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The crabtree effect exerted on the citric acid cycle oxidation of glucose carbons in Ehrlich ascites tumor cells
ARCHIVES
The
OF
BIOCHEMISTRY
Crabtree
AND
Effect
Glucose SHIGERU Biochemistry
Exerted Carbons
TSUIKI, Division,
BIOPHYSICS
126, 436-443
on the Citric in Ehrlich
TADAYOSHI
November
Acid
Ascites
SUKENO,
the Research Institute University, Received
(1968)
Cycle Tumor
30, 1967; accepted
Leprosy January
of
Cells
HISAKO
AND
for Tuberculosis, Sendai, Japan
Oxidation
and
TAKEDA Tohoku
Cancer,
23, 1968
In mouse Ehrlich ascites tumor cells, the oxidation of glucose carbons via the citric acid cycle is inhibited profoundly when the Crabtree effect is in operation. This is shown by the fact that either glucose exhaustion or dinitrophenol addition, both of which are known to eliminate the Crabtree effect, results in a many-fold stimulation of glucose C-2, C-3 + C-4, and C-6 oxidation to CO%. Under these conditions, the percentage increase in total oxygen consumption is much less. The steadystate levels of the citric acid cycle intermediates derived from glucose were determined under a variety of metabolic conditions. The stimulation of glucose carbon oxidation by either of glucose exhaustion and dinitrophenol addition is accompanied by a marked rise in the level of citrate. The level of pyruvate is elevated much less or rather unaltered. These results are interpreted as indicating that under the operation of the Crabtree effect, the glucose carbon oxidation via the citric acid cycle is inhibited primarily, if not entirely, by a decreased activity of pyruvate to acetyl CoA oxidation. Apparently, the pyruvate oxidation is inhibited to a much greater degree than the subsequent, oxidation of acetyl CoA via the citric acid cycle.
Previous workers (l-3) have concluded that in Ehrlich ascites tumor cells, the Crabtree effect (an inhibition of respiration in the presence of glucose) is exerted on the oxidation of endogenous substrates but not on the oxidation of glucose. The conclusion is based mainly upon the observation that the respiratory inhibition impairs the oxidation of 14C-fatty acids to 14C02 but not the oxidation of U-14C-glucose t’o 14COr . Studies with 1-14C-pyruvate (4-6), however, demonstrated that the 14C02 production decreases upon the addition of glucose and it has been suggested by Ram et al. (5) that the decrease in 14C02production is associated with the respiratory inhibition induced by glucose but not due to an isotopic dilution by glucase-derived pyruvate. Since glucose can be oxidized in the citric acid cycle only after being converted into pyruvate, the oxidation of glucose carbons in the citric acid cycle should be impaired by the elicitation of the Crabtree effect if the Crabtree effect inhibits the oxidation of exogenous pyruvate. 436
In order to settle these problems and also to provide a better insight into the mechanism of the Crabtree effect, the oxidation of glucose carbons in Ehrlich ascites tumor cells was compared before and after the elimination of the Crabtree effect. The Crabtree effect was eliminated either by the exhaustion of glucose or by the addition of dinitrophenol (7-11). Careful consideration was given to distinguish between the two sites of glucose carbon oxidation: (1) the citric acid cycle; (2) the pentose cycle. The results obtained demonstrate clearly that under the operation of the Crabtree effect, the citric acid cycle oxidation of glucose carbons is in a profoundly inhibited state, due primarily to a decreased activity of pyruvate to acetyl CoA oxidation. MATERIALS The
tumor
used
AND in
the
METHODS present
work
was
a
hyperdiploid Ehrlich ascites carcinoma strain carried in mice were harvested
of the &-strain. from the peritoneal
The
tumor cells cavity of the
GLUCOSE
CARBON
OXIDATION
mice 7-10 days following inoculation. The cells were washed twice in physiological saline and then once in a Krebs-Ringer phosphate buffer (Ca++-free), pH 7.4, containing 85 mM phosphate (4). Incubation was conducted in Warburg flasks at 37” for 30 minutes. The gas phase was air. The final incubation mixture contained in 2 ml of the Krebs-Ringer phosphate buffer described above: cells equivalent to 10 mg in dry weight, radioactive glucose and various additions indicated in tables and figures. The use of high phosphate medium rendered the tumor glycolysis rapid (thereby giving rise to an extensive and stable Crabtree effect) but prevented almost completely lowering of pH due to accumulation of lactic acid. After temperature equilibration for 10 minutes, the reaction was initiated by the addition of rad.ioactive glucose. The reaction was terminated by adding 0.2 ml of 40% trichloroacetic acid or, when the citric acid cycle intermediates were being determined, 0.2 ml of 12N H804. Ox&en consumption was measured manometrically. Glucose was assayed according to the method of Somogyi (12) and lactic acid aecording to the method of Barker and Summerson (13). Radioact,ive COZ was determined by the procedure described by Tsuiki and Kikuchi (14). All the analytical results reported are per 10 mg (dry weight) cells per 30 minutes unless otherwise specified. For the isolation of individual organic acids from the reaction mixture, the paper-chromatographic procedure described previously (14, 15) was employed with slight modifications. After the addition of H&SOd, the reaction mixture was centrifuged and 0.5 rmole each of pyruvate and cu-ketoglutarate and 10 Fmoles each of citrate, succinate, fumarate and malate were added to the supernatant as carrier. A 0.5-ml portion of the supernatant was treated with 2,4-dinitrophenylhydrazine and the resulting keto acid hydrazones were purified by extraction with ethyl acetate. The separation of individual keto acid hydrazones was then performed by paper chromatography using butanol-ethanol-water (5:1:4, by volume) as solvent. The hydrazones were eluted from the paper with methanol. Another 0.5-ml portion of the supernatant was mixed with l--g of dried silica (loo-150 mesh). The mixture was packed into a tube of l-cm diamet.er and eluted with 300 ml of ether which had been equilibrated with 0.5 N HaSOd. The eluate was evaporated in z)ucuo and the residue was loaded on paper after being dissolved in a small volume of ammoniacal water. The separation of individual organic acids was then performed by two dimensional paper-chromatography using
IN
ASCITES
437
TUMOR
I
IO
20
30
TIHE (MINUTES) FIG. 1. Course of incorporation of 1% from 6-i4C--glucose into malate in Ehrlich ascites tumor cells. The cells were incubated with 6-l%glucose (10 mu) for the different periods indicated.
