Acetyl Coenzyme A Synthetase and the Regulation of Lipid Synthesis from Acetate in Cultured Cells*

The regulation of lipid synthesis from acetate in cultures of L cells in response to changes in exogenous lipid supply has been studied, and the enzyme acetyl-CoA synthetase has been investigated in reference to its possible role in the regulation of lipid biosynthesis from acetate. When serum lipid was removed from monolayers of L cells, a relatively rapid stimulation of [14C]acetate incorporation into lipid was observed within 2 hours. This stimulation of incorporation was observed in both sterol and fatty acid fractions. Conversely, an inhibition of [14C]acetate incorporation was observed within 1 to 2 hours when cells cultured in lipid-free medium were transferred to serumsupplemented medium. As with the stimulation, this inhibition was observed both in sterol and fatty acid fractions of the cell lipid. The data indicated a coordination of fatty acid and cholesterol metabolism in the cells. Inhibition of [14C]acetate incorporation was observed in sterol as well as free fatty acid and glycerolipid fractions in cultures grown in lipid-free medium and transferred to fatty acid-supplemented medium; and similarly, cultures transferred to medium supplemented with cholesterol showed inhibition not only of acetate incorporation into sterol but also into fatty acid and glycerolipid fractions. Cycloheximide, actinomycin D and mitomycin C seemed to have no influence on the early stimulation or inhibition of [r4C]acetate incorporation into total lipid. Acetyl-CoA synthetase activity was assayed in homogenates of L cells cultured in the presence of serum-supplemented or lipid-free medium and a 5-fold decrease in enzyme activity was observed in homogenates of cultures grown in the presence of exogenous lipid. When cells grown in serum-supplemented medium were transferred to serum-free medium, a stimulation of enzyme activity occurred within 2 to 3 hours which reached a maximum by 6 hours. Conversely, when cells were cultured in lipid-free medium and transferred to serum-supplemented medium, inhibition of enzyme activity occurred within the same time course.


SUMMARY
The regulation of lipid synthesis from acetate in cultures of L cells in response to changes in exogenous lipid supply has been studied, and the enzyme acetyl-CoA synthetase has been investigated in reference to its possible role in the regulation of lipid biosynthesis from acetate.
When serum lipid was removed from monolayers of L cells, a relatively rapid stimulation of [14C]acetate incorporation into lipid was observed within 2 hours. This stimulation of incorporation was observed in both sterol and fatty acid fractions. Conversely, an inhibition of [14C]acetate incorporation was observed within 1 to 2 hours when cells cultured in lipid-free medium were transferred to serumsupplemented medium. As with the stimulation, this inhibition was observed both in sterol and fatty acid fractions of the cell lipid. The data indicated a coordination of fatty acid and cholesterol metabolism in the cells. Inhibition of [14C]acetate incorporation was observed in sterol as well as free fatty acid and glycerolipid fractions in cultures grown in lipid-free medium and transferred to fatty acid-supplemented medium; and similarly, cultures transferred to medium supplemented with cholesterol showed inhibition not only of acetate incorporation into sterol but also into fatty acid and glycerolipid fractions. Cycloheximide, actinomycin D and mitomycin C seemed to have no influence on the early stimulation or inhibition of [r4C]acetate incorporation into total lipid. Acetyl-CoA synthetase activity was assayed in homogenates of L cells cultured in the presence of serum-supplemented or lipid-free medium and a 5-fold decrease in enzyme activity was observed in homogenates of cultures grown in the presence of exogenous lipid. When cells grown in serum-supplemented medium were transferred to serum-free medium, a stimulation of enzyme activity occurred within 2 to 3 hours which reached a maximum by 6 hours. Conversely, when cells were cultured in lipid-free medium and transferred to serum-supplemented medium, inhibition of enzyme activity occurred within the same time course. * This work was supported by Grants HL-16058, AM-14526, HL-05062, and RR-107 from the National Institutes of Health.
