Long-term regulation by theophylline of fatty acid synthetase, acetyl-CoA carboxylase and lipid synthesis in cultured glial cells

195

Biochimica et Biophysics Acto, 431 (1976) 195-205 @ Elsevier Scientific Publishing Company, Amsterdam

- Printed in The Netherlands

BBA 56773

LONG-TERM REGULATION BY THEOPHYLLINE OF FATTY ACID SYNTHETASE, ACETYL-CoA CARBOXYLASE AND LIPID SYNTHESIS IN CULTURED GLIAL CELLS

JOSEPH J. VOLPE * and JAYNE C. MARASA Departments of Pediatrics and Neurology and Neurosurgery (Neurology), Washington University School of Medicine, St. Louis, MO. 63110 (U.S.A.) (Received

November

17th, 1975)

summary The long-term regulation of fatty acid synthetase and acetyl-CoA carboxylase and of fatty acid and sterol synthesis was studied in C-6 glial cells in culture. When theophylline (10T3 M) was added to the culture medium of these cells, rates of lipid synthesis from acetate and activities of synthetase and carboxylase became distinctly lower than in cells that were untreated. This effect appeared after approximately 12 h, and after 48 h enzymatic activities were reduced approx. 2-fold and rates of lipid synthesis from acetate 3- to 4-fold. The likelihood that the decrease in fatty acid synthesis from acetate was caused by the decrease in activities of fatty acid synthetase and acetyl-CoA carboxylase was established by several observations. These indicated that the locus of the effect probably did not reside at the level of acetate uptake into the cell, alterations in acetate pool sizes or conversion of acetate to acetyl-CoA. Moreover, de novo fatty acid synthesis was found to be the predominant pathway in these glial cells, whether treated with theophylline or not. The mechanism of the effect of theophylline on fatty acid synthetase was shown by immunochemical techniques to involve an alteration in content of enzyme rather than in catalytic efficiency. The change in content of fatty acid synthetase was shown by isotopic-immunochemical experiments to involve a decrease in synthesis of the enzyme. The mechanism whereby theophylline leads to a decrease in lipogenesis and in the synthesis of fatty acid synthetase may not be mediated entirely by inhibition of phosphodiesterase and an increase in cyclic AMP levels, because dibutyryl cyclic AMP (10V3 M) only partially reproduced the effect. Abbreviations: Cyclic AMP, adenosine 3’.5’-monophosphate: dibutyryl cyclic AMP, N6, O’-dibutyryl cyclic AMP. * To whom reprint requests should be addressed, at St. Louis Children’s Hospital, 500 South Kingshighway, St. Louis, Missouri 63110. U.S.A.

196

These data establish a role for theophylline and cyclic AMP in the regulation of lipogenesis and the activities of fatty acid synthetase and acetyl-CoA carboxylase in C-6 glial cells, which have features of glia found in developing nervous tissue. In addition, the data may be of particular clinical importance in view of the recent interest in the use of theophylline in the therapy of apneic spells in human newborns.

Introduction The de novo synthesis of fatty acids in mammals proceeds from acetyl-CoA by the sequential action of two enzyme complexes, acetyl-CoA carboxylase and fatty acid synthetase, which catalyze the following reactions, respectively: Acetyl-CoA

