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Studies of Fatty Acid Oxidation IX. The Effects of Uncoupling Agents on the Oxidation of Fatty Acids by Transplantable Tumors D. B. ELLISANDP. G. SCHOLEFIELD* (McGill-Montreal GeneralHospital Research Institute, Montreal, P.Q., Canada) SUMMARY Oxidation of decanoate-1-C14 and palmitate-l-C14 by slices or the ascitic forms of the Ehrlich carcinoma or Sarcoma 37 was relatively resistant to loss of adenosine triphosphate (ATP) produced by uncoupling agents, such as dinitrophenol (DNP) or the fatty acids themselves. However, the rate of incorporation of palmitate-l-C14 into phospholipides was decreased in the presence of DNP. Mutually inhibitory effects among fatty acids occurred. Such effects were shown to be unlikely to result from isotopie dilution, competition among the fatty acids, or to uncoupling effects. The observed inhibitions of the oxidation of decanoate-1-C14 and palmitate-l-C14 are interpreted in terms of the availability of acyl-CoA under various conditions and the effects of such acyl-CoA derivatives on the metabolism of fatty acids. At the beginning of the sequence of reactions involved in the biological oxidation of fatty acids, adenosine triphosphate (ATP) is required, being necessary for the conversion of the various fatty acids to their coenzyme A esters (18). Such par ticipation of ATP in fatty acid oxidation is appar ently essential and is confirmed by the finding (8) that oxidation of fatty acids by liver mito chondria is greatly inhibited by dinitrophenol (DNP). The concentrations of DNP required are those which also lead to an uncoupling of oxidation from phosphorylation (8, 17). Oxidation of pyruvate by liver mitochondria is inhibited by DNP, and the inhibition may be, at least partially, reversed on addition of "priming" agents such as malate (14, 16, 20). The inhibition of fatty acid oxidation by liver mitochondria in the presence of fumara te on addition of DNP shows that "prim ing" agents do not reverse the inhibitory effects on oxidation of butyrate-1-C14, laurate-1-C14, and pal mitate-l-C14. In many cases the presence of a second fatty acid causes a decrease in the produc tion of C14O2from these fatty acids, particularly when the second fatty acid has a chain length of eight carbon atoms or more. Such fatty acids are known to uncouple oxidation from phosphory lation in mitochondrial preparations (21), in as cites cells (7, 22), and in tumor slices (11). The failure of DNP to inhibit fatty acid oxidation in such preparations has therefore been considered further, as well as the interactions among fatty acids in tumor slices and ascitic forms of tumors The effects of changes in the level of ATP pro duced by these agents on the incorporation of fatty acids into phospholipides have also been investigated. fatty acid oxidation (8). Previous experiments (23) had shown that addi tion of DNP to ascites hepatoma 98/15 leads to a stimulation of the rate of oxygen uptake and to a stimulation rather than an inhibition of the * National Cancer Institute of Canada Associate Professor of Biochemistry, McGill University. Received for publication September 25, 1961. MATERIALS AND METHODS ANIMALS All animals used were male Swiss white mice weighing 20-25 gm., purchased from Carworth Farms, New York, U.S.A. TISSUEPREPARATIONS Tumors, ascites cells, tissue slices, and the in cubation technic were as previously described (11). 305 Downloaded from cancerres.aacrjournals.org on August 10, 2017. © 1962 American Association for Cancer Research. Cancer Research 306 The incubation medium was a calcium-free KrebsRinger solution containing 145 HIM NaCl, 5.8 IHM KC1, 1.5 mM KH2PO4, and 1.5 nut MgS04 ("salts solution"), the final volume in the vessel being 3 ml. Slices were incubated in an atmosphere of oxygen and ascites cells in an atmosphere of air. The medium was buffered with 10 mM phos phate, pH 7.4, but when elevated rates of aerobic glycolysis were anticipated the final concentration of phosphate buffer was increased to 20 mM. In studies of fatty acid oxidation the standard incubation time was 90 minutes. ASSAY OF C14-LABELED COMPOUNDS Carbon dioxide.