studies of fatty acid oxidation ix. the effects of ... · phate, ph 7.4, but when elevated rates of...

Studies of Fatty Acid Oxidation

IX. The Effects of Uncoupling Agents on the Oxidationof Fatty Acids by Transplantable Tumors

D. B. ELLISANDP. G. SCHOLEFIELD*

(McGill-Montreal GeneralHospital Research Institute, Montreal, P.Q., Canada)

SUMMARYOxidation 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 tri-phosphate (ATP) produced by uncoupling agents, such as dinitrophenol (DNP) or thefatty acids themselves. However, the rate of incorporation of palmitate-l-C14 intophospholipides was decreased in the presence of DNP.

Mutually inhibitory effects among fatty acids occurred. Such effects were shown tobe unlikely to result from isotopie dilution, competition among the fatty acids, or touncoupling effects.

The observed inhibitions of the oxidation of decanoate-1-C14 and palmitate-l-C14are interpreted in terms of the availability of acyl-CoA under various conditions andthe effects of such acyl-CoA derivatives on the metabolism of fatty acids.

At the beginning of the sequence of reactionsinvolved in the biological oxidation of fatty acids,adenosine triphosphate (ATP) is required, beingnecessary for the conversion of the various fattyacids to their coenzyme A esters (18). Such participation of ATP in fatty acid oxidation is apparently essential and is confirmed by the finding(8) that oxidation of fatty acids by liver mitochondria is greatly inhibited by dinitrophenol(DNP). The concentrations of DNP required arethose which also lead to an uncoupling of oxidationfrom phosphorylation (8, 17). Oxidation of pyru-vate 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 acidoxidation by liver mitochondria in the presenceof fumara te on addition of DNP shows that "priming" agents do not reverse the inhibitory effects on

fatty acid oxidation (8).Previous experiments (23) had shown that addi

tion of DNP to ascites hepatoma 98/15 leadsto a stimulation of the rate of oxygen uptake andto a stimulation rather than an inhibition of the

* National Cancer Institute of Canada Associate Professorof Biochemistry, McGill University.

Received for publication September 25, 1961.

oxidation of butyrate-1-C14, laurate-1-C14, and palmitate-l-C14. In many cases the presence of asecond fatty acid causes a decrease in the production of C14O2from these fatty acids, particularlywhen the second fatty acid has a chain lengthof eight carbon atoms or more. Such fatty acidsare known to uncouple oxidation from phosphorylation in mitochondrial preparations (21), in ascites cells (7, 22), and in tumor slices (11). Thefailure of DNP to inhibit fatty acid oxidation insuch preparations has therefore been consideredfurther, as well as the interactions among fattyacids in tumor slices and ascitic forms of tumorsThe effects of changes in the level of ATP produced by these agents on the incorporation offatty acids into phospholipides have also beeninvestigated.

MATERIALS AND METHODSANIMALS

All animals used were male Swiss white miceweighing 20-25 gm., purchased from CarworthFarms, New York, U.S.A.

TISSUEPREPARATIONSTumors, ascites cells, tissue slices, and the in

cubation technic were as previously described (11).

305

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306 Cancer Research Vol. 22, April 1962

The incubation medium was a calcium-free Krebs-Ringer solution containing 145 HIM NaCl, 5.8IHM 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 atmosphereof oxygen and ascites cells in an atmosphere ofair. The medium was buffered with 10 mM phosphate, pH 7.4, but when elevated rates of aerobicglycolysis were anticipated the final concentrationof phosphate buffer was increased to 20 mM.In studies of fatty acid oxidation the standardincubation time was 90 minutes.

ASSAY OF C14-LABELED COMPOUNDS

Carbon dioxide.â€”C14-labeled substrates were

added to Warburg flasks containing appropriatemedia 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 placedin the center well, the vessels gassed, if necessary,and the zero time was taken as the moment whenflasks plus manometers were placed in the incubation bath. At the end of the incubation period0.2 ml. 30 per cent trichloroacetic acid (TCA)was added from the side-arm to stop the reactionand liberate trapped carbon dioxide. After a further incubation period of 30 minutes the filterpapers were removed with washings to centrifugetubes containing carrier sodium carbonate equivalent to 10 mg. BaCOs. The carbonate was thenprecipitated as BaCOa, washed twice with water,once with acetone, and transferred in acetone totared planchets which were then dried and weighedagain. The radioactivity on the planchets wasassayed with a thin-window gas-flow counter fora sufficient time to give a counting error of lessthan Â±5 per cent. The net counts/minute werecorrected to infinite thinness and referred backto AmÃ³les substrate oxidized by dividing by thespecific activity (counts/min/^mole) of the substrate added.

