ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS 136, 112-121 (1976)

Fatty Acid Synthesis in Developing Mouse Liver’

STUART SMITH AND S. ABRAHAM

The Bruce Lyon Memorial Research Laboratory, Children’s Hospital Medical Cenfer of Northern California, Oakland, California 94609

Received July 11, 1969; accepted October 9, 1969

Fatty acid synthesis in developing mouse liver has been studied by measuring in- corporation of labeled acetate and pyruvate into fatty acids by tissue slices (lipogenic capacity) in conjunction with the determination of the activities of several enzymes involved in lipogenesis.

Hepatic lipogenic capacity, which is normally low in suckling pups, can be prema- turely increased by weaning onto a fat-free diet on or after Day 16 postpartum. Fur- thermore, pups ingesting a linoleate-deficient milk during Days 6 to 15 postpartum showed greater hepatic lipogenic capacities than pups ingesting a linoleate-rich milk. The data have been interpreted to indicate that hepatic lipogenesis in suckling and weanling mice can be regulated by the dietary linoleate content.

The increase in hepatic lipogenic capacity in mouse pups weaned onto a fat-free diet and the accompanying increase in the activities of citrate cleavage enzyme, acetyl CoA carboxylase, fatty acid synthetase, and malic enzyme are dependent on the synthesis of new protein and new RNA. The increases in activities of these en- zymes at weaning follow different time courses; fatty acid synthetase was observed to reach maximum activity earliest.

In neonatal rat liver the activities of en- zymes involved in gluconeogenesis are higher than in either fetal or adult liver (1). Conversely certain lipogenic and glycolytic enzymes have lower activities in neonatal liver than in fetal or adult liver (l-3). Vernon and Walker have indicated that many of the metabolic changes which occur when rats progress through the fetal, suck- ling, and weaning stages of development can be explained in terms of the known adaptive behavior of some enzymes in adult liver to changes in diet (1).

The observation of Ballard and Hanson (2) that in fetal liver, lipogenesis from glu- cose decreases dramatically in the 4-day period immediately before parturition could suggest that the low fatty acid synthesis in suckling rats is not a direct result of the milk diet. Weber et al. (4) have shown with

1 This work was supported by grants from the National Science Foundation (GB-6672) and the American Cancer Society (P-1821).

rats that the activity of certain hepatic glycolytic enzymes increased during the period 18-23 days postpartum whether or not the pups remained with the mother. These workers ruled out the possibility that the increase in enzyme levels was brought about by a change from milk to laboratory chow.

Ballard and Hanson (2) have shown that in the liver of developing rats the activity of citrate cleavage enzyme parallels changes in lipogenic capacity when measurements were made at intervals of several days. The theory that the activity of citrate cleavage enzyme might be the control point in lipo- genesis (5) has been challenged by Foster and Srere (6) who showed that in fasted and refed rats hepatic lipogenic capacity increased 12-24 hr before any change in citrate cleavage activity could be detected.

In an attempt to clarify the possible importance of diet in regulating lipogenesis in the developing animal we set out to

112

FATTY ACID SYNTHESIS IN DEVELOPING LIVER 113

determine whether premature weaning of young animals onto diets low in fat could lead to increased hepatic lipogenesis. In addition we have examined on a short-term basis whether at the time of weaning there is any direct correlation between increased hepatic lipogenic capacity and the activity of any of the enzymes concerned with lipo- genesis. To compare the effectiveness of intra- and extramitochondrially produced acetyl CoA as a precursor for lipogenesis in liver slices, fatty acid synthesis was meas- ured using both acetate 1-“C and pyruvate 2J4C as substrates. Production of 14COz from pyruvate l-14C (via mitochondrial pyruvate dehydrogenase) in liver slices was regarded as an index of intramitochondrial acetyl CoA availability. The activity of citrate cleavage enzyme (ATP-citrate oxalo- acetate-lyase (CoA acetylating and ATP dephosphorylating), EC 4.1.3 .S), a key enzyme in the transfer of acetyl CoA from the intra- to extramitochondrial space, was measured. M:alic enzyme (L-malate: NADP oxidoreductase (decarboxylating) , EC 1.1. 1.40), believed to be important in the re- plenishment of intramitochondrial oxaloace- tate and glucose 6-phosphate dehydrogenase (n-glucose 6-phosphate: NADP oxidore- ductase EC 1.1.1.49) were also assayed. The latter two enzymes provide a potential supply of NADPH for lipogenesis. Acetyl CoA carboxylase (Acetyl CoA: COZ ligase (ADP), EC 6.4.1.2) and fatty acid syn- thetase are required for the conversion of acetyl CoA to fatty acid and were also assayed.

