Download Ketone Body, Glucose, Lactic Acid, and Amino

(CANCER RESEARCH 43, 3497-3503,
August 1983]
Ketone Body, Glucose, Lactic Acid, and Amino Acid Utilization by Tumors in
Vivo in Fasted Rats1
Leonard A. Sauer2 and Robert T. Dauchy
Mary Imogens Basse«Hospital, Cooperstown, New York 13326
ABSTRACT
Arteriovenous differences for acetoacetate, ß-hydroxybutyrate, glucose, lactic acid, and glutamine and other amino acids
were measured across Morris hepatomas 5123C, 7777, and
7288CTCF and Walker sarcocarcinoma 256 in vivo in rats fasted
for 2 days. The acetoacetate and ß-hydroxybutyrate concentra
tions in arterial whole blood of fasted tumor-bearing rats were
0.52 ±0.06 and 1.82 ±0.19 HIM (S.E., n = 38), respectively.
Both ketone bodies were utilized by the tumors, and the rates
of utilization were directly related to the rates of supply. The
mean utilization rates for acetoacetate and 0-hydroxybutyrate
were 13.9 ±2.9 (range, 0 to 64; n = 30) and 24.7 ±4.4 (range,
0 to 145; n = 38) nmol/min/g tumor wet weight, respectively.
Eight of the tumors produced acetoacetate, presumably from
utilized ß-hydroxybutyrate. An average of 52% of the acetoace
tate and 30% of the 0-hydroxybutyrate carried in the arterial
blood was removed during one pass through the tumors. The
concentrations of glucose and glutamine in the arterial whole
blood of fasted tumor-bearing rats (n = 38) were 6.55 ±0.3 and
0.76 ±0.02 HIM, respectively; both of these substrates were
utilized at rates that were directly proportional to the rates of
supply. The mean rates of glucose and glutamine utilization for
all tumors in fasted rats were 101 ±11 (range, 3 to 313) and
8.2 ±1.1 (range, 0 to 25.1) nmol/min/g tumor wet weight,
respectively. Thirty-six % of the glucose and 25% of the gluta
mine supplied to the tumors was utilized. Comparison (by linear
regression and analysis of covariance) of the rates of supply and
utilization of glucose and glutamine in tumors growing in fasted
versus fed rats indicated that these substrates are utilized more
efficiently by tumors growing in fasted animals. Lactic acid was
either produced or utilized, depending on the arterial whole-blood
concentration. Production or utilization occurred, respectively,
when the arterial lactate concentration was less or greater than
1 to 3 HIM. The arterial whole-blood amino acids (except gluta
mine) were utilized at rates that ranged from 1 to 4 nmol/min/g
tumor wet weight. The results indicate that energy production
for tumor growth in fasted rats is supported, in part, by an
increased availability of ketone bodies, by an increased efficiency
of utilization of glucose and glutamine, and, under certain circum
stances, by utilization of lactic acid.
INTRODUCTION
Tumors have the remarkable ability to sustain a rapid rate of
growth during starvation or semistarvation of the host (2, 16).
The tumor growth occurs as the host tissues, especially fat and
skeletal muscle, decrease in weight (4, 5), and it is obvious that
1Supported
by Grant CA 27809 from the USPHS, NIH, and the Stephen C.
Clark Research Fund of the Mary Imogene Bassett Hospital.
2 To whom requests for reprints should be addressed.
Received January 28, 1983; accepted April 21,1983.
the nutrient needs of the tumor are adequately supplied and that
they are extracted from the diminishing resources of the host.