—C14-labeled substrates were added to Warburg flasks containing appropriate media and chilled by being packed in cracked ice. Ascites cells or tumor slices were then added, 0.2 ml. 20 per cent KOH and filter paper placed in the center well, the vessels gassed, if necessary, and the zero time was taken as the moment when flasks plus manometers were placed in the incuba tion bath. At the end of the incubation period 0.2 ml. 30 per cent trichloroacetic acid (TCA) was added from the side-arm to stop the reaction and liberate trapped carbon dioxide. After a fur ther incubation period of 30 minutes the filter papers were removed with washings to centrifuge tubes containing carrier sodium carbonate equiva lent to 10 mg. BaCOs. The carbonate was then precipitated as BaCOa, washed twice with water, once with acetone, and transferred in acetone to tared planchets which were then dried and weighed again. The radioactivity on the planchets was assayed with a thin-window gas-flow counter for a sufficient time to give a counting error of less than ±5 per cent. The net counts/minute were corrected to infinite thinness and referred back to Amóles substrate oxidized by dividing by the specific activity (counts/min/^mole) of the sub strate added. Palmitafe-l-Cli incorporation into phospholipides.—The residue remaining after precipitation of ascites cells with TCA (see above) was washed twice with 5 ml. 5 per cent TCA and extracted for 30 minutes at 50°C. with 5 ml. 95 per cent ethanol. The residue was further extracted with 5 ml. ethanol-ether (1:1 v/v) for 20 minutes at 40°C. The combined extracts were reduced to dryness under nitrogen, and the ensuing residue was dissolved in 1 ml. petroleum ether. The phospholipides were precipitated from this solution according to the method of Sinclair and Dolan (26) by adding 7 ml. dry acetone and 1 drop saturated alcoholic magnesium chloride solution. The precipitated phospholipides were washed with Vol. 22, April 1962 cold acetone, resuspended in moist ether, plated, and counted as described above. Alcohol-soluble and alcohol-insoluble glycine.— The uptake of glycine-1-C14 into the alcohol-solu ble fraction of tumor cells (i.e., into the amino acid pool) and the incorporation of glycine-1-C14 into the alcohol-insoluble fraction (mainly into proteins) were estimated as described previously (11). ESTIMATION OF INORGANICPHOSPHATE, PHOS PHATE ESTERS, AND THE LEVEL OF RADIO ACTIVITYIN THESE FRACTIONS When P32 was used, approximately 20 /nc. was placed in the side-arm of Warburg vessels, and the reaction was begun by tipping the labeled phosphate, together with appropriate inhibitors, after attainment of thermal equilibrium. At the end of the incubation period the vessels were placed on cracked ice and the contents (slices plus medium) poured into 5 ml. ice-cold salts solution, centrifuged, the supernatant was dis carded, and the residue washed with a further 8 ml. ice-cold salts solution. The supernatant was carefully removed, and the sides of the centri fuge tubes were dried with paper tissues. Five ml. 5 per cent TCA was then added to each tube, and the slices were homogenized. Ascites cells were washed once with 8 ml. salts solution. All operations thus far were carried out at 2°-4° C., and the resulting suspensions were allowed to stand for a further 30 minutes at this temperature to complete the extraction of the TCA-soluble materials. The nucleotides were separated from the TCA extract by treatment with approximately 50 mg. Norit A (purified by successive treatments with pyridine, HC1, and distilled water) for 10 minutes at 2°C. according to the method of Crane and Lipmann (6). The supernatant, together with two washings, was made up to 20 ml., and 3.4 ml. was used for estimation of inorganic phosphate by Bartlett's modification (4) of the method of Fiske and SubbaRow (12). There was little differ ence between the results obtained by the method of Fiske and SubbaRow (12) and that of Bartlett (4), which involves heating in acid, suggesting that the amount of non-nucleotide easily hydrolyzable phosphate esters (such as creatine phosphate) was small compared with the amounts of phos phate present. The results are therefore referred to as levels of inorganic phosphate. The labile phosphate of the nucleotide fraction was measured by treatment of the washed char coal with 4 ml. N HC1 in a boiling water bath, cooling, centrifuging, and assaying inorganic phos- Downloaded from cancerres.aacrjournals.org