chemical Research, Los Angeles, California; or Sigma Chemical Company, St. Louis, Missouri, and were of the highest purity available. W-La- beled compounds were purchased from New Eng- land Nuclear Corporation, Boston, Massachusetts. Actinomycin D and puromycin were gifts from Dr. R. B. Painter, Laboratory of Radiobiology, University of California, San Francisco. W- Labeled pyruvate was stored frozen at -20” in an equimolar solution of HCl as recommended by Von Korff (7).

Animals and their treatment. Mice of the C3H strain were used. During the lactating period, dams were fed Purina Mouse Chow and water ad Zibitum unless otherwise stated. Litters of at least five pups, both males and females, which showed normal weight gain (approximately 0.4 g per day} during the suckling period were selected from our colony for study. All adults used in these experi- ments were females. The commercial chow diet contained 50% carbohydrate, 11% fat, and 20% protein; the high-fat diet contained 50yo glucose, 15% corn oil, and 22% casein; the fat-free diet contained 50% glucose and 22% casein. A more complete dietary analysis has been reported else- where (8).

Animals were killed by cervical dislocation. Tissue slices (9) and homogenates (10) were pre- pared as described previously. To obtain suf&ient tissue for both slice experiments and enzyme as- says, it w&s necessary to pool livers from two pups. Adult livers were not pooled.

Enzyme a.ctivities measured under opti- mum conditions are interpreted as an indi- cation of the maximum potential of the lipogenic pathway. We have therefore at- tempted to correlate changes in enzyme activities with actual changes in hepatic lipogenic capacity by comparing data from enzyme studies with data obtained by incorporating labeled precursors into fatty acids in tissue slices.

Incubation of tissue slices. One hundred milli- grams of liver slices (0.4~mm thick) were incubated in 2.0 ml Krebs-Henseleit bicarbonate buffer (11) containing either labeled acetate (2 pmoles) or pyruvate (5 pmoles). After incubating for 3 hr at 37” in an atmosphere of 95% oxygen-5% carbon dioxide, yields of radioactive carbon dioxide (12) and fatty acids (13) were determined. Under these conditions, carbon dioxide production and fatty acid synthesis were found to be directly propor- tional to time over the 3-hr incubation period.

EXPERIMENTAL PROCEDURE

Enzyme assays. All assays were performed at 30” on the “100,009 g” supernatant fractions ob- tained from tissues homogenized in 0.25 M sucrose. Acetyl CoA carboxylase, fatty acid synthetase, malic enzyme, and glucose 6-phosphate dehydro- genase were assayed as described previously (8). Citrate cleavage enzyme was assayed by the method of Srere (14). Enzyme activities were proportional to protein concentration and incuba- tion time.

Protein determinations. The method of Lowry et al. (15) was used except for determination of protein content of tissue slices when the biuret method of Gornall et al. (16) was employed. De-

Materials. Substrates, cofactors, and auxiliary enzymes required in the enzyme assays were bought from either Boehringer and Soehne, Mann- heim, Germany; California Corporation for Bio-

114 SMITH AND

PUPS

Age. days ADULTS

FIG. 1. Incorporation of acetate and pyruvate into fatty acids by liver slices from suckling and weanling mice. Open symbols represent incorpora- tion of acetate 1-W (0) and pyruvate 2-l% (0) into fatty acids by liver slices prepared from suck- ling mice. Solid symbols represent acetate l-i% (0) and pyruvate 2-W (m) incorporation into fatty acids by liver slices prepared from pups which had been removed from the dam and sup- plied with the fat-free diet for 3 days prior to sacrifice. Each point is the mean of determinations on 3-19 liver pools. Standard errors are indicated wherever possible. Chow-fed adults, 80 days old were transferred to the fat-free diet 3 days before sacrifice.

fatted human serum albumin (17) was used as standard.

Lipid analysis. The procedures for obtaining mouse milk, isolation of milk and tissue lipids, methylation, and subsequent fractionation of methyl esters by gas chromatography was de- scribed in detail previously (8).