The host-derived metabolic fuels utilized by the tumor in the
fasted animal are not known. Arterial blood glucose, the putative
major fuel for fast-growing tumors, is decreased in concentration
during fasting, and, as we (26) and others (9) have shown, the
rate at which tumors utilize glucose in vivo is directly dependent
on the rate of supply. Consequently, arterial glucose is unlikely
to be the only fuel utilized by a tumor during fasting. Fenselau
ef al. (6) made the important observation that succinylCoA:acetoacetyl-CoA transferase, the initial enzyme for utiliza
tion of acetoacetate by peripheral tissues, is increased in activity
with increased hepatoma growth rate. ß-Hydroxybutyrate dehydrogenase is present but relative to liver is reduced in activity in
Morris hepatomas (20). These in vitro results suggested that
ketone bodies may be important fuels for tumor growth in vivo
in the fasted or semifasted animal. In this study, we measured
the arteriovenous differences for the ketone bodies, lactic acid,
glucose, and amino acids across 4 rat tumors in fasted rats. We
found that both acetoacetate and /3-hydroxybutyrate were uti
lized by rat tumors in vivo and that the rates of utilization were
directly proportional to the rates of supply. Lactate was utilized
at arterial lactate levels greater than 1 to 3 mw. Evidence is also
presented which suggests that arterial glucose and glutamine
are utilized more efficiently by tumors growing in fasted rats
relative to tumors growing in fed rats. These properties of the
tumor enhance access to available nutrients and may help ex
plain the rapid tumor growth during fasting.
MATERIALS AND METHODS
Animals, Tumors, and Reagents. Adult male and female Buffalo and
HarÃ-anSprague-Dawley rats were obtained from colonies established
here. The rats were fed a standard laboratory chow (Charles River Rat,
Mouse, Hamster Formula; Agway, Inc., Syracuse, N. Y.) and water ad
libitum and were subjected to alternate 12-hr periods of dark and light.
Tumor implantation, growth of "tissue-isolated" tumors, preparation of
the animal for tumor harvest, and collection of arterial and venous blood
samples were as described previously (26). The Morris hepatomas were
originally obtained from the late Dr. Harold P. Morris, Morris Hepatoma
Program, Howard University Cancer Center, Washington, D. C. The
Walker sarcocarcinoma 256 was obtained from the E. G. and G. Mason
Research Institute, Worcester, Mass. These tumors have been carried
in this laboratory for about 4 years. The mean tumor blood flow rate was
107 ±4 /al/min (n = 38). Food was removed from fasted animals at 8
a.m. after the overnight feeding period, and the tumors were harvested
48 hr later.
Blood Sample Preparation and Assay. Perchloric and trichloroacetic
acid extracts of arterial and venous whole blood were prepared as
described previously (26). Acetoacetate (18) and fj-hydroxybutyrate (33)
were assayed fluorometrically by enzymatic methods using an Eppendorf
photometer with fluorometric attachment. Acetoacetate was measured
immediately after neutralization of the perchloric acid extract. Glucose,
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L. A. Sauer and R. T. Dauchy
lactic acid, glutamine, and amino acids were assayed as described
previously (26).
Expression and Evaluation of Results. For interpretation of the
arteriovenous difference measurements, we assume that the tumor is in
a steady state with regard to nutrient supply and metabolism and that
this steady state continues during the period of sample collection, usually
about 5 min. Utilization or production of a nutrient by the tumor occurs
when the arteriovenous difference is positive or negative, respectively.
Utilization (or use) of a nutrient includes inter alia oxidation to CO2 and/
or conversion via biosynthesis to form protoplasm and secretions. Only
the overall process of utilization was measured in these experiments.
Utilization and production rates are given in nmol substrate utilized or
produced per min per g tumor, wet weight and were calculated from the
arteriovenous differences and the tumor blood flow rate (¿Ã-l/min/g).
Supply rates are expressed as nmol/min/g tumor, wet weight and were
calculated from the arterial whole-blood concentration and the tumor
blood flow rate. Errors in assay of amino acids and other substrates and
in measurement of tumor blood flow rates and the effects of these errors
on calculations of utilization and production rates are the same as those
described earlier (26). Data are presented as mean ±S.E. Groups of
results were compared by linear regression and analysis of covariance
(27). The same caveats are in effect here with regard to interpretation of
data by linear regression as were in effect in the previous report (26).