ethanol-ammonia-water (80:5: 15, by volume) in the first direction and pentanol-formic acidwater (19:1:6, by volume) in the second. The organic acids were eluted from the paper with ammoniacal water after being located by means of autoradiography. The eluates were evaporated in vacua and the residue dissolved in 15 ml of a liquid scintillator described by Bray (16). The radioactivity was then determined with a Packard liquid scintillation counter. U-i4C-, l-i%and B-i%-glucose were purchased from Daiichi Pure Chemicals Co., Tokyo. 2-l%glucose was obtained from the Radiochemical Centre, Amersham, England and 3,4-i4C-glucose from New England Nuclear Corp., Boston. RESULTS
The previous workers emproyed U-Wglucose as substrate (1-3). In these experiments, therefore, 14C02 was produced not only in the citric acid cycle, but also in the pentose cycle which is not directly related to the mitochondrial oxidative process. To distinguish these two sites of glucose carbon oxidation, the present studies employed 6J4C-glucose as substrate when the extent of glucose carbon oxidation in the citric acid cycle was being determined. For the estimation of the levels of the citric acid cycle intermediates derived from glucose, the cells were incubated with U-14C-glucose and the radioactivity of each intermediate was determined after isolation. As illustrated in Fig. 1, the incorporation of 14C from 6-14C-glucose into malate reached a
TSUIKI.
43s
SUKENO,
plateau as early as 10 minutes after the glucose addition. In most of the experiments described below, the determination was conducted after 30 minutes of incubation. Hence, the values reported must have represented steady state levels. Each of these experiments was repeated several times giving essentially the same results. However, only the data from individual representative experiments are given. Figure 2 shows that when Ehrlich ascites tumor cells were incubated in the presence of 5 or 20 Nmolesof 6-14C-glucose,no difference in metabolic rates due to the difference in glucose concentration was observed during the first 30 minutes of incubation. However, thereafter, in the “low glucose” system but not in the “high glucose” system, exhaustion of glucose was followed by the elimination of the Crabtree effect. This was known by an increased oxygen consumption accompanied by a marked stimulation of 14C02 production. Thus in the “low glucose” system, the 14C0, produced during the second 30 minutes was almost 430% that produced in the first 30 minutes (Table I). Since the corresponding value for oxygen uptake was only 160%, the Crabtree effect appears to inhibit the citric acid cycle oxidation of glucose carbons more profoundly than the oxidation of endogenoussubstrates. Table I also presents the data from analogous experiments carried out with U-14C-, l-Y& 2-14C- and 3, 4-14C-glucose as substrate. With the latter two, the stim-
TIMElYlMJTE.9
Course of respiratory and glycolytic activities of Ehrlich ascites tumor cells at two different initial 6-‘%-glucose concentrations. The tumor cells were incubated with 2.5 (0) or 10 mM (0) 6-l%-glucose for the periods indicated. FIG.
2.
AND
TAKEDA TABLE
EFFECT
TION
I
0~ GLUCOSE EXHAUSTION ON THE OXIDAOF DIFFERENT CARBONS OF GLUCOSE IN EHRLICH ASCITES TUMOR CELLP mpatoms oxidized
Duration of incubation (minutes)
Total C C-l c-2 c-3 + c-4 C-6
of carbon to Cot
(t30
30-60
(A)
(B)
202
361 60 39 162 30
147 8 33 7
B/.4
(74
179 41 488 491 430
QEhrlich ascites tumor cells were incubated with U-l%-, l-l%-, 2-l%, 3,4-l%- or 6-l%-glucose under the standard conditions for 30 or 60 minutes and the amount of WOZ formed during the incubation was determined. The concentration of labeled glucose added was 5 pmoles/2 ml.
ulation of 14C02production observed in the second 30 minutes was even greater than that observed for 6J4C-glucose oxidation. On the contrary, the 14C02 production from 1-14C-glucosewas decreased 60% in the second 30 minutes as compared to the first 30 minutes. This occurred because glucose oxidation in the pentose cycle ceasedearly in the second 30 minutes owing to the exhaustion of glucose. The 14C02 production from U-14C-glucose was increased in the second 30 minutes but to an extent much lower than those found for 2-14C-,3, 4-14C-and 6-14C-glucoseoxidation. The lower degree of stimulation observed for U-14C-glucose oxidation can be explained by the fact that during the first, 30 minutes, a significant portion of 14C02was formed in the pentose cycle, the contribution of which became almost negligible in the second 30 minutes. The finding that C-3 (+ C-4) and C-6 oxidation was almost equally stimulated upon the elimination of the Crabtree effect suggested that under the operation of the Crabtree effect, glucose carbon oxidation may be limited by the activity of pyruvate to acetyl CoA oxidation. In order to explore this possibility, the tumor cells were incubated with 5 or 20 pmoles of U-14Cglucose for two different lengths of time. Of the four systems listed in Table II, the
GLUCOSE
CARBON
OXIDATION
IN
TABLE EFFECT Duration
OF GLUCOSE
of incubation
Glucose
present
initially
Glucose
present
after
‘4C (mfiatoms) pyruvate citrate: succinate
found
EXHAUSTION (minutes)
:
bmoles)
:
incubation
ASCITES
439
TUMOR
II
ON THE LEVEL
OF CITRIC
ACID
CYCLE
INTERMEDIATEP
20
bmoles)
:
40
5.0
20.0
5.0
1.2
15.9
-
689.2 6.8 9.2
683.3 8.2 9.6
661.5 19.6 32.5
20.0 12.7
in
: :
824.2 8.4 10.2
a The four vessels each containing 10 mg (dry weight) tumor cells in 2 ml of the Krebs-Ringer phate buffer described in the text were divided into two pairs. One vessel of each pair received of W4C-glucose and the other 20 pmoles. One pair of vessels were incubated for 20 minutes other 40 minutes. TABLE
III
EFFECT OF DINITROPHENOL CONCENTRATION THE METABOLISM OF 6-14C-G~u~os~ IN EHRLICH ASCITES TUMOR CELLS” Dini trophenol concentration bM)
0 0.10 0 0.05 0.10 0.15 a The
“CO2 produc-
Glucase
Oxygen uptake (fimoles)
Glucose uptake (pmoles)
Lactate production moles)
(p~$es)
+ + + +
2.49 2.45 1.11 1.82 2.56 2.61
6.00 7.42 9.37 10.71
9.61 13.01 15.71 17.69
0.005 0.035 0.058 0.056
6.“C-
concentration
of 6-*4C-glucose
ON
was 10 rnM.
