Regulation of mouse hepatic ATP-citrate lyase activity by dietary fat evidence for the presence of inactive enzyme

101

Biochimica et Biophysica Acta 714 101-113

Elsevier BiomedicalPress BBA 21025 REGULATION OF MOUSE HEPATIC ATP-CITRATE LYASE ACTIVITY BY DIETARY FAT EVIDENCE FOR THE PRESENCE OF INACTIVE ENZYME ROBERT S. SCHWARTZand S. ABRAHAM * Bruce Lyon Memorial Research Laboratory, Children's Hospital Medical Center, 51st and Grove Streets, Oakland, CA 94609 (u.s.A.)

(Received May 20th, 1981) (Revised manuscript received August 21st, 1981)

Key words: A TP-citrate lyase; Dietary fat; Enzyme reglulation; (Mouse liver)

The induction of ATP-citmte lyase activity in mouse liver by dietary carbohydrate (glucose) is markedly reduced by including in the diet a source of polyunsaturated fatty acids. Within 72 h after changing from a standard mouse chow diet to a high carbohydrate diet containing 15% (w/w) of hydrogenated cottonseed oil (as a source of saturated fatty acids), the activity of mouse liver ATP-citrate lyase per milligram cytosolic protein was approx. 3-fold higher than that from mice fed a similar diet containing 15% (w/w) of corn oil. The rate of synthesis of ATP-citrate lyase relative to that for total protein and the rate of degradation of the enzyme were similar for both dietary groups. Elevated levels of enzyme activity in the hydrogenated cottonseed oil-fed livers were not accompanied by a similar increase in the amount of enzyme protein. To explain such fmdings, we propose that the activity of hepatic ATP-citrate lyase is regulated by dietary polyunsaturated fatty acids through a mechanism involving the conversion of a catalytically active form of the enzyme to a catalytically inactive form. A reversal of this conversion (inactive-active) is evident within 72 h of removing the mice from the corn oil diet and placing them on the hydrogenated cottonseed oil diet. Furthermore, the conversion appears to be independent of the in vivo rate of synthesis of the enzyme.

Introduction ATP-citrate lyase (EC 4.1.3.8), which is involved in the process of transfering acetyl groups across mitochondrial membranes [1 ] and is, therefore, considered a lipogenic enzyme, undergoes adaptive changes in response to dietary [2-4] and hormonal manipulation [3]. For example, Bartley and Abraham [2] have demonstrated that the specific activity of ATP-citrate lyase was 3- to 10-fold higher in the livers of both rats and mice fed a high carbohydrate diet containing saturated fat than in animals maintained on a similar diet containing polyunsaturated fat.

* To whom correspondence should be addressed.

Gibson et al. [5] showed that the increase in hepatic ATP-citrate lyase activity observed upon refeeding a high carbohydrate fat-free diet to starved rats was accompanied by an increase in the synthetic rate of the enzyme. Spence et al. [6] however, found that the decrease in ATP-citrate lyase activity they observed when cultured rat hepatocytes were treated with glucagon or dibutyryl-cyclic AMP, could not be explained entirely by a decrease in enzyme synthesis. It seems likely, therefore, that the mechanism whereby ATP-citrate lyase activity is regulated by dietary or hormonal manipulation may be unique for each treatment. The purpose of the present communication was to shed light on the mechanism whereby dietary polyunsaturated fatty acids inhibited the activity of hepa-

0304-4165/82/0000-0000[$02.75 © 1982 Elsevier BiomedicalPress

102 tic ATP-citrate lyase in carbohydrate-fed mice. Here we present experiments which show a marked increase in the amount of immunologically reactive, but catalytically inactive enzyme in the livers of mice fed a glucose diet containing 15% (w/w) corn oil. Furthermore, we showed that in the livers of animals fed identical diets in which the corn oil was replaced by hydrogenated cottonseed oil, conversion of a less active form of the enzyme to a more active form resulted. This conversion occurred between 1 and 3 days after the mice were fed the diets and appeared to be independent of the rate at which the enzyme was synthesized in vivo.

Experimental Procedures Materials L-[4,5-aH]Leucine (specific activity 76 Ci/mol) was obtained from Amersham, Arlington Heights, IL; CoASH, ATP, NADH and fat-free bovine serum albumin Fraction V were from Sigma Chemical Co., St. Louis, MO. Malate dehydrogenase was obtained from Calbiochem-Behring Corp., La Jolla, CA; sheepanti-rabbit y-globulin from Antibodies Inc., Davis, CA; Protosol from New England Nuclear, Boston, MA. Vitamin-free casein, glucose, salt mixture XIV, cellulose and vitamin mix were purchased from ICN Nutritional Biochemicals, Cleveland, OH. Hydrogenated cottonseed oil flakes were a gift from PVO International, Richmond, CA; corn oil was purchased in the market place, and defatted liver powder was obtained from Viobin Corp., Monticello, IL. Freund's complete and incomplete adjuvant was obtained from Difco Laboratories, Detroit, MI. Coomassie Brilliant Blue R-250 was purchased from Bio-Rad Laboratories, Richmond, CA. Animals and diets Male BALB/c mice were purchased from Simonsen Laboratories, Gilroy, CA. Mice weighed 2 0 - 2 5 g when received and were maintained on Purina Mouse Chow 9F diet (containing 9% lipid) for at least 7 days before the start of each experiment. They were housed in metal cages, three mice per cage, in a room with 12 h/12 h controlled lighting (light from 8 a.m. to 8 pm). All mice had free access to food and water at all times. At the beginning of an experimental period,

TABLE I FATTY ACID COMPOSITIONOI: THE DIETS Samples of the hydrogenated cottonseed oil and corn oil diets were saponified and analyzed by gas liquid chromatography as previously described [2]. Results are expressed as percentage of total fatty acids. Fatty acid

Hydrogenated cottonseed oil

Corn oil

14:0 16 : 0 16:1 18 : 0 18 : lo:9 18 : 2w6 18 : 3oo3 Unidentified long chain fatty acids above 18 : 3

2 27 ! 64 5 0 0

0 12 0 2 24 55 1

1

5

random groups of mice were switched from the Purina Mouse Chow diet to one of the purified diets containing either 15% (w/w) hydrogenated cottonseed oil or 15% (w/w) corn oil. The composition of these diets was the same as that reported previously [7]. The fatty acid composition of the purified diets are reported in Table I.