Palmitafe-l-Cli incorporation into phospholip-ides.â€”The residue remaining after precipitation

of ascites cells with TCA (see above) was washedtwice with 5 ml. 5 per cent TCA and extractedfor 30 minutes at 50Â°C. with 5 ml. 95 per cent

ethanol. The residue was further extracted with5 ml. ethanol-ether (1:1 v/v) for 20 minutesat 40Â°C. The combined extracts were reduced

to dryness under nitrogen, and the ensuing residuewas dissolved in 1 ml. petroleum ether. The phos-pholipides were precipitated from this solutionaccording to the method of Sinclair and Dolan(26) by adding 7 ml. dry acetone and 1 dropsaturated alcoholic magnesium chloride solution.The precipitated phospholipides were washed with

cold acetone, resuspended in moist ether, plated,and counted as described above.

Alcohol-soluble and alcohol-insoluble glycine.â€”The uptake of glycine-1-C14 into the alcohol-solu

ble fraction of tumor cells (i.e., into the aminoacid pool) and the incorporation of glycine-1-C14into the alcohol-insoluble fraction (mainly intoproteins) were estimated as described previously(11).

ESTIMATIONOF INORGANICPHOSPHATE, PHOSPHATE ESTERS, AND THE LEVEL OF RADIO

ACTIVITYIN THESE FRACTIONSWhen P32 was used, approximately 20 /nc. was

placed in the side-arm of Warburg vessels, andthe reaction was begun by tipping the labeledphosphate, together with appropriate inhibitors,after attainment of thermal equilibrium. At theend of the incubation period the vessels wereplaced on cracked ice and the contents (slicesplus medium) poured into 5 ml. ice-cold saltssolution, centrifuged, the supernatant was discarded, and the residue washed with a further8 ml. ice-cold salts solution. The supernatantwas carefully removed, and the sides of the centrifuge tubes were dried with paper tissues. Fiveml. 5 per cent TCA was then added to eachtube, and the slices were homogenized. Ascitescells were washed once with 8 ml. salts solution.All operations thus far were carried out at 2Â°-4Â°C.,

and the resulting suspensions were allowed tostand for a further 30 minutes at this temperatureto 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 withpyridine, HC1, and distilled water) for 10 minutesat 2Â°C. according to the method of Crane and

Lipmann (6). The supernatant, together with twowashings, was made up to 20 ml., and 3.4 ml.was used for estimation of inorganic phosphateby Bartlett's modification (4) of the method of

Fiske and SubbaRow (12). There was little difference between the results obtained by the methodof Fiske and SubbaRow (12) and that of Bartlett(4), which involves heating in acid, suggestingthat the amount of non-nucleotide easily hydrolyz-able phosphate esters (such as creatine phosphate)was small compared with the amounts of phosphate present. The results are therefore referredto as levels of inorganic phosphate.

The labile phosphate of the nucleotide fractionwas measured by treatment of the washed charcoal with 4 ml. N HC1 in a boiling water bath,cooling, centrifuging, and assaying inorganic phos-



ELLIS AND SCHOLEFIELDâ€”Fatty Acid Oxidation by TumorÂ» 307

phate on a 2-ml. aliquot of the supernatant bythe method of Bartlett (4). This fraction is termedthe 7-minute nucleotide phosphate fraction.

Aliquots (usually 200 jul.) of these two fractionswere plated and counted as described above. Theinorganic phosphate fraction was neutralized onthe planchet with NH4OH, and the HC1 solutionof hydrolyzed nucleotide was neutralized with0.2 ml. N NaOH. One drop of a 2 per cent solution of cetyltrimethylammonium bromide was alsoadded to produce even films on the planchets,which were dried under an infrared lamp beforebeing counted.