RESULTS AND DISCUSSION
Ketone Body Utilization. Ketone bodies were utilized by 37
of the 38 tumors examined. The rates of utilization by Morris
hepatomas 5123C, 7777, and 7288CTCF and Walker sarcocarcinoma 256 were directly dependent on the rate of supply (Chart
1, A and B). The mean rate of acetoacetate and /3-hydroxybutyrate utilization for all tumors was 13.9 ±2.9 (n = 30) and 24.7
±4.4 (n = 38) nmol/min/g tumor, wet weight, respectively. Eight
of the tumors produced acetoacetate at slow rates (not shown),
presumably from /3-hydroxybutyrate utilized (see below). The
fastest rate of acetoacetate utilization (64 nmol/min/g tumor,
wet weight) was observed in a hepatoma 7288CTCF, and the
fastest rate of /3-hydroxybutyrate utilization (145 nmol/min/g
tumor, wet weight) occurred in a hepatoma 5123C (not shown
in Chart 10). The rate of /3-hydroxybutyrate supply to the latter
WALKER 256
7288CTCF
7777
IOO
SUPPLY,
O
IOO
200
nmol/min/g
Chart 1. Regression of the rate of acetoacetate and /J-hydroxybutyrate utiliza
tion on the rate of supply in rat tumors in fasted rats in vivo. Each point represents
the determination for a single tumor.
, least-squares fit to the data points. A,
acetoacetate [y = 0.53x - 0.92, n = 30, r = 0.961 (p < 0.05)]; B, #-hydroxybutyrate
[y = 0.30X - 0.4, n = 38, r = 0.869 (p < 0.05)].
3498
tumor was 403 nmol/min/g tumor, wet weight, a value that
agrees reasonably well with the predicted supply rate (482 nmol/
min/g tumor, wet weight) based on the linear regression analysis.
It is interesting that, ¡nthe Morris hepatomas, the activity of
succinyl-CoA:acetoacetyl-CoA
transferase is increased (6) and
that the activity of /3-hydroxybutyrate dehydrogenase is de
creased (20) during tumor progression. Of the hepatomas stud
ied here, the CoA transferase activity has been reported to be
most active in hepatoma 7288CTCF (6). Total enzyme activity
of the homogenate was 5.1 //mol/min/g tumor, wet weight
(measured in the direction of acetoacetate formation). Assuming
an equivalent reaction rate in the direction of acetoacetyl-CoA
formation, the fastest acetoacetate utilization rate observed in a
hepatoma 7288CTCF was less than 5% of the enzyme activity
measured in vitro. The mean /3-hydroxybutyrate dehydrogenase
activity of hepatoma 5123C has been reported to be 2.5 ¿¿mol/
min/g tumor, wet weight (20). The maximum rate of /3-hydroxy
butyrate utilization by hepatoma 5123C in this study was 145
nmol/min/g tumor, wet weight, a rate that is less than 10% of
the maximum in vitro enzyme activity. It is worth noting that the
in vitro enzyme activity determinations were made at saturating
levels of substrates and other cofactors and were performed
with isolated hepatoma mitochondria solubilized by either deter
gent treatment (6) or sonication (20). The in vitro enzyme activity
measurements indicate that the specific enzyme content of the
hepatoma mitochondria is more than adequate to explain the
observed ketone body utilization rates and that the potential
exists for even greater utilization rates than those measured
here. The apparent straight-line relationship between ketone
body utilization and supply illustrated in Chart 1 indicates that
the rate of transport of the substrates into the tumor cells and
the availability of required cofactors in the mitochondria are also
adequate. The enzyme phenotype for mitochondrial energy pro
duction that develops during tumor progression in hepatomas
results from both the synthesis of new enzymes, e.g., CoA
transferase, and the persistence of constitutive enzymes, e.g.,
0-hydroxybutyrate dehydrogenase. Though adequate for the
needs of the tumor, the final enzyme activities in the tumor
mitochondria may be low relative to the activities found in mito
chondria of the parent organ or in mitochondria of other special
ized organs as with, for example, the /3-hydroxybutyrate dehy
drogenase in liver (20, 32), kidney, and brain (32), and CoA
transferase in kidney (32) and heart muscle (6, 32). The NAD(P)dependent malic enzyme (25) and glutaminase (17), 2 other
mitochondrial enzymes newly synthesized by Morris hepatomas
during tumor progression, are additional examples. Both of these
enzymes are much higher in activity in mitochondria from small
intestinal mucosa (22, 24), a tissue in which glutamine is known
to be a major respiratory fuel (34).
