicron triglycerides by lipoprotein lipase, diffuse or are transported across the cell membranes into the cell where they are esterified sequentially forming diglycerides and triglycerides. The pathway is known to be active in intestinal mucosa 17s and adipose tissue 324 of some species. Also, there is a considerable body of evidence that it can operate in mammary gland although its quantitative significance is a matter for debate. In the experiments of West et al, 4°5 described earlier (see Section II), it was estimated that at least 80% of the chylomicron triglycerides in the plasma of lactating goats were hydrolyzed to glycerol and free fatty acids and the results were consistent with complete hydrolysis before uptake by the mammary gland, although this could not be proved conclusively because of re-esterification of the glycerol and fatty acids in the mammary gland. On the other hand, Mendelson and Scow, 2~6 in similar experiments with perfused rat mammary gland, calculated that a high proportion of the glycerol taken up by the gland was in the form of monoglyJ.P.L.R. 1 7 4

r

384

J.H. Moore and W. W. Christie

oerides. Although monoglycerides can be formed in vitro when lipoprotein lipase, is incubated with chylomicron triglycerides (see Section II.C above), no evidence appears to have been obtained for their formation in vivo in ruminants. There have been a number of attempts to use monoglycerides as substrates for triglyceride biosynthesis in mammary tissue preparations with rather variable results. For example, monoglycerides did not serve as acceptors for acyl-CoA esters in some experiments with homogenates of rat 88 and goat 33° mammary gland. On the other hand, with a microsomal fraction of guinea pig mammary gland in t',itro, 245 monoolein served as an acceptor for fatty acids provided that ATP and CoA were present as cofactors for fatty acid activation (the cofactor requirements confirmed that a lipase-catalyzed exchange reaction was , not operative although lipases were present). Similar activity was demonstrated in sow mammary homogenates 4t and low levels of activity were detected in goat 41 and bovine 2°3 mammary tissue. Part of the problem in determining whether monoglycerides could serve as acceptors in vitro may have been due to practical difficulties. For example, lipases including monoglyceride lipases 41'24s have been detected in mammary tissue which could hydrolyze the monoglyceride to free glycerol and this could, in turn, serve as an acceptor for triglyceride biosynthesis by the sn-glycerol-3-phosphate pathway after activation by glycerolkinase. Although 2-monoglycerides are the main product of lipoprotein lipase hydrolysis, 1-monoglycerides were used as acceptors in many of the above experiments and may have given misleading results. 2-Monoglycerides are highly labile and readily isomerize to the l(3}-acyl derivative so that even those experiments in which the former were nominally added to the incubation medium may not have been entirely reliable. Glyceryl ethers have frequently been used as acceptors in vitro and in vivo to mimic monoglycerides and to obviate these problems. Goat mammary preparations 4~ and freshly-secreted milk from goats ~3 were capable of esterifying 2-monoglyceryl ethers much more rapidly than the corresponding l-monoglyceryl ethers in vitro. Similarly, 2-monoglyceryl ethers infused arterially into lactating goats were rapidly absorbed and esterified by the mammary gland although trace amounts only of l-monoglyceryl ethers were recovered in the mammary tissue: no glyceryl triethers were absorbed 44 (unfortunately, full experimental details of this work never appear to have been published). It has also been argued that evidence for the existence of the monoglyceride pathway in the mammary gland can be adduced from considerations of the structures of ruminant milk fat triglycerides. The higher-melting triglycerides, i.e. those that contain much of the C16 and C18 fatty acids derived from the plasma triglycerides, were shown to have a much higher proportion of palmitic acid in position sn-2 (up to 65%) than did the lower-melting triglycerides which contained the short-chain fatty acids synthesized de novo in the tissue. 22'9° Plasma triglycerides in ruminants also contained a high proportion of palmitic acid in position sn-2 (see Christie 74b) and it has been suggested that 2-monoglycerides with this distinctive structure, formed as a result of lipoprotein lipase hydrolysis, diffused across the cell membrane and were re-esterified with the longer-chain fatty acids that were released during hydrolysis by acyltransferases located in the vicinity of the cell membrane. 22 No such acyltransferases have yet been identified, however. When labeled palmitic acid was infused intravenously into goats, the specific activity of the palmitic acid incorporated into position sn-2 of the higher-melting milk triglycerides was lower than that in the total triglycerides which suggested that a nonradioactive pool of palmitic acid, assumed to be that in position sn-2 of the plasma triglycerides, was diluting that in this position of the milk triglycerides.9L248 An equally plausible explanation, however, might be that this pool of unlabeled palmitic acid was that synthesized de novo in the cell from acetate since Kinsella ~94 has reported that endogenously synthesized palmitic acid is concentrated in position sn-2 of bovine milk triglycerides. A key experiment that might settle the point would be to infuse chylomicrons containing synthetic triglycerides with labeled fatty acids specifically in position sn-2 into the mammary gland and to follow the incorporation of the label into each position of the milk triglycerides. In adipose and intestinal tissue of the hamster, monoglycerides were found

