Short-Term Effects of Free Fatty Acids on the Regulation of Fatty Acid Biosynthesis in Ehrlich Ascites Tumor Cells

[CANCER RESEARCH 34, 3355â€”3362,December1974] Short-Term Effects of Free Fatty Acids on the Regulation of Fatty Acid Biosynthesis in Ehrlich Ascites Tumor Cells

Richard McGee and Arthur A. Spector Departments ofBiochemistry [RM, AS] and InternalMedicine fAS] , Universityoflowa, Iowa City, Iowa 52242

SUMMARY observed (1 1, 13, 15, 16). For example, when isolated hepatocytes or cultured skin fibroblasts are incubated in media Fatty acid biosynthesis in Ehrlich ascites tumor cells was containing long-chain fatty acids complexed to BSA,@ fatty @ studied in vitro by measuring the incorporation H20 into acid synthesis is inhibited as compared with control incuba saponifiable lipids. Glucose was found to be a much better tions containing no added free fatty acids (1 1, 15 , 16, 23). substrate for fatty acid synthesis than acetate, @3-hydroxy The degree of inhibition depends on both the concentration butyrate, or amino acids. Enzyme assays using Ehrlich cell and structure of the added fatty acid (1 1, 15, 16, 23). cytosol preparations demonstrated the presence of the de novo Although the mechanism of short-term regulation has not been biosynthetic enzymes, acetyl coenzyme A carboxylase and established conclusively, several investigators have suggested fatty acid synthetase. The distribution of 3H between various that it involves inhibition of acetyl-CoA carboxylase by either fatty acids separated using gas-liquid chromatography sug free fatty acids or fatty acyl-CoA (9, 11, 13, 23, 24). gested, however, that both de novo synthesis and chain Fatty acid biosynthesis in normal liver is regulated by the elongation occur in intact Ehrlich cells. The existence of an dietary intake (2, 4, 6, 7, 10, 20, 22). By contrast, fatty acid elongation pathway was confirmed by demonstrating that biosynthesis in rat and mouse hepatomas is unaffected by the considerable amounts of radioactivity were present in fatty same dietary changes (19, 20, 26, 27). These findings have led acids longer than palniitate when cells were incubated with to a widely held view that the usual mechanism regulating palmitate-l-' â€˜C.Total fatty acid synthesis was inhibited when fatty acid production in nonmalignant tissues is absent in stearate, palmitate, oleate, or linoleate was added to incuba turmors. In the original studies with hepatomas, however, no tion media containing bovine serum albumin. Stearate pro distinction was made between short- and long-term control duced the largest effect, as much as 85% inhibition under processes. More recent work definitely indicates that hepa certain conditions. By contrast, myristate had little effect on tomas do not possess the long-term control mechanism (19), and laurate actually stimulated total fatty acid synthesis. In but no information is available as yet concerning short-term agreement with these observations, both laurate and lauroyl regulation in either hepatomas or any other tumor. coenzyme A stimulated acetyl coenzyme A carboxylase in an Ehrlich ascites tumor cells are suspended in a lipid-rich Ehrlich cell homogenate, while stearate and stearoyl coenzyme plasma during growth in the mouse peritoneal cavity (29). The A inhibited the enzyme. These findings indicate that, as in ascites plasma contains free fatty acids and lipoproteins, the nonmalignant cells, fatty acid synthesis in the Ehrlich cell is latter composed predominantly of very-low-density lipopro subject to short-term regulation by extracellular free fatty teins that contain large quantities of triglycerides (3, 31). acids. Recent studies indicate that Ehrlich cells can utilize both the free fatty acids and the triglycerides present in the ascites fluid (3, 29â€”31). On the other hand, in vitro studies have INTRODUCTION demonstrated that Ehrlich cells contain the CO2 -dependent de novo biosynthetic pathway for fatty acids (25). Therefore, it The de novo biosynthesis of fatty acids in mammalian was of interest to determine whether fatty acid production in tissues is regulated by at least 2 control mechanisms. One these tumor cells might be subject to any regulation by the involves changes in the actual amounts of the biosynthetic lipids present in the peritoneal fluid. Our results indicate that enzymes, acetyl-CoA carboxylase and the fatty acid synthetase the short-term regulatory mechanism for fatty acid biosyn complex. The content of these enzymes in the liver depends on the nutritional status and the dietary intake of fat and thesis, which has been described in nonmalignant cells, also is carbohydrate (4, 6). Changes in the levels of these enzymes operative in the Ehrlich cell. occur relatively slowly during a 2- or 3-day period, and this form of regulation is known as long-term control (2, 4, 6, 7, MATERIALS AND METhODS 10, 20, 22). A short-term control process that operates rapidly and without any changes in enzyme levels also has been Ehrlich Ascites Tumor Cells. Ehrlich cells were harvested from male CBA mice 11 or 12 days after i.p. injection of 0.4

1 This work was supported by Research Grants HL 14,781 and HL 14,388 from the National Heart and Lung Institute and ResearchGrant 2 The abbreviations used are: BSA, bovine serum albumin; GLC, 71-895 from the American Heart Association. gas-liquid chromatography; LDH, lactate dehydrogenase; U, uniformly ReceivedMay6, 1974; accepted September 9, 1974. labeled compound; iT,molar ratio of fatty acid to BSA.