Arterial whole-blood concentrations of acetoacetate and ßhydroxybutyrate in several 2-day-fasted control Buffalo rats and
tumor-bearing Buffalo and Sprague-Dawley rats are listed in
Table 1. Total arterial whole-blood ketone body concentrations
for control and tumor-bearing animals were 3.75 ± 0.85 RIM
(range, 2.0 to 8.7; n = 7) and 2.33 ±0.22 ITIM(range, 0.4 to 7.1 ;
n = 38), respectively, suggesting that the presence of the tumor
decreased the ketogenic response of the host to starvation. The
fat stores of tumor-bearing rats are known to decrease during
tumor growth (4, 5), and the lower arterial whole-blood ketone
body concentrations in tumor-bearing animals probably result, in
part, from a decreased availability of substrate for ketogenesis.
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Tumor Metabolism
in Vivo
Table 1
Arterial whole-blood concentrations and in vivo utilization or production rates for ketone bodies, glucose, láclate, ano glutamina by tumors in rats tasted tor 2 days
AcetoacetateTumorA.
tion(nmol/min/gtumor,wet tion(nmol/min/gtumor.wet tion(nmol/min/gtumor,wet tion(nmol/min/gtumor.wet
tion(nmol/min/gtumor,wet
tion(mu)3.03±0.661.220.721.940.611.882.422.010.232.230.791.060.992.332.313.22Utiliza
tration(ttlM)4.83±0.364.889.95.627.336.965.316.506.376.288.867.328.087.635.617.38Utiliza
tration(rtiM)0.72±0.050.500.550.880.710.630.800.880.670.540.770.
tration(mu)1.24±0.360.951.11.082.581.6
ment69709778112113929310481987479105107eArterialconcentra
tion
(rriM)0.72±0.13"1.010.570.930.240.710.660.330.201.310.060.140.220.200.110.36Utiliza
wt)644566222445282e31e21e6e0-HydroxybutyrateArterialconcentra
wt)30536538702949222015242829GlucoseArterialconcen
wt)16247176921261241147465187313187959396GlutamineArterialconcen
wt)151136717981161017776LáclateArterialconcen
wt)1891411175126810556
Control"B.
Lactate-producing
tumorsHepatoma7288CTCFHepatoma
123CWalker5
carcinosar-coma
256C.
Lactate-utilizing
tumorsHepatoma7288CTCFHepatoma
123CWalker5
carcinosar-coma256Experi
"Buffato rats (n = 7).
6 Mean ±S.E.
c These represent production rates.
d These represent utilization rates.
8 Fasted for 96 hr.
The acetoacetate:/î-hydroxybutyrate ratios in the arterial blood
of the 38 animals examined ranged from 0 to 1.42. In some
animals, significant differences were noted between the acetoacetate:0-hydroxybutyrate
ratio in the carotid arterial blood and
the ratio in the tumor venous blood. Assuming that these ratios
are measures of the oxidation-reduction state in the animal
carcass and in the tumor, respectively, we may conclude that
the tumor is able to maintain an oxidation-reduction state differ
ent from that of the rest of the animal. An interesting relationship
was noticed between the arterial and venous acetoacetate:/?hydroxybutyrate ratio differences across the tumor and whether
a tumor was a lactate producer or a lactate utilizer. For the
purposes of this discussion, we divided the tumors into 3 groups.
Group 1 tumors were termed strong lactate producers and
showed production rates of greater than 20 nmol lactate pro
duced per min per g tumor, wet weight. Group 2 tumors either
produced or utilized less than 20 nmol lactate per min per g
tumor, and Group 3 tumors were strong lactate utilizers (greater
than 20 nmol lactate utilized per min per g tumor). The acetoacetate:0-hydroxybutyrate
ratios in the carotid arterial whole
blood for animals carrying Group 1, 2, and 3 tumors were 0.56
±0.08 (n = 15), 0.36 ±0.09 (n = 8), and 0.12 ±0.02 (n = 11),
respectively. The ratios in the tumor venous whole blood for
Group 1, 2, and 3 tumors were 0.34 ±0.04, 0.39 ±0.14, and
0.23 ±0.05, respectively. The arterial whole-blood lactic acid
concentrations for the animals carrying Group 1,2, and 3 tumors
were 1.27 ±0.11,1.64 ±0.31, and 3.45 ±0.42 mw, respectively.