$ To whom reprint requests should be addressed at Clinical Research Center, Philadelphia General Hospital, Philadelphia, Pa. 19104.
Cycloheximide, actinomycin D, and mitomycin C had no influence on the stimulation or inhibition of enzyme activity observed in response to changes in exogenous lipids. Inhibition of enzyme activity also was observed in cells cultured in lipid-free medium which were transferred to medium containing either fatty acid or cholesterol, indicating fatty acid and cholesterol might be part of the components of serum lipid that can influence the activity of acetyl-CoA synthetase. However, when various concentrations of serum lipid were added to the reaction mixture in vitro, there was no influence on enzyme activity. The data suggest that the enzyme acetyl-CoA synthetase may play a role in the regulation of lipid biosynthesis from acetate in cultured cells.
Cells in culture have provided an easily manipulatable system for the study of the regulation of lipid metabolism at the cellular level. It was observed that cells cultured in the presence of serum derived most of their lipid from the serum in the growth medium (I), while cells cultured in the absence of exogenous lipid synthesized cell lipids from carbon sources such as acetate and glucose (2). More recent studies have centered around attempts to localize the enzymatic sites of regulation of lipid biosynthesis.
In investigations of the regulation of fatty acid synthesis in skin fibroblasts, Jacobs et al. (3) showed that there was induction and repression of the enzyme acetyl-CoA carboxylase over a 2-day time course. Recently, Alberts et al. (4) have demonstrated a similar regulation of the synthesis of the enzyme fatty acid synthetase in cultures of L and HeLa cells. In the case of sterol metabolism, the work of Williams and Avigan (5) and Rothblat et al. (6) suggested that there is a regulatory point in the path of cholesterol synthesis at the site of mevalonic acid. It was demonstrated by Watson (7) in hepatoma cells and Brown et al. (8) in skin fibroblast cultures that there were alterations in the activity of 3-hydroxy-a-methylglutaryl (HMG)-CoA reductase within 4 hours after changes in exogenous cholesterol and that the rise in enzyme activity required de novo synthesis.
In considering lipid biosynthesis from acetate, however, several lines of evidence indicate that there might be other 7912 points of regulation.
In their study of the regulation of fatty acid biosynthesis in cultured fibroblasts, Jacobs et al. (3) noticed a rapid inhibition and stimulation of [14C]acetate incorporation whose time course did not parallel the observed changes in the enzyme acetyl-CoA carboxylase.
In further studies of the short term inhibition of fatty acid synthesis from acetate (9), these investigators found that the inhibition could not be correlated with intracellular changes in citrate, acetyl-CoA or free fatty acid levels. This rapid regulation of fatty acid synthesis has also been observed in viva (10,11). A second suggestion for alternative control mechanisms comes from previous studies (2) which indicated a coordinate inhibition of [14C]acetate incorporation into fatty acid, sterol, and amino acids, suggesting a regulation at the level of 2-carbon metabolism.
Coordinate inhibition of both fatty acid and sterol synthesis by exogenous cholesterol also was observed in some of the studies of Kandutsch and Chen (12).
In vivo studies have suggested that one possible site for coordination of fatty acid and sterol biosynthesis from acetate is at the level of the enzyme acetyl-CoA synthetase (acetate:CoA ligase (AMP), EC 6.2.1.1). Steiner and Cahill (13) first suggested the role of this enzyme in the control of fatty acid synthesis from studies on brown adipose tissue homogenates. Barth et al. (14) have reported changes in acetyl-CoA synthetase in several rat tissues in response to dietary manipulation; carbohydrate feeding after fasting led to a 170% elevation of enzyme activity in liver and 550% elevation in epididymal fat pads. Murthy and Steiner (15) have reported alterations in this enzyme which parallel changes in rates of hepatic lipogenesis in the rat induced by feeding and fasting.
They also observed in vitro inhibition of enzyme activity in mitochondrial supernatants upon addition of oleate. A regulatory role for this enzyme also is supported by observations of changes in cellular acetate levels in relation to altered lipid metabolism (16,17).