DECEMBER 1974 3355

ml of a I : 10 dilution of tumor cells in sterile 0.1 54 M NaCl. hydrogen flame ionization detector and identified by compari At the time of sacrifice, each mouse contained between 5 X son with known standards. When fractions from the chromato 108 and 1 X l0@ tumor cells suspended in 5 to 10 ml of gram were to be collected for measurement of radioactivity ascites plasma. Cells were separated from the plasma by content, the column effluent was diverted from the detector centrifugation at 600 X g for 5 mm at 4Â°.Contaminating to a heated collecting port. Each fraction of the chromatogram erythrocytes were removed through mild hemolysis and was collected separately by attaching a 10-cm length of Teflon extensive washing with cold Krebs-Ringer bicarbonate buffer. tubing of 1.6-mm inside diameter to the collecting port and The cells were then resuspended in this buffer and kept at 0Â° allowing the effluent gas to bubble into 6 ml of the for not more than 30 mm prior to incubation. The toluene:Triton scintillator solution contained in a counting concentration of the suspended cells was determined by vial. The Teflon tubing was then cut into small pieces and counting a 1:100 dilution in a clinical hemocytometer. placed in the counting vial along with an additional 12 ml of Preparation of Incubation Media. The BSA used in these scintillator solution. Through the use of radioactive fatty acid experiments was defatted according to the procedure of Chen standards, it was determined that the efficiency of this (5). Fatty acids were added to solutions of BSA in buffer using collection method was between 85 and 90% and that the the Celite method (32). Media to which fatty acids had been retention time for a given methyl ester was the same when added were readjusted to pH 7.4. Fatty acid concentrations in measured with the flame ionization detector or collection of these solutions were determined by the titration method of the column effluent. Retention times for duplicate samples Trout et aL (36). Protein concentrations were measured by the were found to be reproducible to Â±0.02mm over a given 24-hr method of Lowry et aL (18) with defatted, lyophilized BSA as period. a standard. Unless indicated otherwise, the incubation media Measurement of Fatty Acid-synthetic Enzymes in Ehrlich contained 11 mM D-glucose. Isotopic tracers were added as Cell Cytosol Preparations. Washed Ehrlich cells were sus indicated in the legend of each chart and table. pended in 0.25 M sucrose at a concentration of about 200 mg, In Vitro Incubations. Metabolic experiments were carried wet weight, per ml sucrose. A Paar cell disruption bomb was out in rubber-stoppered glass flasks with a gas phase of 95% used to rupture the cells (37). The cell suspension was exposed 02 :5% CO2. Each flask contained 2 ml of the bicarbonate to 1000 psi N2 for 20 mm at 0Â°and then rapidly returned to buffer to which appropriate substrates had been added. After atmospheric pressure. The resulting homogenate was centri 1.0 ml of washed tumor cells (1.0 to 1.5 X 108 cells) were fuged successively at 600, 12,000, and 20,000 X g, each for 15 added to the flasks by injection, the contents were incubated mm, and then at 105 ,000 X g for 60 mm. All centrifugations at 37Â°with shaking in a temperature-controlled water bath. weredoneat 4Â°.Thesupernatantmaterialservedasthesource The reactions were terminated by rapid transfer of the of the Ehrlich cell de novo fatty acid-synthetic enzymes. incubation contents into 30 ml of cold 0.154 M NaCl in Acetyl-CoAcarboxylasewasmeasuredbythe formationof @ centrifuge tubes, followed by centrifugation at 600 X g for 5 acid-stable â€˜C from CO3 using the methods of mm. After the supernatant was removed, the cells were Gregolin et aL (12) and Moss et aL (21). Glucose 6-phosphate resuspended and washed 2 additional times with cold NaCl dehydrogenase was assayed by the method of Lobs and Wailer solution. (17). Fatty acid synthetase assays were carried out using the Total lipids were extracted from the tumor cells by a spectrophotometric assay system described by Goodridge (1 1). modification of the procedure of Folch et aL (8). The lipids To determine which fatty acids were synthesized, 0.2 jiCi of extracted into the chloroform phase were saponified at 65Â°for malony-CoA-l ,3-' 4C was incubated with the standard synthe 1 hr in 2 ml of ethanol containing 0.12 ml of 33% KOH. tase assay system for 15 mm at 25Â°.The incubations were Nonsaponifiable lipid was removed by extracting the alkaline terminated by the addition of 1 drop of 33% KOH. This saponified mixture 3 times with hexane. After acidification ensured that any fatty acyl-CoA that was formed during the the fatty acids were isolated by 3 extractions with hexane. incubation would be hydrolyzed. After acidification, fatty The radioactivity present in the saponifiable lipid fraction was acids were isolated through 3 extractions with hexane. Before either assayed directly or subjected to further fractionation. methylation, 0.5 mg each of myristate, palmitate, and stearate Radioactivity measurements were made in a refrigerated were added to the synthesized fatty acids to serve as carriers Packard Tri-Carb 2425 liquid scintillation spectrometer using a and to facifitate location of methyl esters on the gas-liquid toluene:Triton X-100 (4: 1, v,v) scintillator solution contain chromatogram. After methylation, the radioactivity present in ing PPO, 5.5 g/liter and POPOP, 0.1 g/liter. Correction for individual fatty acids was determined as described above. quenching was made using the automatic external standard LDH Release from Ehrlich Cells. Washed Ehrlich cells were channels ratio method. incubated at 37Â°in the bicarbonate buffer. After incubation, Separation of Individual Fatty Acids by GLC. Fatty acids the contents of the flasks were cooled rapidly to 0Â°and then contained in the saponifiable fraction of the Ehrlich cell lipid sedimented at 800 X g for 10 mm. The supernatant solution extract were methylated by incubation with 2 ml of 14% was then analyzed spectrophotometrically for LDH activity boron trifluoride in methanol for 10 mm at 85Â°.After the (14). In order to determine the total LDH activity present in addition of 2 ml of water, the fatty acid methyl esters were the cells, the contents of several flasks were sonically disrupted recovered by 3 extractions with hexane. The methyl esters for 3 mm in ice using a Branson Sonifier cell disruptor were separated using a Barber-Colman gas-liquid chromato equipped with a microtip and operating at maximum intensity. graph with a column containing 10% ethylene glycol succinate The homogenates were centrifuged at 800 X g for 10 mm, and on 80 to 100 mesh Chromosorb WAW with N2 as the carrier the supernatant solution was assayed for LDH activity after gas. Individual methyl ester peaks were detected with a being diluted 1:50 with bicarbonate buffer. The LDH assays