Preparation of liver cytosol and measurement of ATPcitrate lyase activity Mice were killed by cervical dislocation and the livers were quickly removed, weighed and placed in ice-cold 0.25 M sucrose. The livers were then homogenized in a Potter-Elvehjem homogenizer with 3 vol. ice-cold 0.25 M sucrose. Homogenates were centrifuged for 1 h at 100000 × g and the clear supernatant fractions (cytosol) were separated and used for all enzyme assays and subsequent experiments. All preparative procedures were carried out at 0-4°C. ATP-citrate lyase (EC 4.1.3.8) was assayed according to published methods [8]. 1 unit of ATP-citrate lyase is defined as the amount of enzyme necessary to catalyze the oxidation of 1 nmol NADH/min at 30°C. Specific enzyme activity is defined as units of enzyme activity per mg cytosolic protein. Protein determination Soluble protein was estimated by the microbiuret

103 method [9], using fatty acid-free bovine serum albumin as standard.

Preparation of antisera specific for mouse liver A TPcitrate lyase ATP-citrate lyase was purified to homogeneity, as evidenced by a single protein band of M r 110000 upon SDS-polyacrylamide gel electrophoresis (data not shown), from the livers of mice previously fed a 50% glucose fat-free diet for a minimum of 7 days, according to the procedure of Linn and Srere [10]. The purified enzyme had a specific activity of 6000 units/mg protein. To prepare anti-ATP citrate lyase antibody, 125 ~tl aliquots (containing 250 /.tg pure enzyme) were mixed with an equal volume of Freund's complete adjuvant and injected subcutaneously into a 3.2 kg male New Zealand rabbit. 2 weeks later, two boosters, each containing 125 /.Lg antigen in an equal volume of Freund's incomplete adjuvant, were injected subcutaneously at 10 day intervals. 1 week following the last injection the rabbit was bled from the marginal ear vein and the serum was collected. Pooled serum samples containing a high titer for antiATP-citrate lyase antibodies, as determined by Ouchterlony double diffusion analysis [11 ], were subjected to precipitation with ammonium sulfate. The protein which precipitated between 0-40% saturation at 0-4°C was dissolved in 0.02 M sodium phosphate, pH 7.2, 0.15 M NaC1 and stored in aliquots at -70°C. Synthesis and degradation of A TP-citrate lyase Synthesis of ATP-citrate lyase was determined by the following procedure. Mice fed the two experimental diets for specific time periods of from 1 to 11 days were injected intraperitoneally with 0.1 ml containing 100 laCi L-[4,5,3H]leucine in water and then killed after 1h. From each liver, cytosol was prepared. To determine total protein synthesis, 10 aliquots of cytosol were spotted on Whatman 3 MM filter paper discs (Whatman Ltd., U.K.), protein was precipitated with hot trichloroacetic acid and the disc were washed according to the procedure of Mans and Novelli [12]. Protein on the filter discs was then assayed for radioactivity in 10 ml scintillation fluid containing 2 vols. 0.5% Omniflour (New England Nuclear, Boston, MA) in toluene and 1 vol. 2-ethoxyethanol (Mallinckrodt,

St; Louis, MO) in a liquid scintillation spectrometer. To determine ATP-citrate lyase synthesis, 0.2 ml aliquots of the cytosol were incubated for 1 h at 37°C in a total volume of 1 ml in buffer 1 (10 mM sodium phosphate, pH 7.2, 150 mM NaC1, 1 mM disodium EDTA, 1 mM phenylmethylsulfonylflouride, 5 mM Lleucine, 1% Triton-X 100, 1% sodium deoxycholate) and an amount of anti-ATP-citrate lyase antibody sufficient to neutralize at least 1.5-times the enzyme activity present. Following incubation, an amount of sheep anti-rabbit 7-globulin equal to 4-times the antiATP-citrate lyase protein was added and the mixture was allowed to form a precipitate overnight at 4°C. The precipitate was collected by centrifugation at 4°C through a 1 M sucrose cushion in buffer 1 at 1000 X g for 30 min and washed three times (or until the washings contained background levels of tritium). The resultant supernatant fractions were routinely assayed for ATP-citrate lyase activity: none was found, indicating complete immtmoprecipitation of the enzyme. Additional aliquots of these supernatant fractions were subjected to SDS-polyacrylamide gel electrophoresis [13], and in no case did the gels (following washing, fixing and staining) show a protein band which corresponded to ATP-citrate lyase, not did we f'md radioactivity in gels that were sliced corresponding to that of the enzyme. The immunoprecipitated enzyme pellets were dissolved at 90°C in 0.02 M sodium phosphate, pH 7.2 containing 0.1% SDS and aliquots were assayed for radioactivity as given above. Other portions of the washed immunoprecipitates were subjected to SDSpolyacrylamide gel electrophoresis in gels containing 5% acrylamide, essentially as described by Weber and Osborn [13]. Staining of the gels with Coomassie Brillant Blue R-250 and destaining was done as described by Weber and Osborn [13]. Following electrophoresis, the gels were cut into 2 mm slices, and each slice was placed in a glass scintillation vial to which 1 ml 95% (v/v) Protosol was added. Digestion of the slices proceeded for 2 h at 55°C, after which the vials were cooled, 10 ml toleune containing 0.5% (w/v) Omnifluor were then added, together with 0.025 ml glacial acetic acid to neutralize the protosol. The samples were then assayed for radioactivity in a liquid scintillation spectrometer. Separate gels containing pure proteins (fatty acid synthetase (220 000), bovine serum albumin (67 000), ovalbumin (43 000)