In this study, we present data on the kinetics of regulation of lipid metabolism from acetate in cultures of L cells in response to changes in exogenous lipid supply.
The enzyme acetyl-CoA synthetase in these cultures also is investigated in reference to its possible role in the regulation of lipid biosynthesis from acetate. A preliminary report of these studies has been presented previously (18). [W]Acetate Incorporation into Cell Lipids-Studies were conducted to determine the time course of stimulation or inhibition of lipid synthesis from acetate in cells in culture upon changes in exogenous lipid.

Cells
These experiments were carried out both with the 2071 strain of L cells cultured in serum-free or serumsupplemented medium and with the L-929 cells which were cultured in the absence of exogenous lipid by supplementing the medium with DLSP.
Essentially similar results were obtained with the two strains of L cells. Therefore, the results described here are mainly from the L-929 cells since their ease of handling allowed a greater number of experiments to be conducted.
For the determination of kinetics of stimulation of ['%]acetate incorporation into cell lipids upon removal of serum lipids, replicate cultures were grown to confluency in serum-supplemented medium.
At zero time (To) the monolayers were rinsed and lipid-free medium added.
Control experiments established that acetate incorporation into lipid was linear for up to 6 hours under the culture and assay conditions utilized (Fig. 1). Lipid synthesis was evaluated at various time points with l-hour pulses of [14C]acetate. The results of these experiments are shown in Fig. 2. The data are expressed as per cent of control so that direct comparison can be made between total lipid and sterol and fatty acid subfractions.
When the specific activity PLUS SERUM The data show that when exogenous lipid was removed from monolayers of L cells, there was a relatively rapid stimulation of acetate incorporation into lipid which was observed within 2 hours.
Data are not given for time points greater than 12 hours because of the variables introduced by random cell proliferation.
It should be mentioned that stimulation of [14C]acetate incorporation into lipid occurred whether or not the medium contained DLSP; the magnitude of this stimulation was no greater in DLSP containing medium than in MEM.
The stimulation of incorporation was observed in both sterol and fatty acid fractions (Fig. 2) of the cell lipid and the time courses for these two fractions were relatively similar. Sterol and fatty acid represented greater than 90% of the total radioactivity from acetate incorporated into lipid under these conditions, and the ratio of fatty acid to sterol fractions averaged not present when inhibition of lipid synthesis was being evaluated.
Since a coordinate time course for sterol and fatty acid stimulation and inhibition was observed, experiments were conducted to determine if there was a coordination of fatty acid and cholesterol metabolism.
Cultures were grown in lipid-free medium and at To received medium supplemented with either serum, fatty acid, or cholesterol.
Lipid biosynthesis after 24 hours was assayed by a l-hour pulse of [14C]acetate, and incorporation of radioactivity into total lipid and lipid subfractions was determined.
The results, shown in Table I, indicate that there was a coordinate inhibition of acetate incorporation into lipid by exogenous fatty acid and cholesterol in these cells. When cultures were supplemented with free fatty acids at To, the expected inhibition of acetate into the free fatty acid and glycerolipid fraction was observed, and in addition the incorporation of acetate into sterol was inhibited.
Similarly, cultures which received cholesterol showed an inhibition of acetate not only into sterol, but also into fatty acid and glycerolipid fractions. The time course of the inhibitory effect of cholesterol and fatty acid was determined in experiments similar to those  (Fig. 2 and 3). The data (Fig. 4) where the medium was unchanged at 2'0.
by guest on March 24, 2020 http://www.jbc.org/ Downloaded from indicated that when either cholesterol or fatty acid was added to the medium [l*C]acetate incorporation into both fatty acid and cholesterol was inhibited and the patterns of inhibition were similar and resembled that of whole serum.