Lipid metabolism in the mammary gland of ruminant animals

385

to inhibit esterification by the sn-glycerol-3-phosphate pathway and could be important in the regulation of triglyceride biosynthesis. 324 Other biosynthetic pathways for triglyceride biosynthesis may exist in the mammary cell. Acyltransferases have been detected in guinea pig mammary tissue 2~s and in freshlysecreted milk from goats at7 that will esterify acyl-CoA esters to primary hydroxyl groups such as that in ethanol. Such enzymes could conceivably have been responsible for the synthesis of monoglycerides of high specific activity that were formed when dispersed bovine mammary cells were incubated with labeled glycerol. 1a9 A possibility that phosphatidylcholine could serve as a precursor for triglyceride biosynthesis via diglyceride intermediates has also been discussed, ala B. Phospholipid Biosynthesis

Although phospholipids are quantitatively minor components of milk fat (approximately 1% of the total lipids), they are nonetheless important as essential constituents of the milk fat globule membrane as well as of mammary tissue membranes in general (reviewed by Christie75). It is virtually certain that all the milk phospholipids are synthesized de novo entirely in mammary tissue since intravenously infused 32p. phosphate labeled milk phospholipids more rapidly and more intensely than those of the plasma. 102 In addition, all the milk phospholipids were labeled roughly in proportion to their masses which suggested that they were derived from a common pool of phosphate in the mammary gland. Similar conclusions were drawn from experiments in which labeled glycerol was incubated in vitro with dispersed mammary cells. 1a9 Other evidence that intact phospholipids are not taken up from the plasma is discussed above (Section II). Phosphatidylcholine is the main phospholipid found in milk and mammary tissue membranes and is synthesized in mammary gland as in most tissues principally from sn-l,2-diglycerides, formed by the phosphatidic acid pathway described above, and CDPcholine with the reaction catalyzed by the enzyme, choline-phosphotransferase. t 9 0 ' t 9 6 ' t 9 9 , 3 t 4 It also seems probable that, as in most tissues, there is a selective utilization of particular diglyceride species for phosphatidylcholine synthesis and that this, together with a deacylation-reacylation cycle, plays a part in determining its eventual fatty acid composition and molecular structure. ~6s,3°2 For example, some of the properties of an acyl-CoA:l-acyl-sn-glycerol-3-phosphorylcholine acyltransferase (EC 2.3.1.1) from bovine mammary microsomal preparations have been described. 2°s OleoylCoA was the preferred substrate when 1-01eyl-sn-glycerol-3~-phosphorylcholine was the acceptor while stearoyl- and linoleoyl-CoA were particularly poor substrates. On the other hand, when 1-palmityl-sn-glycerol-3-phosphorylcholine was the acceptor, palmitoyl- and myristoyl-CoA were better substrates. It has been suggested that the enzyme may play a part in a lysophosphatidylcholine-phosphatidylcholine acylation-deacylation cycle possibly involved in the assembly of the milk fat globules within the cell, in the transport of these to the apical membrane and in their eventual extrusion from the cell. t 99.2o5 A small proportion of the milk phosphatidylcholine was apparently synthesized by methylation of phosphatidylethanolamine and by choline exchange. ~99 From the results of various kinetic studies, the existence of two discrete pools of phosphatidylcholine has been postulated. One with a rapid turnover may be involved in intracellular metabolism, membrane replenishment and exchange reactions while the second is used in the formation of the plasma membrane and is secreted slowly with the milk fat globule membrane, x99 Phosphatidylethanolamine is synthesized by a similar mechanism to that for phosphatidylcholine from sn-l,2-diglycerides and CDP-ethanolamine and its formation in mammary tissue has been demonstrated in a number of s t u d i e s . 73'1°2"189"196A99'314 Two distinct kinases have been detected in the high-speed supernatant fraction of mammary tissue that initiate the formation either of phosphorylethanolamine or of phosphorylcholine prior to the formation of the CDP-derivatives. ~74 A small proportion