only one (incubated with 5 pmoles of glucose for 40 minutes) had exhausted glucose at the time of determination and this system exhibited a much higher level of citrate and succinate as; compared to other systems. As the level of pyruvate was not appreciably elevated under these conditions, the rise in citra,te may be taken as evidence that the elimination of the Crabtree effect stimulates the step pyruvate to citrate. This implies that under the operation of the Crabtree effect, the step pyruvate to citrate may .be in an inhibited state. Glucose carbon oxidation under the operation of the Crabtree effect was further studied using dinitrophenol, an uncoupler known by it’s capacity to release the Crabtree effect (7-11). As shown in Table III,
phos5 rmoles and the
with 6-14C-glucose as substrate, increasing concentrations of dinitrophenol progressively stimulated a number of metabolic activities of the tumor cells. The percentage Increase varies, however, for each pathway. Upon the addition of 0.1 mM dinitrophenol, glucose uptake was increased 56 % and lactate production 64%. The 14C02 production was increased 1060%, while oxygen uptake was increased only to the endogenous level. Thus dinitrophenol resembles glucose exhaustion in that glucose carbon oxidation is stimulated much more extensively than is oxygen uptake. Table IV compares the oxidation of different carbons of glucose in the presence of dinitrophenol. The oxidation of all the carbons tested was stimulated by dinitrophenol and the pattern of stimulation was similar to that found in t,he case TABLE
IV
EFFECT OF DINITROPHENOL ON THE OXIDATION OF DIFFERENT CARBONS OF GLUCOSES mcatoms oxidized Dinitrophenol
C-l c-3 C-6
+ c-4
a Ehrlich ascites with l-l%-, 3,4-l%standard conditions formed was determined. glucose, 10 mM; and
of carbon to COz
-
+
105 40 8
203 454 95
-
tumor cells were incubated or 6-W-glucose under the and the amount of i4C02 The concentration: 14Cdinitrophenol, 0.1 my.
440
THUIKI,
SUKEXO,
AND
TAKEDA TABLE
i
0N THE
EFFECTS oF BRSENITE GLUCOSE METABOLISM
OF
TUMOR
ENDOGENOUS AND EHRLICH ASCITES
CELLS’ Addition:
Arsenite concentration mo
01
Pyr 960
Cit 12
KG 10
SIX 11
Fm 6
Ual 12
3. Effect of dinitrophenol on the levels of the citric acid cycle intermediates in Ehrlich ascites tumor cells. The cells were incubated for 30 minutes with 10 mM U-1‘0glucose in the absence (control) and presence of 0.1 mu dinitrophenol. The control levels are given below each intermediate in mpatoms of 1% incorporated. The abbreviations are: Pyr, pyruvate; Cit, citrate; a-ketoglutarate; sue, succinate; Fum, KG, fumarate; and Mal, malate. FIG.
of glucose exhaustion. The stimulation of C-l oxidation observed with dinitrophenol can be attributed solely to an increased citric acid cycle oxidation, since “C-l minus C-6” oxidation did not increase appreciably by the presence of dinitrophenol. These results suggest that dinitropheno1 stimulates glucose carbon oxidation in a manner similar to that found with glucose exhaustion. In order to uncover the points of glucose carbon stimulation observed with dinitrophenol, the levels of the citric acid cycle intermediates were compared between the cells metabolizing U-14C-glucose in the absence and presence of dinitrophenol. In Fig. 3, the ordinate gives the percentage change of each intermediate due to the presence of dinitrophenol. Along the abscissa, the intermediates were arranged in the order of citric acid cycle sequence and below each intermediate, the level found in the absence of dinitrophenol is given as mpatoms of 14C incorporated per 30 minutes per 10 mg (dry weight) cells. Although all the intermediates examined were increased by dinitrophenol, citrate gained the highest increase of almost 200%, while pyruvate was increased much less. It would thus seem that as in the case of glucose exhaustion,
nOne
6.‘Cglucose
6-“C-glucose + DSPb
_____
-I--0 0.05 0.10 0.20
1.95 1.68 0.86 0.70
a The analytical pmoles/30 minutes/l0 b Dinitrophenol. 0.1 rnM.
1.05 0.91 0.82 0.67
0.005 0.006 0.006 0.005
2.12 1.54 1.16 1.07
results are expressed mg (dry weight) cells. Added to a concentration
0.071 0.022 0.009 0.003 in of
the elimination of the Crabtree effect, caused by dinitrophenol is also associated with a stimulation of the step pyruvate to citrate. In Fig. 3, a steep rise was also observed between a-ketoglutarate and succinate. This indicated that another point of stimulation may occur at this step. Further evidence in support of the idea that dinitrophenol stimulates the pyruvate to citrate conversion was obtained when glucose carbon oxidation was examined in the presence of arsenite, a specific inhibitor of pyruvate and ol-ketoglutarate oxidation. The data presented in Table V revealed that arsenite inhibits glucose carbon oxidation in the presence of dinitrophenol but not in its absence. This suggested that in the absence of dinitrophenol, only insignificant amounts of glucose carbons are supplied to the citric acid cycle. The effects of arsenite on the glucose carbon oxidation stimulated by dinitrophenol are illustrated in Fig. 4. Arsenite induced a sharp rise in the levels of pyruvate and a-ketoglutarate and a fall in the levels of citrate and succinate, as would be expected from its inhibitory action on the pyruvate and cyketoglutarate oxidation. The intermediate pattern shown in Fig. 4 is an almost mirror image of the pattern shown in Fig. 3, thus favoring the view that the latter pattern
GLUCOSE
Pyr Cit 9% 41
CARBON
OXIDATION
KG Sue Fm Wal 29
45
17
39
FIG. 4. Effect of arsenite on the levels of the citric acid cycle intermediates in Ehrlich ascites tumor cells. Tlne cells were incubated for 30 min utes with 10 mM U-14C--glucose and 0.1 mMdinitrophenol in the absence (control) and presence of 0.1 my arsenite. The control levels are given below each intermediate in mHatoms of i4C incorporated.
indicates a stimulation as succinate :formation.