In order to begin an investigation of the possible mechanism of regulation of acetate incorporation into lipid in these cells, and in order to coordinate the data on acetate incorporation with that of enzyme activity to be presented below, stimulation and inhibition of acetate incorporation into lipid was evaluated in the presence of certain inhibitors of macromolecule synthesis: cycloheximide was utilized at a concentration of 10 pg per ml which yielded a 95% inhibition of protein synthesis in these cultures within 30 min; actinomycin D was utilized at 5 pg per ml which yielded an inhibition of RNA synthesis of 90% in these cultures within 30 min. Mitomycin C was utilized at a concentration of 100 pg per ml which resulted in a 60% inhibition of DNA synthesis in these cells within 2 hours. For evaluation of their influence on the stimulation of acetate incorporation into lipid observed upon removal of serum lipid, cells were cultured in serum-supplemented medium and replicate cultures received at To either lipid-free medium or lipid-free medium supplemented with one of the indicated inhibitors.
Lipid biosynthesis was evaluated after 6 hours with a l-hour pulse of [l*C]acetate.
The results, shown in Fig. 5, indicate that none of the inhibitors of macromolecule synthesis had any influence on the stimulation of [r*C]acetate incorporation observed after removal of serum lipid.
A similar lack of effect of the inhibitors on inhibition of [nC]acetate incorporation into total lipid was demonstrated in converse experiments (Fig. 5). In this situation, cells were cultured in the presence of delipidized serum protein and at To received serum-supplemented medium with or without the indicated inhibitor.
The data indicate the inhibitors had no effect on the inhibition of acetate incorporation into total lipid in response to addition of serum lipid.
The differences between the magnitude of inhibition observed in the presence of the various inhibitors are not significant since considerable variability was observed in the acetate incorporation in the presence of these inhibitors from experiment to experiment, as indicated by the range of values in Fig. 5.
E$ect of Alterations in Exogenous Lipid on Acetyl-CoA Synthetase Activity in Cell Cultures-The data from the studies on ['*C]acetate incorporation into cell lipid indicated a rapid time course of stimulation and inhibition of lipid biosynthesis from acetate and a coordination of the effects of cholesterol and fatty acid. Since one possible mechanism for this coordinated control could be a regulation at the 2-carbon level of metabolism, attention was focused on studies of the enzyme acetyl-CoA synthetase in these cultures.
Experiments were conducted in parallel fashion to those described above which assessed acetate incorporation. Fig. 6 shows the results of experiments to standardize the assay conditions.
Homogenates of L cells converted acetate to acetyl-CoA at a rate that was linear with time and protein concentration (Fig. 6, A and B). The concentrations of coenzyme A (Fig. 6C) and ATP (Fig. 60) that gave half-maximal velocities were 0.08 and 18 mM, respectively, for enzyme activity in cell homogenates.
The kinetics of enzyme activity vera-us ATP concentrations yielded a sigmoidal curve, suggesting either a possible autocatalytic effect of ATP, or significant ATPase activity despite the presence of fluoride.
Although enzyme activity was assayed by steaming unreacted acetate from the reaction mixture, it was confirmed that the acetate was indeed converted to a,cetyl-CoA by paper chromatography of the reaction mixture according to the method of Huang (25).