104 and chymotrypsin (25 000)) were run simultaneously to allow for the estimation of the molecular weights of the radioactive peaks derived from the gels containing the all-labeled immunoprecipitated ATPcitrate lyase. Degradation of ATP-citrate lyase was measured essentially as described above except that the mice received an injection of 100/.tCi [3H]leucine immediately before being placed on one of the two experimental diets. Groups of mice were killed at various times thereafter from 1 to 11 days, as indicated.

Antibody-antigen titration Titrations were carried out with a constant amount of antibody (100/~g) against varying amounts of cytosolic antigen. Antibody and antigen were incubated in a total volume of 0.1' ml cqntaining 100 mM potassium phosphate, pH 7.0, 10% glycerol, 1 mM MgC12, 0.1 mM disodium EDTA, and 5 mM dithiothreitol for 1 h at 37°C, and were then allowed to stand overnight at 4°C. The immunoprecipitates which formed were collected by centrifugation at 1 0 0 0 0 X g for 20 min at 4°C, and the supernatant fractions were assayed for ATP-citrate lyase activity as above.

Statistical analyses were carried out using the 2tailed Student's t-test. Where a statistical analysis is not presented, no differences between dietary groups were noted at P < 10% probability. Results

Dietary-induced changes in the specific enzyme activity of mouse liver A TP-citrate lyase When mice were switched from the Purina Mouse Chow diet to one containing 15% hydrogenated cottonseed oil, the specific activity of liver ATPcitrate lyase in the cytosols increased approx. 5-fold over the 11-day period (Fig. I). The increase became apparent after 3 days on this diet and continued over the next 8 days, although a slight decrease was ob-

24

21

._= 18

Rocket immunoelectrophoresis This was performed essentially as described by Weeke [14], using a Pharmacia flat bed electrophoresis apparatus. 1% agarose gels (22 × 11 X 0.I cm) containing 2.3% anti-ATP-citrate lyase antibody were formed in barbital/glycine/Tris-HC1 buffer, ionic strength I = 0.02 ~ [14]. A similar buffer, but with I = 0.04 /.t was used as electrode buffer. Cytosols from hydrogenated cottonseed oil-fed or corn oil-fed livers were diluted 3:1 (v/v) with barbital/glycine/ Tris-HC1 buffer, I = 0.08/.t and 8 /.tl samples were applied to wells 3.5 mm in diameter. To confirm the proportionality of rocket height to enzyme protein, each sample was run at several dilutions. For each experiment, all samples were run on the same gel on the same day. A voltage of 4 V/cm was applied and electrophpresis was allowed to proceed overnight at 10°C. At the completion of the run, plates were washed in running tap water, pressed dry and then stained for protein with Coomassie Brilliant Blue R-250 [14]. Rocket heights were measured from the center of each well to the rocket peak.

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5 ~

0

i

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4

6

8

I0

12

Days on diet

Fig. 1. Effect of dietary fat on the specific activity of mouse liver ATP-citrate lyase. Mice maintained on Purina Mouse Chow were allowed to eat ad libitum a high carbohydrate diet containing either 15% hydrogenated cottonseed oil (e e) or 15% corn oil (o o) for from 1 to 11 days. Liver cytosols were prepared on the days indicated and ATP-citrate lyase measured. Each point represents the mean and the vertical bars the S.E. for three animals, assayed individually.

105 TABLE 11 THE EFFECT OF DIETARY FAT ON THE ABILITY OF HEPATIC CYTOSOLS TO INHIBIT PURE ATP-CITRATE LYASE ACTIVITY 100 ul cytosols from livers of mice fed the diets indicated below for the periods of time specified, whose ATP-citrate lyase activities were known, were incubated with pure ATP-citrate lyase (22.5 #g containing 135 units activity) for 15 min at 30°C. Following incubation, the mixtures were assayed for ATP-citrate lyase activity. Each value represents the mean ± S.D. of three animals, determined individually. Days on diet

Diet Hydrogenated cottonseed oil Before incubation (A)

1

3 6 11

143±

1

154± 7 175 ± 22 185 ± 14

After incubation (B)

Corn oil % Change (B-A/B)

Beforeincubation (C)

After incubation (D)

% Change (D-C/D)

143±13

0

146±2

159±2

4

177±18 189 ± 15 192 ± 25

13 7 4

144± 1 146 ± 2 144 _*1

167±3 162 ± 7 166 ± 4

14 10 13

served between the third and sixth day. In contrast, the specific activity of the enzyme from the 15% corn off-fed mice remained unchanged over the entire 11-day period. That the mice were consuming each diet was apparent from the steady gain in body weights (data not shown).