+ CO2 + ATP -+ Malonyl-CoA

Acetyl-CoA + 7 Malonyl-CoA 14 NADP++ 7 CO2 + 6 Hz0

+ ADP + Pi

+ 14 NADPH + 14 Ht+

Palmitic

acid -t 8 CoA +

Re~lation of palmitic acid synthesis has been studied in greatest detail in liver (for review, see ref. 1). Regulation of this process is of particular importance in brain because virtually all of the fatty acids found in the nervous system (except the polyunsaturated fatty acids) can be derived from this compound. Although fatty acid synthetase of mammalian brain has been studied in vivo in considerable detail [Z--5], precise delineation of the important regulatory effecters of this enzyme has been hindered by such factors as the cellular heterogeneity of the tissue, the uncertainties of transport of potential effecters under various experimental circumstances, the difficulties of studying the tissue early in differentiation, and the possibility that different cell types are regulated by different effecters during restricted time periods of development. In an attempt to circumvent some of these problems we have turned to glial cells in culture (C-6, rat atrocytoma [6] ). We have demonstrated previously that fatty acid synthesis in these cells can be regulated by lipid [7] ; this is of particular interest because studies of whole brain in vivo suggested that regulation of palmitic acid synthesis by lipid did not occur in this tissue [3,4]. Theophylline is a methylxanthine, classified in pharmacological terms as a central nervous system stimulant 181. The drug has received wide use experimentally, particularly because of its effect of inhibition of cyclic AMP phosphodiesterase and potentiation of responses mediated by this mononucleotide [9]. Theophylline administered to starved rats that are refed a fat-free diet results in liver in a diminution in the induction of fatty acid synthesis, the activities of fatty acid synthetase and acetyl-CoA carboxylase, and the synthesis of fatty acid synthetase [lo]. The drug also partially inhibits the induction of fatty acid synthetase by insulin in Chang liver cells in culture [ 111. In view of these facts that theophylline has important effects on the central nervous system and on fatty acid synthesis, we undertook the present study with the following objectives: (1) to determine whether theophyll~e has an effect on lipid synthesis and

197 the activities of fatty acid synthetase and acetyl-CoA carboxylase in C-6 glial cells. (2) to define whether the effects on fatty acid synthesis occur primarily in the de novo pathway, (3) to define the mechanism causing the changes in activity of fatty acid synthetase, and (4) to determine whether the effect of theophylline can be reproduced by cyclic AMP. Materials and Methods Materials Acetyl-CoA (P-L Biochemicals), malonyl-CoA (P-L Biochemicals), NADPH (Sigma), dithiothreitol (Sigma), ATP (Sigma) and fatty acid-poor albumin (Miles Laboratories) were obtained from the designated sources. NaH14C03 (specific radioactivity 9.2 Ci/mol), sodium [ 1-14C] acetate (specific radioactivity 54.8 Ci/mol), sodium [2-‘“Cl acetate (specific radioactivity, 58.6 Ci/mol, D[U-‘4C] glucose (specific radioactivity 195 Ci/mol), ~-[4,5-~H] leucine (specific radioactivity 43.9 Ci/mmol) and [ 1-14C] palmitic acid (specific radioactivity 6.59 Ci/mol) were obtained from New England Nuclear. NCS Solubilizer was purchased from Amersham-Searle. Radioactive products were counted in a preblended scintillation fluid (3a70, Research Products International). Calf serum, antibiotics and other reagents for cell culture were obtained from Grand Island Biological Company. Tissue culture flasks were purchased from Falcon. Theophylline and dibutyryl cyclic AMP were obtained from Sigma. Cell culture The cells used in this study, C-6 glial cells, cloned originally from a rat astrocytoma, were obtained from the American Type Culture Collection 3 years ago and maintained in this laboratory since. The various methods of cell culture were described previously [ 71. Cells for each experiment were derived from a single flask; size of each inoculum was identical and based on cell number (determined on a Gentian violet-stained aliquot and counted in a hemocytometer). Inocula were adjusted to give a final concentration of cells transferred of 0.5 - 106/ml in either 25-cm2 or 75-cm2 flasks, as indicated below. In all experiments, the medium was changed every 24 h and, when appropriate, freshly prepared theophylline or dibutyryl cyclic mononucleotide added. Preparation of extracts Cells were harvested and disrupted by freeze-thawing, and the supernatant solution after centrifugation of the disrupted cells at 12 000 X g for 10 min used for assays [7]. Enzyme assays Fatty acid synthetase was determined by the spectrophotometric assay [ 121. One unit of enzyme activity is defined as the amount required to catalyze the oxidation of 1 nmol of NADPH per min at 37°C.