Acetyl-CoA synthetase activity was assayed in homogenates of L-929 cells cultured in the presence of serum or delipidized serum protein, and in homogenates of L-2071 cells cultured in serum-supplemented or serum-free medium. The data shown in Table II  To determine the effect of the inhibitors on under these conditions; actinomycin D (AC!Z'ZiV.D) concentration stimulation of [14C]acetate incorporation, replicate cultures of was 5 rg per ml which inhibits RNA synthesis 95%; cycloheximide L-929 cells were grown in medium supplemented with 20% fetal (CYCLOHEX) was used at 10 pg per ml which gives greater than bovine serum and transferred to lipid-free medium (LF) at To. 95y0 inhibition of protein synthesis in these cells. After incuba-When the effect of these compounds on inhibition of lipid bio-tion for exactly 1 hour, monolayers were rinsed three times and synthesis was assayed, cells were cultivated in medium supple-the cell lipid was extracted and radioactivity was determined as mented with DLSP (2 mg per ml) and transferred to serum-supple- or NCTC-135 supplemented with 10% fetal bovine serum. When cultures reached confluency, the monolayers were rinsed, cells were harvested and homogenized, and the enzyme was assayed as described under "Methods." Values are expressed f the standard error of the mean; they are the average of determinations on four to eight separate cultures. To investigate the mechanism of this regulation, and to compare the activity of the enzyme to the rate of lipid synthesis, the kinetics of stimulation and inhibition of the enzyme activity in response to changes of exogenous lipids were studied in experiments similar to those described in Figs. 2 and 3. The results are shown in Fig. 7. The data indicate that when cells grown in serum-supplemented medium were transferred to serum-free medium at zero time, a stimulation of enzyme activity was observed within 2 to 3 hours and reached a maximum by 6 hours.
Conversely, when cells cultured in lipid-free medium were transferred to serum-supplemented medium, an inhibition of enzyme activity occurred within 3 to 6 hours. The time course of stimulation and inhibition of enzyme activity was ATP (mM) FIG. 6. Assay of acetyl-CoA synthetase activity in cell cultures. Cells were grown to confluency in MEM supplemented with 2 mg per ml of DLSP, and monolayers were harvested after three rinses of CBSS into 0.88 M sucrose containing 0.2yo Triton X-100. Cells were homogenized and enzyme was assayed as described under "Methods" with the following alterations.
A, each assay mixture contained the indicated amount of each substrate and 0.2 ml cell homogenate (800 rg of protein); the reaction was stopped at intervals from 14 to 44 min. B, the reaction mixture contained the indicated amount of substrates and was incubated 20 min; the amount of homogenate was varied from 166 to 909 rg of protein.
C, the reaction was incubated for 20 min with 0.2 ml of homogenate; CoA was varied from 0 to 0.6 mM. D, the reaction mixture contained the reagents as described under "Methods" and was incubated 20 min with 0.2 ml of cell homogenate; ATP was varied from 0 to 60 mM.
::I,, , , I For assay of enzyme inhibition replicate cultures of L-929 cells were grown in medium supplemented with DLSP and transferred to serum-supplemented medium at To. For assay of enzyme stimulation replicate cultures were cultured in medium supplemented with 200/, fetal bovine serum and transferred to medium supplemented with 4 mg per ml of DLSP at To. Enzyme activity was assayed at the indicated times in homogenates of harvested cells as described under "Methods." quite similar to that observed for stimulation and inhibition of acetate incorporation into total lipid. On the other hand, the relative magnitudes of stimulation or inhibition were somewhat less in the case of enzyme activity than they were for lipid biosynthesis from acetate.
In order to evaluate the mechanism of action of serum or individual lipids on enzyme activity, inhibition and stimulation of enzyme was assessed in the presence of inhibitors of macromolecule synthesis described in Fig. 5 Cultures were grown to confluency in medium supplemented with 2 mg per ml of DLSP and the medium was changed at To to similar fresh medium including 10 rg per ml of cycloheximide.
At the indicated times enzyme activity was assayed in homogenates of harvested cells as described under "Methods." conducted in a fashion similar to that described for acetate incorporation.
Cells at To received experimental medium in the presence and absence of the indicated inhibitor. Enzyme activity was determined after 4 to 6 hours. The data, shown in Fig. 8 indicate that the inhibitors had no influence on stimulation or inhibition of enzyme activity in response to changes in exogenous lipid.
The decay rate of the enzyme under these culture conditions was assessed by adding 10 pg per ml of cycloheximide to monolayer cultures of L-929 cells grown in medium supplemented with DLSP.
Enzyme activity was assayed over a 22.hour period in the presence of this inhibitor.
The data (Fig. 9) showed a slow decay of enzyme activity over the time period evaluated, with an apparent half-life of approximately 20 hours under these conditions.