Carbohydrate analysis. The carbohydrate con- tent of milk was measured by the method of Somogyi (18) and Seifter et al. (19). Lactose wae used as the standard.

RESULTS

A direct comparison of data from pups and adults can be made as no difference was found in the protein content of pup (16.3 f 0.4 (8) mg protein per lOO-mg slice, at 10 days postpartum) and adult (17.4 j, 0.6 (6) mg protein per lOO-mg slice, at 80 days postpartum) livers.

Fatty acid synthesis from acetate 1J4C and pyruvate 2-14C by liver slices prepared from the livers of suckling mice remained low until about Day 20 postpartum then rose sharply to the level normally found in female adults fed the chow diet (Fig. 1). When pups were removed from the dam,

ABRAHAM

turn a fat-free diet for 3 days, no change in the level of hepatic fatty acid synthesis was observed (Fig. 1). Those animals weaned on Day 16 postpartum and fed the fat-free diet for 3 days showed rates of hepatic fatty acid synthesis equivalent to the level found in adults fed the chow diet. Pups 17 days of age or older, when weaned onto the fat- free diet showed levels of hepatic fatty acid synthesis as high as was observed in adults fed the same diet (Fig. 1). The specific ac- tivities of citrate cleavage enzyme, fatty acid synthetase, and acetyl CoA carboxylase (Fig. 2) followed a similar trend to that for the overall capacity of the tissue for fatty acid synthesis illustrated in Fig. 1.

In pups which remained with the dam, hepatic fatty acid synthetase, citrate cleav- age, and acetyl CoA carboxylase approached the adult level between Days 20 and 23 postpartum (Fig. 2). In pups which were weaned and given the fat-free diet for 3 days these same enzymes increased in activity at an earlier age and reached levels almost as high as found in adults fed the fat-free diet. This experiment demonstrated clearly that it is possible to prematurely induce a high level of hepatic fatty acid synthesis by weaning onto a fat-free diet. However, as

FIG. 2. Enzyme activities in livers from suck- Iing and weaning mice. Fatty acid synthetase in suckling (0) and weanling (u) mice and citrate cleavage enzyme in suckling (0) and weanling (0) mice are shown. The scale for activity of acetyl CoA carboxylase in suckling (A) and weanling (A) mice is one-tenth that of fatty acid synthetase and citrate cleavage enzyme. See Fig. 1 for further

before Day 16 postpartum, and fed ad l&i- details.

FATTY ACID SYNTHESIS IN DEVELOPING LIVER 115

TABLE I EFFECT OF DIET ON MOUSE MILK COMPOSITION”

Fatty acid

C 8:0 c lo:o c 12:o c 14:o C 16:0 C 16:l C 18:0 C 18:l C 18:2 C 18:3

Grams fat per 100 ml milk

Grams carbohydrate per 100 ml milk

Diet fed

Chow High-fat Fat-free

0.4 0.8 1.0 7.2 6.6 9.9

10.7 9.6 14.5 11.7 11.5 14.7 23.1 22.1 22.7 2.8 2.0 4.2 3.4 2.2 2.1

23.2 18.3 29.8 16.6 25.3 1.1 0.9 1.7 0.6

16.7 25.0 15.7

1.3 1.5 1.4

a Milk samples were obtained either from chow- fed dams or from dams fed the special diets from Day 6 to Day 15 postpartum. Certain trace com- ponents (less than 1%) have been omitted from the table. Fatty acid compositions are reported as percentage by weight. Gas chromatographic analyses were made on single samples. Fat and carbohydrate content was obtained by duplicate analysis of single samples.

pups removed from the dam before Day 16 postpartum ate very little food and lost weight, it was not possible to demonstrate whether the enzymes concerned with fatty acid synthesis could be induced at an earlier age.

Allmann and Gibson (20) have shown that the capacity of liver to synthesize long- chain fatty acids is increased during early linoleate deficiency in 4-week-old mice. Smith et al. (8) have shown that lactating C3H mice fed a fat-free diet for 3 days produce milk containing only trace amounts of linoleate whereas mice fed a corn oil- supplemented diet produce a linoleate-rich milk. If the livers of suckling pups are indeed capable of dietary adaptation, then one might expect to find different hepatic lipo- genie capacities and enzyme levels in ani- mals ingesting either a linoleate-rich or linoleate-deficient milk.