386

.1. H. Moore and W. W. Christie

of the phosphatidylethanolamine in mammary gland was shown to be synthesized by decarboxylation of phosphatidylserine.~ 99 Labeled serine and inositol were found to be incorporated into phosphatidylserine 199 and phosphatidylinositol, 4xs respectively, by bovine mammary tissue in vitro. Some of the properties of cytidinediphosphodiacyl-sn-glycerol:myoinositol transferase, which is located in the microsomal fraction of bovine mammary tissue, have been described. 415 Active phosphatidylinositol phosphohydrolases were also present in the high-speed supernatant and microsomal fractions 2°8 that hydrolyzed the newly-synthesized phosphatidylinositol and may have been responsible for a rapid breakdown of this lipid reported earlier by others. 189"318 The phenomenon may be analogous to the rapid turnover of phosphatidylinositol in other secretory tissues on stimulation by hormones or neural impulses (reviewed by Michel1278) and could be involved in the secretory function of mammary epithelial cells. It has been demonstrated that sphingomyelin is assembled from labeled fatty acids and CDP-choline by mammary tissue preparations in vitro. 73"t02"190"199"318 The longchain base components were apparently synthesized in the tissue since isotopicallylabeled serine was incorporated into sphingomyelin in vitro by dispersed bovine mammary cells. 199 A similar conclusion was drawn from analytical studies by Morrison 291 who found that the long-chain bases of the sphingolipids of milk were very different in nature and composition from those of dietary origin. Also, the long-chain base and fatty acid compositions of sphingomyelin, glucosylceramide and lactosylceramide in milk were very similar which may have implied that they were all derived from a common pool of ceramides. 292 In isotopic studies, Patton et al. 318 found that sphingomyelin was incorporated much more slowly than other phospholipids into the plasma membrane but there appeared to be no mechanism for its selective hydrolysis or removal so that eventually it constituted as high a proportion of the membrane lipids as was phosphatidylcholine or phosphatidylethanolamine. V. CHOLESTEROL AND CHOLESTERYL ESTER METABOLISM IN MAMMARY GLAND Free cholesterol constitutes at most 0.5~/o of the total lipids in milk fat and cholesteryl esters are never more than 0,05~o of the total (see Christie75). Conflicting results have been obtained for their distribution in milk but they are certainly found both in the~ fat globule and in the skim milk fraction. 172,293 Because of its essential role in membrane structure and function, it is probable that much of the unesterified cholesterol is present in the milk fat globule membrane and in other membraneous material in milk. The cholesterol in milk and in mammary gland can be of dietary origin, it can be synthesized in the mammary gland itself and it can be synthesized elsewhere in the body of the animal and transported to the mammary gland in the lipoproteins of the plasma. Ruminants will not normally ingest significant amounts of cholesterol since the plant material, which makes up the major part of their diet, contains little or none although it is possible that they may consume some cholesterol if animal by-products are fed as part of the concentrate fraction of the diet. Rumen protozoa did not appcar to contain significant amounts of cholesterol. 183'29~ lngested cholesterol was hydrogenated in part by rumen microorganisms 9 but some was not affected and eventually a proportion of this was found in the plasma and milk. Similarly, labeled cholesterol placed in the abomasum of a goat was absorbed, transported to the mammary gland and ultimately was found in the milk. 338 In contrast in the rat 7~ and guinea pig, 78 I I 0/o and 20~,0, respectively, of the milk cholesterol were of dietary origin. Labeled cholesterol infused into the plasma of the rat 1°°'33v was rapidly taken up by the mammary gland but appeared more slowly in the milk reaching a maximum activity after about 20 hr. It was suggested ~°° that the time-lag was due to initial incorporation of the cholesterol into the tissue membranes rather than into fat globules and that it was eventually secreted into milk as a constituent of the milk fat globule