Labeled substrates.â€”P32 was obtained fromCharles E. Frosst and Co., Limited, Montreal,and the radioactive fatty acids from Merck andCo., Limited, Montreal.

RESULTSThe metabolism of palmifate-l-Cn in Ehrlich

ascites cells.â€”Addition of fatty acids to ascitescells leads to an inhibition of respiration, but astimulation may (23) or may not (22) occur atlower concentrations. The respiratory activity ofthe Ehrlich ascites cells used in the present experiments was stimulated only slightly by fatty acids.Oxidation of fatty acids by these cells must therefore take place at the expense of the endogenoussubstrates, including the endogenous supply offatty acids. In the preliminary experiments theextent of oxidation of exogenous fatty acid wasdetermined under conditions where there was nosignificant change in the rate of oxygen uptake.Ten Warburg vessels were set up, one containingno palmitate and three containing each of threeconcentrations (0.05, 0.1, and 0.3 IHM)of palmi-tate-l-C14. The rate of oxygen uptake was the samein all vessels. At 30, 60, and 90 minutes, TCAwas tipped into the main compartment of threevessels, one of each palmitate concentration, andthe C14O2produced was estimated as describedabove. The results obtained are presented in Chart1. At a concentration of 0.3 min, palmitate-1-C14was oxidized by ascites cells at a constant ratefor at least 90 minutes. When the concentrationof palmitate-1-C14 was decreased to 0.1 HIMthetime course was again linear, but the rate wassomewhat lower. Further decrease in the concentration of palmitate-1-C14 to 0.05 mM led to anonlinear time course and a lower initial rate ofC14O2production. Such results may be explainedin terms of two hypotheses: (a) that there existswithin the ascites cells a pool of nonradioactivepalmitate or (b) that there is a Kmvalue controllingthe rate of palmitate oxidation. Calculations fromthe initial rates of C14Osproduction show that

the second hypothesis is valid if the Km valuefor palmitate in the process controlling its oxidation is 0.05 HIM. The lowest concentration ofpalmitate added was 0.05 mivi, and hence, asoxidation proceeded, it would be expected thatthe rate of C1402production would decrease withtimeâ€”an effect apparent from Chart 1. Controlof palmitate oxidation could occur by failure to

08

04

Time in minutes

CHART1.â€”The oxidation of palmitate-1-C14 by Khrlichascites carcinoma cells. The cells were incubated with labeledpalmitate under the standard conditions described in the section on "Materials and Methods."

A = 0.05 mMpalmitate-1-C14 present.O = 0.1 mxf palmitate-1-C14 present.â€¢= 0.3 mMpalmitate-1-C14 present.

saturate either the system transporting palmitateinto the ascites cells or the enzyme responsiblefor the initial activation of the palmitate. Theabsence of any lag periods suggests the latter.

The effect of the presence of a metabolic poolof palmitate on the specific activity of the addedpalmitate was determined by the following equation:

Specific activity of added palmitate _ x+ 3 [S]Final specific activity 3 [S]




where x is the number of AmÃ³lesof palmitate inthe metabolic pool of the ascites cells and [S]is the final concentration of palmitate in /LimÃ³les/ml of the 3 ml. of incubation medium. Since theamount of CI4O2produced is proportional to thespecific activity of the palmitate, considerationof a metabolic pool gives rise to an equation whichis of the same form as the Michaelis-Mentenequation. The value of x/3 is therefore 0.05 /Â¿moles(Km = 0.05 HIM) so that x = 0.15 /umole. Thisamount is present in one-eighth ml. of packedcells, and hence is equivalent to 1.2 AmÃ³les/mipacked cells or to a concentration of 1.6 mil,assuming 75 per cent of the cell volume is water.