3356 CANCER RESEARCH VOL. 34

were all performed using 50 p1 of supernatant material in a Table I total volume of 2.5 ml. The total LDH content of Ehrlich cells Utilization of various substrates for fatty acid synthesis was determined both before and after incubation and was The incubations and analyses were preformed as described in the found to be constant. text. All of the media contained 0.1 mM BSA. Each value represents the average of 3 determinations Â±S.E. Glucose Oxidation. Washed Ehrlich cells were incubated in flasks containing center wells designed for CO2 collections ofConcentration3 Incorporation with either glucose-l -â€˜4Cor -6@ C. At the end of the H2 0 @ incubation, â€˜4CO2was trapped by injection of 0.50 ml of 6 N H/l08Substrates(mg/mi)cells/hr)D-Glucose20.532 (Mmole KOH into the center well and 0.3 ml of 4 N H2SO4 into the medium. After the flasks were allowed to continue shaking at 0.008Acetate20.023Â±[email protected] Â± 37Â°for 90 mm, 0.20 ml of the KOH was transferred to 18 ml of a toluene:methanol scintillation solution (35) for measure 0.003Aminoacidsâ€•2.20.042Â±0.001D-Glucose Â± @ ment of radioactivity. Using CO3 we determined that plus amino acidsa2 Â±0.008 @@ this procedure gave quantitative recovery of CO2 from the 2.20.665 bicarbonate-buffered medium. Materials. 3H20, D-glucose-U-'4C, 1-' 4C-fatty acids (pal a Amino acid mixture consisted of a casein hydrolysate, 2 mg/ml, to mitic, stearic, oleic, and linoleic), NaH' 4CO3 , and malonyl-l ,3- which glutamine, 0.1 mg/mI, and asparagine, 0.1 mg/mI, were added. I 4C-CoA were purchased from New England Nuclear, Boston, Table 2 Mass. D-G4 C and D -6@ C were obtained Inhibition offatty acid synthesis by palmitate as measured by from Amersham-Searle Corporation, Chicago, Ill. The BSA 4 C or 3H2 0 used was a Fraction V preparation from Miles Laboratories, Washed Ehrlich cells were incubated with the indicated radioactive Kankakee, Ill. Unlabeled fatty acids and standards for GLC substrates. Incubations were for 2 hr in media containing I 1 mM were provided by the Hormel Institute, Austin, Minn. Packing glucose, 0i mM BSA and palmitate (V as indicated). The results are material for GLC was purchased from Supelco, Inc., Belle expressed as percentage of incorporation relative to incubations in fonte, Pa. All CoA derivatives, dithiothreitol, and NADPH media containing no palmitate (average of 3 determinations Â±S.E.). were obtained from P-L Biochemicals, Milwaukee, Wis. (%)(palmitate:BSA)Glucose-U-' of fatty acid synthesis

VInhibition RESULTS 4CH201.929.6Â±2.413.5Â±3.33.252.4

Substrate Specificities. Table 1 shows the rates of fatty acid @ synthesis that were measured by H incorporation when Â±2.235.3 Â±1.2 various carbon sources were available to the cells. Consider ably higher rates of synthesis were observed with glucose than into cellular saponifiable lipid was essentially linear during a with acetate, j3-hydroxybutyrate, or a casein hydrolysate. A 2-hr incubation in a medium containing BSA and glucose 20% higher rate of synthesis occurred when glucose plus a (Chart 1). The possibility existed that, in the presence of mixture of amino acids was available as compared with glucose defatted BSA, some of the newly synthesized fatty acids might alone. We decided to omit the amino acid mixture in the be released from the cells into the medium (34). Any labeled remainder of this initial study, however, in order to keep the fatty acids released in this manner would not be detected by system as simple as possible so that mechanistic interpretations our usual analytical procedure. In order to investigate this might be more readily apparent. potential source of error, the release of tritiated fatty acids Selection of Radioactive Tracer. The rates of fatty acid was determined in an experiment in which both the cells and @ synthesis were compared when C or 1120 served the medium were assayed for 3H in saponifiable lipids after a as the isotopic tracer. Both isotopes yielded similar values 2-hr incubation with BSA and glucose. The cells contained when glucose was the only carbon source available in the 2.07 Â±0.01 j.zmoles of 3H (mean Â±S.E. of3 determinations), incubation medium. As seen in Table 2, however, different while the medium contained only 0.07 Â±0.01 imole of 3H values were obtained with each isotope when the medium (mean Â±S.E. of 3 determinations). We considered this amount contained unlabeled palmitate in addition to glucose. The of released radioactivity to be insignificant, and therefore fatty inhibition of fatty acid synthesis produced by palmitate acid release was disregarded in the rest of the experiments. @ appeared to be much larger when C served as the Addition of stearate to the incubation medium significantly tracer. Previous work indicated that palmitate is oxidized by depressed the incorporation of 3H into fatty acids by the Ehrlich cells (30, 33) so that one might expect the unlabeled Ehrlich cells (Chart 1), a finding consistent with the decrease acetyl-CoA formed from palmitate oxidation to mix in a noted with palmitate in Table 2. Incorporation of 3H in the common pool with the acetyl-CoA-' 4C produced from presence of stearate was essentially linear with time, and the glucose-U-' 4C. This would cause the specific radioactivity of percentage inhibition produced by stearate was fairly constant the acetyl-CoA pool to be lower in the cells exposed to over the 2-hr incubation period . Similar linearity and palmitate, leading to a falsely low observed rate of fatty acid constancy of percentage inhibition were observed when @ synthesis. Since the use of 1120 as the tracer avoids this palmitate was added in place of stearate. problem, we used this isotope in the present investigation. Chart 2 illustrates the effects of 6 free fatty acids on fatty The Effect of Various Long-Chain Fatty Acids on the Total acid biosynthesis in Ehrlich cells. In these experiments, the Incorporation of3H into Saponifiable Lipid. 3H incorporation concentration of BSA in the medium was constant but the