ls the corn oil-effect due to the presence o f an inhibitor o f hepatic A TP-citrate lyase ? To investigate the possibility that the inhibition caused by dietary corn off of the induction of hepatic ATP-citrate lyase activity by dietary carbohydrate was due to the presence in the cytosols of an inhibitor of the enzyme, the following experiment was performed: When cytosols from both hydrogenated cottonseed oil and corn off-fed mice were incubated with a known amount of pure ATP-citrate lyase, no inhibition of enzyme activity was noted foUowing the incubation (Table II). These results suggest, but do not prove, that the cytosols from the corn off-fed mouse livers did not contain an inhibitor of ATPcitrate lyase activity. Since a 'tightly bound' inhibitor might not dissociate from the enzyme into the reaction mixture, it could escape detection in this system. Synthesis and degradation o f mouse liver A TP-citrate lyase in response to dietary fat feeding To confirm that the material immunoprecipitated

by anti-ATP-citrate lyase antibody was indeed ATPcitrate lyase, immunoprecipitates of cytosols from the corn off or hydrogenated cottonseed off-fed mice (which had been labeled in vivo with L-[4,5)H]leucine) were dissolved and subjected to SDS-polyacrylamide gel electrophoresis. The gels were sliced and slices were assayed for radioactivity. The results obtained for a typical gel are presented in Fig. 2. In general, two regions of radioactivity were observed, one which corresponded to a polypeptide of M r 111 000, and another to a polypeptide o f M r 57 000~ It is interesting to note in this connection that Singh et al. [16] have reported that the pure rat liver enzyme showed fragments with molecular weights of 74000 and 57000 as a result of 'nicking' of the intact subnnit o f M r 125 000. It is therefore possible that the M r 57 000 polypeptide we observed represents a fragment of the intact subunit o f M r 111 000. When pure ATP-citrate lyase (128/.Lg) was added to the all-labeled mouse liver cytosols immediately prior to immunoprecipitation, both of the major peaks (Mr 110000 and M r 57000) that were displayed following SDS-polyacrylamide gel electrophoresis disappeared. Such a result demonstrates that the anti-ATP-citrate lyase antibody used to immunoprecipitate all.labeled cytosolic ATP-citrate lyase recognized both the pure and cytosolic enzymes, and that the two major polypeptides observed following

106 I

I

I

I

SDS-polyacrylamide gel electrophoresis represented authentic ATP-citrate lyase. The monospecificity of the preparation of antiATP-citrate lyase antibody we used was further demonstrated by the formation of a single precipitin line as a result of Ouchterlony double diffusion analysis [11 ] conducted against both the pure enzyme and the mouse liver cytosol (data not shown). The rate of synthesis of mouse liver ATP-citrate lyase relative to that for total protein synthesis (relative synthetic rate) increased approx. 50%

I

57K

12

I0

IIIK

_o x

8

0

I0

20

50

40

50

60

Gel slice number

Fig. 2. SDS-polyacrylamide gel electrophoresis of immunoprecipitated cytosolic ATP-citrate lyase labeled with [3H]leucine in vivo. Animals were injected with L-[4,5-3H] leucine, killed 1 h later and cytosols were prepared from the livers. Where indicated, pure mouse liver ATP-citrate lyase (128 #g) was added to an amount of cytosol calculated to contain 7 ,ug enzyme immediately before the addition of

anti-ATP-citrate lyase ant~ody. Immunoprecipitation of the total ATP-citrate lyase and SDS-polyacrylamide gel electrophoresis analysis of the solubilized immunoprecipitates was carried out. Following electrophoresis, gels were sliced and each slice was assayed for radioactivity following digestion in Protosol. No differences were observed in the pattern of radioactivity found in the ATP-citrate lyase preparation from the livers of the hydrogenated cottonseed oil or the corn oil-fed mice throughout the 11 days of feeding. A typical pattern is presented above (1-day corn oil-fed mouse liver). The molecular weights of the two major peaks are also given and were determined from standards, o o, no addition of pure ATP-citrate lyase; [] t~, addition of 128/Jg pure ATP-citrate lyase.

TABLE III INCORPORATION OF [3H]LEUCINE INTO LIVER ATP-CITRATE LYASE AND SOLUBLE LIVER PROTEINS IN CORN OIL-FED AND HYDROGENATED COTTONSEED OIL-FED MICE Mice received a single intraperitoneal injection of 100 ~Ci L-[4,5-3H]leucine. The mice were killed 1 h later, the livers were rapidly removed, and ATP-citrate lyase was immunoprecipitated. The data are presented at disintegration per minute of total soluble protein or ATP-citrate lyase contained in 0.2 ml cytosol. Each value represent the mean ± S.D. of three animals, determined individually. Diet

Hydrogenated cottonseed oil

Corn oil

Days on diet

[3 H ] leucine incorporated into:

ATP-citreate lyase/total soluble protein (Xl0 -2)

Relative rate of synthesis

Total soluble protein (dpm × 10 -3)

ATP-citrate lyase (dpm × 10 -2)