TABLE 1

THEEFFECTSOFDNP ANDGLUCOSEONTHEMETABOLISMOFPALMITATE-I-C"

BYEHRLICHASCITESCELLS

ADDITIONSDNP(mil)00.040.060.080.1000.040.060.080.10Glucote(mil)000001010101010OXYGEN

UPTAKE(-QO,)13.2

(100)14.1(107)14.8(11Â«)18.6(108)12.7

(96)8.0(61)16.2(128)18.4

(140)16.5(125)15.0

(114)C'<OiPRODUCTION(Â¿1110LE8PALMITATEOXIDIZED/MLPACKED

CELLS)1.67

(100)1.94(116)2.10(126)2

. 16(130)1.95(117)0.84

(51)1.23(74)1.64(99)1.91

(115)1.78(107)PHOSPHOLIPIDES(MMUOLESPALMITATE-1-CÂ»

INCOR

PORATED/MLPACKED

CELLS)470

(100)380(81)320(68)290(62)220(47)740

(158)810(172)800(170)740(158)650

(139)

The cells were incubated at 37Â°C. for 90 minutes in aKrebs-Ringer solution containing 20 mM phosphate buffer,pH 7.4. Concentration of palmitate was 0.8 mM. The C1(O2datain this and subsequent tables are in terms of the amount ofsubstrate oxidized to CO2, calculated on the assumption thatÂ¡illfatty acid carbons are oxidized at the same rate. â€”QoÂ»figures are average values over the 90-minute incubationperiod. Figures in parentheses refer to percentages of controlin the absence of further additions.

A value of 1.6 mM free palmitate is unlikely,and it is therefore presumed that the rate-controlling feature is the Km value for palmitate, whichis 0.05 mM.

From the amounts of CltOÂ¡ produced, the

amount of oxygen corresponding to complete oxidation of the palmitate may be calculated. Itis equal to 20 per cent of the total oxygen uptakeof the ascites cells with 0.05 mM palmitate and to40 per cent with 0.3 m.M,values which are of thesame order as that of 18 per cent quoted for0.13 HIMpalmitate with hepatoma ascites 98/15(23). In further confirmation of the results ofScholefield, Sato, and Weinhouse (23), other fatty

acids (Cio-Cia) have little effect on palmitate-1-C14oxidation until concentrations are added whichalso inhibit respiratory activity. Decanoate (0.2-0.4 HIM)is known to cause effects which are consistent with the suggestion that there occurs anuncoupling of oxidation from phosphorylation (7,11, 22). This should, in turn, have caused acorresponding decrease in the rate of palmitateoxidation, and none was observed. The effectsof DNP on palmitate metabolism in ascites cellswere therefore investigated, and the results obtained are presented in Table 1. They confirmpreviously observed effects of DNP on palmitate-1-C14 oxidation (23) and further indicate thatDNP reverses the inhibitory effect of glucose onfatty acid oxidation. On the other hand, the incorporation of fatty acids into phospholipides, a process which is equally dependent on the formationof an acyl-CoA derivative, is sensitive to the presence of DNP, and this inhibition was largely reversed by glucose (Table 1).

To show that palmitate did not, in some way,interfere with the uncoupling action of DNP,the effects of various combinations of these twoagents on glycine-1-C14 uptake into the aminoacid pool, and glycine-1-C14 incorporation intothe proteins of Ehrlich ascites cells, were investigated. The results obtained are presented in Table2. They demonstrate that palmitate did not influence the inhibitory effects of DNP on these processes. At a concentration (0.04 IHM)which stimulates oxidation of 0.3 nui palmitate-1-C14 by 26per cent (Table 1) there was an inhibition byDNP of glycine incorporation into protein ofnearly 50 per cent in the presence or absence of0.3 mM palmitate (Table 2).

The metabolism of palmitate-1-C1*by tumor slices.â€”Many of the present experiments were performed on slices of the Ehrlich carcinoma andSarcoma 37. Quantitatively similar results wereobtained with these two tumors, but S-37, inour hands, gave a greater yield of non-necroticmaterial, and this tumor was used in most of theexperiments reported.

The effects of variation in the concentrationof palmitate-1-C14 on the respiratory activity ofS-37 slices and the yield of C14O2are presented inTable 3. There was little change in the Qo, value;the yields of C14Ossuggest an apparent Km valuefor palmitate-1-C14 of approximately 0.4 mM, andat a palmitate concentration of 0.5 m\i the contribution of palmitate oxidation to the total oxygen uptake of the slices was less than 10 per cent.

The effects of DNP and glucose on palmitateoxidation were similar to those obtained withascites cells, except that inhibition of respiration



ELLIS AND SCHOLEFIELDâ€”Fatty Acid Oxidation by Tumors 309

by DNP occurred at a slightly lower concentrationof DNP (Table 4). The lackof any specific effectof DNP on fatty acid oxidation and the reversalof the glucose inhibition by DNP were againobserved.