of citrate
as well
DISCUSSION
The present studies have demonstrated that the oxidation of glucose carbons in the citric acid cycle is markedly inhibited by the operation of the Crabtree effect. This is in a sharp contrast to the conclusion reached by previous workers (l-3) that glucose carbon oxidation is rather insensitive to the Crabtree effect. The discrepancy seems to have arisen from the fact that in the present work, the oxidation of individual carbons was, separately studied, whereas the previous workers employed U-14C-glucase as substrate. Hence in the previous studies, the phosphogluconate oxidation participated extensively in the 14C02 production under the operation of the Crabtree effect but not after all the glucose was converted into pyruvate or lactate, thereby neutralizing the stimulation of 14C02 production in the citric acid cycle due to the elimination of the Crabtree effect. High respiratory quotient observed with high glucose concentrations in these studies (1, 2) is also indicative of the phosphogluconate oxidation contributing extensively in total glucose oxidation. Comparison of the effects of dinitrophe-
IN
ASCITES
TUMOR
441
no1 addition with the effects of glucose exhaustion revealed that glucose carbon oxidation is stimulat’ed by both of these conditions in quite a similar manner. It follows then that under the operation of the Crabtree effect, the oxidation of glucose carbons in the citric acid cycle is being inhibited by a decreased activity of pyruvate to citrate conversion. This conclusion is also consistent with the observation of Bloch-Frankenthal and Ram (4) and others (5, 6) that the oxidative decarboxylation of pyruvate added to the t’umor cells is inhibited by the addition of glucose. Although the Crabtree effect apparently inhibits the oxidation of glucose-derived pyruvate (glucose C-3 and C-4 oxidation studied in the present work) much more extensively than it inhibits the oxidation of exogenous pyruvate ( 1-14C-pyruvate oxidation studied by Bloch-Frankenthal and Ram (4)), exogenous pyruvate, present at much higher concentrations than endogenous pyruvate, may reverse the inhibition to some extent by acting as an electron ac. ceptor (17, 18). The formation of citrate from pyruvate (and oxaloacetate) is a two-step reaction requiring citrate synthase in addition to the pyruvate dehydrogenase system. As the citrate synthaserom yeast (19) and rat liver (20, 21) is known to be inhibited by ATP, the possibility cannot readily be ruled out that dinitrophenol stimuIated the activity of this enzyme. However, the finding that the formation of succinate as well as citrate was stimulated by dinitrophenol favors the view that the site of dinitrophenol action is the oxidative decarboxylation of pyruvate rather than the subsequent condensation step. In Ehrlich ascites tumor cells, evidence has accumulated that the addition of glucose inhibits the oxidation of higher fatty acids such as palmitate but not the oxidation of acetate (2, 3, 6, 22). Wenner and CereijoSantalo (23) have shown that the oxidation of succinate to fumarate is not inhibited by the Crabtree effect. The oxidation of 1 ,4-14C-succinate to 14C02 can be stimulated by glucose addition and the stimulating effect has been attributed to the lowering
442
TSUIKI.
SUKENO,
of pH (due to the accumulation of lactic acid), which presumably enhanced the decarboxylation of oxaloacetate to pyruvate (24). If t~his explanation is correct, the part of the citric acid cycle from succinate to oxaloacetate should retain its capacity for being stimulated even under the operation of the Crabtree effect. There thus seem to be good grounds for assuming that under the operation of the Crabtree effect, the oxidation of pyruvate to acetyl CoA is being inhibited rather specifically. This assumption apparently receives support from our own observation that the Crabtree effect inhibits glucose carbon oxidation to a much greater degree than it inhibits oxygen uptake. Ibsen and Fox (25) reported that thoroughly washed Ehrlich ascites tumor cells no longer exhibit the Crabtree effect unless pyruvate or lactate is included in the incubation mixture. This can also be explained well on the basis of the above assumption. The mechanism by which active glycolysis inhibits the pyruvate oxidation is not known at present. Wenner and CereijoSantalo (26) have demonstrated that in Ehrlich-Lettre ascites tumor cells, mitochondrial oxidation of pyruvate is inhibited by t’he addition of a-glycerophosphate. We have shown in a previous paper (27) that in the absence of glucose, the oxidation of pyruvate added to Ehrlich ascites tumor cehs is being inhibited by the activity of succinat,e dehydrogenase and that dinitrophenol or malonate reverses the inhibition. Since mitochondrial a-glycerophosphate dehydrogenase as well as succinate dehydrogenase is a flavin-linked enzyme, the pyruvate oxidation in these tumors appears to be sensitive to the activities of flavin-linked dehydrogenases. A possibihty exists t,hat the pyruvate dehydrogenase of these tumors is inhibited by low concentrations of NADH, as is the a-ketoglutarate dehydrogenase of pig heart (28). The nature and properties of pyruvateoxidizing system may be an important factor in determining the type of metabolism exhibited by tissues. In those tissues where glycolysis is capable of inhibiting the pyruvate oxidation, glycdlysis would be stable,
AND
TAKEDA
since citrate, a powerful inhibitor of phosphofructokinase (18, 29, 30, 31), would be maintained at a low level as long as active glycolysis continues. On the contrary, if pyruvate-oxidizing system is insensitive to glycolysis, glycolysis in such tissues would be unstable and readily inhibited by active respiration, since the citrate level would be kept high as long as active respiration continues. A pronounced Crabtree effect and a high rate of aerobic glycolysis which characterize tumor metabolism may be a conseqrence of the occurrence in tumors of a very activephosphofructokinase (18) and a pyruvate dehydrogenase system susceptible to glycolysis. ACKNOWLEDGMENT
technical assistance of Miss Keiko Shibata is gratefully acknowledged. The
REFERENCES 1. BLOCH-FRANKENTHAL,
L., AND WEINHOUSE, 1082 (1957). MEDES, G., AND WEINHOUSE, S., Cancer Res. 18, 352 (1958). EMMELOT, P., AND NOUT, S. J., Brit. J. Cancer 13, 513 (1959). BLOCH-FRANKENTHAL, L., AND RAM, D., Cancer Res. 19,835 (1959). RAM, D., KALNER, H. S., AND BLOCH-FRANKENTHAL, L., Cancer Res. 93,600 (1963). SAUERMANN, G., Arch. Biochem. Biophys. 104, 208 (1964). RACKER, E., Ann. N.Y. Acad. Sci. 63, 1017 (1956). IBSEN, K. H., COE, E. L., AND MCKEE, R. W., Biochim. Biophys. Acta 30, 384 (1958). KVAMME, E., Acta Physiol. &and. 42, 219 (1958). EMMELOT, P., AND Bos, C. J., Brit. J. Cancer 13, 520 (1959). CHANCE, B., AND HESS, B., J. Biol. Chem. 234, 2421 (1959). SOMOGYI, M., J. Biol. Chem. 196, 19 (1962). BARKER, S. B., AND SUMMERSON, W. H., J. Biol. Chem. 138, 535 (1941). TSUIKI, S., AND KIKUCHI, G., Biochim. Biophys. Aeta 64, 514 (1962). OKUYAMA, M., TSUIKI, S., AND KIKUCHI, G., Biochim. Biophys. Acta 110, 66 (1965). BRAY, G. A., Ancd. Biochem. 1, 279 (1960). WENNER, C. E., AND PAIGEN, K., Arch. Biothem. Biophys. 93, 646 (1961). SUZUKI, R., SATO, K., AND TSUIKI, S., Arch. Biochem. Biophys. 134,596 (1968).