198

Acetyl-CoA carboxylase was assayed by measuring the recovery of acid-stable radioactivity after incubation with NaH14C03 [13] in a modified reaction mixture [ 71. One unit of enzyme activity is defined as the amount required to catalyze the fixation of 1 nmol of Hi4C0; per min at 37°C. Protein was determined by the method of Lowry et al. [ 141 or by a microbiuret procedure [ 151. Fatty acid, s terol and protein

synthesis

Synthesis of fatty acids and sterols was determined by measuring incorporation of radioactivity into these products from sodium [2-14C]acetate (concentration in the medium 0.5 pCi/ml), as described [ 71. In some experiments, the precursor was sodium [l-14C] acetate (see Schmidt decarboxylation) or D[ U-14C] glucose (final specific radioactivity in the medium 7.1 Ci/mol). Experiments in which the rates of protein and lipid synthesis were measured concurrently in separate flasks utilized ~-[4,5-~H] leucine as precursor for determination of protein synthesis. The radioactive amino acid was added to the medium in a concentration of 5 /_&i/ml, duration of pulse was 60 min and protein was isolated by trichloroacetic acid precipitation. Schmidt

decarboxylation

Radioactivity in the carboxyl carbon of fatty acids synthesized from sodium [l-14C] acetate was determined essentially by the adaptation of the Schmidt reaction described by Brady et al. [ 161. Cells in 75-cm2 flasks were incubated for 1 h in medium containing sodium [ 1-14C] acetate (concentration in the medium 1 /.&i/ml). Fatty acids were extracted as described above and unlabelled palmitic acid (10 E.tmol) added. The Schmidt reaction was carried out in 25 ml Erlenmeyer flasks. In lieu of a glass well sealed to the bottom of the flask, a plastic center well was suspended into the flask and inserted into the sealed serum stopper by an attached plastic rod (apparatus supplied by Kontes). The released 14C02 was trapped by 0.3 ml of NCS Solubilizer injected into the plastic well. The plastic well then was placed directly in a scintillation vial, the NCS Solubilizer neutralized with acetic acid, 10 ml of 3a70 added, and the sample counted. Erlenmeyer flasks containing only [ l-14C] palmitic acid were carried concurrently through the whole procedure with all samples, and recoveries of this standard were consistently approx. 90%. Immunological

procedures

The immunochemical techniques utilized antibody prepared against homogeneous rat liver fatty acid synthetase [4]. After O-50% ammonium sulfate precipitation of serum, immunoglobulins were isolated by a batch procedure utilizing DEAE-Sephadex [ 171. Quantitative preciptin analyses [ 4,181 were used to compare the amount of immunoprecipitable enzyme per unit of activity among various extracts. Isotopic-immunochemical analyses were utilized to measure synthesis of fatty acid synthetase, isolated by immunoprecipitation, and total protein, isolated by trichloroacetic acid precipitation [4,7] . Statistical procedures

Statistical

significance

was determined

by Student’s

t test

[ 191. The t test

199

was carried out for all the data for which means f S.E. were calculated, values are presented where uncertainties about statistical significance arise.

and P might

Results Effect of theophylline on fatty acid and sterol synthesis and fatty acid synthetase and acetyl-CoA carboxylase activities The effect on the important lipogenic enzymes, fatty acid synthetase and acetyl-CoA carboxylase, and on the synthesis of fatty acids and sterols from acetate, following exposure of C-6 glials cells to various concentrations of theophylline for 24 and 48 h, is shown in Table I. After 24 h the activities of synthetase and carboxylase were dimished clearly (P< 0.01) with concentrations of theophylline of 10e3 M. After the same interval, this concentration resulted in a greater decrease in rates of fatty acid and sterol synthesis from acetate. After 48 h, effects on enzymatic activities and lipid synthesis were even more striking in the cells grown in 10e3 M theophylline. Modest effects on both the enzymatic activities (P < 0.05) and lipid synthesis (P < 0.01) became apparent in cells grown in lo4 M theophylline for 48 h. The time course of the effect of lob3 M theophylline on synthetase and carboxylase activities and on synthesis of fatty acids and sterols was defined next (Fig. 1). The temporal features of the changes in all parameters were similar. Thus, between 9 and 13 h after exposure to theophylline, a relative decrease in enzymatic activities and in lipid synthesis appeared. The effects became more