In another series of experiments the effectsof other fatty acids (Cg-Cu) on the oxidation of0.2 IBM palmitate-1-C14 by slices of S-37 were

investigated. The results are presented in Table5. Octanoate had no effect on respiration and littleeffect on C14O2 production. Decanoate inhibited

respiration by only 20 per cent at a concentrationof 0.8 mM but inhibited C14O2 production by 59

per cent. With increase in the chain length offatty acid, there was an increase in the inhibitoryeffects, C14C>2production being more sensitive than

total respiratory activity. Further increase in chainlength caused diminishing inhibitory effects. Themost effective inhibitor was laurate (Cn), although

TABLE 2

THE EFFECTSOFDNP ONTHEUPTAKEANDINCORPORATION"OFGLYCINE-I-C"BYEHRLICHASCITESCELLSIN THEABSENCEANDPRESENCEOF0.8 mMPALMI-TATE

ADDITIONSDNP(mM)00.030.060.1000.030.060.10Palmitate(mil)00000.30.30.30.3-Qo.11.812.713.910.612.513.113.09.9Â¿(MOLESGLYCINE-1-C"UPTAKE/MLPACKEDCELLS12.010.99.56.912.810.79.77.1MAMÃ“LESGLYCDÃ•E-1-C14INCORPORATED/ML

PACKEDCELLS362

(100)191(53)118(82)58(15)348(96)189(52)104(29)59( 16)

The cells were incubated at 37Â°C. for 45 minutes in amedium containing 20 mM phosphate, pH 7.4, 2 mM glycine-1-C14(0.2 fie.), and further additions as noted. The figures inparentheses are percentages of the control value in the absenceof added palmitate or DXP.

TABLE 3THEOXIDATIONOFPALMITATE-I-C"

BYSLICESOFSARCOMA37

Concentrationofpalmitate-l-OÂ«(mM)0.10.20.5â€” Qo,4.704.454.51mamÃ³les

labeledCO:produced/gmwet

weight109198283

tridecanoate has about 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 ofpalmitate-1-C14 were investigated, and the results

are presented in Table 6. These substrates (glucose, pyruvate, glutamate, and succinate) hadslight inhibitory effects on palmitate oxidation,but none were as effective as the fatty acids.

The metabolism of decanoate-l-C1* by ascites cellsand tumor slices.â€”The general pattern of interaction between fatty acids in ascites hepatoma

TABLE 4

THEEFFECTSOFDNP ANDGLUCOSEONTHEOXIDATIONorPALMiTATE-l-C14

BYSARCOMA87SLICES

ADDITIONSDNP(mu)00.040.060.0800.040.060.08Glucose(mM)000010101010OXYGENUPTAKEâ€”

dÃ»-.5.7

(100)5.9(108)6.1(107)4.5

(79)3.8(68)6.4(11815.6

(98)5.3( 93)Cl4Ot

PRODUCTION(MpMOLES/GMWET

WEIGHTTUMOB)176

(100)198

(Ili)192(109)155

(88)134(76)207

(118)219(124)205

(116)

The figures quoted are mean values obtained from sixdeterminations. The conditions of incubation were as describedin the section on "Materials and Methods."

Incubation time was 90 minutes. Figures in parenthesesrefer to percentages of values obtained with 0.3 mM palmitate-1-C14in the absence of further additions.

98/15 is that their oxidation is inhibited by fattyacids of greater chain length (23). In agreementwith this pattern it was found that there waslittle or no effect of other fatty acids of shorterchain length on the oxidation of palmitate byascites cells. Similarly, longer chain fatty acidsinhibited the oxidation of decanoate-1-C14 (Table

7) under conditions where oxygen consumptionis not influenced. A linear relationship betweenthe reciprocal of the velocity of oxidation (C14O2

production) and the concentration of inhibitorwas found. This would occur if the inhibitory effects are due to either competitive or noncompetitive inhibition by the second fatty acid (9). Non-competitive inhibitory effects between fatty acidsseem unlikely, and, since increase in the substrate(decanoate) concentration from 0.1 to 0.2 mudid little to reverse these effects, competitiveinhibition seems equally unlikely.

