Download Studies of Fatty Acid Oxidation IX. The Effects of

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
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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-
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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]
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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.
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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-
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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
CÜ2produced/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
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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.
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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.)
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Research.
ELLIS AND SCHOLEFIELD—Fatty Acid Oxidation by Tumors
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Research.
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.
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