CHEMICAL INTERFERENCES IN CELLULAR METABOLISM. CINNAMATE EFFECTS*

BY E. C. WEINBACH, H. J. LOWE, W. R. FRISELL, AND LESLIE HELLERMAN

(From the Department of Physiological Chemistry, The Johns Hopkins University, School of Medicine, Baltimore, Maryland)

(Received for publication, August 10, 1950)

For some time we have been studying in parallel the mechanism and control of enzymatic action and the interference exercised by certain substances with metabolism in selected tissues and cells (1). In this paper we present the results of one of these studies. Interesting effects, especially “shunts” in metabolism, have been observed in slices of various organs of the guinea pig and rat in the presence of the ions of benzoic, cinnamic, and certain other acids. These anions possess the constituent grouping

I I -c=c-coo-

(I)

and previously have been found (1, 2) to interfere with the oxidation of n-amino acids through a reversible competition of the inhibitor and sub- strate for the isolated flavoenzyme, n-amino acid oxidase of kidney. We have considered significant a certain structural similarity in the inhibitors and the iminoketonic acid products of amino acid oxidation.

The subject as it relates to tissues recalls observations previously de- scribed of inhibitions by benzoic acid and related compounds of the oxida- tion of various substrates in kidney and liver preparations. Such sub- strates (3, 4) are butyric, crotonic, and acetic acids (cf. (5, S)), as well as certain L- and n-amino acids (7-9). It has been suggested (9) that the action of certain inhibitors is concerned with intermediate metabolic processes involving various oxidations associated with ammonia formation in liver and kidney.

We have confirmed many of the older observations and in addition have found that in the presence of certain inhibitors the aerobic lactate ac- cumulation is increased to approximately the anaerobic level; in short, there has been observed a reversal of the Pasteur effect as applied to our material.

* This investigation was supported by a research grant from the National Cancer Institute, United States Public Health Service. The authors were assisted by Miss A. E. Sturmfels.

779

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780 CINNAMATE EFFECTS IN METABOLISM

Methods

Oxygen consumption and glycolysis were measured manometrically in Warburg respirometers at 37”. Krebs-Ringer phosphate (pH 7.0) and Ringer-bicarbonate (pH 7.3) buffers (10) were used. Respiration in bi- carbonate buffer was followed by the method of Pardee (11). Protein- free aliquots were obtained for chemical analysis after addition of tri- chloroacetic acid to the vessel contents and centrifugation. Lactate was estimated chemically by the method of Barker and Summerson (12), adapted for the Coleman spectrophotometer, model 6A. Glucose was estimated by the Somogyi-Nelson method (13). Phosphoglyceric acid was hydrolyzed with 10 M sulfuric acid and the phosphate determined by the Fiske-Subbarow method according to the procedure of Lohmann (14, 10). Borsook and Dubnoff’s modification (15) of the Friedemann- Haugen method was used to follow pyruvate utilization. No correction for any accumulation of oxalacetate or or-ketoglutarate was found neces- sary. Ammonia was isolated by the aeration technique (16) and de- termined by the method of Moore and Stein (17).

Rats and guinea pigs, approximately 6 to 8 weeks old, were killed by a blow to the cervical region and the organs were extirpated and placed in cold saline. A few experiments with older animals indicated that age of the animal affected the results negligibly. After slicing by the usual free-hand technique, the tissues were transferred to cold buffer solutions. The diaphragm was quartered rather than sliced. The tissues were weighed on a torsion balance and then placed serially in small beakers containing iced buffer until a sufficient number had been weighed for one bank of Warburg flasks. After being blotted, the tissues were transferred to the manometric flasks. This procedure minimized the amount of preformed metabolites and facilitated simultaneous transfer of the tissues.

In the early experiments with kidney slices, final dry weights were used in calculation of the metabolic quotients. Final dry weights of brain and liver slices produced erratic replicates owing to mechanical loss and diffusion; therefore initial dry weights were employed in subsequent experiments. This procedure yields metabolic quotients lower than those commonly found in the literature (18).