on August 10, 2017. © 1962 American Association for Cancer Research. ELLIS AND SCHOLEFIELD—Fatty Acid Oxidation by Tumor» phate on a 2-ml. aliquot of the supernatant by the method of Bartlett (4). This fraction is termed the 7-minute nucleotide phosphate fraction. Aliquots (usually 200 jul.) of these two fractions were plated and counted as described above. The inorganic phosphate fraction was neutralized on the planchet with NH4OH, and the HC1 solution of hydrolyzed nucleotide was neutralized with 0.2 ml. N NaOH. One drop of a 2 per cent solu tion of cetyltrimethylammonium bromide was also added to produce even films on the planchets, which were dried under an infrared lamp before being counted. Labeled substrates.—P32 was obtained from Charles E. Frosst and Co., Limited, Montreal, and the radioactive fatty acids from Merck and Co., Limited, Montreal. RESULTS The metabolism of palmifate-l-Cn in Ehrlich ascites cells.—Addition of fatty acids to ascites cells leads to an inhibition of respiration, but a stimulation may (23) or may not (22) occur at lower concentrations. The respiratory activity of the Ehrlich ascites cells used in the present experi ments was stimulated only slightly by fatty acids. Oxidation of fatty acids by these cells must there fore take place at the expense of the endogenous substrates, including the endogenous supply of fatty acids. In the preliminary experiments the extent of oxidation of exogenous fatty acid was determined under conditions where there was no significant change in the rate of oxygen uptake. Ten Warburg vessels were set up, one containing no palmitate and three containing each of three concentrations (0.05, 0.1, and 0.3 IHM)of palmitate-l-C14. The rate of oxygen uptake was the same in all vessels. At 30, 60, and 90 minutes, TCA was tipped into the main compartment of three vessels, one of each palmitate concentration, and the C14O2produced was estimated as described above. The results obtained are presented in Chart 1. At a concentration of 0.3 min, palmitate-1-C14 was oxidized by ascites cells at a constant rate for at least 90 minutes. When the concentration of palmitate-1-C14 was decreased to 0.1 HIMthe time course was again linear, but the rate was somewhat lower. Further decrease in the concen tration of palmitate-1-C14 to 0.05 mM led to a nonlinear time course and a lower initial rate of C14O2production. Such results may be explained in terms of two hypotheses: (a) that there exists within the ascites cells a pool of nonradioactive palmitate or (b) that there is a Kmvalue controlling the rate of palmitate oxidation. Calculations from the initial rates of C14Os production show that 307 the second hypothesis is valid if the Km value for palmitate in the process controlling its oxida tion is 0.05 HIM. The lowest concentration of palmitate added was 0.05 mivi, and hence, as oxidation proceeded, it would be expected that the rate of C1402production would decrease with time—an effect apparent from Chart 1. Control of palmitate oxidation could occur by failure to 08 04 Time in minutes CHART1.—The oxidation of palmitate-1-C14 by Khrlich ascites carcinoma cells. The cells were incubated with labeled palmitate under the standard conditions described in the sec tion on "Materials and Methods." A = 0.05 mM palmitate-1-C14 present. O = 0.1 mxf palmitate-1-C14 present. •= 0.3 mM palmitate-1-C14 present. saturate either the system transporting palmitate into the ascites cells or the enzyme responsible for the initial activation of the palmitate. The absence of any lag periods suggests the latter. The effect of the presence of a metabolic pool of palmitate on the specific activity of the added palmitate was determined by the following equa tion: Specific activity of added palmitate _ x+ 3 [S] Final specific activity 3 [S] Downloaded from cancerres.aacrjournals.org on August 10, 2017. © 1962 American Association for Cancer Research. 