TABLE

I

EFFECT OF CONCENTRATION OF THEOPHYLLINE AND FATTY ACID SYNTHETASE AND ACETYL-CoA

ON FATTY ACID AND STEROL CARBOXYLASE ACTIVITIES

SYNTHESIS

Cells were transferred to a series of 24. 25-cm2 flasks and grown in 10% serum for 72 h. At that time, the medium was changed so that all flasks contained no serum. To 12 of the flasks theophylline was added to the culture medium in the concentrations shown. After 24 and 48 h in the appropriate medium, activities of fatty acid synthetase and acetyl-CoA carboxylase were determined in the cells of 3 flasks for each group (i.e. 6 flasks), and synthesis of fatty acids and sterols from [2- 14C] acetate in the cells of 3 separate flasks for each group (i.e. 6 flasks), as described in Methods. Values are expressed as percentage of specific activity of cells from flasks without theophylline. Values for enzymatic specific activity and radioactivity of lipids were means obtained from separate determinations on each of 3 flasks. The 100% values for specific activities in unitslmg protein (mean * S.E.) at 24 and 48 h were. for fatty acid synthetase, 3.88 f 0.20 and 5.73 t 0.35. respectively, and for acetylCoA carboxylase, 1.40 * 0.07 and 2.20 f 0.10, respectively. The 100% values for synthesis in cpm/mg protein X 10e3 (mean f. S.E.) at 24 and 48 h were, for fatty acids, 24.0 + 2.1 and 39.2 C 4.1. respectively, and for sterols. 9.4 f 0.8 and 18.7 ? 1.05. respectively. Similar results were obtained in 3 separate experiments. Time

(M)

Fatty acid synthetase specific activity (% of control)

Acetyl-CoA carboxylase specific activity (% of control)

Fatty acid synthesis (% of control)

Sterol synthesis (% of control)

24 24 24

1o-s lo4 1o-3

100 89 63

100 91 67

100 98 40

100 95 26

48 48 48

1o-5 lo4 ld3

97 85 56

95 85 47

100 75 29

100 78 22

(h)

Theophylline concentration

200

prominent over the next 11 h and then approached a maximum between 24 and 42 h after addition of theophylline, Sterol synthesis was dimished in a similar manner. These effects on synthetase and carboxylase and on lipid synthesis were not accompanied by similar alterations in total protein synthesis. Thus, at several of the times indicated in Fig. 1, L-[4,5-3H]leucine was added to 4 separate flasks, 2 with and 2 without 10V3 M theophylline, and the incorporation of radioactivity into total protein determined, as described in the Methods. No significant difference in protein synthesis between cells grown with or without theophylline was observed at the times studied, i-e. 13, 20, 24 and 42 h after addition of the drug. The magnitude of the decrease in fatty acid synthesis from acetate was greater than that observed for the activities of acetyl-CoA carboxylase and fatty acid synthetase. Thus, after 19 h of growth in theophylline, the rate of fatty acid synthesis was 50% of the rate for cells grown without the drug, whereas the activities of synthetase and carboxylase at this time in cells grown in theophylline were 75% of the activities in control cells. Not until after 42 h of growth in theophylline did the enzymatic activities decrease to 50% of the control values. This raised the possibility that additional factors were operative to cause the larger observed differences in rates of incorporation of acetate into fatty acids, Such factors could include differences in uptake of acetate into the cells, acetate pool sizes, generation of .acetyl-CoA or relative contribution of the chain-elongating systems for synthesis of long chain fatty acids. These possibilities were evaluated further (see next section). Further ~efi~i~io~ of the effect of the~~h~~~ineon lipid s~nthes~ To estimate the role that differences in acetate uptake, pool sizes or activation play in the genesis of the differences in lipid synthesis described above, the effects of theophylline were compared when [ U-14C] glucose, [ 2-14C] acetate, or [ 2-14C] acetate diluted over lOO-fold with unlabeled acetate were used as precursors for lipid synthesis. When cells were grown in the presence of 1O-3 M theophylline for 48 h and [U-‘“1 glucose used as precursor, the rate of fatty acid synthesis was diminished to approximately 25% of control values, A virtually identical result was observed when [2-14C] acetate was used. A coordinate response with the two precursors also was seen when incorporation of radioactivity into sterols was measured. Thus, it is unlikely that differences in acetate uptake or activation to acetyl-CoA play an important role in the genesis of the inhibitor effect of theophylline on lipogenesis. The possibility that differences in acetate pool sizes account for the lower rate of lipid synthesis from acetate was evaluated by diluting the [ 2-14C] acetate approximately 120-fold with unlabelled acetate [lo-’ M). The effect of theophylline was apparent again, and thus isotopic dilution of the labeled precursor within the cell does not appear to underlie the observed differences in incorporation into newly synthesized lipids. To determine whether theophylline causes important differences in the relative contributions of the de novo vs. chain elongation pathways for fatty acid synthesis and thereby cause the observed differences in acetate incorporation into fatty acids, we estimated these contributions by Schmidt decarboxylation of fatty acids synthesized fron [ 1-i4C] acetate by cells grown in serum-free me-