The response to other fatty acids does notappear to be due to loss of ATP, since DNP didnot produce similar effects on decanoate-1-C14 oxi-




dation when either slices or ascites cells wereused (Table 8). At the two highest levels of DNPthere was a greater inhibitory effect on C14O2production than on respiration in the ascites cells,suggesting some response to an uncoupling action.Glucose had little effect on C14O2production, butthe combined effect of DNP and glucose was astimulation of approximately 50 per cent in respiration and nearly 100 per cent in C14O2production.The effects of DNP and glucose in slices weresimilar to the effects quoted for ascites cells,except that glucose stimulated CI4O2 production

from decanoate-1-C14 (35 per cent in the valuesquoted in Table 8) in contrast to its lack ofeffect in ascites cells, and there is no evidencefrom these data of an uncoupling effect of DNPin slices.

It should also be noted that the rates of oxidation of decanoate-1-C14 in the ascitic and solidforms of the Ehrlich carcinoma were similar. Whenpalmitate-1-C14 was used as substrate its rateof oxidation was approximately 10 times as greatin ascites cells as it was in tumor slices (see Tables1 and 3).

TABLE5THEEFFECTSOFOTHERFATTYACIDSONTHEOXIDATIONOF0.2mMPALMITATE-I-C"BYSLICESOFSARCOMA37

CONCENTRATION(mM)

TATTYACIDADDEDC,CÃocâ€žC12câ€žC,4C8C,oCuC12Ci,C,400.10.150.2O.S0.40.4i0.50.60.8Average

â€”Qoj over 90minutes5.14

(100)4.83(100)6.07(100)5.29(100)5.84(100)5

.65 (100)5.13

(97)5.70(97)5.93

(105)6.12(101)5.25

(102)4.40(91)4.67

(88)5.51(94)5.34

(94)5.53

(92)4.27(81)5.44(93)5.68(101)4.28

(89)4.97(85)5.55

(98)4.88(80)5.22

(102)3.61

(68)4.16(86)4.51(74)3.85

(80)mamÃ³les

O*Oj produced/gin wet weight of slices in 90minutes202

(UK))174(100)177(100)182(100)18Â»(100)194

(100)124

(68)119(63)153

(79)142

(80)214

(106)139(80)109

(60)92(49)132

(68)121

(68)72(39)76

(40)112(58)116(67)58

(31)100(52)89

(50)178

(88)30

(17)89

(51)69(39)71

(41)

The figures quoted are mean values from two to eight determinations, those in parentheses referring to percentages of the meanvalues observed in the presence of 0.2 mM paluiitate-1-C" only.

TABLE 6

THEEFFECTSOFOTHERSUBSTRATESONTHEOXIDATIONOFPALMITATE-I-C"

BYSLICESOFSARCOMA37

Additions (10mÂ«)NilGlucosePyruvateGlutamateBuccinate-QO!4.77(100)3.08(

65)4.62(97)5.10(107)5.48

(115)mamÃ³les

labeledCÃœ2produced/gmwetweight/90

minutes201

(100)162(81)157(78)190(95)196( 98)

The figures quoted are mean values obtained from sixdeterminations. The figures in parentheses refer to percentagesof the control values obtained in the presence of 0.2 mMpalmitate-1-C14 only. Conditions of incubation were as described in the section on "Materials and Methods."

To determine the extent of uncoupling in ascitescells, the effects of decanoate and DNP in thepresence and absence of glucose, on the inorganicphosphate and 7-minute nucleotide phosophatelevels, and the incorporation of labeled phosphateinto the latter, were measured. The results arepresented in Table 9. The control levels reportedfor these materials are of the same order as thosequoted by Ibsen, Coe, and McKee (13) and byWu and Racker (27). They remained constantfor periods of 1 hour or more, in contrast tothe findings of Acs and StrÃ¤ub (l), who reporta steady loss of total adenine nucleotides. Theeffects of glucose and DNP are similar to thosepreviously reported (13, 27), and it is now shownthat decanoate produces effects which are parallelto those produced by DNP. There is no question,



ELLIS AXDSCHOLEFIELDâ€”FattyAdd Oxidation by Tumors 311

therefore, that, in the presence of DNP or decano-ate, at the concentrations used, there was a fallin the steady state level of ATP and an evengreater fall in the rate of turnover of phosphatein this compound.

