Download Metabolism of Neoplastic Tissue XII. Effects of Glucose

Metabolism of Neoplastic Tissue
XII. Effects of Glucose Concentration on Respiration
and Glycolysis of Ascites Tumor Cells*
LEAHBLOCH-FRANKENTHAL.!
ANDSIDNEYWEINHOUSE
(The Lankenau Hospital Research Institute and The Institute for Cancer Research, Philadelphia, Pa.)
One of the characteristic metabolic features of
neoplastic cells is the high aerobic and anaerobic
glycolysis, first discovered by Warburg (35).
Though the high lactic acid production has been
verified repeatedly, its significance remains contro
versial (10, 33, 35, 36, 40). Recently, another puz
zling relationship between glycolysis and respira
tion has been uncovered in studies with ascites
tumor cells, namely, an inhibition of respiration in
the presence of high concentrations of glucose. Al
though this effect is most pronounced in ascites
cells, similar inhibitions of respiration of lesser
magnitude were observed in solid tumors by Crabtree (15) and others (7,17). This phenomenon has
recently been studied in ascites tumors by a host
of investigators (6, 9, 11, 20, 21, 24, 26, 31, 34).
One of the stumbling blocks to a better under
standing of these glycolytic-respiratory relation
ships in tumors is our lack of knowledge of the
nature of the respiratory substrates. The fact that
tumor slices can respire for many hours without a
cellular reserve of carbohydrate has been known
for many years (35), and the respiratory quotient
data of Dickens and Simer (16) pointed to noncarbohydrates as possible respiratory substrates.
Studies of fatty acid oxidation (18, 27, 28, 42) also
suggest that these substances may play a domi
nant role in the endogenous respiration of tumors.
As yet, however, the situation prevailing in the
presence of adequate exogenous glucose is uncer
tain. The fact that glucose does not generally in
crease the endogenous respiration of tumor slices
and, in high concentrations, actually inhibits oxy* Aided by grants from the American Cancer Society,
recommended by the Committee on Growth of the National
Research Council, and the National Cancer Institute, National
Institutes of Health. We are grateful to Grace Medes and Alice
Thomas for assistance in some of the experiments.
t Fellow of the Women's Auxiliaries of the Institutes, on
leave from the Cancer Research Laboratories, The Hebrew
University-Hadassah Medical School, Jerusalem, Israel.
Received for publication June 21, 1957.
gen uptake (15) could be cited against the partici
pation of glucose as a respiratory substrate. On the
other hand, the enhanced oxygen uptake of ascites
cells with glucose present at low concentrations
(6, 24) indicates that it is oxidized in ascites cells,
and many studies with Cu-labeled glucose (1, 22,
29, 41, 43) leave no doubt that a wide variety of
tumors have the capability of oxidizing glucose to
completion. Since the use of carbon-14-labeled
substrates makes it possible to distinguish between
exogenous and endogenous respiration, it was felt
that an investigation of glycolysis and respiration,
using this additional tool, might provide a better
insight into these hitherto obscure relationships.
The present report describes experiments on the
effects of wide variations in glucose-C14concentra
tion on the relative participation of exogenous and
endogenous carbon in the respiration of ascites
tumor cells in vitro. It was anticipated that this
study might also help to elucidate the metabo
lism of glucose by these cells as they exist in vivo
in the comparatively low-glucose ascitic fluid (23,
38). Also, ascites cells rather than solid tumor
slices were used, because it was felt that effects of
glucose concentration might be more easily inter
pretable. In solid tumors such effects might be
obscured by uncertainty about the rate of diffu
sion of glucose through multicellular layers of
tissue slices.
MATERIALS
AND METHODS
Preparations of cell suspensions.—The tumor used in these
studies was a strain of Ehrlich ascites cells developed by Lettré
(3) and carried in mice of the Swiss strain. It is distinguished
by a very low accompaniment of erythrocytes. Ascitic fluid
was removed 7-10 days after inoculation and immediately
transferred to an ice-bath. Pooled fluid from several mice was
centrifuged 10 minutes at 8000 r.p.m., the supernatant fluid
was removed, and the cells were resuspended in an equal vol
ume of calcium-free, isotonic, pH 7.4 phosphate-buffered saline
solution. The medium was high in phosphate, being similar to
that employed by Brin and McKee (6). It had the following
composition: sodium phosphate, 0.085 M; NaCl, 0.016 M; KC1,
0.005 M; and MgSO4>0.0018 M. Centrifugation and washing
1082
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BLOCH-FRANKENTHAL
ANDWEINHOUSE—Respirationand Glycolysis of Ascites Tumor 1083
were repeated and the cells finally suspended in 4 times their
volume of the same solution. Homogeneous dispersion was
achieved easily by homogenization for a few seconds with a
very loose-fitting Potter-Elvehjem homogenizer. Procedures
for incubation, collection of respiratory COa with the aid of
carrier carbonate, and radioactivity assay were carried out as
described previously (27, 43). Except where indicated, all
experiments were conducted with 1 ml. of cell suspension, cor
to 79 per cent. This material was diluted as required for the
metabolic experiments. It had the same specific activity as
the glucose from which it was derived.