S., Cancer Res. 17,
2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18.
GLUCOSE
CARBON
OXIDATION
19. HATHAWAY, J. A., AND ATKINSON, I). E., Biochem. Biophys. Res. Commun. 20, 661 (1965). 20. SHEPHERD, D., YATES, D. W., AND GARLAND, P. B., Biochem. J. 97, 3% (1965). 21. SHEPHERD, D., AND GARLAND, P. B., Biochem. Biophys. Res. Commun. 22,89 (1966). 22. EMMELOT, P., AND VALS, G. H. VAN, Brit. J. Cancer 11, 620 (1957). 23. WENNER, C. E., AND CEREIJO-SANTALO, R., Arch. Biochem. Biophys. 98, 67 (1962). 24. WENNER, CJ. E., Arch. Biochem. Biophys. 98, 58 (1962). 25. IBSEN, K. IL, AND Fox, J. P., Arch. Biochem. Biophys. 112, 580 (1965).
IN
ASCITES
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TUMOR
26. WENNER, C. E., AND CEREIJO-SANTALO, R., J. Biol. Chem. 238, 1584 (1963). 27. TAKEDA, H., SUKENO, T., AND TSUIKI, S., Biochem. Biophys. Res. Commun. 29, 90 (1967). 28. GARLAND, P. B., Biochem. J. 92, 10~ (1964). 29. GARLAND, P. B., RANDLE, P. J., AND NEWSHOLME, E. A., Nature 193, 270 (1963). 30. PARMEGGIANI, A., AND BOWMAN, R. H., Biochem. Biophys. Res. Commun. 12, 268 (1963). 31. PASSONNEAU, Biochem. (1963).
J. V., AND LOWRY, Biophys. Res. Commun.
0. 13,
H., 372
The
OF
BIOCHEMISTRY
Crabtree
AND
Effect
Glucose SHIGERU Biochemistry
Exerted Carbons
TSUIKI, Division,
BIOPHYSICS
126, 436-443
on the Citric in Ehrlich
TADAYOSHI
November
Acid
Ascites
SUKENO,
the Research Institute University, Received
(1968)
Cycle Tumor
30, 1967; accepted
Leprosy January
of
Cells
HISAKO
AND
for Tuberculosis, Sendai, Japan
Oxidation
and
TAKEDA Tohoku
Cancer,
23, 1968
In mouse Ehrlich ascites tumor cells, the oxidation of glucose carbons via the citric acid cycle is inhibited profoundly when the Crabtree effect is in operation. This is shown by the fact that either glucose exhaustion or dinitrophenol addition, both of which are known to eliminate the Crabtree effect, results in a many-fold stimulation of glucose C-2, C-3 + C-4, and C-6 oxidation to CO%. Under these conditions, the percentage increase in total oxygen consumption is much less. The steadystate levels of the citric acid cycle intermediates derived from glucose were determined under a variety of metabolic conditions. The stimulation of glucose carbon oxidation by either of glucose exhaustion and dinitrophenol addition is accompanied by a marked rise in the level of citrate. The level of pyruvate is elevated much less or rather unaltered. These results are interpreted as indicating that under the operation of the Crabtree effect, the glucose carbon oxidation via the citric acid cycle is inhibited primarily, if not entirely, by a decreased activity of pyruvate to acetyl CoA oxidation. Apparently, the pyruvate oxidation is inhibited to a much greater degree than the subsequent, oxidation of acetyl CoA via the citric acid cycle.
Previous workers (l-3) have concluded that in Ehrlich ascites tumor cells, the Crabtree effect (an inhibition of respiration in the presence of glucose) is exerted on the oxidation of endogenous substrates but not on the oxidation of glucose. The conclusion is based mainly upon the observation that the respiratory inhibition impairs the oxidation of 14C-fatty acids to 14C02 but not the oxidation of U-14C-glucose t’o 14COr . Studies with 1-14C-pyruvate (4-6), however, demonstrated that the 14C02 production decreases upon the addition of glucose and it has been suggested by Ram et al. (5) that the decrease in 14C02production is associated with the respiratory inhibition induced by glucose but not due to an isotopic dilution by glucase-derived pyruvate. Since glucose can be oxidized in the citric acid cycle only after being converted into pyruvate, the oxidation of glucose carbons in the citric acid cycle should be impaired by the elicitation of the Crabtree effect if the Crabtree effect inhibits the oxidation of exogenous pyruvate. 436
In order to settle these problems and also to provide a better insight into the mechanism of the Crabtree effect, the oxidation of glucose carbons in Ehrlich ascites tumor cells was compared before and after the elimination of the Crabtree effect. The Crabtree effect was eliminated either by the exhaustion of glucose or by the addition of dinitrophenol (7-11). Careful consideration was given to distinguish between the two sites of glucose carbon oxidation: (1) the citric acid cycle; (2) the pentose cycle. The results obtained demonstrate clearly that under the operation of the Crabtree effect, the citric acid cycle oxidation of glucose carbons is in a profoundly inhibited state, due primarily to a decreased activity of pyruvate to acetyl CoA oxidation. MATERIALS The
tumor
used
AND in
the
METHODS present
work
was
a
hyperdiploid Ehrlich ascites carcinoma strain carried in mice were harvested
of the &-strain. from the peritoneal
The
tumor cells cavity of the
GLUCOSE
CARBON
OXIDATION
mice 7-10 days following inoculation. The cells were washed twice in physiological saline and then once in a Krebs-Ringer phosphate buffer (Ca++-free), pH 7.4, containing 85 mM phosphate (4). Incubation was conducted in Warburg flasks at 37” for 30 minutes. The gas phase was air. The final incubation mixture contained in 2 ml of the Krebs-Ringer phosphate buffer described above: cells equivalent to 10 mg in dry weight, radioactive glucose and various additions indicated in tables and figures. The use of high phosphate medium rendered the tumor glycolysis rapid (thereby giving rise to an extensive and stable Crabtree effect) but prevented almost completely lowering of pH due to accumulation of lactic acid. After temperature equilibration for 10 minutes, the reaction was initiated by the addition of rad.ioactive glucose. The reaction was terminated by adding 0.2 ml of 40% trichloroacetic acid or, when the citric acid cycle intermediates were being determined, 0.2 ml of 12N H804. Ox&en consumption was measured manometrically. Glucose was assayed according to the method of Somogyi (12) and lactic acid aecording to the method of Barker and Summerson (13). Radioact,ive COZ was determined by the procedure described by Tsuiki and Kikuchi (14). All the analytical results reported are per 10 mg (dry weight) cells per 30 minutes unless otherwise specified. For the isolation of individual organic acids from the reaction mixture, the paper-chromatographic procedure described previously (14, 15) was employed with slight modifications. After the addition of H&SOd, the reaction mixture was centrifuged and 0.5 rmole each of pyruvate and cu-ketoglutarate and 10 Fmoles each of citrate, succinate, fumarate and malate were added to the supernatant as carrier. A 0.5-ml portion of the supernatant was treated with 2,4-dinitrophenylhydrazine and the resulting keto acid hydrazones were purified by extraction with ethyl acetate. The separation of individual keto acid hydrazones was then performed by paper chromatography using butanol-ethanol-water (5:1:4, by volume) as solvent. The hydrazones were eluted from the paper with methanol. Another 0.5-ml portion of the supernatant was mixed with l--g of dried silica (loo-150 mesh). The mixture was packed into a tube of l-cm diamet.er and eluted with 300 ml of ether which had been equilibrated with 0.5 N HaSOd. The eluate was evaporated in z)ucuo and the residue was loaded on paper after being dissolved in a small volume of ammoniacal water. The separation of individual organic acids was then performed by two dimensional paper-chromatography using
IN
ASCITES
437
TUMOR
I
IO
20
30
TIHE (MINUTES) FIG. 1. Course of incorporation of 1% from 6-i4C--glucose into malate in Ehrlich ascites tumor cells. The cells were incubated with 6-l%glucose (10 mu) for the different periods indicated.