As expected, the arterial lactic acid concentration was decreased
the more oxidized the oxidation-reduction state of the animal
carcass. In animals carrying Group 1 tumors, the tumor was
more reduced (p < 0.05) than the animal carcass, arterial wholeblood lactic acid was low, and the tumors released lactate. Table
1B shows acetoacetate
and /3-hydroxybutyrate
utilization and
lactate production rates and other data for 6 representative
tumors (Experiments 69, 93, 97, 104, 112, and 113) from this
group. Both ketone bodies were utilized by the hepatomas and
by the Walker sarcocarcinoma. In this group, an average of 56%
of the arterial acetoacetate and of 30% of the 0-hydroxybutyrate
was removed during one pass through the tumor. Group 3
tumors were more oxidized (p < 0.05) than the animal carcass,
the arterial whole-blood lactic acid concentration was high, and
the tumors utilized arterial lactic acid. Data for 6 representative
experiments from this tumor group are shown in Table 1C.
Arterial whole-blood acetoacetate concentrations were lower in
these animals, and, as expected, 8 of these tumors released
acetoacetate (see Experiments 74, 81, 105, and 107) into the
venous blood. Utilization of /3-hydroxybutyrate remained high,
however, and 30% of the 0-hydroxybutyrate supplied to the
tumors was utilized. Group 2 tumors were intermediate between
the tumors in Groups 1 and 3, and the oxidation-reduction states
of the tumor and the host were not different (p > 0.05). Three
representative experiments from this tumor group are shown in
Table 1B. Tumors in Groups 1, 2, and 3 did not differ significantly
in oxidation-reduction state, suggesting that the tumor attempts
to maintain a relatively fixed oxidation-reduction state despite
wide fluctuations in the oxidation-reduction state of the host.
This could provide a stable environment ensuring continued
reductive biosynthesis and growth. We assume that the aceto
acetate and 0-hydroxybutyrate reached equilibrium during pas
sage through the tumor and that the ratio is an accurate measure
of the tumor oxidation-reduction state. The high enzyme activi
ties (see above) of the tumors relative to the ketone body supply
rates to the tumors suggest that this is a reasonable assumption.
It is not clear why more than one-half of the control (5 of 7)
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3499
L A Sauer and R. T. Dauchy
and about half of the tumor-bearing fasted rats had acetoacetate:ß-hydroxybutyrate ratios of less than 0.25. Considering that
certain animals may be more susceptible to respiratory depres
sion and tissue hypoxia during pentobarbital anesthesia, we
routinely supplied all anesthetized animals with oxygen through
a loose-fitting nose cone (26). In other experiments, we found
arterial whole-blood lactate concentrations of 2 mw and greater
and low acetoacetate:/i-hydroxybutyrate
ratios in anesthetized
rats breathing 100% oxygen in a closed system with respiration
assisted via a rodent respirator (data not shown). It does appear,
therefore, that tissue hypoxia is not responsible for the elevated
lactate and ß-hydroxybutyrate levels. The acetoacetate:/S-hydroxybutyrate ratio is decreased in rats by starvation. The animal
carcass, particularly skeletal muscle, becomes more reduced as
a response to fasting (8) and may explain the low acetoacetate:/3hydroxybutyrate ratios and high lactic acid concentrations ob
served. A more reduced oxidation-reduction state in muscle
during fasting might help conserve muscle protein (8). In addition
to the oxidation-reduction state data discussed above, other
metabolic data indicate that the Group 3 tumor is more oxidized
than the rest of the body. For example, lactate, /3-hydroxybutyrate, glucose, and glutamine were all utilized (and presumably
oxidized) at substantial rates in these tumors (Table 1C). It seems
very unlikely, therefore, that the tumors listed in Table 1C were
anoxic. Gullino ef al. (10) have shown that a decrease in oxygen
availability decreases the rate of glucose utilization by tumors in
vivo.