Calculations.—Therelative specific activity of the respira
tory C02 was calculated from the formula:
Rei. sp. act. = counts/min BaCO3 X 100/counts/inin
of substrate as BaCO3.
The conversion of substrates to CO2 was estimated in terms
TABLE 1
TIMECOURSE
OFOXYGEN
CONSUMPTION
ANDGLUCOSE
OXIDATION
IN ASCITES
TUMOR
CELLS
Each vessel contained 1 ml. of cell suspension containing 0.20 ml. of packed cells (corresponding to approximately
20 mg. dry wt.), 1.8 ml. of phosphate saline medium, and 0.2 ml. of water or glucose solution. Experiments were run
at 37.5°C. with air in the gas phase. At hourly intervals, one flask of each concentration was acidified with 0.5 ml.
of 10 per cent HzSO4tipped in from the side-arm, and after 15 minutes for complete absorption of respiratory CO*
the flasks were removed and the COs isolated and assayed for radioactivity. Oxygen uptake was measured manometrically at 30-minute intervals after temperature equilibration for 10 minutes.
Oxygen uptakes are given in /amólesand CO2in /¿atomsglucose carbon, calculated as described in the text.
GLUCOSECONCENTRATION,
MOLAR
00,uptake(Minóles)12.523.131.537.80.001OÃ-uptake(/¿moles)12.323.132.439.3C0¡(/¿atoms
TIMEor
INCUBATION
Hours
3
4
Hourly period
First
Second
Third
Fourth
C)3.57.111.417.3
C)4.27.310.210.90.00250¡uptake(/¿moles)10.721.431.539.8COi(/¿atoms
C)4.48.412.716.10.01Ozuptake(/¿moles)5.812.020.027.8CO,(/¿atoms
12.5
10.6
8.4
6.3
12.3
10.8
9.3
69
4.2
8.1
2.9
0.7
10.7
10.7
10.1
8.3
4.4
4.0
4.3
3.4
5.8
6.2
8.0
7.8
3.5
3.6
4.3
5.9
responding to 0.20 ml. of packed cells. Under the conditions
employed, the dry weight of these cells determined by washing
with water, centrifuging, and drying at 110°
C. was approxi
mately 20 mg.
Labeled substrates.—Uniformly OMabeled glucose was ob
tained from the Nuclear Instrument and Chemical Company,
Chicago. It was diluted for use in these experiments to a
specific activity of approximately 0.2 /uc/mg. For radioactivity
assay, an aliquot was oxidized to CO2 by the persulfate proce
dure (8) and converted to barium carbonate. Counting was
carried out with an ultra-thin window counter in 7.5 sq. cm.
plancheta, and the values obtained were corrected for selfabsorption to an "infinitely-thick" layer. The glucose activi
ties thus determined were between 90,000 and 180,000 counts/
min.
Glucose was determined colorimetrically, according to the
anthrone method of Roe (32), and lactic acid was determined
colorimetrically according to Barker and Summerson (2).
Uniformly C14-labeled lactic acid was prepared by the
method of Brin (5) from the labeled glucose, with the same
ascites tumor cells, as follows. Uniformly labeled glucose, 54.8
mg., was divided equally between two large Warburg vessels,
each containing 1 ml. of packed ascites cells suspended in 15
ml. of the high phosphate medium. After aerobic incubation
for about 2 hours, the contents were pooled, acidified to pH l
with sulfuric acid, and centrifuged 10 minutes at 3000 r.p.m.
The supernatant fluid was shaken with an excess of silver
sulfate for removal of chloride ions, and after filtration the
solution was extracted continuously for 24 hours with washed
ethyl ether. The ether extract was removed, and extraction
was continued with fresh ether for an additional 60 hours. The
pooled extracts were evaporated, the residue was taken up in
a little water, made slightly alkaline with NaaCOs, boiled for
30 minutes to hydrolyze any lactide, and an aliquot was re
moved for lactic acid assay. The yield was 43.2 mg., equivalent
CHABT1.—Timecourse of oxygen consumption and glucose
oxidation in ascites tumor suspensions. Experimental details
are given in the text and heading of Table 1. Solid circles are
oxygen uptake, given in Amóles,and open circles are finióles
of carbon dioxide derived from labeled glucose.