DECEMBER 1974 3357

R.McGee andA. A. Spector

fatty acid concentration was varied. The data are expressed in Table 3 terms of the molar ratio of fatty acid to albumin (1). Chart 2 Effect ofconcentration ofBSA or a palmitate.BSA complex on @ shows that each fatty acid affected H incorporation to a 3H incorporation into saponifwble lipids somewhat different degree. In all cases, the most dramatic Incubations were for 2 hr in media containing 11 mM glucose. The molar ratio of palmitate to BSA in flasks containing palmitate was held effects were observed between i@0 and 1, well within the constant at 2.4. Each value represents the average of 3 determinations Â± physiological range of i@values (30). With the saturated fatty S.E. acids the degree of inhibition increased as the chain length @ increased. Also, with the 18-carbon fatty acids, the extent of cells)Without of H20(j@mole H/10@ inhibition decreased as the number of double bonds increased. BSA The general shape of each of the curves is similar (except for (mM)Incorporationpalmitate0.05 palmitateWith laurate), with inhibition approaching a maximum value at relatively low values of iY. As shown in Table 3 , increases in fatty acid concentration 0.10 0.619Â±0.013 0.461Â±0.011 0.200.612Â±0.013 0.626 Â±0.0060.531Â±0.014 0.376 Â±0.008 inhibited fatty acid biosynthesis even when i@did not change. In this set of experiments, the BSA and fatty acid concentrations were raised concomitantly so that Â£@remained constant at 2.4. A 28% reduction in fatty acid biosynthesis was produced by a 4-fold increase in the concentration of the I I palmitate:albumin complex. By contrast, no change in the S -a synthetic rate was produced by corresponding increases in the .@ MS 1.5 U concentration of fatty acid-poor BSA. S 0 Cell Viability. It was possible that the reductions in fatty

@ â€¢55* acid biosynthesis produced when free fatty acids were added

@ 0 STEARATE/I$A to the incubation might be due to a nonspecific toxic effect on

*. 1.0 the cells. In order to evaluate this possibility, the release of I LDH from the cells into media containing BSA was compared in the presence and absence of stearate. As seen in Table 4, less than 2% of the cellular LDH activity was released from the cells during the 1st mm of incubation. There was very little increase in the amount of LDH released between 1 mm and 2 hr of incubation. Moreover, there was no increase in the amount of LDH released when stearate was present in the medium. This finding, together with the fact that albumin bound fatty acids in these concentrations do not reduce the TIME (MIN) oxygen consumption of Ehrlich cells (33), make it appear @ Chart 1. Time dependence of H incorporation into saponifiable unlikely that the reduction in fatty acid synthesis was due to a lipids in the absence and presence of stearate. Incubations were carried nonspecific toxic effect of fatty acids on the cells. out in media containing 11 mM glucose and 0.2 mM BSA. Where Effect of Fatty Acids on Glucose Oxidation. A 2nd possible indicated, 0.15 mM stearate was also present (&7 0.75). Each point explanation for the observed inhibitions was that the represents the average of 3 determinations; bars, S.E. long-chain fatty acids inhibit glycolysis and thus decrease the

I C., II. @ 0 Chart 2. Incorporation of H into saponifiable lipids in the I presence of exogenous fatty acids. Conditions of incubation 0 I- were the same as listed in Chart 1. The time of incubation was 4 g 2 hr. Each point represents the average of 3 determinations; 0 S. bars, S.E. Points without error bars had a standard error too g 0 small to be shown on the graph. The fatty acids are abbreviated U I as carbon number: degree of unsaturation, i.e., 12:0, laurate; 14:0, myristate; 16:0, palmitate; 18:0, stearate; 18:1, oleate; SM 18:2, linoleate.

1.0 2.0 FATTY ACID TO ALBUMIN MOLAR RATIO (P)