Lactating mice were therefore fed either the high fat or fat-free diet from Day 6 to Day 15 postpartum. Pups suckling a chow-

fed mother during this period normally show no change in lipogenic capacity (Figs. 1 and 2). On Day 15 postpartum, milk obtained from the dam fed the high-fat diet con-’ tained about 70% more fat than milk from the dam fed the fat-free diet (Table I). The most striking difference in milk fat composi- tion was in the linoleate content. Milk from the high-fat-fed dam contained 6.3 g lino- leate per 100 ml of milk, whereas that from the dam fed the fat-free diet contained 0.17 g linoleate per 100 ml.

The total lipid contents of pup livers from the two experimental groups were identical, about 5 % (Table II). The linole- ate content of liver from pups receiving the linoleate-rich milk was 1.6 g per 100 g liver (wet weight) and the oleate content was 0.57 g per 100 g liver. In contrast the liver linoleate content of pups ingesting the linoleate-deficient milk was 0.38 g per 100 g liver and the oleate content was 1.70 g per 100 g liver (Table II). Liver slices from pups receiving the linoleate-deficient milk incorporated two to three times more acetate and pyruvate into fatty acids than did liver slices from pups ingesting the linoleate-rich milk. Production of 14C02 from these sub- strates was about the same (Table III).

The activities of fatty acid synthetase and citrate cleavage enzyme were higher in

TABLE II EFFECT OF LINOLEATE-RICH AND LINOLEATE-

DEFICIENT MILK ON HEPATIC LIPID

COMPOSITION OF SUCKLING Pups6

Fatty acid [High-fat Fat-free

c 12:o 0.2 0.3 c 14:o 1.4 1.9 C 16:0 23.9 27.0 C 16:l 1.4 4.0 C 18:0 14.4 13.9 C 18:l 10.4 30.9 C 18:2 29.0 6.9 C 18:3 0.0 0.8 C 20:4 19.2 14.4

7. Lipid (wet wt.) 5.5 5.5

0 Livers were obtained from pups which had suckled dams fed the special diets from Day 6 to Day 15 postpartum. Fatty acid compositions are reported as percentage by weight. Data were obtained from analysis of single samples.

116 SMITH AND ABRAHAM

TABLE III EFFECT OF LINOLEATE-RICH AND LINOLEATE-DEFICIENT MILK ON

HEPATIC FATTY ACID SYNTHESIS IN SUCKLING Pupsa

Type of milk fed

Substrate Liioleate-rich Liioleate-deficient

Fatty acids CO2 Fatty acids COP

Pyruvate 1-W - 3980 f 65 - 4240 f 150 Pyruvate 2-14c 7.0 f 0.5 995 f 15 20.5 f 4.5 1145 f 60 Acetate 1-W 12.4 f 3.0 524 f 6.0 22.4 f 3.2 490 f 20

Q Livers were obtained from pups which had suckled dams fed the special diets from Day 6 to Day 15 postpartum. Activities are expressed as mfimoles substrate converted per 100 mg slice per 3 hr. Values are means of measurements on three separate liver pools, with standard errors.

TABLE IV EFFECT OF LINOLEATE-RICK AND LINOLEATE-DEFICIENT MILK ON

HEPATIC ENZYME ACTIVITIES IN SUCKLING PUPS’

EnWDle Type of milk fed

Linoleate-rich Liioleate-deficient

Acetyl CoA carboxylase 0.1 f 0.06 0.1 f 0.01 Fatty acid synthetase 2.1 f 0.2 5.4 f 0.2 Citrate cleavage enzyme 0.0 3.3 f 0.3 Malie enzyme 3.1 f 0.2 2.3 f 0.3

0 Livers were obtained from pups which had suckled dams fed the special diets from Day 6 to Day 15 postpartum. Activities are expressed as mpmoles substrate utilized per mg protein per minute. Values are means of determinations on three separate liver pools, with standard errors.

pups suckling dams fed the fat-free diet than in pups suckling the high-fat-fed dams (Table IV). Acetyl CoA carboxylase and malic enzyme activities showed no significant differences in the two groups of pups (Table IV).