1 3 6 11

18.1~4.3 22.3±3.3 19.0±0.2 19.5±3.9

9.9±3.9 18.2~4.0 8.9±1.5 9.2±4.3

5.5 8.2 4.7 4.7

1.0 1.5 0.8 0.8

1 3 6 11

19.1±2.1 22.1±3.6 23.7±2.6 21.5±2.3

12.6±4.9 22.3±1.7 22.4±5.4 22.3±8.9

6.6 10.1 9.4 10.4

1.0 1.5 1.4 1.6

107

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25

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12.5

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2

4

6

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Days on diet

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Fig. 3. Rates of degradation of ATP-citrate lyase and soluble protein from the livers of mice consuming 15% hydrogenated cottonseed oil (¢ =) or 15% corn oil (o o) diets. Animals were injected with L-[4,5-aH]leucine and killed at the times indicated. Three mice were killed at each time point. Radioactivity in hepatic ATP-citrate lyase protein was determined after immunoprecipitation and that in total soluble liver protein after trichloroacetic acid precipitation. Results are expressed as the percentage of initial radioactivity remaining on the days indicated in (A) ATP-citrate lyase and (B) total soluble protein contained in 0.2 ml liver cytosol. The lines represent the best fit to the data as determined by least squares analysis. Half4ives (t I/2) were calculated and are presented for each dietary group. Each value represents the mean -+ S.D. for three animals, determined individually.

between the 1st and 3rd day of feeding in both the hydrogenated cottonseed oil-fed and corn oil-fed animals (Table III). Between the 3rd and 1 lth day of feeding, the relative rates of synthesis declined to about those found at the 1-day level in the hydrogenated cottonseed oil-fed mice while those in the corn oil-fed group remained unchanged. The half-life of ATP-citrate lyase calculated for the hydrogenated cottonseed oil-fed mice was found to be 3.8 days, which was very similar to the half-life of 4.4 days for the enzyme from corn oil-fed mice (Fig. 3). These values are very different from those reported by Gibson et al. [5] for rat liver (14 h) and Spence et al. [6] for cultured rat hepatocytes (18 h) and may be ascribed to differences either in species or dietary treatments. It is interesting to note that the half-lives calculated for total liver protein were similar for both hydrogenated cottonseed oil-fed (3.9 days) and corn oil-fed (5.6 days) mice (Fig. 3B). These values were also similar to those found for the half-lives of hepa-

tic ATP-citrate lyase in the same groups of animals. Since the values for the turnover rates of ATP-citrate lyase in the hydrogenated cottonseed oil-fed and corn oil-fed mouse livers were essentially the same, the differences observed in the activities of the enzyme between the two dietary groups could not be explained simply in terms of differences in rates of enzyme synthesis and degradation. We, therefore, investigated the possibility that the enzyme underwent some form of catalytic activation-inactivation as a result of dietary treatments. Thus, additional experiments were designed to demonstrate whether differences in the catalytic efficiency of the enzyme present in the liver cytosols from hydrogenated cottonseed oil and corn oil-fed mice could be found. Immunotitration o f liver A TP-citrate lyase from mice fed the hydrogenated cottonseed oil and the corn oil diets The anti-ATP-citrate lyase preparation we used was capable of precipitating all of the ATP-citrate

108 [2

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TABLE IV

t

THE EH:ECT OF DIETARY EAT ON CHANGES IN ATPCITRATE LYASE ENZYME AND RADIOACTIVITY FROM LIVER CYTOSOLS STUDIED DURING ENZYME SYNTHESIS

I0

Mice were fed the two diets for the periods of time recorded below after which they were injected with 100 #Ci L-[4,53H]leucine and killed 1 h later. ATP-citrate lyase contained in 0.2 ml hepatic cytosol was immunoprecipitated after enzyme activity was measured. The results are expressed as the ratio of dpm immunoprecipitated ATP-citrate lyase per unit ATP-citrate lyase enzymatic activity. Each value represents the mean _+S.D. of three animals determined individually.

o

~

4

~

o

o

c

0

4 8 12 16 Units ATP-Cilroi'e lyase added

20

Fig. 4. Immunotitration of liver ATP-citrate lyase from mice fed the hydrogenated cottonseed oil (. *) or corn oil (o o) diets. Anti-mouse ATP-citrate lyase ant~ody (100/sg) was mixed with varying amounts of cytosols containing known activities of ATP-citrate lyase from livers of mice fed either the hydrogenated cottonseed oil (7 days) or corn oil diets (15 days). The mixtures were incubated in a total volume of 0.1 ml at 37°C for 1 h. The ATP-citrate lyase immunoprecipitates were collected by centrifugation and the activity of ATP-citrate lyase remaining in the supernatant fractions were assayed. The values were plotted and the lines drawn represent to the best fit to the data as determined by least squares analysis. Each value represents the average of two determinations. lyase enzyme activity from cytosols prepared from the livers of either the corn oil or hydrogenated cottonseed oil-fed mice. At equivalence, however, 100/xg antibody precipitated only 1.2 units enzyme activity from the corn oil-fed group, whereas the same amount of antibody precipitated 3.9 units of enzyme activity from the hydrogenated cottonseed oil-fed group (Fig. 4). Thus, it was apparent from this experiment that identical amounts of ATP-citrate lyase protein from hydrogenated cottonseed oil-fed mouse rivers contained at least three times more catalytic activity than those from corn oil-fed mouse rivers.