3358 CANCER RESEARCH VOL. 34

Table 4 demonstrated that Ehrlich cells possess the CO2 -dependent, Release ofLDH during incubation ofEhrlich cells cytoplasmic de novo fatty acid-synthetic pathway. Incubation media contained 11 mM glucose and either 0.2 mMBSA Composition of Synthesized Fatty Acids. The composition or 0.2 mM BSA containing 0.4 mM stearate. Each value represents the of the fatty acids synthesized by the intact Ehrlich cells and average of 3 determinations Â±S.E. made with 3 separate incubation flasks. The total LDH activity in the cells of each incubation was the 105 ,000 X g supematant fraction of the cell homogenate @ equivalent to 13.0 Â±0.2 mmoles of NAD reduced per mm (mean Â± was compared by GLC. H20 was used as the radioactive S.E. of 5 determinations). substrate for the intact cell incubations, whereas malonyl-CoA 1,3-' 4C served as the labeled substrate with the 105 ,000 X g of released incubation to medium supernatant fraction. In initial experiments individual fatty (%)BSA11.2Â±0.1Stearate:BSA11.5MediumTime (mm)LDH acids were collected separately, but in subsequent work only the larger pooled fractions listed in Table 7 were collected. The 1st fraction, <16:0, contained those methyl esters with 0.1BSA1201.8Â±0.1Stearate:BSA1201.7Â± Â± retention times shorter than that of palmitate. In that 0.1 fraction, most of the mass and almost all of the radioactivity was found in mynstate. The 2nd and 3rd fractions, 16:0 + availability of acetyl-CoA for fatty acid synthesis. To examine 16:1 and 18:0 + 18:1, were collected as indicated for 2 @ this possibility, cells were incubated with either glucose-i -â€˜C reasons. First, in both cases the saturated fatty acids contained or -6@ C, and the oxidation of these substrates to almost all of the radioactivity. Second, any radioactivity in 1 4 CO2 was measured (Table 5). Glycolysis, as measured by 16:1 or 18:1 represented net synthesis of 16:0 or 18:0, the oxidation of gl-64 C, was not inhibited even at very respectively, followed by desaturation. Since this study did high concentrations of stearate. From the palmitate data in not specifically deal with desaturation processes, we were Table 2, one might predict some dilution of the acetyl-CoA interested in evaluating only net synthetic activity. The last S 4C generated from glucose-6.' 4C due to stearate oxidation, fraction, >18 :1, contained all of the methyl esters with but even this was not observed. This difference between retention times longer than that of oleate. This included at palmitate and stearate probably is due to the fact that Ehrlich cells oxidize stearate much more slowly than plamitate when Table 5 the medium contains glucose (R. McGee and A. Spector, Effect of stearate on glucose oxidation unpublished observations). The oxidation of glucose-i .@â€˜C The incubation media contained 11 mM glucose, 0.2 mM BSA, @ was inhibited slightly by stearate with inhibition increasing as varying amounts of stearate, and either cose4 C or glucose..6-' C. @ The production of â€˜CÂ°2was measured during a 2-hr incubation, with the stearate concentration was raised. Since glycolysis was not each value representing the mean Â±S.E.of 3 determinations. inhibited, we interpret this reduction to indicate that the pentose phosphate pathway was slightly inhibited in the cells)cose-64(j@mo1es/l0' presence of stearate (20% at 17= 4.0). Acetyl-CoA Carboxylase, Glucose 6-Phosphate Dehydrogen Molar ratio (stearate:BSA)OxidationC0 Ccose4 ase, and Fatty Acid Synthetase Activities. In order to determine whether the de novo fatty acid-synthetic pathway Â±0.02 Â±0.01 was present in Ehrlich cells, the activities of acetyl-CoA 0.4 2.45 Â±0.05 4.15Â±0.05 carboxylase, glucose 6-phosphate dehydrogenase, and fatty 1.0 2.40Â±0.01 4.11Â±0.03 2.0 2.44Â±0.02 4.06Â±0.01 acid synthetase were measured in a 105 ,000 X g supernatant 4.02.46 2.50 Â±0.024.33 4.00 Â±0.02 of a cell homogenate (Fable 6). For comparison the rates of fatty acid synthesis and the pentose phosphate pathway also were measured in intact cells. The homogenate data have been Table 6 normalized to 108 cells to facilitate comparisons. With the Enzymatic activities related to fatty acid synthesis in intact cells and homogenate preparations intact cells, fatty acid synthesis was calculated by assuming Methods for measurement of the enzymatic activities in the 105,000 @ that 1.66 H atoms are incorporated for every 2-carbon x g supernatantandtheintactcellsaredescribedinâ€œMaterialsand fragment (38). Pentose phosphate pathway activity was Methods.â€•The values are means Â±S.E. of the number of determina calculated from the data in Table 4, using the difference tions given in parentheses. All activities are expressed as nmoles/10@ @ between C oxidation and -&@ C oxidation cells/hr. There are 15.3 Â±0.7 (8) mg of 105,000 X g supernatant and assuming that 2 moles NADPH are produced per mole of protein in 10@Ehrlich cells. giucose oxidized. The enzyme activities represent maximum UnitsEnzyme(nmoles)ActivityIntact rates with saturating substrate concentrations. The enzyme activities are similar in magnitude to those reported for rat liver and hepatomas (19, 26). These activities are more than cellsFatty (66)Pentoseacid synthesis2-carbon units260 Â±19 adequate to account for the observed rates of fatty acid 3)HomogenateAcetyl-CoAphosphate pathwayNADPH1 ,870 Â±30( synthesis in the intact Ehrlich cells. Furthermore, the calculated rate of NADPH production through the pentose (10)Fatty carboxylaseMalonyl-CoA550 Â±60 phosphate pathway alone is adequate to supply the reducing 110(10)Glucoseacid synthetase2-carbon units1,260 Â± equivalents needed for fatty acid synthesis in the intact cells. (11)dehydrogenase6-phosphateNADPH35,800 Â±3,100 These results confirm the findings of Pedersen et aL (25) who