201

Fig. 1. Fatty acid synthetase and acetyl-CoA carboxylase specific activities and fatty acid and stem1 synthesis as a function of duration of growth in 10e3 M theophylline. Experimental details were similar to those described in the legend to Table I. Cells were studied at the times indicated. Values are expressed as percentage of specific activity of cells from flasks without theophylline. See legend to Table I. Similar results were obtained in 3 separate experiments. (0) Fatty acid synthetase; (a) acetyl-CoA carboxylase; (0) fatty acid synthesis; (0) sterol synthesis. Fig. 2. Quantitative precipitin reactions of fatty acid synthetase of cells grown with or without theophylline. Experimental detaii were similar to those described in the legend to Table I. Cells were grown with or without 10T3 M theophylline for 48 h. The enzyme extracts were 22-38% ammonium sulfate fractions. Specific activities of fatty acid synthetase in units/mg protein (means f S.E.) were for cells grown in low3 M theophylline (0) 10.2 ? 0.9 and for cells grown in the absence of low3 M theophylline (0) 19.4 + 1.1.

dium with or without theophylline (10e3 M) for 24 h. The % of radioactivity in the carboxyl carbon of fatty acids synthesized by cells grown with theophylline was 21.7 + 3.1, and by cells grown without theophylline, 16.7 f 4.2. Thus, the data indicate that the predominant pathway for incorporation of acetyl-CoA units into fatty acids in C-6 glial cells grown in the presence or absence of theophylline was de novo synthesis. Moreover, there was no marked difference from control cells in the relative contributions of the de novo vs. chain elongation pathways when cells were grown in the presence of theophylline. Mechanism of the effect of theophylline on fatty acid synthetase Because the data presented above indicated that the primary effect of theophylline on fatty acid synthesis was exerted at the level of the enzymes directly involved in de novo synthesis, it became of particular importance to determine the mechanism of this enzymatic effect. To determine whether the inhibitory effect of theophylline on fatty acid synthetase reflected a change in content of enzyme rather than in catalytic efficiency, we utilized immunochemical techniques. Quantitative precipitin analyses of extracts from cells grown with or without theophylline for 48 h revealed identical equivalence points for both groups (Fig. 2). This observation indicated that there was an equal amount of immunoprecipitable enzyme per unit of activity in extracts of both groups. Thus, the differences in specific activity must reflect differences in content of enzyme. To determine the role of enzyme synthesis in the production of the lower content of enzyme observed in cells grown in the presence of theophylline, we performed an isotopic-immunochemical experiment (Table II). A nearly 2-fold

202 TABLE EFFECT

II OF THEOPHYLLINE

ON SYNTHESIS

OF FATTY

ACID SYNTHETASE

Cells were transferred to 8. 75-cmZ flasks and grown in 10% serum for 72 h. Medium was changed so that all flasks contained no serum and one-half of these, low3 M theophylline. After 36 h, an isotopic-immunochemical analysis was carried out. as described in Methods, Values for fatty acid synthetase are means + S.E. obtained from separate determinations on each of 4 flasks. Extracts were pooled from all 4 flasks for the determinations of specific radioactivity, and these values are means of duplicate determinations of two levels of extract from each pooled sample (duplicate numbers differed by less than 5% and values were linear). Similar results were obtained in a second experiment. Additions

Fatty acid synthetase specific activity (units/mg protein)

Specific

radioactivity

Fatty acid synthetase (A) (cpmlmg protein, X 10m3)