Immunoprecipitation o f 3H-labeled A TP-citrate lyase from hydrogenated cottonseed oil and corn oil-fed mouse livers In the experiments in which mice were injected

Days on diets

Diet Hydrogenated cotton seed oil

Corn oil (B)

1 3 6 11

352 _+26 129_+ 17a 91 -+52 a 54-+41a

455 _+116 420-+ 41 443 -+ 132 389-+ 96

% Difference (B-A/B)

23 69 79 86

a Difference between this value and that for corn oil-fed animals is statistically significant at P < 1%. with L-[4,5-3H]leucine, 1 unit ATP-citrate lyase activity contained approx. 400 dpm in the corn oil-fed group. This value did not change significantly during the 11-day feeding period (Table IV). In contrast, the radioactivity contained in 1 unit ATP-citrate lyase activity from hydrogenated cottonseed oil-fed mice continued to decline over the same feeding period (from 350 to 50 dpm). Prior to being placed on the experimental diets, all animals were maintained on a Purina mouse chow diet which contained approx. 5% polyunsaturated fatty acid (linoleic acid). This may explain why both hydrogenated cottonseed oil and corn oil-fed mice gave similar dpm[unit ATP-citrate lyase activity ratios after only 1 day of feeding (Table IV). The data in Table III and Fig. 3A demonstrated that the rates of synthesis of ATP-citrate lyase relative to that of total protein synthesis and the rates of degradation of the enzyme were similar for both hydrogenated cottonseed oil-fed and corn oil-fed animals. It is, therefore, likely that a true increase in the catalytic activity of the enzyme from hydrogenated cottonseed oil-fed mice had occurred, since the similar rates of enzyme

109 TABLE V EFFECTS OF DIETARY FAT ON CHANGES IN ATPCITRATE LYASE FROM LIVER CYTOSOLS STUDIED DURING ENZYME DEGRADATION Mice were injected with 100 ~tCi L-[4,5-aH]leucine and immediately placed on one of the two diets for the times indicated below. The ATP-citrate lyase enzyme activity and radioactivity were determined as given in Table III and the text. The radioactivity present in the immunoprecipitated ATP-citrate lyase from the hepatic cytosols from either dietary group after 6 days were not sufficiently high to allow for accurate determination and are therefore not included in the table. The results are expressed as the ratio of dpm immunoprecipitated ATP-citrate lyase per unit ATP-citrate lyase enzyme activity. Each value represents the mean ± S.D. for three animals, determined individually. Days on diets

Diet

% difference (B-A/B)

Hydrogenated cotton seed oil

Corn oil (B)

1

132 ± 18

121 _* 14

9

3 6

66_+41 59 _*23

87_* 7 62 _*16

24 5

turnover suggested that the pool of labeled enzyme was similar for both groups. It was possible that the appearance of active ATPcitrate lyase in the hydrogenated cottonseed oil-fed mice livers represented the synthesis of a new species of 'active enzyme' or was the result of an ailosteric activation of pre-existing inactivate enzyme. The results presented in Table V suggested that the activation of ATP-citrate lyase in hydrogenated cottonseed oil-fed animals most likely represented new 'active enzyme' synthesis since the ratio of immunoprecipitated radioactivity/unit enzyme activity did not decrease at a faster rate with hydrogenated cottonseed off feeding (in comparison to corn off feeding) as would be expected if pre-existing inactive enzyme present in the hydrogenated cottonseed oilfed livers had been activated. Furthermore, it is generally recognized that allosteric events occur within several hours of dietary manipulation [17], whereas mechanisms involving new protein synthesis usually require relatively longer periods of time to become apparent. Thus, if hydrogenated cottonseed oil feeding were to trigger an activation of preexisting inactive enzyme, one might expect the effect

to occur within 24 h of hydrogenated cottonseed oil feeding. However, the effect was not apparent for at least 72 h, a finding which precludes an explanation based on allosteric activation of existing enzyme activity.

Evidence for the existence of material reactive with enzymatically inactive anti-A TP-citrate lyase Another experiment designed to detect the presence of inactive ATP-citrate lyase was based on a competition for anti-ATP citrate lyase antibody binding sites between pure ATP-citrate lyase (from the livers of mice fed a high carbohydrate fat-free diet) and ATP-citrate lyase from the livers of hydro. genated cottonseed oil and corn off-fed mice (Table VI). When liver cytosols from the corn off-fed mice were incubated with antibody treated pure ATPcitrate lyase, about 30% more ATP-citrate activity was recovered after the incubation than was originally added. In contrast, for the hydrogenated cottonseed oil-fed mice on diet for 3 days or longer, the amount of ATP-citrate lyase activity recovered after the incubation was nearly identical to the activity originally added. These results indicated that in the cytosols from the corn off-fed animals as opposed to those from the hydrogenated cottonseed oil-fed animals, there was a greater amount of ATP-citrate lyase protein associated with a given level of ATP-citrate lyase activity. These results also indicated that the catalytic activity of the enzyme from hydrogenated cottonseed off-fed and fat-free fed mouse livers were identical.

Determination of mouse liver ATP-citrate lyase specific activity using rocket immunoelectrophoresis for quantitation of enzyme protein Another method which allows for the quantitation of individual enzymes where the enzyme is present in a mixture of proteins is rocket immunoelectrophoresis. Thus, we subjected cytosols from both hydrogenated cottonseed off-fed and corn oil-fed mouse livers to rocket immunoelectrophoresis (Table VII). ATP-citrate lyase in the cytosols from hydrogenated cottonseed oil-fed mouse livers contained approx. twice as many units of enzyme activity per unit rocket height than did the ATP-citrate lyase from the corn oil-fed mouse livers. Although this difference was not observed until 3 days after the initiation of

110 TABLE VI THE EFFECT OF DIETARY FAT ON THE COMPETITION BETWEEN PURIFIED ATP-CITRATE LYASE AND THAT IN HEPATIC CYTOSOL FOR ANTIBODY BINDING SITES Purified mouse liver ATP-citrate lyase (17.5 units) was mixed with an amount of anti-ATP-citrate lyase antibody to inactivate 60% of the original enzyme activity. An aliquot of this mixture containing 5 units ATP-citrate lyase activity was then incubated for 15 min at 37°C with 0.5 mg cytosol from either corn oil-fed or hydrogenated cottonseed oil-fed mouse livers with the known amounts of ATP-citrate lyase activity given below. Following incubation, the mixtures were assayed for total ATP-citrate lyase activity. The results are presented as units of enzyme activity before and after incubation with cytosols. Days on diet