DECEMBER 1974 3359

Table 7 biosynthesis responds rapidly to added free fatty acids in the Distribution ofradioactivity among fatty acids synthesized by intact same manner as in nonmalignant cells (1 1, 15, 16). One Ehrlich cells and a cytosol fraction of cell homogenates possible explanation for the apparent differences between Incubations with intact cells were for 2 hr in media containing 11 hepatomas and Ehrlich cells is that these tumors differ in mM glucose, 0.2 mM BSA, and 3H20. Assays with 105,000 X g terms of their metabolic regulatory mechanism. Even if this @ supernatant fraction were performed with malonyl-CoA-1 ,3-' C as the radioactive substrate. Fatty acids were methylated and then separated were true, it would exclude the currently held view that loss of by GLC. The values represent the mean Â±S.E. of 30 determinations control is a general property of tumors. A more likely with intact cells and 8 determinations with the cytosol fraction. More explanation for the apparent discrepancy, however, is that the complete definitions of the fatty acid methyl ester fractions are given in regulation thus far demonstrated in Ehrlich cells concerns only â€œResults.â€• short-term control. By contrast, the regulation that is reported 4 C incorporation to be lost in hepatomas is long-term control, which involves Methyl ester incorporation by cytosol fractions actual levels of the biosynthetic enzymes. The experiments FractionH(%)<16:012.3 (%)1 performed by Sabine et al. (27) were not designed to detect 0.316:0+16:151.6Â±1.388.4Â±0.218:0Â±0.44.3 Â± short-term regulation, and further work with hepatomas is needed to determine whether or not these tumors, like the +0.2>18:115.7 18:120.4 Â±0.55.1 Â± Ehrlich cells, exhibit this control mechanism. Â±1.22.1 Â±0.2 Several pieces of evidence suggest strongly that both de novo synthesis and chain elongation of fatty acids occur in least 7 detectable peaks, the longest retention time being 14 Ehrlich cells. The evidence for de novo synthesis includes the times greater than that of palinitate. The intact cells demonstration by Pedersen et aL (25) of a CO2 requirement @ incorporated appreciable amounts of H into each of the 4 for fatty acid synthesis, the presence of acetyl-CoA car fractions, with about 50% of the radioactivity being recovered boxylase and fatty acid synthetase in Ehrlich cells (Table 6), in the palmitate (16:0 + 16:1) fraction. By contrast, almost and the predominance of palmitate synthesis by both intact 90% of the radioactivity incorporated by the cytosol cells and the cell homogenate (Table 7). Chain elongation is preparation was contained in the palmitate fraction, and only Table 8 very small . amounts were present in the other fractions, Incorporationofpalmitate-1-'â€œCintovariousfattyacidsbyintactcells especially in the >18: 1 fraction. Incubations were performed for 2 hr in media containing 11 mM Ehrlich cells also were incubated with palmitate-l .â€˜4Cfor 2 glucose, 0.2 mM BSA, and palmitate-l-'4C at the indicated molar hr, and the cellular fatty acids were analyzed for radioactivity ratios. Fatty acids from the cells were methylated and then separated by GLC. As shown in Table 8, about 80% of the radioactivity by GLC. Each value represents the mean Â±S.E. of 3 analyses of the remained in the palmitate plus pahnitoleate fraction at both of pooled fatty acids from 2 separate incubations. the molar ratios examined. Essentially no radioactivity was of â€˜â€˜C found in fatty acids shorter than palmitate, but considerable cells/hr)j;(nmolesation/1 0@ quantities of radioactivity were found in the longer fatty acids. Most of the radioactivity was recovered in stearate, but Methyl ester significant quantities also were found in oleate and the longer 2.0<16:0fractionIncorpor =0.4i = fatty acids. These results are consistent with the work of Â±0.2 Â±0.4 Bailey and Dunbar (1) who have shown that Ehrlich cells in 16:0 + 16:1 74.0 Â±0.4 351 Â±1 culture are able toelongate fatty acids. >16:10.3 20.2Â±0.30.4 64.9Â±0.8 Regulation of Acetyl-CoA Carboxylase. The ability of fatty acids and their CoA thioesters to regulate acetyl-CoA Table 9 carboxylase activity was investigated (Table 9). Both laurate Effects offatty acids and fatty acyl-CoA â€˜sonacetyl-CoA and lauroyl-CoA stimulated the enzyme in the 105,000 X g carboxylase activity supernatant of the Ehrlich cell homogenate. Conversely, Acetyl-CoA carboxylase was assayed as described in â€œMaterialsand stearate and stearoyl-CoA inhibited the enzyme. These Methods,â€• using the 105,000 X g supernatant of an Ehrlich cell observations are consistent with the observation that laurate homogenate as the source of enzyme. Fatty acids and acyl-CoA's were stimulates and stearate inhibits fatty acid synthesis in the added as BSA complexes. The BSA concentration was 8.7 @M.Values are the mean Â±S.E.of the number of assays indicated in parentheses. intact Ehrlich cell (Chart 2). of controlNone0.615InhibitorConcentration(SM)Activity (nmoles/mg/min)% DISCUSSION (6)LaurateS Â±0.005 A lack of dietary control of fatty acid biosynthesis has been Â±0.012 (3) 112Lauroyl-CoAS 250.625 0.686Â±0.022(3)102 observed in every rat and mouse hepatoma examined to date. This has led Sabine and Chaikoff (27) to suggest that either 126Stearate5 250.675Â±0.009(3)0.774â€•110 inhibitors of the fatty acid-synthetic enzymes or regulators of Â±0.003 (3) the synthesis of these enzymes cannot enter hepatoma cells. 79Stearoyl-CoA5 250.471 0.485 Â±0.049 (3)76 Furthermore, Sabine et a!. (26) suggest that the loss of Â±0.011 (3) 250.455 0.300 Â±0.004 (3)74 49 enzymatic control is representative of a â€œfundamental aberra tionâ€• in tumor cell metabolism. In Ehrlich cells, however, a Mean of 2 determinations.