Lipid metabolism in the mammary gland of ruminant animals

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membrane. It was also reported 337 that all the serum lipoproteins could supply cholesterol directly to the mammary gland but exchange of cholesterol between lipoprotein classes and lipoprotein catabolism were not adequately considered in interpreting the experimental results. Eighty percent of the milk cholesterol in the rat 72 and 50--70% of that in the guinea pig 78 were found to be derived from the plasma. No data of this kind appear to be available for ruminants but Sabine 3'~9 has pointed out that such values have little meaning as they can be influenced markedly by a number of hormonal and dietary factors. The remaining cholesterol in milk is synthesized in the mammary gland from acetate as the primary precursor. 325 Synthesis of cholesteryl esters is also known to occur in the mammary gland 33'186'2°7'25°'316 but the intracellular site of this process does not appear to be known. Cholesteryl esters of much higher specific activity than that of any other lipid class were found in the milk of goats within 2 hr of infusing labeled palmitic acid into the mammary teat canal. 25°'a~6 Yet within a few hours, this specific activity had dropped to a value comparable with that of the other lipid classes. The function of this small but metabolically-active pool of cholesteryl esters is not known. In milk, cholesteryl esters were found mainly in the milk fat globule and tended to accumulate in this in a manner paralleling triglyceride accumulation. ~86 VI. T H E S E C R E T I O N

OF MILK FAT

The biochemical and histological evidence for the synthesis of milk fat triglycerides in the rough endoplasmic reticulum of the mammary epithelial cell is discussed above (see Section IV.A). It is also possible that the triglycerides are assembled into milk fat globules within the rough endoplasmic reticulum by some as yet undefined mechanism and electron microscopic evidence has been put forward in support of this view. For example, material of similar electron-density to lipid has been reported in cisternae with the rough endoplasmic reticulum 3s° and in modified areas of rough endoplasmic reticulum resembling smooth endoplasmic reticulum) 48 Wooding413 has questioned the interpretation of the electron micrographs, however. Fat droplets were reportedly present throughout the cytoplasm of secretory cells often in close proximity to rough endoplasmic reticulum but not within it and the smaller droplets, in fact, were found near the basal plasmalemma. '*°7 Autoradiographic evidence showed that the larger lipid droplets were actively adding triglyceride to their bulk, as° but it is not known whether this occurred by accretion of individual molecules to a single droplet or by condensation of a number of small droplets formed perhaps in the endoplasmic reticulum. Within the cell, fat droplets were not found bounded by a membrane in contrast to the Golgi vesicles, for example, which contain the proteins for secretion.4°7 Milk fat is secreted in the form of droplets from 0.1 to 20 #m in diameter consisting largely of triglycerides and surrounded by a continuous unit membrane, the milk fat globule membrane. The droplet is enclosed by the membrane during the secretory process for which two mechanisms have been suggested. The first was propounded initially by Bargmann and Knoop 24 in 1959 and since then has found considerable support especially from Patton and co-workers while the second was proposed more recently by Wooding.4°7 The evidence for both theories was obtained by electron microscopy and unfortunately, little biochemical evidence is yet available to support one theory over the other. Bargmann and Knoop's proposed mechanism has been discussed in some detail in a number of papers and review articles. 24'185,236'3z°.312,313,319 Briefly, it is suggested that the large fat droplets are transported to the surface of the cell where they push their way into the apical membrane and, in so doing, become enveloped by the membrane until eventually this is pinched off leaving intact membranes around the droplets and at the same time sealing the apical membrane of the cell. Patton and Fowkes 3~z calculated that the London-Van der Waal's forces that might be operating as a fat droplet approached the plasmalemma could exert a sufficient pressure at a distance of 2 nm to drive out the aqueous phase and to cause the envelopment of

