substance or substances the glycogen of the heart is derived. The

Transcription

1 : THE SOURCE OF THE HEART GLYCOGEN. By J. YULE BOGUE, C. LOVATT EVANS, and R. A. GREGORY.' From the Department of Physiology, Biochemistry, and Pharmacology, University College, London. (Rece ived for publication 14th April 1937.) THE object of the experiments to be described was to find from what substance or substances the glycogen of the heart is derived. The recent observation [AMcGinty and Miller, 1933; Evans, Grande, and Hsu, 1935 a] that the heart uses considerable amounts of lactate when that substance is available in the circulating blood, and, moreover, uses it in preference to, and in larger amount than, glucose, at once opened several questions regarding the fate of the lactate which disappeared. It might, for instance, be oxidised forthwith; or it might be converted into glycogen, as seems to occur in some other tissues, thereby replacing glycogen which had previously been lost, or perhaps continuously replenishing glycogen which was being constantly used up in any way in the tissue. It seems certain that, in hearts under conditions which are reasonably normal, the glycogen content is steady, and in the bleeder dogs after C and E anaesthesia the heart glycogen averages about 0-7 g./100 g. [Evans et al., 1935 b]. This glycogen content is maintained for periods of several hours in hearts isolated from the body by the establishment of heart-lung preparations [Visscher and Mulder, 1930] or of isolated heart-oxygenator preparations [Bogue et al., 1935], provided the hearts so isolated are kept working under conditions as nearly as possible corresponding to those to which they would be subjected in vivo. It would seem, then, that under these usual conditions the heart glycogen is either not drawn upon at all, and so plays no part in the causation of the cardiac contraction, or else is replaced as fast as it is withdrawn. If the former alternative is the truth, the heart glycogen content should remain unaltered for long periods irrespective of any alterations in the composition of the blood; if the latter, it should be possible to lower the heart glycogen by withholding the material from which it is normally replenished. It seems unlikely in any case that the metabolism of the heart is a purely carbohydrate one, but we may make the not improbable assumption 1 Sharpey Scholar.

2 28 Bogue, Evans, and Gregory that at least 50 per cent. of the oxygen utilisation of the heart represents usage for the combustion of carbohydrate. If, then, we take the oxygen usage of the dog's heart working under the usual conditions of a heart-lung preparation as being about 350 c.c./100 g./hr. it may be calculated that the heart glycogen, if it were the carbohydrate combusted and was drawn upon but not replenished, would be quite exhausted within three hours. There are three possible sources from which the heart glycogen, if used, could be recouped, viz. blood sugar, blood lactate, or substances such as protein or fat in the cardiac muscle itself. It should be easy to decide by withdrawing the first two, separately or together, whether these are the responsible substances; the third group, the muscle constituents, it would be difficult to test in this manner. Again, were it possible in any circumstances, by deprivation of precursor or in any other way, to deplete the heart of its glycogen, it should be possible also, by either withholding or providing a given substance, to ascertain whether that substance were able to function as a glycogen former or not. We have attempted to attack the problem in all of these ways. It has already been shown in a previous paper [Bogue et al., 1935] that when both blood glucose and blood lactate are allowed to fall to a very low level the heart glycogen soon begins to show diminution, and we have deemed further experiments in this direction unnecessary for the present. These experiments supplied presumptive evidence that when both glucose and lactate were available the glycogen was either not drawn upon or else was replaced as used. In some of the experiments [Bogue et al., 1935, Expts. 483, 498] the blood sugar was allowed to fall to a low concentration and no further additions of glucose were made to the blood, but the lactate concentrations were kept up by additions of sodium lactate. In these experiments also there was a reduction in the glycogen content, though this appeared to be a somewhat smaller one than was obtained when both glucose and lactate were kept as low as possible to the end. These experiments were not conclusive and required some extension, though the tentative conclusion drawn from them was that lactate was used to supply energy, while glucose was probably convertible into glycogen. In the present experiments, in order to ensure and to accelerate the exhaustion of heart glycogen we availed ourselves of the fact that administration of adrenaline, especially if combined with heavy mechanical work, rapidly reduces blood sugar and lactate and lowers the heart glycogen [Bogue et al., 1935; Chang, 1937]. Repeated trials having satisfied us that adrenaline invariably does produce these effects, we proceeded in further experiments to test the effect of following the period of intensive driving by adrenaline, conjoined with heavy work, by a period of "recovery" without adrenaline and with relatively