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of paterna of substrate carbon converted, calculated from the
formula:
ft atoms substrate C oxidized =
A noteworthy finding in this experiment was the
relative constancy in the ratio of glucose oxidation
to the oxygen consumption. For each concentra
tion, roughly the same proportion of glucose car
counts/min BaCOa X /amólesBaCOj
counts/ min substrate as BaCOa
bon was present in the respiratory CO2. With
0.001 M glucose, the substrate accounted for 34
RESULTS
per cent of the oxygen consumption, and this
Table 1 and Chart 1 illustrate the typical res ratio was maintained until the last hour when,
piratory behavior of the Ehrlich ascites tumor at owing presumably to exhaustion of substrate, only
various initial glucose concentrations, over a 4- 10 per cent of the oxygen uptake was so accounted.
hour period. In this experiment, suspensions of At 0.0025 M, a constant proportion of approxi
cells were incubated in Warburg vessels in air at mately 40 per cent was maintained throughout the
37°C., and flasks were removed at successive hour
entire incubation period. At 0.01 M, the ratio re
ly intervals. Oxygen consumption was measured mained essentially constant at about 58 per cent
manómetrically, and glucose oxidation was deter
for 3 hours, then increased during the last hour to
mined by radioactivity assay of the respiratory
75 per cent.
CO2. On the whole the oxygen uptake data were
It is evident that glucose is readily oxidized by
in close agreement with those of Kun et al. (24) this tumor, and when present in adequate quanti
and Brin and McKee (6). At 0.001 Mand 0.0025 M, ties it can provide a substantial proportion of the
oxygen uptake was almost identical with the en carbon used in respiration. Nevertheless, even at
dogenous, but at 0.01 Mthe Crabtree Effect result
the highest glucose concentration, a considerable
ed in a considerable decrease in oxygen consump
proportion of respiration involves the oxidation of
tion. At the lowest glucose concentration, 0.001 M, endogenous carbon. This endogenous respiration
as with no substrate, oxygen uptake proceeded is decreased, but by no means eliminated, in the
during the 4 hours with slight but steadily dimin
presence of exogenous glucose.
ishing velocity, whereas with 0.0025 M it was con
To gain a better conception of the relative par
stant. At 0.01 M it was decreased almost 50 per ticipation of endogenous and exogenous carbon
cent at the earliest period of measurement but in during respiration at different glucose concentra
creased steadily thereafter, and after 4 hours it tions, a number of "large scale" experiments were
was about 25 per cent lower than the endogenous conducted. Using approximately 200 mg. dry
oxygen uptake. The observed oxygen uptakes cor weight of cells suspended in 12 ml. of phosphate
respond to a Qo, (/¿litersoxygen/mg dry wt/hr) of saline medium, in Warburg flasks of 125-ml.
10-13 at low concentrations and about 6-7 at capacity, sufficient CO2 can be collected in 2 hours
of incubation for isolation and radioactivity assay
0.01 M or above.
It can be seen from Table 1 and Chart 1 that in without the addition of carrier carbonate. This
general glucose oxidation closely paralleled the allows a direct determination of the relative spe
oxygen uptake. At 0.001 M, oxidation diminished cific activity of the respiratory C02 and thus pro
gradually for 3 hours, then dropped markedly dur
vides a direct measure of the proportion of exog
ing the 4th hour. This can plausibly be attributed
enous carbon in the total CO2 without reference
to substrate exhaustion, since at this time approxi
to oxygen uptake. The typical results of one such
mately 60 per cent of the added glucose was con experiment are given in Table 2. The total oxygen
verted to CO2. The oxidation at 0.0025 Mparalleled uptakes and total CO2 yields were approximately
the oxygen uptake throughout, being linear until equal for the three lower concentrations. However,
the 4th hour, when it dropped slightly. At 0.01 M, the proportion of exogenous glucose carbon in the
glucose oxidation, again paralleling the oxygen up
CO2 increased from 4.4 per cent at 0.0002 M to 46
take, proceeded at an ever-increasing rate and was per cent at 0.004 M. Undoubtedly, part of the rea
almost twice as fast at the 4th hour as at the 1st. son for the lower proportions of glucose carbon in
As pointed out by Brin and McKee (6), the res
the respiratory CO2 at the lower glucose concen
piratory inhibition is overcome as glucose disap
trations was exhaustion of substrate. The 11.6
pears, and the present experiment shows that the /¿atomsof glucose carbon oxidized in the presence
of 2 /¿molesof glucose represent 11.6/12.0 = 95
same effect is exerted on glucose oxidation.
per cent of the total glucose carbon added; and
In Table 1, numerical data on oxygen consump
tion and conversion of glucose carbon to COs for with 10 /¿molesglucose present, the 36.6 /¿atoms
this experiment are listed. In the lower section of oxidized represent 36.6/60 = 61 per cent of the
this table, these data are given in the form of total.
At the two highest concentrations, however,
hourly rates.