Lactic Acid Production and Utilization. In a previous study
(26) performed with fed tumor-bearing rats, we found that tumors
in vivo could either produce or utilize lactic acid. Which of these
processes occurred depended on the arterial lactic acid concen
tration. Production of lactate was most often observed at lactic
acid concentrations below 2 mM, and lactate utilization generally
occurred at lactate levels above 2 mM. In the range of 1 to 3 mM
arterial lactate, tumors were often found that neither removed
nor added (despite the fact that glucose utilization had occurred)
significant amounts of lactate to the blood. Chart 2 shows that
an identical relationship was observed in the tumors growing in
the 38 fasted rats studied here. The data for both the fasted and
fed animals (see Ref. 26) were combined in Chart 2 to illustrate
the identical nature of the results obtained and to emphasize the
frequency with which in vivo lactate utilization occurred. A total
of 71 tumors were sampled; 24 were found to utilize arterial
whole-blood lactate. Since lactic acid is a metabolic end product,
it can reenter metabolism only through oxidation via lactic dehydrogenase; therefore, the capacity for NADH plus pyruvate
oxidation in these tumors must be high. For example, assuming
that 1 and 2 mol of pyruvic acid were formed, respectively, from
each mol of arterial lactate and glucose utilized, the hepatoma
7288CTCF listed in Table 1C (Experiment 98) may have oxidized
more than 650 nmol of pyruvate per min per g of tumor. These
oxidations occurred simultaneously with the utilization of 10 and
20 nmol/min/g of tumor of glutamine and ß-hydroxybutyrate,
respectively. Unlike normal liver, the pyruvate dehydrogenase
activity of fast-growing Morris hepatomas is not decreased by
fasting (11).
Several of the tumors that produced lactate at low rates (Group
2 tumors) were high-glucose utilizers (see Table 1B, Experiments
70, 78, and 92). The great potential for lactate production was
not expressed, presumably because the oxidation-reduction
states of tumor and host were similar and because arterial
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Chart 2. Lactic acid production and utilization by rat tumors in vivo plotted
against the arterial lactic acid concentration. Each point represents the determina
tion for a single tumor. Fed animals had free access to food, and fasted animals
were fasted for 48 hr.
lactate-derived and glucose-derived NADH and pyruvate gener
ated intracellularly were being utilized at the same time, and only
a small amount of lactate was released into the tumor vein.
Windmueller and Spaeth (34) have shown in rat small intestinal
mucosa that arterial lactate may be utilized at the same time that
glucose-derived lactate is released into the venous blood. When
the arterial lactate concentration was greater than 1.2 to 1.6 mw,
net lactate utilization occurred simultaneously with glucose utili
zation (34). In tumor, as in small intestinal mucosa, it would
appear that the arterial lactic acid concentration plays an impor
tant role in determining if intracellular lactate (as NADH and
pyruvate) is oxidized or is formed and released.
More than 50 years ago, Warburg (29) described the produc
tion of large amounts of lactic acid from glucose by tumor slices
incubated aerobically. Since then, numerous other in vitro ex
periments confirmed this finding, and a high rate of aerobic
glycolysis became an acknowledged biochemical hallmark of
tumor tissues (15). More recently, however, high rates of aerobic
glycolysis were described in normal tissues (3, 13, 19, 21, 23),
and then, following the development of the Morris hepatomas, it
was discovered that slow-growing tumors were poor lactate
producers (1). Although these findings did alter the view that
aerobic glycolysis was associated only with malignant cells, the
idea that a high rate of lactate production is a property of a fastgrowing tumor cell has persisted and is supported by in vitro
experiments (31). We believe, however, that this idea can no
longer be considered as an accurate representation of glucose
and lactate metabolism by tumors in vivo. In the total of 71
tumors examined in vivo (Chart 2), about 50% either utilized
arterial lactate or released only a small amount of lactic acid into
the venous blood. These tumors included Morris hepatoma
7288CTCF, a fast-growing hepatoma, and Walker sarcocarcinoma 256, an undifferentiated tumor. High arterial lactate con
centrations inhibited release of lactate even though glucose had
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Tumor Metabolism
been utilized (Table 1C). On the other hand, low arterial lactate
concentrations promoted lactate release (Table 1B). Since in
vitro experiments of glucose oxidation and lactate production
are most often performed in the absence of added lactate, high
rates of lactate formation are expected (Chart 2). There are no
discrepancies between results obtained in vitro and the in vivo
results shown in Chart 2 if it is recognized that, in the presence
of high arterial lactate, tumor tissues may remove lactate from
the arterial blood. The uptake, metabolism, and oxidation of
lactate utilized by tumors in vitro have been described by Spen
cer and Lehninger (28), Katz et al. (14), and Weinhouse (30),
respectively.