To determine whether the increased hepatic lipogenic capacity observed at weaning was associated with enzyme syn- thesis, mice were given injections of actino- mycin D or puromycin during the weaning period. Oxidation of pyruvate and acetate to COZ by liver slices was slightly elevated in the case of the actinomycin D-treated animals and slightly depressed in experi- ments with puromycin-treated animals when compared to controls (Table V). In contrast, fatty acid synthesis in the control group of animals was considerably higher than in animals treated with actinomycin D or puromycin. In line with these observa- tions was the fact that the activities of citrate cleavage enzyme, fatty acid syn-

thetase, malic enzyme, and acetyl CoA carboxylase were much higher in the controls than in the experimentally treated groups (Table VI). These experiments suggest that inhibition of RNA and protein syn- thesis for 2 days completely abolishes the rise in lipogenic capacity associated with weaning, but has little effect on maintenance of citric acid cycle function.

An attempt was also made to determine whether at the weaning stage there was any correlation, on a short-term basis, between increased hepatic fatty acid-synthesizing capacity and the activity of certain en- zymes. Pups were therefore weaned on Day 17 postpartum by transfer to the fat-free diet for various lengths of time. Fatty acid synthesis from acetate and pyruvate was measured in liver slices and the activities of certain enzymes concerned with lipogenesis were determined. The results are shown in Figs. 3 and 4. Very little change in fatty acid synthesis from acetate or pyruvate

FATTY ACID SYNTHESIS IN DEVELOPING LIVER 117

TABLE V EFFECT OF ACTINOMYCIN D AND PUROMYCIN ON HEPATIC METABOLISM OF

ACETATE AND PYRUVATE IN RECENTLY WEANED PUPS’

Substrate

Pyruvate 1-W Pyruvate 2-W! Acetate l-r4C

Control Actimmycin D-treated Puromycin-treated

Fatty acid co, Fatty acid co2 Fatty acid co2

- 3380 - 3890 - 2950 70 700 5 890 10 450

110 140 4 209 14 84

0 Mice were weaned on Day 17 postpartum and were fed the fat-free diet ad lib&m for 2 days. Mice were given four intraperitoneal injections at 12-hr intervals. The control animals were given injections of 0.2 ml NaCl (0.9%), the second group 1 fig actinomycin D in 0.2 ml NaCl (0.9y0), and the third group 1 mg puromycin in 0.2 ml NaCl (0.9%). Each value represents the mean of determinations on two liver pools. Data are expressed as mctmoles substrate converted per 100 mg slices per 3 hr.

TABLE VI EFFECT OF ACTINOMYCIN D AND PUROMYCIN ON

HEPATIC ENZYME ACTIVITIES IN RECENTLY WEANED Pupsa

Acetyl CoA carboxylase 0.6 0.1 0.1 Fatty acid synthetase 19.3 0.7 2.0 Citrate cleavage 14.8 2.1 2.6 Malic enzyme 17.0 2.1 1.6

a Experimental conditions are described in Table V. Enzyme activities are expressed as mpmoles substrate utilized per mg protein per minute. Data represent means of determinations on three liver pools.

was observed during the first 29 hr after removal of the pups from the dams. During this time the pups ate very little food and usually lost weight (average loss about 2% of body weight). Fatty acid synthesis from pyruvate and acetate showed a dramatic increase during the second day after wean- ing, reaching a plateau between 48 and 72 hr after transfer to solid food (Fig. 3). On the average, mice increased their body weight by about 10% after 72 hr on the fat- free diet. Of the enzyme activities measured, fatty acid synthetase and citrate cleavage enzyme showed the largest increase in ac- tivity, about B-fold. The activity of fatty acid synthetase reached a maximum value before any of the other enzymes. Acetyl CoA carboxylase and malic enzyme activities continued to increase during the third day

after weaning during which time there was no increase in the hepatic lipogenic capacity. Glucose 6-phosphate dehydrogenase showed a comparatively small increase in activity, approximately 2-fold during the weaning period (Fig. 4).

DISCUSSION

Weber et al. (4) showed that the activity of hepatic glucokinase and of pyruvate

Hourr waonad

FIG. 3. Synthesis of fatty acids from acetate and pyruvate in weanbng mice. On Day 17 post- partum mice were removed from the dam and supplied with the fat-free diet for the periods in- dicated. Incorporations of acetate 1-W (0) and pyruvate 2-W (m) into fatty acids are expressed relative to the values obtained from 17-day-old suckling mice which are arbitrarily given the value of unity. At 17 days postpartum the actual values were 9.0 nmmoles pyruvate incorporated per 100 mg slice per 3 hr and 9.2 mpmoles acetate incorporated per 109 mg slice per 3 hr. Each point represents the mean of determinations on 6-15 liver pools.