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ANDWEINHOUSE—Respirationand Glycolysis of Ascites Tumor 1085
substrate exhaustion was not a factor, and under
these conditions the relative specific activities
were the same, at about 45 per cent. This surpris
ing constancy in relative specific activity of res
piratory CÛ2,in the face of a fivefold change in
glucose concentration, accompanied by an over-all
inhibition of 32 per cent in total oxygen consump
tion, has been observed repeatedly. These results
make it evident that, under the conditions em-
appeared in 5 hour and was accounted for almost
completely as lactic acid. Thereafter, the lactic
acid disappeared rapidly; at 0.001 M it was utilized
completely by 3 hours. With glucose at 0.01 M,
glycolysis was much more rapid in the bicarbonate
medium. In 2 hours all the glucose had disap
peared, at which time lactate had reached its
maximum. In the phosphate buffer, glycolysis was
rapid during the 1st hour, then leveled off. Glucose
TABLE 2
GLUCOSE
OXIDATIONIN ASCITESCELLSUSPENSIONS
In this experiment 284 mg. dry weight of cells were suspended in phosphate saline solution of the following com
position: NaCl, 0.13 M; KC1, 0.006 M; MgSO4, 0.002 M; and sodium phosphate buffer, pH 7.4, 0.02 M. Incubation
was carried out for 2 hours at 38°C. with oxygen in the gas phase.
GLUCOSE
Concentra
added
tion
(Minóles)
(Molar)
2
0.00017
10
0.00083
50
0.0042
250
0.021
Amount
RESPIRATORY
COi
sp.act.(per
OXÕGEXUPTAKE(vmoles)292284Ã-fi-1178Amount(/jmoles)264265266223Rei.
cent)4.413.844.246.1Glucoseconverted(/jatoms
C)11.636.6118IOSRESPIRATORYQUOTIENT0.900.931.011.25QO,Gil/mg/hr)11.5
ployed, the inhibition of respiration was exerted
on both the exogenous and endogenous respiration.
The direct determination of respiratory carbon
dioxide in this experiment allows an independent
calculation of the respiratory quotients, and these
are shown in column 7. With increasing glucose
concentration, this value increased from 0.90 to
1.25. These are in the same range as those reported
by Kun et al. (24) from manometric data. The
R.Q. of greater than 1 at the high glucose concen
trations has been observed repeatedly. It suggests
the rapid occurrence of reductive processes, simul
taneously with respiration, possibly the conse
quence of synthetic activities of growing tumor
cells. The reason for these high R.Q.'s deserves
further attention.1
Glucose disappearance and lactate formation.—
The preceding data indicate that both oxygen
consumption and glucose oxidation proceed at
very steady rates for at least 4 hours, as long as
sufficient substrate is provided. This observation
is surprising when considered against the wide
variations occurring in the glucose and lactic acid
levels during this time. Chart 2 shows typical
changes in these substances during incubation in
1234
0
HOURS
a 0.01 Msodium bicarbonate or a 0.01 Mphosphate
medium. At glucose concentrations of 0.001 and
CHART2.—Time course of glucose disappearance and lac
0.0025 M, the behavior in either medium was iden
tate formation in ascites tumor suspensions. Open circles are
tical, and only those for phosphate are given. glucose disappearance in mg., in 0.01 Mphosphate buffer, and
Under the conditions employed, glucose had dis- solid circles are lactate formation in mg., in phosphate buffer.
1In subsequent experiments (Medes and Weinhouse, un
published) it has been established that lipogenesis from glu
cose is not sufficiently rapid to account for the high R.Q.
Crossed circles and half-shaded circles are, respectively, glu
cose disappearance and lactate formation in 0.01 Mbicarbonate
buffer (given only for 0.01 Mglucose). Otherwise, experimental
conditions and saline components were as described in Table 1.
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disappearance was likewise more rapid during the
1st hour, then slowed to a steady rate thereafter.
As noted by other investigators (6, 24), no lactate
was formed in the absence of glucose; and the
small amount of lactate originally present quickly
disappeared.
Initial rates of glucose catabolism.—The relative
constancy of oxygen consumption and glucose oxi
dation over prolonged periods in the face of a rapid
conversón of glucose to lactic acid is in accord with
the generally accepted view that oxidation of both
substances to C02 proceeds via a common inter
mediate such as pyruvic acid. It suggests further
that glucose oxidation in these ascites cells may
not be highly dependent on the substrate concen
tration. This supposition receives support from the
2 of Table 3 points to a somewhat similar situation
for the oxidation of lactic acid. Its oxidation was
somewhat
more concentration-dependent
than
that of glucose, there being a threefold variation
in lactate oxidation over the entire 100-fold con
centration range from 0.0002 to 0.02 M.
This provides a plausible explanation for the
otherwise paradoxical observation
that, whereas
glucose oxidation at intermediate levels, measured
over long periods, increased somewhat with in
creasing concentrations,
no such concentrationdependence was observed in the short-term experi
ments illustrated in experiment 1 of Table 3. In
the latter, glucose oxidation was measured over
intervals sufficiently short so that lactate levels
did not differ greatly.