Total protein (B) (cpm/mg protein. x 165)

Relative rate of synthesis (100 A/B)

None

8.46

9.34

1.63

5.73

Theophylline. 1O-3 M

4.46

5.61

1.83

3.01

lower synthetase specific activity was observed in cells grown in the presence of the drug. The incorporation of ~-[4,5-~H] leucine into the enzyme was also considerably lower in these cells, whereas the incorporation of the radioactive amino acid into total protein was slightly higher. The most meaningful way to interpret these data is to relate the incorporation of radioactivity into the enzyme to the incorporation of radioactivity into total protein. This controls in large part for such factors as variations in uptake of leucine or size of precursor pools. This relative rate of synthesis was nearly 2-fold lower in the cells grown in the presence of theophylline, and this difference corresponds closely to that observed for specific activity. Thus, these data indicate that a decrease in synthetase synthesis causes the decrease in content of the enzyme in cells grown in the presence of theophylline.

TABLE

III

EFFECT OF DIBUTYRYL CYCLIC AMP ON FATTY ACID AND STEROL ACID SYNTHETASE AND ACETYL-CoA CARBOXYLASE ACTIVITIES

SYNTHESIS

AND FATTY

Experimental details were similar to those described In the legend to Table I. To one-half of the flasks dibutyryl cyclic AMP was added in the concentrations indicated and incubations were for 24 or 48 h. Values are expressed as percentage of control, i.e. cells from flasks without the mononucleotide. and were derived as described in the legend to Table I. The 100% values for enzymatic specific activity and radioactivity of lipids were similar to those recorded in the legend to Table I. Time (hl

Dibutyryl cyclic AMP concentration

Fatty acid synthetase specific activity (% of control)

Acetyl-CoA carboxylase specific activity (% of control)

Fatty acid synthesis (% of control)

Sterol synthesis (96 of control)

96 77 91 76

85 76 81 75

85 78 73 49

80 68 81 42

(Ml 24 24 48 48

10-4 10-3 10-4 10-g

203

Effect of dibutyryl cyclic AMP on fatty acid and sterol synthesis and fatty acid synthe tase and acetyl-CoA carboxylase activities In view of the inhibitory effect of methylxanthines on the phosphodiesterase that catalyzes the degradation of cyclic AMP, we evaluated the effect of the dibutyryl derivative of cyclic AMP on lipid synthesis and on the activities of fatty acid synthetase and acetyl-CoA carboxylase (Table III). The most significant effects on the lipogenic enzymes occurred with concentrations of 10e3 M. A modest, 25% decrease of specific activities (P < 0.01) occurred after 24 h of growth in the cyclic mononucleotide and did not become more prominent after an additional 24 h. After 48 h, lipid synthesis from acetate was decreased more than 50%, although not so impressively as when the same concentration of theophylline was used (compare Table I). Discussion This study has dealt with the long-term regulation of fatty acid synthesis in cultured glial cells and, specifically, the role of theophylline in this regulation. Cells exposed to this drug exhibit distinctly lower rates of synthesis of fatty acids and sterols and activities of the important lipogenic enzymes, fatty acid synthetase and acetyl-CoA carboxylase, when compared to cells grown in the absence of the drug. The effect becomes apparent approximately 12 h after addition of theophylline to the culture medium. The decrease in lipid synthesis from acetate is apparently not related to differences in acetate uptake or conversion of acetate to acetyl-CoA, since a similar decrease was observed with glucose as lipid precursor. Production of a similar inhibitory effect when large amounts of unlabelled acetate were added to the labeled acetate indicate that major differences in acetate pool sizes do not contribute to the results. Moreover, the predominant pathway of fatty acid synthesis in these cells, cultured either in the presence or absence of theophylline, was shown to be the de novo parthway. In view of the findings recorded above, it is highly probable that the major effect of theophylline on fatty acid synthesis is exerted at the level of fatty acid synthetase and acetyl-CoA carboxylase. Thus, it is of particular interest that, although quite distinct, the theophylline-induced decrease in the activity of these enzymes is not so marked as the decrease in the rate of fatty acid synthesis. If either synthetase or carboxylase were rate-limiting for fatty acid synthesis under these circumstances, it might be expected that the extent of the decrease in enzymatic activity of one or the other would predict the extent of the decrease in fatty acid synthesis. One possible explanation for the failure of the extent of the decrease in activity of either enzyme to predict the extent of the decrease in fatty acid synthesis is that neither synthetase nor carboxylase alone is rate-limiting under these experimental conditions and that the larger decrease in fatty acid synthesis results because the effects on the two enzymes are additive. Indeed, the quantitative and temporal changes of synthetase and carboxylase under a variety of conditions concerned with the long-term regulation of lipogenesis in vivo and in cell culture are closely coordinate [ 11. Thus the possibility must be considered that both enzymes may be involved concurrently in the control of the rate of fatty acid synthesis under certain circum-