308 Cancer Research Vol. 22, April 1962 acids (Cio-Cia) have little effect on palmitate-1-C14 oxidation until concentrations are added which also inhibit respiratory activity. Decanoate (0.20.4 HIM)is known to cause effects which are con sistent with the suggestion that there occurs an uncoupling of oxidation from phosphorylation (7, 11, 22). This should, in turn, have caused a corresponding decrease in the rate of palmitate oxidation, and none was observed. The effects of DNP on palmitate metabolism in ascites cells were therefore investigated, and the results ob tained are presented in Table 1. They confirm previously observed effects of DNP on palmitate1-C14 oxidation (23) and further indicate that DNP reverses the inhibitory effect of glucose on TABLE 1 fatty acid oxidation. On the other hand, the incor poration of fatty acids into phospholipides, a proc THEEFFECTS OFDNP ANDGLUCOSE ONTHE METABOLISM OFPALMITATE-I-C" ess which is equally dependent on the formation of an acyl-CoA derivative, is sensitive to the pres BYEHRLICH ASCITES CELLS ence of DNP, and this inhibition was largely re versed by glucose (Table 1). ADDI TIONSDNP(mil)00.040.060.080.1000.040.060.080.10Glucote(mil)000001010101010OXYGEN To show that palmitate did not, in some way, UPTAKE(-QO,)13.2 interfere with the uncoupling action of DNP, INCOR the effects of various combinations of these two TED/MLPACKED PORA CELLS)470 CELLS)1.67 agents on glycine-1-C14 uptake into the amino acid pool, and glycine-1-C14 incorporation into (100)1.94 (100)380 (100)14.1 (116)2.10 81)320 ( (107)14.8(11«)18.6(108)12.7 the proteins of Ehrlich ascites cells, were investi (126)2 68)290 ( gated. The results obtained are presented in Table 62)220 ( (130)1.95 . 16 2. They demonstrate that palmitate did not influ 96)8.0( ( (117)0.84 47)740 ( (158)810 61)16.2(128)18.4 51)1.23( ence the inhibitory effects of DNP on these proc 74)1.64( (172)800 esses. At a concentration (0.04 IHM)which stimu (170)740 (140)16.5 99)1.91( lates oxidation of 0.3 nui palmitate-1-C14 by 26 (158)650 (125)15.0 (115)1.78 (139) (114)C'<OiPRODUCTION(¿1110LE8PALMITATEOXIDIZED/MLPACKED (107)PHOSPHOLIPIDES(MMUOLESPALMITATE-1-C» per cent (Table 1) there was an inhibition by DNP of glycine incorporation into protein of The cells were incubated at 37°C. for 90 minutes in a nearly 50 per cent in the presence or absence of Krebs-Ringer solution containing 20 mM phosphate buffer, pH 7.4. Concentration of palmitate was 0.8 mM. The C1(O2data 0.3 mM palmitate (Table 2). The metabolism of palmitate-1-C1*by tumor slices. in this and subsequent tables are in terms of the amount of substrate oxidized to CO2, calculated on the assumption that —Many of the present experiments were per ¡illfatty acid carbons are oxidized at the same rate. —Qo» formed on slices of the Ehrlich carcinoma and figures are average values over the 90-minute incubation period. Figures in parentheses refer to percentages of control Sarcoma 37. Quantitatively similar results were in the absence of further additions. obtained with these two tumors, but S-37, in our hands, gave a greater yield of non-necrotic A value of 1.6 mM free palmitate is unlikely, material, and this tumor was used in most of the and it is therefore presumed that the rate-control experiments reported. ling feature is the Km value for palmitate, which The effects of variation in the concentration is 0.05 mM. of palmitate-1-C14 on the respiratory activity of From the amounts of CltO¡ produced, the S-37 slices and the yield of C14O2are presented in amount of oxygen corresponding to complete oxi Table 3. There was little change in the Qo, value; dation of the palmitate may be calculated. It the yields of C14Ossuggest an apparent Km value is equal to 20 per cent of the total oxygen uptake for palmitate-1-C14 of approximately 0.4 mM, and of the ascites cells with 0.05 mM palmitate and to at a palmitate concentration of 0.5 m\i the con 40 per cent with 0.3 m.M,values which are of the tribution of palmitate oxidation to the total oxy same order as that of 18 per cent quoted for gen uptake of the slices was less than 10 per cent. The effects of DNP and glucose on palmitate 0.13 HIMpalmitate with hepatoma ascites 98/15 (23). In further confirmation of the results of oxidation were similar to those obtained with Scholefield, Sato, and Weinhouse (23), other fatty ascites cells, except that inhibition of respiration where x is the number of Amólesof palmitate in the metabolic pool of the ascites cells and [S] is the final concentration of palmitate in /Limóles/ ml of the 3 ml. of incubation medium. Since the amount of CI4O2produced is proportional to the specific activity of the palmitate, consideration of a metabolic pool gives rise to an