Diet Hydrogenated cottonseed oil

1

3 6 11

Corn oil

Before incubation (A)

After incubation (B)

% Change (B-A/B)

Before incubation (C)

After incubation (D)

% Change (D-C/D)

9.6 9.2 22.2 28.9

10.1 9.4 24.0 30.5

5 2 8 5

8.0 7.8 7.7 8.3

7.8 10.0 11.9 12.0

22 35 31

TABLE VII THE EFFECT OF DIETARY FAT ON THE QUANTITY OF ATP-CITRATE LYASE PROTEIN IN MOUSE LIVER CYTOSOLS Cytosols prepared from the livers of mice consuming either the hydrogenated cottonseed oil or the corn oil-containing diets for the indicated times were made 25% (v/v) in rocket immunoelectrophoresis buffer. 8-/A aliquots were subject to electrophoresis in 1% agarose gel containing 2.3% (v/v) antimouse liver ATP-citrate lyase antibody. Following electrophoresis, gels were washed, dried and stained. Results are expressed as units of ATP-citrate lyase activity/rocket height (cm). Each figure represents the mean and the standard deviation of three determinations per sample. All samples were run on the same gel and on the same day. Several dilutions of the samples were run and the rocket heights were measured to insure proportionality of rocket height to enzyme protein. Days on diet

1

3 6 11

Diet (×10 -2) Hydrogenated cottonseed oil (A)

Corn oil (B)

52 _+10 96± 6a 96± 6 a 118± 10a

56 ± 46± 46± 59±

% Difference (B-A/B)

4

7

11 6 0

109 109 100

a Difference between this value and that for corn oil-fed animals is statistically significant at P < 1%.

3

the diets, it persisted for the remaining 8 days o f the feeding period. Such a pattern was similar to that observed in the experiments where we studied immunotitration (Fig. 4) and immunoprecipitation (Table IV) o f hepatic ATP-citrate lyase. Since in our experiments, rocket height was shown to be directly proportional to enzyme protein content it was apparent that the catalytic efficiency o f ATP-citrate lyase had increased during the fig'st 3 days of hydrogenated cottonseed oil feeding, whereas it had remained unchanged in the corn oil-fed mice. Discussion The catalytic efficiency of some hepatic enzymes concerned with lipogenesis may be regulated by the dietary status of the animal [ 1 8 - 2 1 ] . Recently, Ashcraft et al. [18] have demonstrated, in avian liver, an in vivo transformation o f the enzymatically active polymeric form o f acetyl-CoA carboxylase into the enzymatically inactive protomeric form when birds maintained on a high carbohydrate diet were subjected to short term fasting or were intubated with safflower oil. A similar observation was made by Clarke and Hillard [19] with cultured chick hepatocytes. These workers showed that the additon o f linoleate or dibutyryl-cyclic AMP to the culture medium

111 resulted in an increase in the amount of activity released from the cells upon subsequent treatment with digitonin. Clarke and Hfflard [19] interpreted these results as demonstrating a conversion of the catalytically active filamentous form of the enzyme to the catalytically inactive protomeric form. Both Kelly et al. [20] and Hizi and Yagff [21] have provided evidence that rat liver glucose-6-phosphate dehydrogenase is regulated by diet through changes in the catalytic efficiency of the enzyme. Thus for example, when Kelley et al. [20] took rats which had been previously starved, and re-fed them a fat-free diet containing 70% sucrose, there was a 9-fold increase in hepatic enzyme activity but no increase in the amount of immunoprecipitable enzyme protein. When starved rats were instead transferred to a high-fat diet (approx. 70% fat), there was no change in either enzyme activity or in the amount of immunoprecipatable enzyme protein. This, and other evidence, led Kelley et al. [20] to suggest that the hepatic enzyme must have occurred in both an active and inactive form. Hizi and Yagil [21], likewise, found for mice that the large increase in hepatic glucose-6-phosphate dehydrogenase specific activity observed when animals were switched from a high-fat (25% corn off) to a fat-free diet did not involve new enzyme synthesis. They suggested that a form of enzyme which was either completely or partially inactive existed in the livers of the mice fed the fatcontaining diets. The results of the experiments we presented here have led us to conclude that feeding polyunsaturated fatty acids to mice resulted in a decrease in the catalytic efficiency of hepatic ATP-citrate lyase when compared to mice fed similar diets containing only saturated fatty acids. This conclusion was based on the following findings: 1. At immunoequivalence, a given amount of antiATP-citrate lyase antibody precipitated only one-half the amount of cytosolic enzyme activity from the corn off-fed mouse livers as from the hydrogenated cottonseed off-fed livers. 2. In those experiments in which the enzyme was pulse-labeled in vivo with L-[4,5-aH]leucine the value for the ratio of immunoprecipitable hepatic ATPcitrate lyase radioactivity/unit enzyme activity was approx. 7-fold higher for corn oil-fed than for hydrogenated cottonseed off-fed mice.