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@ suggested by the incorporation of considerable amounts H acyl-CoA. Taken together, these observations suggest that into fatty acids longer than palmitate in intact cells (Table 7), defective control of fatty acid biosynthesis in tumors involves the elongation of palmitate-l-' 4C in intact cells (Table 8), and the regulation of enzyme production, not short-term modula the demonstration by Bailey and Dunbar (1) that oleate is tion of enzyme activity. elongated by Ehrlich cells in culture. From the data in Table 7 one can estimate the relative proportion of the total fatty acid synthesis contributed by the de novo and elongation path REFERENCES ways. As has been shown with several different purified fatty acid synthetases (28), the Ehrlich cell enzyme produced 1. Bailey, 3. M., and Dunbar, L. M. Essential Fatty Acid Require predominantly palmitic acid. With this particle-free system, ments ofCells in Tissue Culture. Exptl. MoL Pathol., 18: 142â€”161, only about 0.2% of the â€˜4Crecovered in fatty acids was 1973. found in acids shorter than myristate (Table 7). For this 2. Bartley, J. C., and Abraham, S. Hepatic Lipogenesis in Fasted, @ reason, the H incorporated into myristate in intact cells Refed Rats and Mice: Response to Dietary Fats of Differing Fatty probably occurs through de novo synthesis of myristate, not Acid Composition. Biochim. Biophys. Acta, 380: 258â€”266,1972. through de novo synthesis of laurate followed by chain 3. Brenneman, D. E., and Spector, A. A. Utilization of Ascites Plasma elongation to myristate. Likewise, most of the 3H in the Very Low Density Lipoprotein Triglycerides by Ehrlich Cells. J. pahnitate plus palmitoleate fraction of intact cells probably Lipid Res., 15: 309â€”316,1974. 4. Burton, D. W., Collins, J. M., Kennan, A. L., and Porter, J. W. The arises from de novo synthesis, for palmitate was the major Effects of Nutritional and Hormonal Factors on the Fatty Acid product of the synthetase in the particle-free system. Since Synthetase Level of Rat Liver. J. Biol. Chem., 244: 4510â€”4516, about 5% of the fatty acid made by the synthetase in the 1969. particle-free system is stearate, at least a portion of the â€˜Hin 5. Chen, R. F. Removal of Fatty Acids from Serum Albumin by the stearate plus oleate fraction of the intact cells probably Charcoal Treatment. J. Biol. Chem., 242: 163â€”181,1967. also is incorporated through de novo synthesis. On the basis of 6. Craig, M. C., Dugan, R. E., Muesing, R. A., Slakey, L. L., and @ the above reasoning, we estimate that 75 to 85% of the H Porter, J. W. Comparative Effects of Dietary Regimens on the incorporated into Ehrlich cell fatty acids in the absence of Levels of Enzymes Regulating the Synthesis of Fatty Acids and exogenous fatty acids occurs through de novo biosynthesis. Cholesterolin Rat Liver.Arch. Biochem.Biophys.,151: 128â€”136, 1972. Therefore, in the intact Ehrlich cell, measurement of the 7. Craig, M. C., Nepokoeff, C. M., Laksmann, M. R., and Porter, J. W. incorporation of 3H into the total saponifiable lipids is a Effects of Dietary Change on the Rates of Synthesis and moderately good estimate of de novo fatty acid synthesis. Degradation of Rat Liver Fatty Acid Synthetase. Arch. Biochem. Since the addition of the 16- and 18-carbon fatty acids to the Biophys.,151:619â€”630,1972. incubation medium inhibited total fatty acid synthesis by as 8. Folch, J., Lees, M., and Sloane-Stanley,G. H. S. A Simple Method much as 85%, much ,f this decrease must be due to inhibition for the Isolation and Purification of Total Lipids from Animal of de novo synthesis. Furthermore, since free fatty acids are Tissues. J. Biol. Chem., 226: 497â€”509, 1957. present in the ascites plasma in which the Ehrlich cells are 9. Goodridge, A. G. Regulation of the Activity of Acetyl Coenzyme bathed (29â€”31), de novo synthesis probably is at least A Carboxylase by Palmityl Coenzyme A and Citrate. J. Biol. Chem.,247:6946â€”6952,1972. partially inhibited during the growth of the cells in the mouse 10. Goodridge, A. G. On the Relationship between Fatty Acid peritoneal cavity. Synthesis and the Total Activities of Acetyl Coenzyme A Unlike the longer fatty acids, laurate actually stimulated Carboxylase and Fatty Acid Synthetase in the LIver of Prenatal total fatty acid synthesis (Chart 2). This could result from and Early Postnatal Chicks. J. Biol. Chem., 248: 1932â€”1938, chain elongation of the exogenously supplied laurate. Alterna 1973. tively, stimulation may be due to activation of acetyl-CoA 11. Goodridge, A. G. Regulation of Fatty Acid Synthesis in Isolated carboxylase (Table 9). Hepatocytesâ€”Evidence for a Physiological Role for Long Chain Some striking similarities exist in fatty acid synthesis and its Fatty Acyl CoA and Citrate. J. BioL Chem., 248: 4318â€”4326, short-term control in Ehrlich cells, hepatocytes, and skin 1973. fIbroblasts. The levels of acetyl-CoA carboxylase and fatty 12. Gregolin, C., Ryder, E., and Lane, M. D. Liver Acetyl Coenzyme A Carboxylase. I. Isolation and Catalytic Properties. J. Biol. Chem., acid synthetase in Ehrlich cells (Table 6) are similar to those in 243:4227â€”4235,1968. hepatocytes and fibroblasts (12, 16). Rates of fatty acid 13. Guynn, R. W., Veloso, D., and Veech, R. L. The Concentration of synthesis in the intact Ehrlich cells (Table 6) also are similar to Malonyl-Coenzyme A and the Control of Fatty Acid Synthesis in those reported in hepatocytes (1 1). The relative ability of Vivo.J. BioLChem.,247: 7325â€”7331,1972. fatty acids to inhibit fatty acid biosynthesis in hepatocytes 14. Hsieh, W. T., and Vestling, C. S., Lactate Dehydrogenase from Rat and fibroblasts is stearate > palmitate@oleate > linoleate (1 1, Liver. Biochem. Prep., 2: 69â€”75,1966. 16), the same as in Ehrlich cells (Chart 2). Finally, the degree 15. Jacobs, R. A., and Majerus, P. W. The Regulation of Fatty Acid of inhibition produced by stearate and palmitate in Ehrlich Synthesis in Human Skin Fibroblasts. Inhibition of Fatty Acid cells (Chart 2) is about the same as in hepatocytes (1 1). Synthesis by Free Fatty Acids. J. BioL Chem., 248: 8392â€”8401, Therefore, the Ehrlich cell possesses a mechanism for 1973. 16. Jacobs, R. A., Sly, W. S., and Majerus, P. W. The Regulation of short-term control that is similar or identical to that of Fatty Acid Biosynthesis in Human Skin Fibroblasts. J. BioL Chem., nonmalignant cells. This is consistent with the findings of 248: 1268â€”1276,1973. Sabine and Chaikoff (27) and Majerus et al. (19) that the 17. LÃ¶hr,G. W., and Waller, H. D. Glucose-6-Phosphate Dehydrogen acetyl-CoA carboxylase of hepatomas, like the enzyme in ase. In: H. W. Bergmeyer (ed.), Methods of Enzymatic Analysis, normal liver, can be regulated by fatty acids and fatty pp. 744â€”747.New York: Academic Press, Inc., 1963.