ethanol-ammonia-water (80:5: 15, by volume) in the first direction and pentanol-formic acidwater (19:1:6, by volume) in the second. The organic acids were eluted from the paper with ammoniacal water after being located by means of autoradiography. The eluates were evaporated in vacua and the residue dissolved in 15 ml of a liquid scintillator described by Bray (16). The radioactivity was then determined with a Packard liquid scintillation counter. U-i4C-, l-i%and B-i%-glucose were purchased from Daiichi Pure Chemicals Co., Tokyo. 2-l%glucose was obtained from the Radiochemical Centre, Amersham, England and 3,4-i4C-glucose from New England Nuclear Corp., Boston. RESULTS
The previous workers emproyed U-Wglucose as substrate (1-3). In these experiments, therefore, 14C02 was produced not only in the citric acid cycle, but also in the pentose cycle which is not directly related to the mitochondrial oxidative process. To distinguish these two sites of glucose carbon oxidation, the present studies employed 6J4C-glucose as substrate when the extent of glucose carbon oxidation in the citric acid cycle was being determined. For the estimation of the levels of the citric acid cycle intermediates derived from glucose, the cells were incubated with U-14C-glucose and the radioactivity of each intermediate was determined after isolation. As illustrated in Fig. 1, the incorporation of 14C from 6-14C-glucose into malate reached a
TSUIKI.
43s
SUKENO,
plateau as early as 10 minutes after the glucose addition. In most of the experiments described below, the determination was conducted after 30 minutes of incubation. Hence, the values reported must have represented steady state levels. Each of these experiments was repeated several times giving essentially the same results. However, only the data from individual representative experiments are given. Figure 2 shows that when Ehrlich ascites tumor cells were incubated in the presence of 5 or 20 Nmolesof 6-14C-glucose,no difference in metabolic rates due to the difference in glucose concentration was observed during the first 30 minutes of incubation. However, thereafter, in the “low glucose” system but not in the “high glucose” system, exhaustion of glucose was followed by the elimination of the Crabtree effect. This was known by an increased oxygen consumption accompanied by a marked stimulation of 14C02 production. Thus in the “low glucose” system, the 14C0, produced during the second 30 minutes was almost 430% that produced in the first 30 minutes (Table I). Since the corresponding value for oxygen uptake was only 160%, the Crabtree effect appears to inhibit the citric acid cycle oxidation of glucose carbons more profoundly than the oxidation of endogenoussubstrates. Table I also presents the data from analogous experiments carried out with U-14C-, l-Y& 2-14C- and 3, 4-14C-glucose as substrate. With the latter two, the stim-
TIMElYlMJTE.9
Course of respiratory and glycolytic activities of Ehrlich ascites tumor cells at two different initial 6-‘%-glucose concentrations. The tumor cells were incubated with 2.5 (0) or 10 mM (0) 6-l%-glucose for the periods indicated. FIG.
2.
AND
TAKEDA TABLE
EFFECT
TION
I
0~ GLUCOSE EXHAUSTION ON THE OXIDAOF DIFFERENT CARBONS OF GLUCOSE IN EHRLICH ASCITES TUMOR CELLP mpatoms oxidized
Duration of incubation (minutes)
Total C C-l c-2 c-3 + c-4 C-6
of carbon to Cot
(t30
30-60
(A)
(B)
202
361 60 39 162 30
147 8 33 7
B/.4
(74
179 41 488 491 430
QEhrlich ascites tumor cells were incubated with U-l%-, l-l%-, 2-l%, 3,4-l%- or 6-l%-glucose under the standard conditions for 30 or 60 minutes and the amount of WOZ formed during the incubation was determined. The concentration of labeled glucose added was 5 pmoles/2 ml.
ulation of 14C02production observed in the second 30 minutes was even greater than that observed for 6J4C-glucose oxidation. On the contrary, the 14C02 production from 1-14C-glucosewas decreased 60% in the second 30 minutes as compared to the first 30 minutes. This occurred because glucose oxidation in the pentose cycle ceasedearly in the second 30 minutes owing to the exhaustion of glucose. The 14C02 production from U-14C-glucose was increased in the second 30 minutes but to an extent much lower than those found for 2-14C-,3, 4-14C-and 6-14C-glucoseoxidation. The lower degree of stimulation observed for U-14C-glucose oxidation can be explained by the fact that during the first, 30 minutes, a significant portion of 14C02was formed in the pentose cycle, the contribution of which became almost negligible in the second 30 minutes. The finding that C-3 (+ C-4) and C-6 oxidation was almost equally stimulated upon the elimination of the Crabtree effect suggested that under the operation of the Crabtree effect, glucose carbon oxidation may be limited by the activity of pyruvate to acetyl CoA oxidation. In order to explore this possibility, the tumor cells were incubated with 5 or 20 pmoles of U-14Cglucose for two different lengths of time. Of the four systems listed in Table II, the
GLUCOSE
CARBON
OXIDATION
IN
TABLE EFFECT Duration
OF GLUCOSE
of incubation
Glucose
present
initially
Glucose
present
after
‘4C (mfiatoms) pyruvate citrate: succinate
found
EXHAUSTION (minutes)
:
bmoles)
:
incubation
ASCITES
439
TUMOR
II
ON THE LEVEL
OF CITRIC
ACID
CYCLE
INTERMEDIATEP
20
bmoles)
:
40
5.0
20.0
5.0
1.2
15.9
-
689.2 6.8 9.2
683.3 8.2 9.6
661.5 19.6 32.5
20.0 12.7
in
: :
824.2 8.4 10.2
a The four vessels each containing 10 mg (dry weight) tumor cells in 2 ml of the Krebs-Ringer phate buffer described in the text were divided into two pairs. One vessel of each pair received of W4C-glucose and the other 20 pmoles. One pair of vessels were incubated for 20 minutes other 40 minutes. TABLE
III
EFFECT OF DINITROPHENOL CONCENTRATION THE METABOLISM OF 6-14C-G~u~os~ IN EHRLICH ASCITES TUMOR CELLS” Dini trophenol concentration bM)
0 0.10 0 0.05 0.10 0.15 a The
“CO2 produc-
Glucase
Oxygen uptake (fimoles)
Glucose uptake (pmoles)
Lactate production moles)
(p~$es)
+ + + +
2.49 2.45 1.11 1.82 2.56 2.61
6.00 7.42 9.37 10.71
9.61 13.01 15.71 17.69
0.005 0.035 0.058 0.056
6.“C-
concentration
of 6-*4C-glucose
ON
was 10 rnM.