DISCUSSIONThe subject of the present investigation has

been the relative lack of sensitivity of fatty acidoxidation in tumor slices and ascites cells to theinhibitory effects of uncoupling agents. It is suggested that the explanation of this resistance to

loss of ATP from the cell lies in a Km valuefor ATP in the initial activation process, whichis such that adequate production of the coenzymeA esters of fatty acids occurs when little ATPis present. There is, in fact, a definite decrease inthe total ATP of these tissues on addition ofDNP or fatty acids themselves, since:

a) These agents bring about a decrease in thetotal easily hydrolyzable nucleotide phosphatecontent of the tumors. The actual fall in ATPmay be greater, because complete conversion ofATP to adenosine diphosphate (ADP) would cause

TABLE 7

THE EFFECTSOFOTHERFATTYACIDSONTHEOXIDATIONOFo.l mMDECANOATEBYSAKCOMA87 ASCITESCELLS

Fatty acid

added(mM)00.020.050.090.100.150.3CnOÃ•A

r\A""/OlUJ243

(77)199(63)152(48)104

(33)CuÂ«Â«H181

(57)103(33)74(23)34*(11)a,*gg|(100)266

(58)108(24)42*(9)Cn87Â°1(100)215

(61)102(29)54(15)34

(10)CÂ»370

Vinoi338;(100)241

(68)165(47)103(29)67

(19)Ci.Â«}<Â«Â»>265(58)198

(43)

The values quoted are from typical experiments and refer to m/xmolesdecanoa te oxidized/hr/ml packed cells. Thefigures 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 EFFECTSOFDINITROPHENOLANDGLUCOSEONTHEOXIDATIONOFo.l mMDECANOATE-I-C"BY

ASCITESCELLSANDTUMORSLICES

TABLE 9

THEEFFECTSOFPOTASSIUMDECANOATEANDGLUCOSEONINORGANICANDNUCLEOTIDEPHOSPHATE

LEVELSINEHRLICHASCITESCELLS

ADDITIONDNP(mil)00.030.040.050.060.080.1000.030.040.050.060.080.10Glucose(mil)000000010101010101010EHRLICHASCITES-Qo.10.211.810.49.57.76.314.115.815.213.6mamÃ³lesC"O2produced/

ml packedcells430530450260210460690800850730EHRLICH

CARCINOMA-Qo,4.95.55.65.55.13.55.46.16.66.1mpmolesC'<OÂ¡

pro-

duced/gmwetweightof

tissue365445475450390500620650670610

ADDITIONSDeca

noate(mM)00.40.81.200.40.81.2DNP(mM)0.050.05Glucose(mM)000010101010010â€”

Qo,12.612.710.49.58.48.89.38.215.818.2MMOLES

PHOSPHATE/ML

CELLSPÂ¡5.86.17.58.46.14.94.73.87.64.77-min.nucleotidephosphate2.92.82.41.53.23.13.02.81.93.0COUNTS/MIN/MLPACKED

CELLSOrCHARCOAL-ADSORBEDNDCLEOTIDK316.8X10'12.97.93518.918.918.014.96.617.1

The incubation was carried out for 90 minutes, in air forthe ascites cells and in oxygen for the slices.

The cells were incubated in air for 30 minutes at 37Â°C.Phosphate buffer (pH 7.4) was present at a final concentrationof 10 mM and a specific radioactivity of 8.8 X 10* counts/min/pmole phosphate.




a decrease of only 50 per cent in this phosphatefraction.

6) The presence of these agents causes an evengreater decrease in the amount of radioactivityincorporated into the total nucleotide fractionon incubation of the tissues with P32-labeled phosphate.

c) The rate of glycine inci.. ^ration into proteins and the extent of its uptake into the metabolic pool of the tissues (both being ATP-requiringreactions) are markedly decreased in the presenceof DNP or fatty acids.

d) Addition of DNP causes a decrease in therate of incorporation of palmitate into phospholip-ides, an effect which is reversed by the presenceof glucose. The effect of glucose alone is to inhibitpalmitate oxidation (by acting as a preferentialsubstrate) but to stimulate phospholipide synthesis (presumably by supplying the triÃ³semoietyfor glycerol formation). In these experiments DXPhas little effect on palmitate oxidation and actuallyreverses the inhibitory effect of glucose.