To better understand the initial rate of lactic
TABLE 3
acid formation as a function of glucose concentra
EFFECTOFGLUCOSE
CONCENTRATION
ONINITIALRATES tion, under conditions where the glucose concen
OFGLUCOSEANDLACTATEOXIDATIONANDON
trations remain reasonably constant, experiments
were conducted with only one-tenth or one-twen
LACTATEPRODUCTION
IN ASCITESCELLS
Experiments were started by tipping in glucose or lactate
tieth the quantity of cells, viz., 2 mg. or 1 mg. dry
from the side-arm at zero time (after an equilibration period of weight. The results are seen in experiments 3 and
15 minutes) and shaking aerobically for 18 minutes at 38°C.
(except for experiment 4, shaken for 10 minutes). Experiment 1 4 of Table 3. Under these conditions, ample sub
was conducted with glucose-U-C14, experiment 2 with L-Iac- strate was available at all concentrations,
thus
tate-U-C", and experiments 3 and 4 with unlabeled glucose.
yielding valid data for estimation of initial gly
Values for COj are given in /¿atomssubstrate carbon converted.
colysis rates. In experiment 3, glycolysis was con
Values for lactic acid are given in ¿imoles
lactate and are cor
rected for endogenous lactate. Experiments 1 and 2 had 20 mg. centration-dependent
from 0.0001 to 0.0005 M,
dry wt. of cells per vessel; experiment 3, 2 mg/vessel; and ex
increasing
5-fold
over
this
5-fold range. At higher
periment 4, 1 mg/vessel. Experimental conditions otherwise
concentrations,
however,
concentration-depend
were as given in Table 1.
4:*GLUCOSETO
ence
decreased;
there
was
only
a 50 per cent in
3:GLUCOSETO
*:LACTATETO
1:GLUCOSETO
SUBSTRATECONCENTRA
crease in glycolysis over the 10-fold concentration
LACTATE(jiatoma LACTATE(^atoma range of 0.001 to 0.01 M.3 In experiment
CO2(patema
COj(/latoma
TION(Molar)0.00010.00020.00050.0010.00250.010.02FJCP.
4, with a
C)0.390.962.103.153.844.68EXP.
C)1.091.331.521.761.57
C)0.650.891.161.361.582.10FJCP.
C)0.250.370.460.420.450.45EXP.
different ascites tumor, concentration-dependence
for glycolysis was even less over the same range.
Kun et al. (24) similarly found initial glycolytic
rates of ascites tumor cells to be the same for glu
cose at 0.05 or 0.005 M. However, these results dif
fer somewhat from those of Warburg (35, p. 136),
* This experiment was carried out with TA-3 ascites tumor
who observed a rather marked concentration-de
cells.
pendence for anaerobic glycolysis in slices of the
rat carcinoma at 0.002-0.003 M
data in Tables 1 and 2 which indicate that under Flexner-Jobling
conditions where glucose oxidation was not affect
glucose.
ed by substrate availability, the absolute conver
At all concentrations
tested, lactate formation
sion of glucose carbon to CÛ2was not influenced was considerably more rapid than either glucose
greatly by its initial concentration. This observa
or lactate oxidation. At 0.001 M for example, the
tion has been made repeatedly in similar experi
ments2 and has been verified in more decisive 1.05 jumólesof lactate formed in 13 minutes by 2
short-term experiments designed to estimate initial mg. of cells corresponds to a Qu,,.,. of 54. This
rate provides lactate carbon about 8 times more
rates of glycolysis and glucose oxidation.
As shown in Table 3, through a 100-fold range rapidly than it can be oxidized. Apparently, when
in glucose concentration from 0.0001 to 0.01 M,the glucose is present in ample quantities, even at the
conversion of glucose carbon to COj in a 13-minute
incubation period varied only slightly. Experiment
1G. Medes and S. Weinhouse, unpublished.
1It would have been desirable to measure glucose oxidation
by radioactivity assay in this experiment, but the yield would
have been too low for measurements using the radioactive
glucose presently available.
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BLOCH-FRANKENTHAL
ANDWEINHOUSE—Respirationand Glycolysis of Ascites Tumor 1087
lowest concentrations, lactate is formed far more
rapidly than it can be oxidized to C02Calculation of the Michaelis constant for oxida
tion of glucose, by plotting 1/V versus 1/S accord
ing to Lineweaver and Burk (25), gave a Km value
for the data in experiment 1 of 1.5 X 10~4 M and
for lactic acid oxidation (experiment 2) the value
was about 3 X 10~4M. The Km for lactate forma
tion from glucose, estimated in the same manner
from the data of experiment 3, gave a value of
about 5 X 10~4 M. For experiment 4, the Km
value was indeterminate, because at the lowest
concentration measured, namely 0.0001 M, glycolysis was more than half maximal.