11s SMITH AND ABRAHAM

How weaned

FIG. 4. Enzyme activities in weanling mice. See Fig. 3 for details. The activities of fatty acid synthetase (w, 1.2 mpmoles/mg protein/min), citrate cleavage enzyme (0, 1.0 mpmoles/mg pro- tein/min), acetyl CoA carboxylase (A, 0.15 mpmoles/mg protein/min), malic enzyme ((>, 4.2 wmoles/mg protein/min) and glucose B-phos- phate dehydrogenase (X, 7.1 mFmoles/mg pro- tein/min) in 17-day-old suckling mice were arbi- trarily assigned the value of unity.

kinase in young rats increased during Days 18 to 23 postpartum whether or not the pups remained with the dam. On the basis of this observation these workers ruled out the possibility that the increases in enzyme activities were induced by a dietary change. However, no mention was made of any precautions taken to ensure that the young pups had no access to the dam’s food. Data presented here show that when young mice are left with the dam an increase in the hepatic lipogenic capacity of the pup is observed between Day 20 and Day 23 postpartum. During this period these young mice ate the diet put in the cage for the dam. It is possible therefore that the increase in hepatic fatty acid synthesis we observed might have been induced by a dietary change rather than a genetically controlled develop- mental factor.

Weaning young mice onto a fat-free diet on or after Day 16 postpartum resulted in a premature increase in both hepatic lipo- genie capacity and the activities of fatty acid synthetase, citrate cleavage enzyme, and acetyl CoA carboxylase above the levels found in adult animals fed a chow diet. As has been pointed out by Walker and Eaton (al), it is extremely difficult to test the

effect of synthetic diets on young rats be- fore Day 15 postpartum because of the problems involved in feeding such immature animals. We, as well, found it impossible to successfully wean young mice onto pelleted, powdered, or fluid diets before Day 16 post- partum. Although Walker and Eaton (21) administered a high glucose diet by stomach tube to rat pups during the day and re- turned the pups to the lactating dam over- night, they could demonstrate no induction of hepatic glucokinase before Day 17 post- partum.

In a previous study (8) we showed that feeding lactating mice diets of varying fat contents changed the fat composition of the milk. A practical approach to testing responses of hepatic lipogenesis to dietary manipulation in very immature animals therefore seemed to be in changing the composition of the milk secreted by the dam. Thus, by feeding the lactating mice either a high-fat or fat-free diet, we were able to effectively feed the suckling mice milk of differing fat content.

Pups ingesting the relatively low fat (16 g/100 ml milk), low linoleate (1% of the fatty acids) milk showed low levels of hepatic linoleate compared with pups in- gesting the high fat (25 g/100 ml milk), high linoleate (25% of the fatty acids) milk. According to Allmann and Gibson (20), the capacity of mouse liver to synthesize fatty acids is enhanced during early linoleate deficiency. Furthermore, Sabine et nl. (22), after feeding various fat diets to mice, showed that only diets high in linoleate suppressed hepatic fatty acid synthesis. Our present data indicate that hepatic fatty acid synthesis in suckling mice can be influ- enced by diet since pups ingesting the low- fat, low-linoleate milk between Days 6 and 15 postpartum showed rates of hepatic fatty acid synthesis twice as high as pups ingest- ing the high-fat, high-linoleate milk. While there was no significant difference in the activities of acetyl CoA carboxylase and malic enzyme in these two experimental groups, the activities of fatty acid synthetase and citrate cleavage enzyme were higher in the livers of pups ingesting the relatively low-fat, low-linoleate milk. Significantly

FATTY ACID SYNTHESIS IN DEVELOPING LIVER 119

perhaps, these latter two enzymes were the ones found to increase to the greatest extent when pups were weaned from Day 17 postpartum onto fat-free diets (Fig. 4).

This study illustrates clearly that the course of hepatic enzymic differentiation can be modified by dietary changes. We would emphasize, however, that no con- sideration has been given to other possible factors, such as hormonal effects, and we do not therefore exclude the possibility that the natural development of certain liver func- tions may involve stimuli other than those of dietary origin.