The results of the inhibitor experiments and the long half-life suggested that acetyl-CoA synthetase might be regulated at the level of enzyme activity.
It was thus of interest to determine the effects of cholesterol and fatty acid on the enzyme. This was first evaluated by adding these compounds to the medium over monolayers of cells and then assaying enzyme activity in cell homogenates.
Cells were cultured in medium supplemented with DLSP and, at To, were transferred to medium supplemented with either fetal calf serum, fatty acid, or cholesterol. After 6 hours, enzyme activity was determined in homogenates  Cells were subcultivated at 1:4 ratios in the indicated medium. The DLSP concentration was 2 mg per ml and fetal bovine serum was 20%. Fatty acid (sodium palmitate) was added at 30 rg per ml on albumin carrier (1.7 mg of albumin per ml) as described under "Methods." Cholesterol was present at 30 pg per ml on DLSP carrier (2 mg per ml) as described under "Methods." Confluent monolayers were rinsed and harvested and the enzyme was assayed in cell homogenates as described under "Methods." The data show the range of three determinations. The data (Table III) indicate that the cultures supplemented with calf serum displayed an inhibition of enzyme activity to about 300/, of the control value.
Cultures receiving either fatty acid or cholesterol also showed inhibition, but the extent of inhibition was less than that produced by total serum. The results suggested that fatty acid and cholesterol might be part of the components of serum that can influence the activity of acetyl-CoA synthetase. However, in cell homogenates, when various concentrations of serum lipid from 5 to 40% were added to the reaction mixture, there was no influence on enzyme activity (Table IV).
Preliminary experiments using isolated serum lipids added to the reaction mixture also showed no inhibitory effect.

Acetate Incorporation
into Lipids-Alterations of exogenous lipid levels produced rapid compensatory changes in the rate of [i4C]acetate incorporation into cellular lipid. Total cell lipid was measured in these experiments to investigate possible coordinate regulation of sterol and fatty acid synthesis. When incorporation of [i4C]acetate into sterol and fatty acids were measured separately, similar changes were observed which paralleled those of total lipid under the culture conditions employed.
The inhibition and stimulation of sterol synthesis  (7), Brown et al. (8), and Kandutsch and Chen (12). The stimulation of fatty acid synthesis from acetate observed upon changes in serum lipid supply was similar to that observed by Jacobs et al. (3). These workers found, however, an even more rapid inhibition of [r4C]acetate incorporation into fatty acid which occurred within 10 min of addition of serum or fatty acid to the medium.
It is important to note that the present study deals with the early control events which occur within 12 hours after change in external lipid source. Additional changes in rates of acetate incorporation, especially into fatty acids, occur during the next several days; these could be due to induction or repression of several enzymes (3,4) and also to multiple effects related to induction of cell division in the culture.
A coordinate effect between fatty acid and sterol metabolism has been demonstrated in these studies. That is, fatty acids added to the culture medium resulted in inhibition of sterol synthesis from acetate and, conversely, cholesterol in the medium inhibited incorporation of [i4C]acetate into both fatty acids and glycerolipids.
Although the inhibition of sterol synthesis from acetate by fatty acid could result partly from dilution of intracellular acetyl-CoA pools through oxidation of fatty acid, this dilution effect could not be applicable to the converse observation of inhibition of acetate incorporation into fatty acid by cholesterol since cholesterol is not converted to acetate in animal cells. Similar coordination has been observed previously in long term growth experiments (2). An inhibition of fatty acid synthesis by cholesterol was observed in some of the experiments of Kandutsch and Chen (12), who investigated the effect of various sterol analogs on sterol synthesis in L cells and liver cell cultures.
In our hands, inhibition of fatty acid synthesis by sterol was quite reproducible, but always of lesser magnitude (around 40 to 60%) as compared to the magnitude of inhibition (85 to 90%) when fatty acid or total serum are added (Table I and Figs. 4 and 5). This suggests that the effects of sterol on fatty acid synthesis may be influenced by other variables such as growth conditions or be mediated by only one of the possible control points in Fig. 10.