Glucose Utilization. The mean arterial whole-blood glucose
concentrations in fasted control and tumor-bearing rats were
4.83 ±0.36 mw (n = 8) (Table 1) and 6.55 ±0.3 mw (n = 38),
respectively. The same relative differences were observed in
control and tumor-bearing fed rats (26), suggesting that the
presence of the tumor increases the arterial whole-blood glucose
concentration of the fasted host. All 38 of the tumors growing in
the fasted rats utilized glucose; the rate of glucose utilization for
each tumor plotted against the rate of glucose supply to the
tumor via the arterial blood is shown in Chart 3. Chart 3 also
shows regressions of the rates of glucose utilization on the rates
of glucose supply for tumors growing in the fasted rats (
)
and for tumors growing in fed animals (
, data taken from
Ref. 26). The same apparent direct dependence of the glucose
utilization rate on the rate of supply observed in vivo ¡ntumors
growing in fed rats (9, 26) was observed for tumors growing in
fasted rats. The results for the 2 animal groups were not identical,
however, as shown by the shift to the left of the regression line
for tumors grown in the fasted rats. Comparison of the 2 regres
sion lines by analysis of covariance (27) indicated that the differ
ence between the regression lines (and, hence, between the
sample populations) is significant (p < 0.05). At any rate of
glucose supply, tumors growing in fasted rats increased their
glucose utilization rates by about 50 nmol/min/g tumor over
those of tumors growing in fed rats. This apparent increase in
efficiency of glucose utilization by tumors in fasted animals may
in Vivo
compensate for the decreased arterial glucose concentration
observed in fasted (6.55 mw, n = 38) versus fed (7.4 ±0.4 mw,
n = 26; Ref. 26) tumor-bearing rats.
Amino Acid Utilization. Glutamine, the most abundant amino
acid in rat arterial blood, was the most rapidly utilized. The rate
of glutamine utilization by tumors growing in fasted rats appeared
to be directly dependent on the rate of glutamine supply to the
tumor. An identical relationship was noted previously for tumors
growing in fed rats (26). Chart 4 shows the rates of glutamine
utilization by 38 tumors growing in fasted rats plotted against
the rates of glutamine supply to the tumors. The regression line
for these data points is shown by the solid line. The dashed line
is the regression line for the data obtained for tumors growing in
fed animals (data not shown; see Ref. 26). As was noted for the
relationship between glucose utilization and supply by tumors
growing in fed and fasted animals (Chart 3), the regression line
for glutamine utilization on glutamine supply for fasted rats was
shifted to the left of that for fed rats. These results suggest that
tumors growing in fasted rats are able to increase the efficiency
of the processes by which arterial glutamine and glucose are
utilized. Since the rate-limiting steps for glucose and glutamine
utilizations in vivo are not known, no decisions can be made
about the mechanisms of these apparent activations of substrate
uptake.
Arterial whole-blood amino acid levels in fasted control and
tumor-bearing Buffalo rats are listed in Table 2. The concentra
tions of threonine, serine, glycine, and alanine were increased,
and the concentration of lysine was decreased in the blood of
the hepatoma 7288CTCF-bearing animals. Serine was the only
amino acid the concentration of which was increased in the blood
of hepatoma 5123C-bearing rats. Amino acid utilization and
production by tumors growing in fasted rats (not shown) were
similar to those observed for tumors growing in fed rats (26).