Usually, substrates were added from the side arm at zero time in the form of their sodium salts adjusted to pH 7. Pyruvate solutions were prepared by careful neutralization at 0” of highly purified pyruvic acid (19). Solutions of 3-phosphoglycerate were prepared by treatment of barium phosphoglycerate with excess sodium sulfate in slightly acid solu- tion and removal of barium sulfate, followed by adjustment to pH 7.4 with sodium hydroxide. Phosphoryl-enolpyruvic acid,l obtained as the

1 We are indebted to Dr. Wendell T. Caraway for the preparation of this com- pound.

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WEINBACH, LOWE, FRISELL, AND HELLERMAN 781

silver-barium salt by the method of Baer and Fischer (20), was freed of silver by solution in a slight excess of 0.2 M hydrochloric acid and centrifu- gation. The solution was freed of barium by the addition of a slight excess of sodium sulfate, removal of barium sulfate, and adjustment to pH 7.4 with sodium hydroxide. When substrates were present initially, or when their utilization was followed analytically, zero time concentrates were determined. Except as noted, each vessel contained 2.0 ml. of buffer, a final concentration of 0.01 M inhibitor, and 0.015 M substrate, made up to a total volume of 2.4 ml.

EXPERIMENTAL

In the studies of interference by cinnamate and related inhibitors two effects are evident: (1) the respiration is suppressed, and (2) the rate of aerobic lactate production is increased, approaching in some tissues the anaerobic level.

These effects in various tissues, held in different buffer media in the presence and absence of added dextrose, are shown in Table I. Without added substrate, the inhibition observed in the presence of cinnamate is approximately 50 per cent for guinea pig and rat kidney (cf. (7, 9)). Table I shows also that cinnamate greatly enhances the production of lactate in the presence of added glucose. This effect is most marked for guinea pig tissues, particularly kidney cortex and brain, which, of the tissues studied here, have the largest capacity for glycolysis. However, the per cent increase in lactate production is of the same relative magni- tude in all the tissues investigated, in the sense that this approaches the anaerobic lactate production observed in such tissues. In the absence of added glucose (endogenous metabolism), there is no significant increase in the lactate level after the addition of cinnamate or related inhibitors.

The course of glycolysis for kidney cortex of the guinea pig under various conditions is plotted in Fig. 1. Curves I, II, and III represent similar experiments accomplished in an oxygen environment in bicarbon- ate, phosphate, and bicarbonate plus phosphate buffers, respectively. Curve I illustrates the normally low “aerobic” lactate level for kidney slices, while Curve II indicates an almost linear lactate accumulation in phosphate buffer. When the mixture of the two buffers is employed, there is a rapid initial lactate accumulation which ceases after the first 30 minutes (Curve III). Curve IV is similar to Curve II and illustrates the relationship of reduced oxygen partial pressure to the rate of lactate accumulation in phosphate. Curve V represents the anaerobic (95 per cent N,-5 per cent CO,) lactate accumulation. Of paramount interest for this investigation is the effect exhibited by Curve VI: in the presence of cinnamate and bicarbonate (cf. Curve I) the aerobic lactate production is increased to the anaerobic level.

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WEINBACE, LOWE, FRISELL, AND HELLERMAN 783

Ionic Efects; Reversibility-In view of the slight solubility of mag- nesium cinnamate (21) it seemed possible that cinnamate in general might influence oxidation by removal of a divalent cation (22). Inhibitions in the usual phosphate buffer media (cf. (23)) have been attributed to the removal of calcium ions from the medium as calcium phosphate or acid phosphate (24). To evaluate this in reference to our observations, slices of guinea pig kidney were incubated for 1 hour, either in phosphate or in bicarbonate medium with added cinnamate and dextrose, after which the tissues were removed to cinnamate-free bicarbonate medium (plus dex-

BUFFER GAS PHASE I I I I

A BICARBONATE 95%0,,5%CO, @ “t PHOSPHATE -- - SAME

I - 1 I I 1 I I I J 0 20 b/?li 80 100 120

U T E”“s

FIG. 1. Course of glycolysis for slices of guinea pig kidney cortex under VU conditions (see the text).