From data quoted by Kornberg and Pricer(15) the concentration of ATP corresponding tothe Km value for conversion of palmitateto its hydroxamate is something less than 0.5m\i. The results of Drysdale and Lardy (10)indicate that 0.025 MmoleATP/0.7 ml (0.04r-0.07mm) causes half maximal rate of oxidation ofcaprylate in a soluble enzyme system. In thepresent experiments the level of ATP found inthe absence of uncoupling agents is approximately1.5 mu (assuming little ADP to be present).Loss of much of the ATP may therefore stillprovide enough acyl-CoA for maximal rate offatty acid oxidation but not enough for phospholipide synthesis (see Table 1).

Another alternative is that production of ATPis not essential and that the coenzyme A estersare formed by a mechanism not involving ATP.It has been pointed out by Pritchard and Tove(19) that transacylation of coenzyme A estersmay occur. If this suggestion is extended to includetransfer from succinyl coenzyme A, then the sequence may be

a-ketoglutaric acid â€”Â»succinyl-CoA

succinyl-CoA + fatty acid â€”Â»acyl-CoA

+ succinate .

On addition of DNP the formation of succinyl-CoA from the operation of the citric acid cyclewill still occur, and hence acyl-CoA formationmay carry on. Alternatively, the succinyl-CoA

may react with ADP and inorganic phosphate toyield ATP even in the presence of DNP, and thismay be sufficient to permit acyl-CoA formationvia the thiokinases.

The actions of fatty acids as uncoupling agentsare undoubtedly similar to that of DNP, butinhibitions are obtained (see, for example, Table7) at levels of fatty acid which can have littleeffect on coupled phosphorylation. As noted inthe text, these effects are unlikely to be due tosimple competition between the free fatty acids;but competition between their coenzyme A derivatives remains a possibility (2, 3). Similar effectsare obtained on addition of benzoate or palmitateto rat liver mitochondria oxidizing butyrate-1-C14(2), and it is suggested that the inhibition ofC14U2production occurs as a result of competitionbetween the CoA esters of these acids and labeledacetyl-CoA.

Finally, it should be pointed out that uncoupling agents do not equally influence all those reactions which are coupled to the metabolism ofATP in tumors. Dinitrophenol, at a concentrationof 0.05 min, stimulates aerobic glycolysis by ascitescells several-fold (5, 24). It stimulates anaerobicglycolysis (22, 24), respiration (25), and, as shownabove, fatty acid oxidation by 25-50 per cent.The extent of glycine-1-C14 uptake into the freeamino acid pool of tumor slices is not influencedby 0.05 HIM DNP, but the uptake of glycineby ascites cells is decreased by 25 per cent, andits incorporation into the proteins of both typesof tumor is decreased by 60 per cent (11). Preliminary unpublished experiments suggest thatthis sensitivity of glycine incorporation to DNPmay be related to the sensitivity of glutaminesynthetase to loss of ATP due to the presence ofDNP. The incorporation of palmitate-1-C14 into

phospholipides is inhibited to the extent of 25per cent by 0.05 mM DNP. Similarly, the incorporation of adenine-8-C14 into the acid-soluble

nucleotides of ascites cells is inhibited by 0.05mM DNP to the extent of 25 per cent, but itsincorporation into nucleic acids is inhibited by40 per cent.1

ACKNOWLEDGMENTSIt is a pleasure to acknowledge the continued interest of

Professor J. H. Quastel, F.R.S., and to thank the NationalCancer Institute of Canada for a grant-in-aid. \Ve are also mostgrateful to the National Research Council of Canada for financial assistance.

1D. B. Ellis and P. G. Scholefield, The effects of adenine andglucose on synthesis of nucleotides by Ehrlich ascites carcinomacells in ritro. (In preparation.)



ELLIS AND SCHOLEFIELDâ€”Fatty Acid Oxidation by Tumors 313

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1962;22:305-313. Cancer Res D. B. Ellis and P. G. Scholefield TumorsAgents on the Oxidation of Fatty Acids by Transplantable Studies of Fatty Acid Oxidation: IX. The Effects of Uncoupling

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