equation which is of the same form as the Michaelis-Menten equation. The value of x/3 is therefore 0.05 /¿moles (Km = 0.05 HIM) so that x = 0.15 /umole. This amount is present in one-eighth ml. of packed cells, and hence is equivalent to 1.2 Amóles/mi packed cells or to a concentration of 1.6 mil, assuming 75 per cent of the cell volume is water. Downloaded from cancerres.aacrjournals.org on August 10, 2017. © 1962 American Association for Cancer Research. ELLIS AND SCHOLEFIELD—Fatty Acid Oxidation by Tumors by DNP occurred at a slightly lower concentration of DNP (Table 4). The lackof any specific effect of DNP on fatty acid oxidation and the reversal of the glucose inhibition by DNP were again observed. In another series of experiments the effects of other fatty acids (Cg-Cu) on the oxidation of 0.2 IBM palmitate-1-C14 by slices of S-37 were investigated. The results are presented in Table 5. Octanoate had no effect on respiration and little effect on C14O2 production. Decanoate inhibited respiration by only 20 per cent at a concentration of 0.8 mM but inhibited C14O2 production by 59 per cent. With increase in the chain length of fatty acid, there was an increase in the inhibitory effects, C14C>2 production being more sensitive than total respiratory activity. Further increase in chain length caused diminishing inhibitory effects. The most effective inhibitor was laurate (Cn), although tridecanoate has about 309 the same effect on C14O2 production. Such results raise the question of the extent of isotopie dilution. The effects of other substrates, at a concentration of 10 mat, on the oxidation of palmitate-1-C14 were investigated, and the results are presented in Table 6. These substrates (glu cose, pyruvate, glutamate, and succinate) had slight inhibitory effects on palmitate oxidation, but none were as effective as the fatty acids. The metabolism of decanoate-l-C1* by ascites cells and tumor slices.—The general pattern of inter action between fatty acids in ascites hepatoma TABLE 4 THEEFFECTS OFDNP ANDGLUCOSE ONTHE OXIDATION orPALMiTATE-l-C14 BYSARCOMA 87SLICES ADDITIONSDNP(mu)00.040.060.0800.040.060.08Glucose(mM)000010101010OXYGENUPTAKE— PRODUCTION(MpMOLES/GMWET TABLE 2 dû-.5.7 THE EFFECTSOFDNP ONTHE UPTAKEANDINCORPO RATION" OFGLYCINE-I-C" BYEHRLICH ASCITES CELLS IN THEABSENCE ANDPRESENCE OF0.8 mM PALMITATE TUMOB)176 WEIGHT (100)198 (100)5.9 (Ili)192 (108)6.1 (109)155 (107)4.5 88)134 ( 79)3.8( ( 76)207 ( 68)6.4 ADDITIONSDNP(mM)00.030.060.1000.030.060.10Palmitate(mil)00000.30.30.30.3-Qo.11.812.713.910.612.513.113.09.9¿(MOLESGLYCINE(118)219 (11815.6 1-C"UPTAKE/MLPACKEDCELLS12.010.99.56.912.810.79.77.1MAMÓLESGLYCDÕE-1-C14INCORPORATED/ML (124)205 98)5.3 ( (116) ( 93)Cl4Ot PACKEDCELLS362 (100)191 53)118 ( 82)58 ( 15)348 ( 96)189 ( 52)104 ( 29)59 ( ( 16) The cells were incubated at 37°C. for 45 minutes in a medium containing 20 mM phosphate, pH 7.4, 2 mM glycine1-C14(0.2 fie.), and further additions as noted. The figures in parentheses are percentages of the control value in the absence of added palmitate or DXP. TABLE 3 THEOXIDATION OFPALMITATE-I-C" BYSLICES OFSARCOMA 37 Concentration ofpalmitatel-O«(mM)0.10.20.5— CO:produced/gmwet labeled Qo,4.704.454.51mamóles weight109198283 The figures quoted are mean values obtained from six determinations. The conditions of incubation were as described in the section on "Materials and Methods." Incubation time was 90 minutes. Figures in parentheses refer to percentages of values obtained with 0.3 mM palmitate1-C14in the absence of further additions. 98/15 is that their oxidation is inhibited by fatty acids of greater chain length (23). In agreement with this pattern it was found that there was little or no effect of other fatty acids of shorter chain length on the oxidation of palmitate by ascites cells. Similarly, longer chain fatty acids inhibited the oxidation of decanoate-1-C14 (Table 7) under conditions where oxygen consumption is not influenced. A linear relationship between the reciprocal of the velocity of oxidation (C14O2 production) and the concentration of inhibitor was found. This would occur if the inhibitory ef fects are due to either competitive or noncompeti tive inhibition by the second fatty acid (9). Noncompetitive inhibitory effects between fatty acids seem unlikely, and, since increase in the substrate (decanoate) concentration from 0.1 to 0.2 mu did little to reverse these effects, competitive inhibition seems equally unlikely. The response to other fatty acids does not appear to be due to loss of ATP, since DNP did not produce similar effects on decanoate-1-C14 oxi- Downloaded from cancerres.aacrjournals.org on August 10, 2017. © 1962 American Association for Cancer Research. 