-ious

trose) for a 2nd hour (Table II). Manipulation of slices from the usual bicarbonate medium to another portion of the same buffer for the 2nd hour resulted in no increase in lactate production, while transfers from phosphate to bicarbonate did not prevent accelerated production of lac- tate (Fig. 1, Curve II). On the other hand, the transfers from cin- namate-buffer to cinnamate-free media resulted in a marked decrease in lactate production, approximately to the normal value (control, after transfer), coincidental with the restoration of the level of respiratory activity. The withdrawal of calcium and magnesium ions from the media did not appear to affect significantly these results.

Nature of Inhibition-It was considered that the increased lactate output might arise either as a result of decreased oxidative utilization of

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784 CINNAMATE EFFECTS IN METABOLISM

pyruvate, intermediate in glucose metabolism, or as a result of an ac- celerated glycolysis, whether initiated directly or indirectly. Presumably the effect first named could occur by interference with the condensation of pyruvate (or a product therefrom) with oxalacetate, or, by inhibition of one or more of the reactions in the tricarboxylic acid cycle. Cinnamate apparently does not compete with cocarboxylase2 if this sub-

TABLE II Reversibility of Inhibitor Effect; Buffer Composition

Slices of guinea pig kidney. All calculations based on the initial dry weights, calculated from the initial wet weights. Each vessel contained 2.0 ml. of buffer; inhibitor added from the side arm at zero time; final volume 2.4 ml. The flasks were incubated 1 hour, and the tissues removed, washed in fresh buffer, and placed in the appropriate flasks. After the usual gassing and equilibration, they were in- cubated an additional hour. Glucose present in all flasks. All lactate values cor- rected for accumulation to zero time.

No. of determi- nations

Medium, 1st hr.

Bicarbonate*

Bicarbonate + cinnamatet

Phosphate

Y

(t:i- 8.8)

36.0

(K13.0)

Medium, 2nd hr.

Bicarbonate*

Same minus MgClz ‘I “ CaCL ‘I “ I‘ and

MgClz Bicarbonatet Same minus MgClz

‘I “ CaClz “ “ MgClz and CaClz

Bicarbonate

Lactate per ng. dry weight

per hr. after transfer)

Y

9.3

9 10 13

14 20 20 18

15.4

* Qg:, 1st hour, 8.5; 2nd hour, 11.0. t Qg:, 1st hour, 5.2; 2nd hour, 11.0.

stance is cell-permeable under the conditions, since addition (cf. (25)) of this coenzyme (0.005 M) does not prevent the accumulation of lactate. Employing a variety of substrates and metabolic intermediates, including several associated with the cycle, we did not observe inhibitions of suf- ficient magnitude to account for the characteristic metabolic effects of cinnamate and related inhibitors. This contrasts with results obtained in parallel tests with fluoroacetate (see below).

Thus, with no added substrate, cinnamate (Table III) reduces the

2 We are grateful to Merck and Company, Inc., for a gift of cocarboxylase.

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WEINBACH, LOWE, FRISELL, AND HELPIERMAN 785

respiratory rate, Qo,, from 11.6 to 5.2, a decrease of 6.4. In the presence of added substrate, the decrement in Qo, rarely is greater than 6.4, and in most cases is less. This might suggest that the “endogenous respiration” remains more or less operative; then the addition of certain substrates

TABLE III

Comparative Effect of Cinnamate and Fluoroacetate*

Slices of guinea pig kidney. Each vessel contained 2.0 ml. of Krebs phosphate buffer; substrates present initially; inhibitors tipped in from the side arm at sero time; final volume 2.4 ml. Metabolic quotients based on the jinal dry weight.

-

I Respiration (Qoe)

Substrate 0.01 M

None (endogenous)

Dextrose.

Pyruvate .

Lactate.............

Acetate.............

Citrate.

Succinate. .

Fumarate...........

Malate.