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J . H . Moore and W. W. Christie

the fat droplet and its extrusion into the alveolus. In support of this hypothesis, the lipid composition of the milk fat globule membrane was shown to be virtually identical to that of the plasma membrane (see ChristieT5). Also, a number of enzymes that are characteristic of plasma membrane have been found in the milk fat globule membrane.97.319 It did, however, appear that some rearrangement of the membrane constituents occurred on milk fat secretion as considerable morphological changes were noted by electron microscopy. 23"28"t58,383 Wooding4°7 found some elements of these proposals to be incompatible with his own observations. For example, he reported the presence of a layer of electron-dense material, probably derived from the cytoplasm, between the fat droplet and the milk fat globule membrane and suggested that the plasmalemma and the fat droplet could never approach sufficiently closely for the London-Van der Waal's forces to exert sufficient effect. In addition, 1-5~ of the milk fat globules in freshly-secreted goat milk included so-called "crescents" of cytoplasm containing recognizable subcellular organeUes such as endoplasmic reticulum or mitochondria and it was argued that milk fat secretion might be an apocrine process. 414 It was noted 4°7 that, in the apex of

FIG. 5. Electron micrograph of a section through a "cytoplasmic droplet" or "christiesome''7~' (reproduced by kind permission of Dr. F. B. P. Wooding).

Lipid metabolism in the mammary gland of ruminant animals

389

the cell, the fat droplets had numerous peripheral vesicles which were probably derived from the Golgi body. The progressive fusion of these vesicles apparently resulted in the extrusion of the fat droplet surrounded by a unit membrane formed partly from the vesicles and partly from the plasmalemma. Later, it was suggested that in some circumstances a complete milk fat globule membrane could be formed by fusion of the Golgi vesicles without participation of the plasmalemma.*t° In support of this hypothesis, enzymes characteristic of endoplasmic reticulum and the Goigi apparatus have been found in milk fat globule membrane suggesting a complex origin. 97'267'26s'319 For example, galactosyltransferase and other enzymes normally present only in the Golgi apparatus were found in human milk fat globule membrane. 26s The presence of large cytoplasmic fragments with distinctive flotation properties, which render them readily isolable in freshly-secreted goat milk, 76 may also lend support to Wooding's theories. The fragments contain a fat droplet, rough endoplasmic reticulum in sheets, vesicles or swollen cisternae, the occasional mitochondrion or pieces of Golgi apparatus but no nucleus while the whole is bounded by a membrane probably identical to the milk fat globule membrane. A typical so-called "cytoplasmic droplet" or "christiesome,,412 is illustrated in Fig. 5. The particles contain essentially all the lipid biosynthetic activity reported to be present in freshly-secreted goat m i l k . 73"74'251'39't When milk fat globules are secreted into the alveolus, they may lose some of their membranes by a process of vesiculation leaving a poorly-defined secondary membrane enclosing the milk-fat globule.4°7-4°9'.11 VII. R E L A T E D

REVIEW ARTICLES

A number of review articles have appeared in recent years of relevance to lipid metabolism in the mammary gland of ruminant animals. 26'a°'39's4's6,s9'92'1°s' 115--I 17,120,125,181,233,234,277,282,283,285,313,315,320,347,354,359,368

(Received 23 October 1978)

VIII. R E F E R E N C E S 1. 2. 3. 4. 5. 6, 7, 8. 9. 10. I1. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29.

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