310 Cancer Research dation when either slices or ascites cells were used (Table 8). At the two highest levels of DNP there was a greater inhibitory effect on C14O2 production than on respiration in the ascites cells, suggesting some response to an uncoupling action. Glucose had little effect on C14O2production, but the combined effect of DNP and glucose was a stimulation of approximately 50 per cent in respi ration and nearly 100 per cent in C14O2production. The effects of DNP and glucose in slices were similar to the effects quoted for ascites cells, except that glucose stimulated CI4O2 production Vol. 22, April 1962 from decanoate-1-C14 (35 per cent in the values quoted in Table 8) in contrast to its lack of effect in ascites cells, and there is no evidence from these data of an uncoupling effect of DNP in slices. It should also be noted that the rates of oxida tion of decanoate-1-C14 in the ascitic and solid forms of the Ehrlich carcinoma were similar. When palmitate-1-C14 was used as substrate its rate of oxidation was approximately 10 times as great in ascites cells as it was in tumor slices (see Tables 1 and 3). TABLE5 THEEFFECTS OFOTHERFATTYACIDSONTHEOXIDATION OF0.2mMPALMITATE-I-C" BYSLICESOFSARCOMA 37 CONCENTRATION (mM) TATTYACIDADDEDC,CÃ-oc„C12c„C,4C8C,oCuC12Ci,C,400.10.150.2O.S0.40.4i0.50.60.8Average minutes5.14 —Qoj over 90 (100)4.83 (102)4.40 (102)3.61 (100)6.07 (80)mamóles (89)4.97(85)5.55 (91)4.67 (100)5.29 (92)4.27 (74)3.85 (100)5.84 (97)5.70(97)5.93 (68)4.16(86)4.51 (88)5.51 (81)5.44(93)5.68(101)4.28 (100)5 (94)5.34 . 65 (100)5.13 (105)6.12(101)5.25 (98)4.88(80)5.22 (94)5.53 minutes202 O*Oj produced/gin wet weight of slices in 90 (UK))174 (100)177 (100)182 (100)18» (68)119 (100)194 (63)153 (100)124 (79)142 (106)139 (80)109 (80)214 (68)72 (60)92 (39)76 (40)112 (31)100 (49)132 (68)121 (58)116(67)58 (52)89 (88)30 (50)178 (51)69 (39)71 (41) (17)89 The figures quoted are mean values from two to eight determinations, those in parentheses referring to percentages of the mean values observed in the presence of 0.2 mM paluiitate-1-C" only. TABLE 6 To determine the extent of uncoupling in ascites cells, the effects of decanoate and DNP in the presence and absence of glucose, on the inorganic phosphate and 7-minute nucleotide phosophate levels, and the incorporation of labeled phosphate into the latter, were measured. The results are CÃœ2produced/gm labeled Additions m«)NilGlucosePyruvateGlutamateBuccinate-QO!4.77(100)3.08( (10 wetweight/90 presented in Table 9. The control levels reported minutes201 for these materials are of the same order as those quoted by Ibsen, Coe, and McKee (13) and by (100)162 65)4.62 81)157 ( Wu and Racker (27). They remained constant 97)5.10(107)5.48 ( 78)190 ( for periods of 1 hour or more, in contrast to 95)196 ( the findings of Acs and Sträub (l), who report (115)mamóles ( 98) a steady loss of total adenine nucleotides. The The figures quoted are mean values obtained from six effects of glucose and DNP are similar to those determinations. The figures in parentheses refer to percentages previously reported (13, 27), and it is now shown of the control values obtained in the presence of 0.2 mM that decanoate produces effects which are parallel palmitate-1-C14 only. Conditions of incubation were as de scribed in the section on "Materials and Methods." to those produced by DNP. There is no question, THEEFFECTS OFOTHERSUBSTRATES ONTHE OXIDATION OFPALMITATE-I-C" BYSLICES OFSARCOMA 37 Downloaded from cancerres.aacrjournals.org on August 10, 2017. © 1962 American Association for Cancer Research. ELLIS AXDSCHOLEFIELD—Fatty Add Oxidation by Tumors therefore, that, in the presence of DNP or decanoate, at the concentrations used, there was a fall in the steady state level of ATP and an even greater fall in the rate of turnover of phosphate in this compound. DISCUSSION The subject of the present investigation has been the relative lack of sensitivity of fatty acid