The supply of acetyl CoA from pyruvate can be estimated as equivalent to the amount of 14C02 produced from pyruvate l-14C. In all the conditions investigated, since “CO2 production from pyruvate 1J4C is in great excess over the amount of fatty acids pro- duced from pyruvate 2-14C, there appears to be an abundant supply of intramito- chondrial acetyl CoA. Synthesis of fatty acids from pyruvate 2-14C occurred at ap- proximately the same rate as that from acetate l-l% under all conditions, indicat- ing that transport of pyruvate into the mitochondria and transport of acetyl CoA (as citrate presumably) out of the mito- chondria does not limit the rate of fatty acid synthesis. Therefore it seems likely that under our conditions, the rate-control- ling step in fatty acid synthesis occurs after transfer of acetyl CoA out of the mitochon- dria. Regen and Terre11 (23), using perfused rat liver found that the incorporation of pyruvate 3-14C and acetate 2J4C into choles- terol relative to ketone bodies was essentially the same. They, too, concluded that trans- port of acetyl groups across the mitochondria occurred rapidly compared to the turnover of the acetyl CoA pool.

The abolishment of the rise in hepatic lipogenic capacity and enzyme activities by puromycin and actinomycin D is strong evidence that increases in the amount of certain liver enzymes are closely associated with the increased capacity of the tissue for fatty acid synthesis. We therefore care- fully considered the status of each of the enzymes to determine whether any one of them might be implicated in a primary

mechanism for control of hepatic lipogenesis in the developing liver.

The activity of acetyl CoA carboxylase was found to be considerably lower than fatty acid synthetase, citrate cleavage en- zyme, and malic enzyme under all condi- tions examined. The highest activity of this carboxylase in livers of C3H mice (1.3 mpmoles/mg protein/mm) is considerably lower than that reported by Greenspan and Lowenstein (24) for the hepatic enzyme in Sprague-Dawley rats (30 mpmoles/mg pro- tein/mm) and C57BL/6J mice (5.1 mp- moles/mg protein/min). Greenspan and Lowenstein (24) reported that activation of acetyl CoA carboxylase by MgZ+ and cit- rate was strongly inhibited by the pres- ence of ATP in the preincubation medium. Since our preincubation medium rou- tinely contained Mg2+, citrate, and ATP, we also measured activity in the assay system described by Greenspan and Lowenstein (24). Activities measured in this way were identical with activities measured by our assay system (unpublished observations). Even at this low level, the activity of acetyl CoA carboxylase was always con- siderably more than sufficient to account for the observed rate of fatty acid synthe- sis in liver slices from the same animal.2

Comparison of rates of incorporation of acetyl CoA and malonyl CoA into fatty acids and direct measurements of acetyl CoA carboxylase activity in tissue homo- genates have lent support to the theory that acetyl CoA carboxylase is the rate-limiting enzyme of fatty acid synthesis (25-32). In the present study however, we have ob- served two conditions where changes in hepatic lipogenic capacity were not paral- leled by changes in activity of acetyl CoA

2 For example: Incorporation of acetate into fatty acids equals 110 m~moles/lOO mg slice/3 hr at 37” (Control group, TableV). Since tissue slices contain 16.3 mg protein per 100 mg wet weight, this is equivalent to 0.61 mpmoles/l6.3 mg slice protein/min or 0.038 mpmoles/mg slice protein/ min at 37”. Acetyl CoA carboxylase activity equals 0.6 -moles/mg “100,000 g” protein/min at 30” (Control group, Table VI). Since the ‘WO,OOO g” protein accounts for approximately 35% of the total tissue protein, this is equivalent to 0.21 mpmoles/mg slice protein/min at 30”.

120 SMITH AND ABRAHAM

carboxylase (Figs. 3 and 4, Tables III and IV). We conclude, therefore, that carboxyla- tion of acetyl CoA in the liver cell proceeds at a rate considerably below its optimum and that the amount of this enzyme present in the liver cell is not the rate-controlling factor in fatty acid synthesis.