only one (incubated with 5 pmoles of glucose for 40 minutes) had exhausted glucose at the time of determination and this system exhibited a much higher level of citrate and succinate as; compared to other systems. As the level of pyruvate was not appreciably elevated under these conditions, the rise in citra,te may be taken as evidence that the elimination of the Crabtree effect stimulates the step pyruvate to citrate. This implies that under the operation of the Crabtree effect, the step pyruvate to citrate may .be in an inhibited state. Glucose carbon oxidation under the operation of the Crabtree effect was further studied using dinitrophenol, an uncoupler known by it’s capacity to release the Crabtree effect (7-11). As shown in Table III,
phos5 rmoles and the
with 6-14C-glucose as substrate, increasing concentrations of dinitrophenol progressively stimulated a number of metabolic activities of the tumor cells. The percentage Increase varies, however, for each pathway. Upon the addition of 0.1 mM dinitrophenol, glucose uptake was increased 56 % and lactate production 64%. The 14C02 production was increased 1060%, while oxygen uptake was increased only to the endogenous level. Thus dinitrophenol resembles glucose exhaustion in that glucose carbon oxidation is stimulated much more extensively than is oxygen uptake. Table IV compares the oxidation of different carbons of glucose in the presence of dinitrophenol. The oxidation of all the carbons tested was stimulated by dinitrophenol and the pattern of stimulation was similar to that found in t,he case TABLE
IV
EFFECT OF DINITROPHENOL ON THE OXIDATION OF DIFFERENT CARBONS OF GLUCOSES mcatoms oxidized Dinitrophenol
C-l c-3 C-6
+ c-4
a Ehrlich ascites with l-l%-, 3,4-l%standard conditions formed was determined. glucose, 10 mM; and
of carbon to COz
-
+
105 40 8
203 454 95
-
tumor cells were incubated or 6-W-glucose under the and the amount of i4C02 The concentration: 14Cdinitrophenol, 0.1 my.
440
THUIKI,
SUKEXO,
AND
TAKEDA TABLE
i
0N THE
EFFECTS oF BRSENITE GLUCOSE METABOLISM
OF
TUMOR
ENDOGENOUS AND EHRLICH ASCITES
CELLS’ Addition:
Arsenite concentration mo
01
Pyr 960
Cit 12
KG 10
SIX 11
Fm 6
Ual 12
3. Effect of dinitrophenol on the levels of the citric acid cycle intermediates in Ehrlich ascites tumor cells. The cells were incubated for 30 minutes with 10 mM U-1‘0glucose in the absence (control) and presence of 0.1 mu dinitrophenol. The control levels are given below each intermediate in mpatoms of 1% incorporated. The abbreviations are: Pyr, pyruvate; Cit, citrate; a-ketoglutarate; sue, succinate; Fum, KG, fumarate; and Mal, malate. FIG.
of glucose exhaustion. The stimulation of C-l oxidation observed with dinitrophenol can be attributed solely to an increased citric acid cycle oxidation, since “C-l minus C-6” oxidation did not increase appreciably by the presence of dinitrophenol. These results suggest that dinitropheno1 stimulates glucose carbon oxidation in a manner similar to that found with glucose exhaustion. In order to uncover the points of glucose carbon stimulation observed with dinitrophenol, the levels of the citric acid cycle intermediates were compared between the cells metabolizing U-14C-glucose in the absence and presence of dinitrophenol. In Fig. 3, the ordinate gives the percentage change of each intermediate due to the presence of dinitrophenol. Along the abscissa, the intermediates were arranged in the order of citric acid cycle sequence and below each intermediate, the level found in the absence of dinitrophenol is given as mpatoms of 14C incorporated per 30 minutes per 10 mg (dry weight) cells. Although all the intermediates examined were increased by dinitrophenol, citrate gained the highest increase of almost 200%, while pyruvate was increased much less. It would thus seem that as in the case of glucose exhaustion,
nOne
6.‘Cglucose
6-“C-glucose + DSPb
_____
-I--0 0.05 0.10 0.20
1.95 1.68 0.86 0.70
a The analytical pmoles/30 minutes/l0 b Dinitrophenol. 0.1 rnM.
1.05 0.91 0.82 0.67
0.005 0.006 0.006 0.005
2.12 1.54 1.16 1.07
results are expressed mg (dry weight) cells. Added to a concentration
0.071 0.022 0.009 0.003 in of
the elimination of the Crabtree effect, caused by dinitrophenol is also associated with a stimulation of the step pyruvate to citrate. In Fig. 3, a steep rise was also observed between a-ketoglutarate and succinate. This indicated that another point of stimulation may occur at this step. Further evidence in support of the idea that dinitrophenol stimulates the pyruvate to citrate conversion was obtained when glucose carbon oxidation was examined in the presence of arsenite, a specific inhibitor of pyruvate and ol-ketoglutarate oxidation. The data presented in Table V revealed that arsenite inhibits glucose carbon oxidation in the presence of dinitrophenol but not in its absence. This suggested that in the absence of dinitrophenol, only insignificant amounts of glucose carbons are supplied to the citric acid cycle. The effects of arsenite on the glucose carbon oxidation stimulated by dinitrophenol are illustrated in Fig. 4. Arsenite induced a sharp rise in the levels of pyruvate and a-ketoglutarate and a fall in the levels of citrate and succinate, as would be expected from its inhibitory action on the pyruvate and cyketoglutarate oxidation. The intermediate pattern shown in Fig. 4 is an almost mirror image of the pattern shown in Fig. 3, thus favoring the view that the latter pattern
GLUCOSE
Pyr Cit 9% 41
CARBON
OXIDATION
KG Sue Fm Wal 29
45
17
39
FIG. 4. Effect of arsenite on the levels of the citric acid cycle intermediates in Ehrlich ascites tumor cells. Tlne cells were incubated for 30 min utes with 10 mM U-14C--glucose and 0.1 mMdinitrophenol in the absence (control) and presence of 0.1 my arsenite. The control levels are given below each intermediate in mHatoms of i4C incorporated.