Each of the amino acids measured was utilized by hepatoma
5123C (n = 9); the utilization rates ranged from 0 to 3 nmol/min/
g tumor, except for the utilization rates of lysine and glutamine,
which were 5.5 and 8.2, respectively. Similar rates of amino acid
utilization were noted in the faster-growing tumors, hepatoma
7288CTCF (n = 9) and Walker sarcocarcinoma 256 (n = 14).
V = WALKER 256
«7288CTCF
A
o = 7777
= 5I23C
o
400
GLUCOSE
SUPPLY,
800
0
nmol/min/g
GLUTAMINE SUPPLY, nmol/min/g
Chart 3. Regression of the rate of glucose utilization on the rate of glucose
supply in rat tumors in fasted rats in vivo. Each point represents the determination
for a single tumor.
, least-squares fit to the data points shown for the tumors
growing in fasted animals [y = 0.38x - 4.7, n = 38, r = 0.864 (p < 0.05)];
, least-squares fit for data from tumors growing in fed rats taken from Ref.
26 (y = 0.36x - 36.9). Comparison by analysis of covariance indicated that the 2
regression lines were different (p < 0.05).
AUGUST
40
80
Chart 4. Regression of the rate of glutamine utilization on the rate of glutamine
supply in rat tumors in fasted rats in vivo. Each point represents the determination
for a single tumor.
, least-squares fit to the data points shown for the tumors
growing in fasted rats [y = 0.23x + 0.30. n = 38, r = 0.631 (p < 0.05)];
,
least-squares fit for data from tumors growing in fed rats taken from Ref. 26 (y =
0.24x - 4.4). Comparison by analysis of covariance indicated that the 2 regression
lines were different (p < 0.05).
1983
Downloaded from cancerres.aacrjournals.org on April 29, 2017. © 1983 American Association for Cancer Research.
3501
L A. Sauer and R. T. Dauchy
Table 2
Amino acid levels in arterial whole blood of normal and tumor-bearing fasted
Buffalo rats
Whole-blood samples were removed from the carotid arteries of anesthetized
animals, and amino acid analyses were performed as described in 'Materials and
Methods/
2-dayhepatomafasted
rats(i/mol/liter)49 bearing rats "
acidAspartic
Amino
Oimol/liter)37
hepatoma-bearing
rats Ornol/liter)45
of substrates (glucose and glutamine). An increased efficiency of
glucose and glutamine utilization during fasting is important,
because it suggests that the signals generated in the host during
fasting which cause hypometabolic changes in some host tissues
will promote a hypermetabolic state in the tumor.
ACKNOWLEDGMENTS
acidThreonineSerineGlutamic579±
3110±
±
6133±18C231
±
Specialthanks are due Dr. Walter Nagelfor numeroushelpfuldiscussions during
5153±
13198
this research and for careful reading of the manuscript. Dr. Lorann Stallones also
7e234±
16C283
±
±10239
provided valuable help and advice in the statistical analyses.
acidGlutamineGlycineAlanineIsoleucineLeucineTyrosinePhenylalanineLysineAmmoniaNormal
16724±
13775±
31807
±
50241
±
37255±
±64351
23e224
±
15148
±
±29200
27°76
±
1068
±
REFERENCES
±2064±
799±
795
7119±
±
1255
±
1060±
±
1559
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649±
450
862±
3516±
4513±
7442±
2. Buzby, G. P., Mullen, J. L, Stein, T. P., Miller, E. E., Hobbs, C. L. and Rosato,
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±27
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Again, glutamine and lysine were the most rapidly utilized. Low
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In this paper, we have shown that tumors growing in fasted
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blood. To our knowledge, this is the first demonstration of the
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strates) are infused into the tumor arterial blood followed by
isolation, identification, and measurement of C02 and other
products in the tumor venous blood are required to determine
the extent of substrate oxidation in vivo. Despite our present
lack of information on the extent of the oxidation of these
substrates, it seems likely that the utilization of the ketone bodies
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3L. A. Sauer and R. T. Dauchy, manuscript in preparation.
3502
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AUGUST 1983
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3503
Ketone Body, Glucose, Lactic Acid, and Amino Acid Utilization
by Tumors in Vivo in Fasted Rats
Leonard A. Sauer and Robert T. Dauchy
Cancer Res 1983;43:3497-3503.
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