In this study changes in the activity of citrate cleavage enzyme parallel quite closely changes in hepatic lipogenic capac- ity. When young mice were weaned onto a fat-free diet, citrate-cleavage activity in- creased at about the same time as the over- all lipogenic capacity (Figs. 3 and 4). We did not observe a lag in the induction of citrate- cleavage activity behind the increase in lipogenic capacity as described by Foster and Srere in the case of fasted and refed rats (6). As stated earlier, under the condi- tions of our experiments, acetate and pyru- vate were equally effective as precursors for fatty acid synthesis and therefore citrate cleavage activity appears to always be main- tained at a level so as not to limit the rate of fatty acid synthesis. Glucose 6-phosphate dehydrogenase activity increased 2-fold after weaning mice onto a fat-free diet for 3 days (Fig. 4). The relatively small magni- tude of this change makes it unlikely that supply of reduced pyridine nucleotide by this enzyme is of rate-controlling significance under these conditions. Although malic en- zyme increased in activity after weaning, the change did not follow the same time course as the change in overall lipogenic capacity (Figs. 3 and 4). Furthermore, the activity of malic enzyme in livers of mice receiving the linoleate-deficient milk was not higher than in livers of mice receiving the linoleate-rich milk whereas the lipogenic capacity was higher in the former group of animals. Although the activity of fatty acid synthetase is always much higher than that of acetyl CoA carboxylase, changes in fatty acid synthetase activity correlate remark- ably well with changes in the capacity of liver slices for fatty acid synthesis. On this basis it is tempting to propose that changes in the level of fatty acid synthetase activity might directly influence lipogenic capacity. However, we consider that measurement of enzyme activities in tissue homogenate fractions is more a measure of the total

amount of that enzyme present in the tissue than a measure of the actual activity in the tissue where such factors as concentration of inhibitors and activators may exert considerable influence. This is especially important in conditions where metabolic throughput measured in tissue slices (e.g., acetate or pyruvate incorporation into fatty acids) is lower than the activity of any of the enzymes measured. Nevertheless, it is evident in this study that one of the earliest changes observed under conditions where hepatic lipogenic capacity is altered is in the level of fatty acid synthetase enzyme present.

The primary mechanism responsible for triggering changes in lipogenic activity is still not fully understood. Allmann and Gibson (20) suggested that the increase in fatty acid synthesis observed after ingestion of a fat-free diet may involve either (a) reduction in the concentration of inhibitors of lipogenesis, such as free long-chain fatty acids or fatty acyl CoA’s, or (b) an increase in the level of enzyme protein. More re- cently, Muto and Gibson (33) have demon- strated a correlation between free linoleate and arachidonate levels and hepatic fatty acid synthetase activity. We have shown here that the increased capacity of the neonatal liver for fatty acid synthesis brought about by feeding low-fat, low-lino- leate diets, involves both synthesis of new enzyme and reduction in total liver linoleate content. The experiments described in Tables I and IV showed that hepatic lipo- genie capacity of pups ingesting a linoleate- deficient milk was twice as high as in pups receiving a linoleate-rich milk. Since the linoleate-rich milk contained more total fat than the linoleate-deficient milk, it might be argued that the difference in hepatic lipogenic capacity is a result of the different quantities of fat in the diet rather than the linoleate content specifically. However, milk from chow-fed dams con- tains the same amount of fat as milk from dams fed the fat-free diet. The major dif- ference in the two types of milk is in the linoleate content, 16.6% in the chow-fed dam, 1.1% in the fat-free-fed dam. In pups suckling the fat-free-fed dam, hepatic fatty acid synthesis from acetate (22 mpmoles

FATTY ACID SYNTHESIS IN DEVELOPING LIVER 121

per 100 mg slice per 3 hr) and pyruvate (20 mpmoles per 100 mg slice per 3 hr) was significantly higher than from acetate (4 rrqumoles per 100 mg slice per 3 hr) and pyruvate (6 wmoles per 100 mg slice per 3 hr) in pups of similar age suckling a chow- fed dam (Table III and Fig. 1). Since the carbohydrate content of milk from chow- fed, high-fat-fed, and fat-free-fed dams is identical, it is unlikely to play any signifi- cant role in the induction of hepatic lipo- genesis under these conditions. We would therefore support the theory that in the mouse, regulation of hepatic lipogenesis by dietary fat is specifically due to the linoleate content (20, 22). The fact that fatty acid synthetase is inhibited fairly nonspecifically by free long-chain fatty acids (31) would tend to speak against the idea that free linoleate levels might directly control the activity of fatty acid synthetase. The pos- sibility that free linoleate is involved in repression and derepression of enzyme syn- thesis however, cannot be ruled out.

ACKNOWLEDGMENTS

The authors thank Mrs. Shirley Barber, Miss Inger Holst, Mrs. Maureen Reiner, and Miss Carol Robertson for their excellent technical as- sistance.

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