indicates a stimulation as succinate :formation.
of citrate
as well
DISCUSSION
The present studies have demonstrated that the oxidation of glucose carbons in the citric acid cycle is markedly inhibited by the operation of the Crabtree effect. This is in a sharp contrast to the conclusion reached by previous workers (l-3) that glucose carbon oxidation is rather insensitive to the Crabtree effect. The discrepancy seems to have arisen from the fact that in the present work, the oxidation of individual carbons was, separately studied, whereas the previous workers employed U-14C-glucase as substrate. Hence in the previous studies, the phosphogluconate oxidation participated extensively in the 14C02 production under the operation of the Crabtree effect but not after all the glucose was converted into pyruvate or lactate, thereby neutralizing the stimulation of 14C02 production in the citric acid cycle due to the elimination of the Crabtree effect. High respiratory quotient observed with high glucose concentrations in these studies (1, 2) is also indicative of the phosphogluconate oxidation contributing extensively in total glucose oxidation. Comparison of the effects of dinitrophe-
IN
ASCITES
TUMOR
441
no1 addition with the effects of glucose exhaustion revealed that glucose carbon oxidation is stimulat’ed by both of these conditions in quite a similar manner. It follows then that under the operation of the Crabtree effect, the oxidation of glucose carbons in the citric acid cycle is being inhibited by a decreased activity of pyruvate to citrate conversion. This conclusion is also consistent with the observation of Bloch-Frankenthal and Ram (4) and others (5, 6) that the oxidative decarboxylation of pyruvate added to the t’umor cells is inhibited by the addition of glucose. Although the Crabtree effect apparently inhibits the oxidation of glucose-derived pyruvate (glucose C-3 and C-4 oxidation studied in the present work) much more extensively than it inhibits the oxidation of exogenous pyruvate ( 1-14C-pyruvate oxidation studied by Bloch-Frankenthal and Ram (4)), exogenous pyruvate, present at much higher concentrations than endogenous pyruvate, may reverse the inhibition to some extent by acting as an electron ac. ceptor (17, 18). The formation of citrate from pyruvate (and oxaloacetate) is a two-step reaction requiring citrate synthase in addition to the pyruvate dehydrogenase system. As the citrate synthaserom yeast (19) and rat liver (20, 21) is known to be inhibited by ATP, the possibility cannot readily be ruled out that dinitrophenol stimuIated the activity of this enzyme. However, the finding that the formation of succinate as well as citrate was stimulated by dinitrophenol favors the view that the site of dinitrophenol action is the oxidative decarboxylation of pyruvate rather than the subsequent condensation step. In Ehrlich ascites tumor cells, evidence has accumulated that the addition of glucose inhibits the oxidation of higher fatty acids such as palmitate but not the oxidation of acetate (2, 3, 6, 22). Wenner and CereijoSantalo (23) have shown that the oxidation of succinate to fumarate is not inhibited by the Crabtree effect. The oxidation of 1 ,4-14C-succinate to 14C02 can be stimulated by glucose addition and the stimulating effect has been attributed to the lowering
442
TSUIKI.
SUKENO,
of pH (due to the accumulation of lactic acid), which presumably enhanced the decarboxylation of oxaloacetate to pyruvate (24). If t~his explanation is correct, the part of the citric acid cycle from succinate to oxaloacetate should retain its capacity for being stimulated even under the operation of the Crabtree effect. There thus seem to be good grounds for assuming that under the operation of the Crabtree effect, the oxidation of pyruvate to acetyl CoA is being inhibited rather specifically. This assumption apparently receives support from our own observation that the Crabtree effect inhibits glucose carbon oxidation to a much greater degree than it inhibits oxygen uptake. Ibsen and Fox (25) reported that thoroughly washed Ehrlich ascites tumor cells no longer exhibit the Crabtree effect unless pyruvate or lactate is included in the incubation mixture. This can also be explained well on the basis of the above assumption. The mechanism by which active glycolysis inhibits the pyruvate oxidation is not known at present. Wenner and CereijoSantalo (26) have demonstrated that in Ehrlich-Lettre ascites tumor cells, mitochondrial oxidation of pyruvate is inhibited by t’he addition of a-glycerophosphate. We have shown in a previous paper (27) that in the absence of glucose, the oxidation of pyruvate added to Ehrlich ascites tumor cehs is being inhibited by the activity of succinat,e dehydrogenase and that dinitrophenol or malonate reverses the inhibition. Since mitochondrial a-glycerophosphate dehydrogenase as well as succinate dehydrogenase is a flavin-linked enzyme, the pyruvate oxidation in these tumors appears to be sensitive to the activities of flavin-linked dehydrogenases. A possibihty exists t,hat the pyruvate dehydrogenase of these tumors is inhibited by low concentrations of NADH, as is the a-ketoglutarate dehydrogenase of pig heart (28). The nature and properties of pyruvateoxidizing system may be an important factor in determining the type of metabolism exhibited by tissues. In those tissues where glycolysis is capable of inhibiting the pyruvate oxidation, glycdlysis would be stable,
AND
TAKEDA
since citrate, a powerful inhibitor of phosphofructokinase (18, 29, 30, 31), would be maintained at a low level as long as active glycolysis continues. On the contrary, if pyruvate-oxidizing system is insensitive to glycolysis, glycolysis in such tissues would be unstable and readily inhibited by active respiration, since the citrate level would be kept high as long as active respiration continues. A pronounced Crabtree effect and a high rate of aerobic glycolysis which characterize tumor metabolism may be a conseqrence of the occurrence in tumors of a very activephosphofructokinase (18) and a pyruvate dehydrogenase system susceptible to glycolysis. ACKNOWLEDGMENT
technical assistance of Miss Keiko Shibata is gratefully acknowledged. The
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GLUCOSE
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OXIDATION
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IN
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