--

-

2--x-f mina- tions

14

18

12

6

9

6

13

7

9

Control

11.6 (7.7-13.5) 13.8

(10.4-14.0) 18.0

(14.0-19.2) 13.1

(12.1-14.1) 20.8

(18.8-23.4) 23.6

(19.0-26.8) 30.4

(22.4-39.9) 19.2

(17.0-20.4) 20.3

(16.c27.0)

r .-

12

21

12

6

6

3

7

3

6

-7

--

-

Cinnamate, 0.01 M

5.2 (3.9- 6.3)

(2 9.9) 15.0 (9.8-16.1)

(:::- 6.7) 14.8

(12.0-16.9) 18.4

30.0 (23.7-38.4) 19.5

16.9 (15.3-19.4)

-7

1 i&E* mina- tions

11

6

6

3

6

3

3

3

6

(ii- 6.8)

(Z- 5.0)

&O.O) 6.0

(X- 8.0) 10.6

25.4

13.9

12.7 (10.0-16.0)

* Parallel experiments performed in the presence of low concentration of NaF indicated that none of the observed effects were due to contamination of the fluoro- acetate solutions with inorganic fluoride; zirconium-aliearin assay of the stock sodium fluoroacetate indicated that the concentration of fluoride ion in any vessel containing 0.01 M fluoroacetate could not have exceeded 0.0004 M.

would contribute in part additively to the cellular respiration (cj. (26)). If so, the inhibitions observed in the presence of these particular added substrates might result largely from the effect of cinnamate on the respira- tion of endogenous substrates.

Fluoroacetate, which has been reported to block acetate (27) or pyru- vate (28) utilization, and, even more significantly, intermediate citrate function (2931), was used for comparative purposes. Without the ad-

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786 CINNAMATE EFFECT@ IN MEZlABBLISM

dition of any of the substrates named in Table III, fluoroacetate reduces the respiratory rate (endogenous) from 11.6 to 5.3. With an added substrate, fluoroacetate effects a decrease in Qo2, amounting essentially to 6.3 or more. In accord with the observations of other investigators, this suggests that fluoroacetate with certain substrates may inhibit the total respiration.

TABLE IV

Utilization of Glycolytic Intermediates Slices of guinea pig kidney. All calculations based on the initial dry weights,

calculated from the initial wet weights. Each flask contained 2.0 ml. of Krebs- Ringer bicarbonate. Substrate and inhibitor tipped in from the side arm at zero time; initial concentrations determined by chemical analysis of flask contents, removed at zero time ( :SC :e the substrate column).

Substrate

-f*

Dextrose, 6000

3-Phosphoglycerate, 15501

Pyruvate, 140$

“ 140 + 0.01 M NaF

Phosphoryl-enolpyru- vate, 3409

-

I

1

.-

6

3

12

7

3

Control

Substrate utilized

7 w w. $er hr.

32.6 (29.4-53.0) 26.4

(&I .9) 7.4

(5.6- 9.0) 20.3

(15.5-25.3)

(i:Y-5.8)

(Z-5.8)

(:::-a.l)

0.01 M cinnamate

Substrate utilized

Y *e* m. $er hr.

f̂ w mg. per hr.

66.8 36.0 (63.5-90.0) (29.6-43.7) 29.4 1.0

6.8 (4.8- 8.6)

(76:Y-10.9) 18.9

(13.7-24.4)

2.2 (0.9- 4.4)

s- 5.5)

(:::- 2.5)

* In each vessel (see the text). t Removal of 3-phosphoglyceric acid through a competing enzymatic hydrolysis

was not followed; however, QS: in the presence of this substrate was 12.7 (cf. en- dogenous 0:: = 9.6).

$ Determinations in a phosphate-buffered medium were not significantly dif-

ferent . J Determined by the method of Lohmann and Meyerhof (10).

Utilization of Glucose, Pyruvate, and Phosphoryl-enolpyruvate-The utilization of added glucose, pyruvate, phosphoryl-enolpyruvate, and phosphog1ycerat.e were followed analytically (Table IV). Three slices of guinea pig kidney were used per vessel to insure a measurable utilization. In the presence of cinnamate, the rate of glucose disappearance, as an- ticipated from other data, was increased significantly; the lactate produc- tion was concomitantly enhanced.

