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

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Short-Term Effects of Free Fatty Acids on the Regulation of Fatty Acid Biosynthesis in Ehrlich Ascites Tumor Cells [CANCER RESEARCH 34, 3355—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-' ‘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, 29—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 Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 1974 American Association for Cancer Research. R. McGee andA. A. Spector 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 4°.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 0° 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 ±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 0°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 37°with shaking in a temperature-controlled water bath. weredoneat 4°.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 ‘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 65°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.
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