It is interesting that Beck (4) obtained a Km of
similar magnitude for glycolysis in homogenates
of normal and neoplastic leukocytes, namely,
2 X 10~4 M. He likewise found Km values for
other glycolytic enzymes in leukocytes to fall in
the range of 10~4to 10~6M, and, though he did not
report values for hexokinase, others (14) have re
ported values in this same range. Beck (4) has
pointed out that one cannot determine the ratelimiting step of an enzyme-sequence in a simple
manner from substrate saturation data alone.
However, as a rough approximation, one may as
sume that a low degree of cell permeability toward
glucose would result in a marked gradient in its
concentration between intra- and intercellular
fluid, and hence to an apparent high Km for gly
colysis in intact cells. On this basis, the low ob
served value, which is in the range of most or all
of the individual intracellular enzymes, indicates
that these ascites cells are highly permeable to
glucose and that there is relatively little resistance
to transfer of glucose across the membrane.4
DISCUSSION
This study leaves no doubt of the capacity of
this ascites tumor to oxidize glucose to C02. De
pending on the glucose concentration and time of
incubation, up to 60 or more per cent of the total
respiration can be accounted for by glucose oxida
tion. At intermediate concentrations, correspond
ing to levels expected in the blood, about 35-40 per
cent of the total respiration is so derived. A signifi
cant observation was the linearity in oxygen con*Cori in a recent review (D. Green, Currents in Biochemi
cal Research, pp. 198-214, New York: Interscience, 1956) has
reported results of unpublished work by Field, Heimberg, and
Crane which indicates that the same situation prevails for
anaerobic glycolysis in ascites tumor cells. These investigators
obtained Km values of 1 X 10~6M for glycolysis, a figure of
the same order as Km for ascites cell hexokinase.
More recently, Crane, Field, and Cori, J. Biol. Chem., 224:
649, 1957, reported similar Km values for the rate of penetra
tion of nonutilizable sugars into Ehrlich ascites tumor cells.
sumption (and in glucose oxidation when substrate
was available), despite the wide fluctuation in
glucose and lactic acid concentrations. This result
is in accord with our present conception of glucose
metabolism in tumors (4, 43), which envisages a
preponderant catabolism of glucose to pyruvic
acid, followed by a rapid reduction of the latter to
lactic acid, while a relatively minor proportion of
pyruvate is drawn off for oxidation through the
citric acid cycle.
The pyruvate destined for oxidation would con
tribute to the pool of acetyl coenzyme A being
supplied through catabolism of endogenous sub
strates such as fatty acids, and presumably the
greater the availability of pyruvate the greater
will be the proportion of glucose-derived acetyl
groups. This would account for the somewhat in
creased percentage of glucose carbon in the respir
atory CO2 with increasing concentrations in the
medium.
All investigators who have studied lactate for
mation in ascites tumors have been impressed by
the high rates of aerobic and anaerobic glycolysis,
which are of the order of 2 or 3 times those of solid
tumor slices. Warburg (36) believes that this high
glycolysis represents the behavior of "pure" tumor
cells, whose metabolism is characterized by a wide
disparity between glycolysis and respiration (oxy
gen uptake), in contradistinction to solid tumors
whose lower glycolytic rates are attributed to rela
tively large amounts of noncancer substance. The
present results suggest an alternate explanation.
The relatively low order of concentration-depend
ence for glycolysis, observed in the present study,
demonstrates an unusual degree of permeability of
these cells toward glucose, similar to their behav
ior toward certain amino acids, observed by Christensen and co-workers (12).
These findings suggest that perhaps the general
ly high glycolysis of solid as well as ascites tumor
cells, which as yet does not appear to be associated
with deviations in enzyme or coenzyme levels,
may have its origin in a membrane highly perme
able to sugars. The lower glycolytic rates of the
solid tumor slices could be attributed to limita
tions in diffusion of glucose through multicellular
layers, in contrast with the ascites cells which are
in contact with the medium over their whole sur
face. Further studies are being directed to the
evaluation of this hypothesis by studying the rela
tionship between glucose concentration and its
utilization in a variety of ascites and solid tumors
and homogenates prepared therefrom.