It is interesting that the stimulation of [i4C]acetate incorporation into lipid observed in L cell cultures occurred when cells were transferred both to serum-free medium and to medium supplemented with delipidized serum proteins and that no greater magnitude of stimulation was observed in the presence of DLSP.
Williams and Avigan (5) have investigated the effect of DLSP on the stimulation of sterol and fatty acid synthesis, and their data indicate a stimulatory effect of DLSP which is more pronounced in fibroblast cultures.
The results of the studies of stimulation and inhibition of acetate incorporation into total lipid in the presence of inhibitors of macromolecule synthesis must be interpreted with great caution since it is well established that these inhibitors do not have single clearly defined actions in animal cells (27). In fact, the increased variability observed in the data in these experiments is probably a reflection of the complex action of these compounds.
However, the fact that their presence seemed to have no effect on the time course of the early stimulation and inhibition of enzyme levels is an indication that the regulation is occurring at the level of enzyme activity; in cases of control where changes in absolute amounts of enzyme protein levels have been confirmed, these inhibitors did influence the course of events. For example, Brown et al. (8) report that cycloheximide inhibits the st.imulation of HMG-CoA reductase in cultured fibroblasts, and the data of Raff (28) indicate that, when glucose is used as a precursor of fatty acid, cycloheximide prevents stimulation of fatty acid synthesis, reflecting the long term regulatory steps at the level of acetyl-CoA carboxylase (3) and fatty acid synthetase (4).
Acetyl-CoA Synthetase Activity-No other studies have been reported on the activity of this enzyme in cultured cells. Acetyl-CoA synthetase has been studied, however, in whole animals (29,30). The Km observed for the enzyme present in cell homogenates corresponds quite well with that obtained for more purified preparations.
Londesborough et al. (31) report a K, of 30 mM for ATP and 1 mM for CoA in a purified enzyme from ox heart mitochondria, and Klein and Jahnke (32) report saturation of the yeast acetyl-CoA synthetase by 0.2 mM CoA and 8 miw ATP.
Several lines of evidence suggest that the enzyme may have a role in the regulation of acetate incorporation into lipid in cultured cells. In the first place, the time course of stimulation and inhibition of enzyme activity parallels that of acetate incorporation into lipid. Second, both responses are insensitive to inhibitors of macromolecule synthesis. Third, both acetate incorporation and enzyme activity are inhibited by addition of fatty acid and cholesterol to the medium.
Finally, calculations of the amount of carbon converted to lipid based on the observed values for enzyme activity and lipid synthesis from acetate indicate that the enzyme in cells cultured in the presence of serum could be the rate-limiting step of conversion of acetate into cell lipid.
Several studies conducted on preparations from whole animals support a possible regulatory role for this enzyme (13,17,32). Steiner and Cahill (13) found that, upon feeding high fat diet, lipogenesis from acetate was decreased in brown adipose tissue homogenates, but lipogenesis from acetyl-CoA remained constant. Murthy and Steiner (15) found that activity of acetyl-CoA synthetase decreased in livers of starved or alloxan diabetic rats. cell cultures and the lack of effect of inhibitors on macromolecule synthesis suggested that lipids might be acting as allosteric effecters of this enzyme. The sigmoidal shape of the response of the activity to changing ATP concentration also would be consistent with it being allosterically controlled. However, no effect was observed in these studies when serum lipid was added directly to the homogenates during assay of enzyme activity. Preliminary attempts were made in this study to add individual lipids to the reaction mixture with negative results. It is interesting that Murthy and Steiner (15) report inhibitory effect of sodium oleate (1 to 4 mM) on enzyme activity in vitro. It is very difficult, however, to separate this observation from a detergent effect of the lipid.