oxidation in tumor slices and ascites cells to the inhibitory effects of uncoupling agents. It is sug gested that the explanation of this resistance to 311 loss of ATP from the cell lies in a Km value for ATP in the initial activation process, which is such that adequate production of the coenzyme A esters of fatty acids occurs when little ATP is present. There is, in fact, a definite decrease in the total ATP of these tissues on addition of DNP or fatty acids themselves, since: a) These agents bring about a decrease in the total easily hydrolyzable nucleotide phosphate content of the tumors. The actual fall in ATP may be greater, because complete conversion of ATP to adenosine diphosphate (ADP) would cause TABLE 7 THE EFFECTSOFOTHERFATTYACIDSONTHEOXIDATION OFo.l mM DECANOATE BYSAKCOMA 87 ASCITESCELLS Fatty acid added (mM)00.020.050.090.100.150.3CnOÕA noi338;(100)241 Vi r\A""/OlUJ243 (77)199 (63)152 (48)104 (68)165 (61)102 (58)108 (57)103 (29)54 (47)103 (24)42*(9)Cn87°1(100)215 (33)74 (15)34 (29)67 (23)34*(11)a,*gg|(100)266 (58)198 (33)Cu««H181 (10)C»370 (19)Ci.«}<«»>265 (43) The values quoted are from typical experiments and refer to m/xmolesdecanoa te oxidized/hr/ml packed cells. The figures in parentheses refer to percentages of the control values obtained in the presence of decanoate only. * In these cases there was a slight inhibition of respiration amounting to not more than 10 per cent. TABLE 8 THE EFFECTSOFDINITROPHENOL ANDGLUCOSE ONTHE OXIDATIONOFo.l mM DECANOATE-I-C"BY ASCITESCELLSANDTUMORSLICES TABLE 9 THEEFFECTS OFPOTASSIUM DECANOATE ANDGLUCOSE ONINORGANIC ANDNUCLEOTIDE PHOSPHATE LEVELS INEHRLICH ASCITES CELLS ADDITIONSDeca CARCINOMA-Qo,4.95.55.65.55.13.55.46.16.66.1mpmolesC'<O¡ ADDITIONDNP(mil)00.030.040.050.060.080.1000.030.040.050.060.080.10Glucose(mil)000000010101010101010EHRLICH ASCITES-Qo.10.211.810.49.57.76.314.115.815.213.6mamólesC"O2produced/ PHOSPHATE/ML CELLSP¡5.86.17.58.46.14.94.73.87.64.77-min.nucleotidephosphate2.92.82.41.53.23.13.02.81.93.0CO OrCHARCOAL-ADSORBEDNDCLEOTIDK316.8X10'12.97.93518 CELLS Qo,12.612.710.49.58.48.89.38.215.818.2MMOLES produced/gm noate(mM)00.40.81.200.40.81.2DNP(mM)0.050.05Glu cose(mM)000010101010010— wet weightof ml packedcells430530450260210460690800850730EHRLICH tissue365445475450390500620650670610 The incubation was carried out for 90 minutes, in air for the ascites cells and in oxygen for the slices. The cells were incubated in air for 30 minutes at 37°C. Phosphate buffer (pH 7.4) was present at a final concentration of 10 mM and a specific radioactivity of 8.8 X 10* counts/ min/pmole phosphate. Downloaded from cancerres.aacrjournals.org on August 10, 2017. © 1962 American Association for Cancer Research. 312 Cancer Research a decrease of only 50 per cent in this phosphate fraction. 6) The presence of these agents causes an even greater decrease in the amount of radioactivity incorporated into the total nucleotide fraction on incubation of the tissues with P32-labeled phos phate. c) The rate of glycine inci.. ^ration into pro teins and the extent of its uptake into the meta bolic pool of the tissues (both being ATP-requiring reactions) are markedly decreased in the presence of DNP or fatty acids. d) Addition of DNP causes a decrease in the rate of incorporation of palmitate into phospholipides, an effect which is reversed by the presence of glucose. The effect of glucose alone is to inhibit palmitate oxidation (by acting as a preferential substrate) but to stimulate phospholipide syn thesis (presumably by supplying the triósemoiety for glycerol formation). In these experiments DXP has little effect on palmitate oxidation and actually reverses the inhibitory effect of glucose. From data quoted by Kornberg and Pricer (15) the concentration of ATP corresponding to the Km value for conversion of palmitate to its hydroxamate is something less than 0.5 m\i. The results of Drysdale and Lardy (10) indicate that 0.025 MmoleATP/0.7 ml (0.04r-0.07 mm) causes half maximal rate of oxidation of caprylate in a soluble enzyme system. In the present experiments the level of ATP found in the absence of uncoupling agents is approximately 1.5 mu (assuming little ADP to be present). Loss of much of the ATP may therefore still provide enough acyl-CoA for maximal rate of fatty acid oxidation but not enough for phos pholipide synthesis (see Table 1). Another alternative is that production of ATP is not essential and that the coenzyme A esters are formed by a mechanism not involving ATP. It has been pointed out by Pritchard and Tove (19) that transacylation of coenzyme A esters may occur. If this suggestion is extended to include transfer from succinyl coenzyme A, then the se quence may be a-ketoglutaric acid —» succinyl-CoA succinyl-CoA + fatty acid —» acyl-CoA + succinate . On addition of DNP the formation of succinylCoA from the operation of the citric acid cycle will still occur, and hence acyl-CoA formation may carry on. Alternatively, the succinyl-CoA Vol. 22, April 1962 may react with ADP and inorganic phosphate to yield ATP even in the presence of DNP, and this may be sufficient to permit acyl-CoA formation via the thiokinases. The actions of fatty acids as uncoupling agents are undoubtedly similar to that of DNP, but inhibitions are obtained (see, for example, Table 7) at levels of fatty acid which can have little effect on coupled phosphorylation. As noted in the text, these effects are unlikely to be due to simple competition between the free fatty acids; but competition between their coenzyme A deriva tives remains a possibility (2, 3). Similar effects are obtained on addition of benzoate or palmitate to rat liver mitochondria oxidizing butyrate-1-C14 (2), and it is suggested that the inhibition of C14U2production occurs as a result of competition between the CoA esters of these acids and labeled acetyl-CoA. Finally, it should be pointed out that uncou pling agents do not equally influence all those reac tions which are coupled to the metabolism of ATP in tumors. Dinitrophenol, at a concentration of 0.05 min, stimulates aerobic glycolysis by ascites cells several-fold (5, 24). It stimulates anaerobic glycolysis (22, 24), respiration (25), and, as shown above, fatty acid oxidation by 25-50 per cent. The extent of glycine-1-C14 uptake into the free amino acid pool of tumor slices is not influenced by 0.05 HIM DNP, but the uptake of glycine by ascites cells is decreased by 25 per cent, and its incorporation into the proteins of both types of tumor is decreased by 60 per cent (11). Pre liminary unpublished experiments suggest that this sensitivity of glycine incorporation to DNP may be related to the sensitivity of glutamine synthetase to loss of ATP due to the presence of DNP. The incorporation of palmitate-1-C14 into phospholipides is inhibited to the extent of 25 per cent by 0.05 mM DNP. Similarly, the incor poration of adenine-8-C14 into the acid-soluble nucleotides of ascites cells is inhibited by 0.05 mM DNP to the extent of 25 per cent, but its incorporation into nucleic acids is inhibited by 40 per cent.1 ACKNOWLEDGMENTS It is a pleasure to acknowledge the continued interest of Professor J. H. Quastel, F.R.S., and to thank the National Cancer Institute of Canada for a grant-in-aid. \Ve are also most grateful to the National Research Council of Canada for finan cial assistance. 1D. B. Ellis and P. G. Scholefield, The effects of adenine and glucose on synthesis of nucleotides by Ehrlich ascites carcinoma cells in ritro. (In preparation.) Downloaded from cancerres.aacrjournals.org on August 10, 2017. © 1962 American Association for Cancer Research. ELLIS AND SCHOLEFIELD—Fatty Acid Oxidation by Tumors REFERENCES 1. Acs, G., and STRÄUB, F. B. Metabolism within Ascitic Cancer Cells. Doklady Akad. Nauk, U.S.S.R., 96:102124, 1954. 2. AVIGAN,J.; QUASTEL,J. 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Studies of Fatty Acid Oxidation: IX. The Effects of Uncoupling Agents on the Oxidation of Fatty Acids by Transplantable Tumors D. B. Ellis and P. G. Scholefield Cancer Res 1962;22:305-313. Updated version E-mail alerts Reprints and Subscriptions Permissions Access the most recent version of this article at: http://cancerres.aacrjournals.org/content/22/3/305 Sign up to receive free email-alerts related to this article or journal. To order reprints of this article or to subscribe to the journal, contact the AACR Publications Department at [email protected]. To request permission to re-use all or part of this article, contact the AACR Publications Department at [email protected]. Downloaded from cancerres.aacrjournals.org on August 10, 2017. © 1962 American Association for Cancer Research.