3. In antibody competition experiments, given amounts of hepatic cytosols from mice fed the corn off diet for periods from 3-11 days when incubated with solutions containing a known amount of pure enzyme and anti-enzyme antibody, resulted in an increase (30%) in post-incubation enzyme activity. In contrast, similar addition of hepatic cytosols from mice fed the hydrogenated cottonseed off diet for the same periods of time did not result in any increase in post-incubation enzyme activity. Liver cytosols from mice fed either the corn oil or hydrogenated cottonseed off diet for only 1 day when added to pure enzyme and anti-enzyme antibody, likewise did not result in any increase in post-incubation enzyme activity. 4. Rocket immunoelectrophoresis performed on hepatic cytosols from mice fed the two diets showed that in the corn off-fed mice, there was approx. 2times more enzyme protein per unit of enzyme activity than in the cytosols from the hydrogenated cottonseed off-fed mice. It is likely that the activation of ATP-citrate lyase in hydrogenated cottonseed off-fed mice we have described here represented the synthesis of a new species of active enzyme and not simply an activation of a pre-existing inactivate form of the enzyme. Newly synthesized ATP-citrate lyase labeled with radioactivity at the start of the dietary regimen did not exhibit a change in the ratio of immunoprecipirated enzyme/unit enzyme activity when animals were fed the hydrogenated cottonseed off diet. In addition, we believe that the time required for the appearance of active enzyme, which we showed to be between 1-3 days of hydrogenated cottonseed off feeding, is consistent with a mechanism involving new protein synthesis and not allosteric activation. Muto and Gibson [4] showed that the adaptive rise in ATP-citrate lyase activity when fasted rats were refed high carbohydrate fat-free diets could be prevented by injecting the animals with puromycin at the onset of the refeeding period. This evidence indicated that the synthesis of new ATP-citrate lyase protein was required for the induction of enzyme activity by dietary carbohydrate. We suggest that, at least for the inhibition by dietary corn off on mouse hepatic ATP-citrate lyase activity, new enzyme synthesis occurs at a constant rate throughout the entire experimental feeding period, and that synthesis of

112 new enzyme protein, although required to see the dietary effect, is not itself a regulatory mechanism. Whether this is true for all conditions whereby the activity of ATP-citrate lyase is effected by dietary or hormonal treatments is not yet clear. Other 'lipogenlc' enzymes respond to dietary fatfeeding through changes not involving catalytic efficiency [22]. For example, we have found that mouse hepatic fatty acid synthetase failed to show differences in catalytic efficiency when compared between corn off-fed and hydrogenated cottonseed oil-fed mice that were subjected to the same experimental conditions as those used here (unpublished data). Such findings are in agreement with Flick et al. [22] who suggested that dietary polyunsaturated fatty acids inhibited rat liver fatty acid synthetase activity by decreasing the content of the enzyme and not by lowering its catalytic efficiency. However, Yu and Burton [23,24] have presented evidence suggesting the presence of enzymatically inactive, immunologically reactive fatty acid synthetase in rat liver during the first 3 h of refeeding a fat-free high carbohydrate diet following a 48-h fast. Thus, different dietary conditions may give rise to more than one regulatory mechanism for each enzyme. Gibson et al. [5] have suggested that ATP-citrate lyase is one of a group of lipogenic enzymes that respond coordinately to nutritional manipulation. The data presented here for ATP-citrate lyase and that of Flick et al. [22] for fatty acid synthetase, as well as our own unpublished observations, suggest that although the activities of the lipogenic enzymes may change in a similar direction to a given nutritional stimulus, the mechanism by which that change is accomplished may be unique for each enzyme. Whether dietary polyunsaturated fat exerts its effect on murine hepatic ATP-citrate lyase directly or indirectly through some intermediate is unknown. Several reports have described the presence of 'factors' isolated from rat liver cytosols which are capable of regulating enzyme activity [29-31 ]. For example, Furuya and Uyeda [25] have purified an 'activation factor' from rat liver which binds more strongly to the de-phosphorylated form of phosphofructokinase than to the phosphorylated form of the enzyme. These workers have also shown that the activity of the phosphorylated enzyme when subjected to inhibitory concentrations of ATP can be restored by

the addition of the 'activation factor'. Abdel-Halim and Porter [29] have described another 'factor' which inhibited rat hepatic acetylCoA carboxylase activity. They reported that when rats were starved for 48 h the amount of the inhibitory factor present in the livers of these animals was minimal whereas when the animals were refed for a similar period of time the amount of inhibitor was maximal. Rat liver ATP-citrate lyase has recently been shown to be capable of undergoing reversible phosphorylation-dephosphorylation [26]. Furthermore, the phosphorylation state can be effected by hormonal manipulation [26-28]. Although experiments performed to date have failed to show any differences between the catalytic efficiencies of the purified phosphorylated or dephosphorylated forms of the enzyme [15], it is possible that a regulatory 'factor', which may bind to and consequently inactivate only one form of the enzyme, is lost during the purification procedure. Osterlund and Bridger [30-31] have isolated a peptide from rat liver which protects ATP-citrate lyase from thermal inactivation. They have proposed that this peptide factor could be involved in the regulation of the intracellular degradation of the enzyme. Our results failed to demonstrate any significant differences in the degradative rates of mouse hepatic ATP-citrate lyase under conditions that effected the activity of the enzyme. We feel therefore that it is unlikely that the peptide factor described by Osterlund and Bridger [30-31] played a significant role in the regulation of mouse hepatic ATP-citrate lyase activity by dietary polyunsaturated fatty acids.

Acknowledgements The excellent technical assistance of Ms. Hope McGrath in performing the gas-liquid chromatographic analysis is gratefully ackvowledged. We are also grateful to Dr. S. Smith of Bruce Lyon Memoral.Research Laboratory for helpful discussions. These studies were supported by National Cancer Institute Grant No. Ca-29767 and Biomedical Research Support Grant No. RR-05467 from the National Institutes of Health, Department of Health, Education and Welfare.

113

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