In the present study a rough parallelism was
observed between the effect of high glucose con
centration on its own oxidation and its effect on
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the over-all oxygen consumption. As seen in Table requires the participation of ATP (30). However,
1, both were inhibited during the early stages, and the mechanism of acylCoA formation from en
both were partially released from this inhibition dogenous lipides is not yet known, nor do we yet
following the disappearance of glucose. It is clear know what other metabolites contribute to the
that the Crabtree Effect is not restricted to the endogenous respiration. The participation of fatty
acids in the Crabtree Effect is now under study.
respiration involving glucose alone, nor to endoge
nous respiration alone, but affects the oxidation
Another factor, namely, intracellular acidifica
tion, owing to a combination of inefficient buffer
of both exogenous and endogenous metabolites.
ing and rapid glycolysis, may also play a part in
The extent of inhibition of each is still not en
the Crabtree Effect, though Chance (11), Racker
tirely clear. In Table 1, for example, oxygen up
take during the 1st hour was inhibited (10.6 —¿ (31), and Brin and McKee (6) discount this
5.8)/10.6 = 45 per cent by changing from 0.0025 possibility.6
All the suggestions which link the Crabtree Ef
to 0.1 M glucose, whereas glucose oxidation was
inhibited only (4.4 —¿
3.5)/4.4 = 20 per cent.
fect with phosphorylation processes, or with intra
Over the whole 4-hour period, oxygen uptake was cellular pH changes, presuppose that it is in some
inhibited (39.8 - 27.8)/39.8 = 30 per cent, but way directly associated with the intensity of glu
glucose oxidation was (17.3 —¿
16.1)/16.0 = 7 per cose metabolism. However, the results of the pres
cent higher over the same increment of glucose ent study show that both glycolysis and respira
concentration. These findings indicate that the tion proceed maximally or nearly so at concentra
tions of the order of 10~4 M, whereas the respira
Crabtree Effect is exerted principally on the en
dogenous metabolism. This is evident also in ex tory inhibition comes into play at concentrations
periment 2, in which, on changing from 0.004 to 50- to 100-fold greater. Inasmuch as the oxidation
0.02 M glucose, oxygen uptake was inhibited of not only glucose but also endogenous metabo
(262 - 178)/262 = 32 per cent, whereas glucose lites is inhibited, and there appears to be no inhibi
oxidation was inhibited only (118 —¿
103)/118 =
tion of glycolysis, one is led to the possibility that
high concentrations of glucose may exert a direct
13 per cent. In experiment 1 of Table 3, no inhibi
inhibitory action on one of the steps of electron
tion at all was noted in glucose oxidation.
transport. This type of effect is in accord with
Using the method of rapid spectrophotometric
observation, Chance and Hess (11) found that Racker's (31) observation that the Crabtree Effect
both glucose oxidation and oxygen uptake are is overcome by dinitrophenol. Racker (31) has re
strongly inhibited, after an initial short period of ported other interesting findings from his labora
respiratory acceleration, on the addition of glucose tory, which offer the possibility of explaining the
to ascites tumor cells. This inhibition is overcome
8Racker (31) reported a pH lowering from 7.5 to 6.6 during
by the addition of agents which uncouple phosactive glycolysis in ascites cells. We have noted a lowering of
phorylation from respiration. They concluded that pH from 7.4 to 5.1 after 4 hours of active glycolysis in a 0.01 M
the regulation of glucose oxidation was exerted phosphate-buffered medium. Although the pH does not fall
below 6.9 with a high phosphate buffer, such as was used in
through the relative availability of phosphate
donors and acceptors.6 Racker (31, 44) has made these studies, a number of other observations point to a pos
intracellular pH effect. Warburg et al. first pointed out
similar observations and has suggested that gly- sible
that glycolysis in tumor slices is more rapid in a bicarbonate
colysis occurring in the extramitochondrial portion than in a phosphate-buffered medium (39), and this has also
of cells is in competition with mitochondrial res been observed in ascites cells in this and other studies (24, 37).
Brin and McKee (6) observed that the inhibition can be largely
piratory processes for the available inorganic phos
by increasing the phosphate content of the medium
phate or adenine nucleotides. Our present results overcome
and suggested a mechanism for this effect based on competition
suggest that if these factors play a role in glucose between glycolytic and respiratory enzymes for inorganic
oxidation, they must probably also exert a similar phosphate. An alternate explanation would be that the high
phosphate medium exerts a better buffering action. Since cer
or even a greater action on endogenous metabo
lites. It is known that fatty acids participate in the tain of the enzymatic steps in electron transport, such as, for
example, those involving the pyridine nucleotides, yield
endogenous respiration of tumors, and it is easy to hydrogen ions, an intracellular acidification might inhibit res
see how adenine nucleotides would influence the piration by shifting the equilibrium of these reactions.
oxidation of fatty acids, since acylation with coIt is interesting in this connection to note that Clowes and
Keltch (13), who measured respiration in ascites tumor cells
enzyme A, the first step in fatty acid activation,
1In a private communication, Chance has questioned
whether the remarkably rapid and marked sequential aceleration and inhibition of respiration, which he observed on the
addition of glucose to Ehrlich ascites cells, is the same effect
observed by the usual manometric technics.
using bicarbonate or glycylglycine buffers, apparently did not
observe the Crabtree Effect. Oxygen consumption, they state,
"was virtually unaffected by the presence or absence of as
similable hexoses." The same is apparently true in experiments
of Warburg and Hiepler (38) in which a bicarbonate buffer
also was used.