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18. Lowry, 0. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. 28. Smith, S. Studies on the Immunological Cross-Reactivity and Protein Measurement with the Folin Phenol Reagent. J. BioL Physical Properties of Fatty Acid Synthetases. Arch. Biochem.

. Chem., 193: 265â€”275, 1951. Biophys., 156: 75 1â€”758,1973. 19. Majerus, P. W., Jacobs, R., Smith, M. B., and Morris, H. P. The 29. Spector, A. A. The Importance of Free Fatty Acids in Tumor Regulation of Fatty Acid Biosynthesis in Rat Hepatomas. J. Biol. Nutrition. Cancer Res., 27: 1580â€”1586, 1967. Chem., 243: 3588â€”3595, 1968. 30. Spector, A. A. Fatty Acid Glyceride and PhospholipidMetabolism. 20. Majerus, P. W., and Kilbum, E. Acetyl Coenzyme A Carboxylase. In: G. H. Rothblat and V. J. Christofalo (eds.), Growth Nutrition The Roles of Synthesis and Degradation in Regulation of Enzymes and Metabolism of Cells in Culture, VoL 1, pp. 257â€”296. New Levels in Rat Liver. J. BioL Chem., 244: 6254â€”6262, 1969. York: Academic Press, Inc., 1972. 21. Moss, J., Yamagishi, M., Kleinschmidt, A. K., and Lane, M. D. 31. Spector, A. A., and Brenneman, D. E. Role of Free Fatty Acid and Acetyl CoA Carboxylaseâ€”Purification and Properties of the Bovine Lipoproteins in the Lipid Nutrition of Tumor Cells.In: R. Wood Adipose Tissue Enzyme. Biochemistry, 11: 3779â€”3786, 1972. (ed.), Tumor Lipids: Biochemistry and Metabolism, pp. 1â€”13. 22. Muto, Y., and Gibson, D. M. Selective Dampening of Lipogenic Champaign, ifi.: American Oil Chemists Society Press, 1973. Enzymes of Liver by Exogenous Polyunsaturated Fatty Acids. 32. Spector, A. A., and Hoak, J. C. An Improved Method for the Biochem. Biophys. Res. Commun., 38: 9â€”15,1970. Addition of Long Chain Free Fatty Acids to Protein Solutions. 23. Nilsson, A., Sundler, R., and Akesson, B. Biosynthesis of Fatty AnaL Biochem., 32: 297â€”302,1969. Acids and Cholesterol in Isolated Rat-Liver Parenchymal Cells 33. Spector, A. A., and Steinberg, D. The Utilization of Unesterified Effect of Albumin-Bound Fatty Acids. European J. Biochem., 39: Palmitate by Ehrlich Ascites Tumor Cells. J. Biol. Chem., 240: 613â€”620,1973. 3747â€”3753, 1965. 24. Numa, S., Ringlemann, F., and Lynen, F. Zur Hemming der 34. Spector, A. A., and Steinberg, D. Releaseof Free Fatty Acidsfrom Acetyl-CoA-Carboxylase durch Fettsaure Coenzyme A-Verbin Ehrlich Ascites Tumor Cells. J. Lipid Res., 7: 649â€”656,1966. dungen. Biochem. Z., 343: 243â€”257, 1965. 35. Spector, A. A., Steinberg, D., and Tanaka, A. Uptake of Free Fatty 25. Pederson, B. N., Bromek, A., and Daehnfeldt, J. L. Extramitochon Acids by Ehrlich Ascites Tumor Cells. J. Biol. Chem., 240: drial Fatty Acid Synthesis in Ehrlich Ascites Tumor Cells 1032â€”1041, 1965. Propagated in Vitro and in Vivo. Proc. Soc. Exptl. Biol. Med., 141: 36. Trout, D. L., Estes, E. H., and Friedberg, S. J. Titration of Free 506â€”509, 1972. Fatty Acids of Plasma: A Study of Current Methods and a New 26. Sabine, J. R., Abraham, S., and Morris, H. P. Defective Dietary Modification. J. Lipid Res., 1: 199â€”202,1960. Control of Fatty Acid Metabolism in Four Transplantable Rat 37. Wallach, D. F. H., and Ullrey, D. Studies on the Surface and Hepatomas: Numbers 5123C, 7793, 7795, 7800. Cancer Res., 28: Cytoplasmic Membranes of Ehrlich Ascitesâ€”Carcinoma Cells. 46â€”51,1968. Biochim. Biophys. Acta, 64: 526â€”539,1962. 27. Sabine, J. R., and Chaikoff, I. L. Control of Fatty Acid Synthesis 38. Windmueller, H. G., and Spaeth, A. E. Perfusion in Situ with in Homogenate Preparations of Mouse Hepatoma BW 7756. Tritium Oxide to Measure Hepatic Lipogenesis and Lipid Secretion. Australian J. Exptl. Biol. Med. Sci., 45: 541 â€”548,1967. J.Biol.Chem.,241:2891â€”2899,1966.

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Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 1974 American Association for Cancer Research. Short-Term Effects of Free Fatty Acids on the Regulation of Fatty Acid Biosynthesis in Ehrlich Ascites Tumor Cells

Richard McGee and Arthur A. Spector

Cancer Res 1974;34:3355-3362.

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