204

stances. However, this consideration must be made with the awareness that under other circumstances either synthetase or carboxylase does appear to be rate-limiting for fatty acid synthesis [ 20,211. The mechanism of the effect of theophylline on fatty acid synthetase was elucidated by immunochemical techniques. The decrease in activity of synthetase induced by growth of cells in theophylline was shown by quantitative precipitin analyses to reflect a decrease in content of the enzyme not an alteration in catalytic efficiency. This decrease in synthetase content was shown by the isotopic-immunochemical experiments to be caused by a decrease in synthesis of the enzyme. Thus the methylxanthine induced an alteration(s) in C-6 glial cells that affects the synthetic machinery for fatty acid synthetase. This is the first direct demonstration in cell culture of a decrease in enzyme synthesis induced by theophylline. The findings have obvious analogies to those that we have made concerning the inhibitory effect of theophylline on the increase in hepatic synthetase synthesis produced by refeeding a starved animal [ 221. The mechanism of the effect of theophylline on the synthesis of fatty acid synthetase may relate only in part to the inhibition of cyclic AMP phosphodiesterase by this drug [ 91. Thus a considerable literature attests to this role of theophylline in increasing cellular levels of cyclic AMP [9] . However, in C-6 glial cells, theophylline does not markedly potentiate the stimulator-y effect of norepinephrine on cyclic AMP levels, whereas certain other methylxanthines, e.g. isobutylmethylxanthine, do [23,24]. Therefore, it may not be surprising that dibutyryl cyclic AMP only partially mimics the effect of theophylline on lipid synthesis and the activities of synthetase and carboxylase. Theophylline has other important physiological effects, apparently not mediated by inhibition of phosphodiesterase, such as alterations in membrane permeability [8, 251. At the present time, the paucity of our knowledge about the biochemical effects of methylxanthines on cultured glial cells prevents further delineation of the mechanism of the inhibition of synthesis of fatty acid synthetase induced by theophylline. Theophylline and dibutyryl cyclic AMP had inhibitory effects on sterol synthesis in C-6 glia that were similar to the effects observed on fatty acid synthesis. A decrease in the conversion of acetate to cholesterol has been observed in liver slices and in intact liver cells incubated in the presence of dibutyryl cyclic AMP [26,27]. The enzymatic locus of the inhibition of sterol synthesis under these circumstances remains unknown. These data concerning the effects of theophylline may be of important clinical significance. Thus, the effectiveness of theophylline in the therapy of neonatal apnea has been reported recently [28,29] , and an intense interest in this approach is developing. A major concern of such therapy is the possibility of a deleterious effect on the developing nervous system. Evidence that C-6 glial cells in culture exhibit properties of oligodendroglia [30,31] as well as astrocytes [32] has led to the conclusion that these cells are related most closely to the less differentiated stem cell that gives rise to these glial types. Thus, data derived with C-6 glial cells probably relate more to the developing than to the mature nervous system. When viewed in this context, the inhibitory effects of theophylline on lipid synthesis in these cells, therefore, may assume additional significance.