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BLOCK-FRANKENTHAL
ANDWEiNHOTJSE—Respiration
and Glycolysis of Ascites Tumor 1089
Crabtree Effect on the basis of alternate metabolic
pathways. At low concentrations, glucose carbons
1 and 6 were oxidized at approximately equal
rates, whereas at concentrations resulting in the
Crabtree Effect glucose carbon 1 was oxidized
more rapidly and carbon 6 less rapidly. Obviously,
more information must be obtained concerning the
nature and interplay of respiratory substrates be
fore this effect is understood.
9. CHANCE,B. Dynamics of Respiratory Pigments of Ascites
Tumor Cells. Trans. N.Y. Acad. Sc., 16:74-75, 1953.
10. CHANCE,B., and CASTOR,L. N. Some Patterns of the
Respiratory Pigments of Ascites Tumors of Mice. Science,
116:200-202, 1952.
11. CHANCE,B., and HESS, B. On the Control of Metabolism
in Ascites Tumor Cell Suspensions. Ann. N.Y. Acad. Sc.,
63:1008-16, 1956.
12. CHHISTENSEN,
H. N. Mode of Transport of Amino Acids
into Cells. In: W. D. MCELROYand H. B. GLASS(eds.),
Amino Acid Metabolism, pp. 63-106. Baltimore: Johns
Hopkins Press, 1955.
18. CLOWES,G. H. A., and KELTCH,A. K. Glucose, Mannose,
and Fructose Metabolism by Ascites Tumor Cells: Effects
of Dinitrocresol (21185). Proc. Soc. Exper. Biol. & Med.,
86:629-34, 1954.
14. COLOWICK,
S. In: 3. B. SÃœMNEH
and K. MYRBACK(eds.),
The Enzymes, 2:131. New York: Academic Press, 1951.
15. CRABTBEE,H. G. Observations on the Carbohydrate
Metabolism of Tumors. Biochem. J., 23:536-45, 1929.
16. DICKENS,F., and SIMEH,F. The Metabolism of Normal
and Tumor Tissue II. The Respiratory Quotient, and the
Relationship of Respiration to Glycolysis. Biochem. J.,
24:1301-26,1930.
17. ELLIOT,K. A. C., and BAKER,Z. The Respiratory Quo
tients of Normal and Tumor Tissue. Biochem. J., 29:243341, 1935.
18. EMMELOT,C., and Bos, C. J. Factors Influencing the
Fatty Acid Oxidation of Tumors Mitochondria with Spe
cial Reference to Changes in Spontaneous Mouse Hepatomas. Experientia, 11:338-54, 1955.
19. ETINOOF,R. N., and GERSHANOWICH,
V. N. The Reverse
Pasteur Effect of Ascitic Cancer Cells of Mice. Biokimiia,
18(6): 668-74, 1953.
20. GATT, S.; KHIMSKY,I.; and RACKER,E. Reconstructed
Systems of Glycolysis and Oxidative Phosphorylation.
Fed. Proc., 15:259, 1956.
21. IBSEN,K.; MCCARTY,B.; and McKEE, R. W. Phosphate
Content of Ehrlich's Ascites Tumor Cells and Its Impor
SUMMARY
The effect of variations in glucose concentration
on respiration and glycolysis in cells of the Ehrlich
ascites tumor was investigated, with the use of
glucose uniformly labeled with C14.Over a 4-hour
incubation period, oxygen uptake and glucose oxi
dation were linear despite a rapid glycolysis, result
ing in replacement of the glucose by lactic acid.
Short-term kinetic studies indicated that initial
rates of glucose and lactic acid oxidation were not
highly concentration-dependent
above 0.001 M,
and glycolysis was maximal at approximately
10~4 M glucose. At all concentrations tested, gly
colysis was much more rapid than glucose or lactate oxidation. The inhibition of respiration at
high glucose concentrations (Crabtree Effect) was
consistently observed and was exerted on both
glucose and on endogenous substrates. It was sug
gested that the unusually high glycolysis of ascites
cells, in comparison with solid tumor slices, may
be owing to the easy accessibility of glucose to
these freely suspended single cells, resulting in a
highly efficient glycolysis, not limited by the intance in Metabolism. Fed. Proc., 15:279, 1956.
tracellular availability of glucose.
22. KIT, S. The Role of the Hexose Monophosphate Shunt in
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Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1957 American Association for Cancer Research.
Metabolism of Neoplastic Tissue: XII. Effects of Glucose
Concentration on Respiration and Glycolysis of Ascites Tumor
Cells
Leah Bloch-Frankenthal and Sidney Weinhouse
Cancer Res 1957;17:1082-1090.
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