When pyruvate was substituted as the added substrate, the rate of its decrease was found to be approximately the same in the presence and in

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WEINBACH, LOWE, FRISELL, AND HELLERMAN 787

the absence of cinnamate. Some lactate was found under these con- ditions. There might have occurred a marginal glycolysis from residual intermediates, although there are other possibilities. However, the rate of pyruvate decrease, when followed in the presence of sodium fluoride, 0.01 M, again was found to be unaffected by cinnamate, with little change in the lactate. It was found also that 3-phosphoglycerate, used as the sole added substrate, apparently was removed at a rate independent of the presence of cinnamate ions. On the basis of the analytical data, phosphoryl-enolpyruvate likewise was removed without formation of lac- tate, but at a rate roughly comparable with the pyruvate decrease, and again unaffected by cinnamate.3 The data argue against the probability that the phosphoenol was hydrolyzed to pyruvate before oxidative re-

TABLE V

Suppression of Amino Acid Metabolism

All values based on the initial dry weight. Each vessel contained 2.0 ml. of buffer; substrate and inhibitor added from the side arm at zero time to make a final volume of 2.4 ml. Rat kidney slices (phosphate); six determinations each.

I I I I Q 02

01 Per cent Q 02

inhibition ??=a Per cent

inhibition

Control (endogenous). 11.7 1.7 “ -I- 0.01 M cinnamate. 5.2 56 0.85 50

n-Aspartate,* 0.01 M. 18.3 3.5 “ + 0.01 M cinnamate.. 5.3 71 0.8 77

* In the case of L-proline, hydroxy-L-proline, and L-glutamate, 0.005 M cin- namate inhibited the respiration by 24, 53, and 37 per cent, respectively, and in- hibited the ammonia formation by 39, 40, 52 per cent, respectively.

moval. It is of interest that these substrates were found to be utilized at the rate approximately of 0.1 PM per mg. of dry weight per hour.

It appears, therefore, that the increased lactate production observed with glucose and these inhibitors is to be correlated with the initiation, directly or indirectly, of increased glycolysis.

Suppression of Oxygen Uptake in Presence of Amino Acids and of Oc- tanoate-Our work with L-amino acids as substrates, to be detailed else- where, indicates that the inhibition by cinnamate and related inhibitors of ammonia formation and of oxygen consumption is of the same order as has been reported (7, 9) for benzoate. Some values are given in Table

8 Our data generally support the idea that cinnamate does not embarrass the utilization of pyruvate in the kidney cortex slices. However, some interference with glucose or pyruvate oxidation in certain other tissues is not precluded. Thus, we have preliminary evidence that there is interference with the utilization of py- ruvate in brain as well as in certain transplanted malignant tumors of the rat.

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788 CINNAMATE EFFECTS IN METABOLISM

V. We have also conducted experiments with octanoate in glycero- phosphate buffer (cf. (3, 4)), observing inhibitions in excess of the sup- pression of the endogenous rate. It would appear probable that such fatty acid anions added to tissue slices are less accessible to oxidative processes than intracellular fatty acid.

E$ect of Highly Oxidizable Substrates-It will be indicated under the “Discussion” that the initiation of aerobic glycolysis by the inhibitors

TABLE VI Prevention of Inhibitor E$ects upon Addition of Various Substrates

Slices of guinea pig kidney.* All values based on the initial dry weight. Each vessel contained 2.0 ml. of Krebs-Ringer bicarbonate; substrates (0.01 M) and in- hibitor added from side arm at zero time to make a final volume of 2.4 ml. All lactate values are corrected for accumulation to zero time. Dextrose and cin- namate, w

No. of de terminations

42 Dextrose

20 ‘I + cinnamate

3 Same + L-aspartate 6 “ + n-glutamate

“ + octanoate “ + acetate IL + succinate “ + fumarate “ + L-malate

hen 1 present, 0.015 and 0.01 M, respectively.

Substrate Lactate Dextrose utilized per mg. tissuet per mg. tissue

Y

(E- 8.8) 36.0

(29.6-43.7) 16.2 23.6

(23.1-26.5) 25.2 24.8 18.2 20.3 19.1

Y

32.6 (29.4-53.0) 66.8

(63.5-90.0)

25.8 (21.2-27.6)

37.4

* A few experiments with rat kidney slices showed similar effects. t Experiments performed in phosphate buffer gave a similar, but less marked,

trend. In addition, the Q8: was restored partially to the control level upon ad- dition of the following substrates: n-glutamate, n-aspartate, and acetate.

might be correlated with an inhibition in the initial stages of oxidation of amino acids or fatty acids or both, effecting thereby a transfer of oxidative activity to the process concerned with oxidation of triose phosphate. If this were true, the introduction simultaneously with glucose of a second efficiently oxidizable substrate that might antagonize the action of the inhibitor, e.g. glutamate, or alternatively of one that would invoke high oxidative activity in the terminal process, e.g. succinate, should act to prevent lactate production. In Table VI data are presented that indicate a marked suppression of lactate production, coincident with decrease in dextrose utilization, when L-glutamate and succinate are so employed.