Further studies are planned to elucidate whether isolated lipids added in vitro are allosteric effecters of the enzyme, or whether a secondary regulator such as a protein kinase might be involved.
Mechanisms of Regulation of Acetate Incorporation into Lipid-Although it disguises the undoubtedly greater complexity of the regulation of cell lipid biosynthesis, Fig. 10 illustrates schematically the control points for lipid synthesis that thus far have been identified and partially characterized in cultured cells. From the available evidence it seems that two major control points are at the first committed steps in fatty acid and sterol biosynthesis (Fig. 10, 1 and 2). It appears that these steps are regulated via the induction and repression of the appropriate enzymes acetyl-CoA carboxylase (3) and HMG-CoA reductase (7,8).
Another control point in fatty acid synthesis via enzyme induction and repression occurs at the level of fatty acid synthetase (Fig. 10, S) (4). From the evidence presented here a fourth point of control which would influence both pathways is via regulation of the acetate activating enzyme (Fig. 10, 4) the mechanism, which is so far unidentified, appears to be at the level of enzyme activity rather than to involve induction and repression of synthesis.
It is also indicated in Fig. 10 that other control points may exist between mevalonic acid and cholesterol (Fig. 10, 6) (33).
It is di,fficult, however, to determine which control point is rate-limiting and most important physiologically. It is possible that, although a coordinate effect between fatty acid and sterol has been observed, separate points of regulation in fatty acid and sterol pathways might be the rate-limiting ones. It is clear in the case of fatty acid synthesis that the time course of regulation of lipid synthesis from acetate observed in our hands and by Jacobs et al. (3,9) could not be accounted for by the kinetics of regulation of acetyl-CoA carboxylase or fatty acid synthetase. It also has been observed in in tivo experiments that acetate incorporation into fatty acid is greatly inhibited in a rapid fashion after a short period of fasting (10,11). The definition of the relative role of various control points in sterol synthesis is more difficult since the induction and repression of HMG-CoA reductase occurs within 4 to 6 hours of change in exogenous lipid (8). Therefore, a stimulation of [*dC]acetate incorporation into sterol would reflect either changes in HMG-CoA reductase or regulation at the level of acetate or both. Similarly, inhibition of [14C]acetate incorporation into sterol could reflect changes in either HMG-CoA reductase or acetate activation and in addition be subjected to dilution effects in the oxidation of fatty acid to acetyl-CoA.
Some indications of the relative importance of acetyl-CoA synthetase and HMG-CoA reductase in regulation of sterol synthesis comes from data of Bates and Rothblat (26). They compared inhibition of sterol synthesis from [Wlacetate and aHzO and observed a greater per cent inhibition of synthesis in L cells using acetate as a precursor, the difference presumably reflecting controls at the level of 2-carbon metabolism.
The significance of various cellular control points may be related to the importance of acetate as a substrate for lipid synthesis.
In cultured cells grown in medium containing 0.1 mg per ml of acetate, it serves as approximately 20% of the carbon source for lipid synthesis.* Acetate is also a substrate for acetate metabolism in tiuo at times of ethanol ingestion, since ethanol is converted to acetate by the liver (34). It is also possible that regulation of acetyl-CoA synthetase is involved in vivo in the metabolism of acetoacetate, which also can serve as its substrate (29). It thus would be important in situations of ketoacidosis when ketone bodies are significant carbon sources to peripheral cells.
Finally, there are several points in addition to acetyl-CoA synthetase at which control of acetate metabolism could occur. One possible site is acetate transport into and out of the cell. It also has been established recently that three pools of acetyl-CoA exist in certain cells (35,36) and that tolbutamide can effect the intracellular flow of acetyl-CoA between the three pools. The final possibility is that there could be a competition intracellularly for coenzyme A so that availability of CoA and its intracellular transport may be a site of coordination and control. Studies are currently in progress using cells in culture to determine which mechanisms are active in control of 2-carbon metabolism and to elucidate the physiological significance of acetyl-CoA synthetase in the regulation of lipogenesis.