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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 Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1957 American Association for Cancer Research. 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. Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1957 American Association for Cancer Research. 1084 Cancer Research 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. Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1957 American Association for Cancer Research. BLOCH-FRANKENTHAL 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. Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1957 American Association for Cancer Research. 1086 Cancer Research 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. Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1957 American Association for Cancer Research. 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 Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1957 American Association for Cancer Research. 1088 Cancer Research 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. Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1957 American Association for Cancer Research. 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. 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