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WEINBACH, LOWE, FRISELL, AND HELLERMAN 789

Certain other substrates listed in Table VI, especially aspartate, fumarate, and malate, likewise suppress the lactate accumulation. The effect of glutamate is not to be attributed to an increased removal of intermediate pyruvate via transamination, since there is a decrease in the utilization of dextrose.

DISCUSSION

The accumulations of lactate observed aerobically in the presence of glucose and cinnamate are to be correlated with factors that increase the rate of glycolysis. In a sense this constitutes a reversal of the Pasteur effect (32-35). The complexities of such effects are recognized (32), and interpretations often have been focused upon carbohydrate interrelation- ships. However, it has been pointed out (35) that there is involved in many tissues, for the provision of energy, the oxidation of metabolites derived from other foodstuffs as well. Indeed, it is the reciprocal rela- tionship of aerobic glycolysis to the availability of energy from non- carbohydrate materials that we wish to stress here.

Cinnamate has been found to inhibit efficiently the rate of acetoacetate formation from butyrate in slices of guinea pig kidney (3, 4), as well as to inhibit the oxidation of fatty acid in a metabolically active preparation (36) of rabbit kidney. Our work, together with that of others (7, 9), demonstrates further that cinnamate blocks ammonia production in kidney slices. The oxidative production of ammonia in rat kidney slices, even in the presence of glucose, may account (37) for 30 per cent of the oxygen consumption, and the low respiratory quotient of kidney tissue in general (35) points to the oxidation of non-carbohydrate substances.4 Our results with other tissues suggest a similar partial dependence of the cell upon the supply of energy from endogenous fats and amino acids. The effect of cinnamate on brain slices might be interpretable in part3 on the basis of suppression of the oxidation of endogenous fatty acid (cf. (3840)), possibly from phospholipide stores (cf. (41)). In liver slices the inhibitory effect of cinnamate is believed to concern largely the fatty acid oxidation. The high glycolytic rate apparently characteristic of most malignant cells may be indicative of a lowered capacity for oxidation of fatty or amino acids; in consistency with this are observations5 that certain highly malignant cells derived in culture from rat fibroblasts display only in greatly diminished degree the shift in metabolism de- scribed in this paper.

4 Consonant with earlier work are our observations from differential manometry of an endogenous R. Q. of 0.5 to 0.7 in the guinea pig kidney slice, as compared with approximately 0.94 in the presence of glucose.

6 Unpublished data, George 0. Gey, L. Hellerman, and coworkers.

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790 CINNAMATE EFFECTS IN METABOLISM

In general it may be concluded that substances such as benzoate, nico- tinate, and especially cinnamate and furylacrylate, suppress the initial stages of oxidation of and the elaboration of acetoacetate from added fatty acids, as well as the rate of oxygen consumption” in mammalian tissue slices. They are especially inhibitory with respect to L-amino acid oxida- tion in kidney slices as well as to n-amino acid oxidases with which they display competitive, reversible action. Hippurate, cyclohexanecarboxyl- at,e, nicotinamide, dihydrocinnamate, cinnamamide, and furylacrylamide inhibit to a lesser degree or not at all. In this connection, the formal resemblance in the general structure (I) of the inhibitors to the inter- mediate products of oxidation (II) of amino acids and to acetoacetate (enol) (III) is suggestive.

7

OH H

I I HN=C-COO- HOC-C=C-COO-

(II) (III)

Of pertinence to the argument is our demonstration of the reversibility of the cinnamate effect in guinea pig kidney.