205

Acknowledgements This investigation was supported by Grant 1 ROl HD-07464-02 from the National Institutes of Health. J.J.V. is a recipient of a Research Career Development Award 1 K4 HD-70,608-02 from the National Institutes of Health. References 1 2 3 4 5 6

Volpe. Brady, Volpe, Volpe, Volpe, Benda,

J.J. and Vagelos, P.R. (1973) Annu. Rev. Biochem. 42. 21-60 R.O. (1960) J. Biol. Chem. 235. 3099-3103 J.J. and Kishimoto. Y.K. (1972) J. Neurochem. 19.737-753 J.J., Lyles. T.O.. Roncari. D.A.K. and Vagelos, P.R. (1973) J. Biol. Chem. 248. J.J. and Vagelos, P.R. (1973) Biochim. Biophys. Acta 326, 293-304 P., Lightbody, J., Sate, G., Levine, L. and Sweet, W. (1968) Science 161. 370-371

2502-2513

7 Volpe. J.J. and Marasa, J.C. (1975) J. Neurochem. 25. 333-340 8 Ritchie. J.M. (1975) in The PharmacoIigicaI Basis of Therapeutics (Goodman, L.E. and Gibnan, A.), pp. 367-378. Macmillan, London 9 Robison, C.A., Butcher. R.W. and Sutherland, E.W. (1971) Cyclic AMP, Academic Press. New York 10 Volpe. J.J. and Vagelos, P.R. (1974) Proc. NatI. Acad. Sci. U.S. 71. 889-893 11 Alberts. A.W.. Ferguson. K.. Hennessy, S. and Vagelos. P.R. (1974) J. Biol. Chem. 249, 5241-5249 12 Martin, D.B.. Homing, M.G. and Vagelos, P.R. (1961) J. Biol. Chem. 236,663-668 13 Martin, D.B. and Vagelos, P.R. (1962) J. Biol. Chem. 237.1787-1792 14 Lowry, O.H.. Rosebrough. N.J.. Farr, A.L. and RandalI. R.J. (1951) J. Biol. Chem. 193. 265-275 15 Munkres, K.O. and Richards, F.M. (1965) Arch. Biochem. Biophys. 109.466479 16 Brady, R.O.. Bradley. R.M. and Trams, E.H. (1960) J. Biol. Chem. 235.3093-3098 17 Baumstark, J.S.. Laffin, R.J. and Bardawil. W.A. (1964) Arch. Biochem. Biophys. 108, 514-521 18 Kabat. E.A. and Meyer. M.M. (1961) Experimental Immunochemistry, Charles C. Thomas, Springfield 19 Snedecor, G.W. (1956) Statistical Methods Applied to Experiments in Agriculture and Biology, Iowa State College Press. Ames 20 Klain, G.J. and Weiaer. P.C. (1973) Biochem. Biophys. Res. Commun. 55. 76-83 21 Guynn, R.W., Veloso. D. and Veech. R.L. (1972) J. Biol. Chem. 247.7325-7331 22 Volpe, J.J. and Marasa. J.C. (1975) Biochim. Biophys. Acta 380.454472 23 Gilman. A.G. and Nirenberg, M. (1971) hoc. NatI. Acad. Sci.. U.S. 68. 2165-2168 24 Schultz. J.. Hamprecht. B. and Daly, J.W. (1972) Proc. NatI. Acad. Sci. U.S. 69.1266-1270 25 Cuthbert, A.W. and Painter. E. (1968) J. Pharm. Pharmacol. 20.492495 26 Bricker. L.A. and Levey. G.S. (1972) J. Biol. Chem. 247.49144915 27 Capuzzi, D.M., Rothman, V. and MargoIis. S. (1974) J. Biol. Chem. 249.1286-1294 28 Shannon, D.C.. Gotay. F.. Stein, I.M.. Rogers, M.C., Todres, D. and MoyIan, F.M.B. (1975) Pediatrics 55.589-594 29 Uauy. R., Shapiro, D.L., Smith, B. and Warshaw. J.B. (1975) Pediatrics 55. 595-598 30 Zanetta. J.P.. Benda. P., Gombos. G. and Morgan. I.G. (1972) J. Neurochem. 19. 881-883 31 Volpe, J.J.. Fujimoto. K., Marasa. J.C. and AgrawaI, H.C. (1975) Biochem. 152. 701-703 32 Benda. P., Someda, K., Messer. J. and Sweet, W.H. (1971) J. Neurosurg. 34.310-323