It is possible to correlate tentatively in terms of enzymatic activity the suppression of oxygen utilization accompanied by lactate production in the presence of these agents. Speculations concerned with the Pasteur effect have invoked numerous mechanisms designed to interpret the ac- celeration of fermentation or glycolysis when partial pressures of oxygen are decreased or when an inhibitory process upon cytochrome oxidase or upon some other element in the aerobic catalytic chain is instituted. The evidence presented in this paper has emphasized a suppression of oxygen consumption, accompanied by elicitation of glycolytic lactate production, through a reversible inhibitory process attributed to the denial (43) of certain substrates to aerobic oxidation. The effect is realized under experi- mental conditions ordinarily highly favorable to the aerobic pattern of metabolism. The release under these conditions of catalytic capacity (cy- tochrome reductase, cytochromes, cytochrome oxidase, O2 route), which normally may be considered to be apportioned among the various systems operating usually through pyridinoenzymes, implies a shunt initiating in- creased phosphorylative oxidation of triose phosphate. Concomitantly, the decline in the turnover of phosphate at the hand of aerobic processes would release phosphate ions to glycolytic coupling (44) and the converse would

6 The specificity of these inhibitions is relative rather than absolute, in the sense that numerous agents in greater or lesser degree induce, under favorable conditions, aerobic glycolysis (for example, see Webb and Elliott (42)). Our special interest in the conjugated anions (e.g., cinnamate) is concerned with their utility in in- vestigating with some precision the mechanism of an inhibitor process.

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WEINBACH, LOWE, FRISELL, AND HELLERMAN 791

be realized during reversal or when inhibition is overcome by the methods illustrated in Table VI. If then the rate of entrance of pyruvate, or a related intermediary, into the terminal aerobic cycle may be assumed to be limited to a maximal level characteristic of the cell (see Table IV) and species, the above sequence of events would account at least in part for the greatly increased conversion of glucose to lactate, operating col- laterally. Mechanical removal of the inhibitor (Table II) should (and does) restore the normal balance.

SUMMARY

1. Cinnamate suppresses the respiration and in the presence of added glucose increases the rate of lactate production to the anaerobic level in several rat and guinea pig tissues. The effect is representative of the action of a number of oc,@-unsaturated carboxylic anions. In guinea pig kidney, particularly, the effect is largely reversible.

2. The evidence available does not point to marked interference by cinnamate with reactions in the tricarboxylic acid cycle or to interference, particularly in kidney cortex, with the oxidative removal of pyruvate or phosphoryl-enolpyruvate.

3. The lactate accumulation observed is paralleled by increased glucose removal and is correlated with factors that increase the rate of glycolysis.

4. Phosphate buffers are less applicable than bicarbonate media in the glycolysis studies, and they display an adverse (irreversible) effect upon certain tissues.

5. The inhibitor ions embarrass oxidation of amino acids and fatty acids in various tissues. Several lines of evidence bear upon the reciprocal relationship of aerobic glycolysis to the availability of energy from non- carbohydrate materials. The observations are interpreted as a reversal of the Pasteur effect instituted by denial of substrates through a competi- tive mechanism.

BIBLIOGRAPHY

1. Hellerman, L., Transactions, 2nd conference, Josiah h!facy, Jr., Foundation, New York, 78-92 (1947).

2. Hellerman, L., Lindsay, A., and Bovarnick, M. R., J. Biol. Chem., 163, 553 (1946).

3. Jowett, M., and Quastel, J. H., Biochem. J., 29, 2143 (1935). 4. Quastel, J. H., and Wheatley, A. H., Biochem. J., 27, 1753 (1933). 5. Griffith, W. H., Proc. Xoc. Exp. Biol. and Med., 37, 279 (1937). 6. Lehmann, J., Skand. Arch. Physiol., 80, 237 (1938). 7. Herner, B., Acta physiol. Scan&, 4, 76 (1942). 8. Klein, J. R., and Kamin, H., J. Biol. Chem., 138, 507 (1941). 9. Herner, B., Acta physiol. &and., 8, suppl. 23 (1944).

10. Umbreit, W. W., Burris, R. H., and Stauffer, J. F., Manometric techniques and tissue metabolism, Minneapolis (1949).

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792 CINNAMATE EFFECTS IN METABOLISM

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