3 The Source of the Heart Glycogen light mechanical work. During this second period we either made no addition to the blood or else added glucose, lactate, or both substances. After two or more hours of recovery the experiment was terminated and the heart glycogen estimated. The results obtained served, we believe, to confirm the previously expressed opinions of Bogue et al. [1935] that the heart glycogen is rapidly formed from glucose but not from lactate. METHODS. In most of the experiments dog's heart-lung preparations, but in some of them heart-oxygenator preparations [Evans, Grande, and Hsu, 1934, modified by Bogue and Gregory, 1937], were used. This latter preparation had the advantage of maintaining a lower lactate level in the blood. The blood volume in the circuits was kept as small as possible (usually about 600 c.c.). Throughout each experiment records were made of aortic and inferior caval pressures; the venous pressure was a valuable index to the physiological state of the heart. Blood glucose and lactate were estimated as in a former paper [Evans, Grande, and Hsu, 1934]. At the close of each experiment glycogen was estimated; in order to avoid errors due to uneven distribution the whole of both ventricles was used, as described by Evans et al. [1935 b], and the mean of four determinations on the tissue stock solution was taken. When adrenaline was given, the usual procedure was to add to the blood one or two portions totalling 0 03 to 0-06 mg. within a few minutes, until the heart rate had reached a maximum of 220 to 230 per minute, after which steady infusion of a 1: 20,000 solution was made at a suitable rate, from 5 to 8 c.c./hr. ( =0.25 to 0 40 mg./hr.) into the blood of the venous reservoir by means of the apparatus described by Burn and Dale [1924] throughout the whole of the adrenaline period. (Ciba synthetic adrenaline was used, and was diluted with 0.1 per cent. acetic acid to ensure stability.) Ventilation of the heart-lung preparations was sometimes with air, sometimes with 95 per cent. 02 and 5 per cent. C02; the results were similar in both series. Heart-oxygenator preparations were all aerated with 95 per cent. 02 and 5 per cent. CO2. Lactic acid, when added, was given as a solution of the sodium salt, recently prepared from pure crystalline d-lactic acid. The strong lactate solution was mixed with some blood, to give about 1 per cent. concentration, and the mixture slowly added to the circulating blood. HEART GLYCOGEN DEPLETION BY ADRENALINE. When a heart-lung preparation is driven by continuously administering adrenaline at the rate of about 0 3 mg./hr., the rate of mechanical work being of the order of 60 kg.-m./hr. (say 600 c.c./min. with arterial 29

4 30 Bogue, Evans, and Gregory pressure of 120 mm. Hg) and with a heart rate of about 230 beats per minute, signs of exhaustion generally become apparent in about FIG. 1 (Expt. 572). Arterial (A.P.) and venous pressures (V.P.) of heart-lung preparation, showing the sudden increase of venous pressure which set in after about 11 hours administration of adrenaline (0-25 mg./hr.), which began at p.m. Aortic pressure, 120 mm. Hg. Output of heart, 750 c.c./min. Glycogen of heart was g. per cent. Time =minutes. two hours. The most reliable indication of impending exhaustion is a rise of venous pressure in the inferior vena cava. This pressure is normally near to zero, but we have arbitrarily called it + 1 cm. H20, at the start; after one to one and a half hours of driving in the abovementioned manner it has usually risen to about 2 cm., and towards the end of the period may show a further very rapid rise (figs. 1 and 2, and Table I.). Such experiments were at once terminated before actual failure occurred so that the glycogen could be determined; this was always found to be very low, viz. 0u019 per cent., and per cent. in the two cases of figs. 1 and 2, and it is perhaps worthy of mention that in one instance (Expt. 572) the heart, immediately it was excised and before it could be dropped into the hot potash, became hard and

5 The Source of the Heart Glycogen resistant to the touch, resembling skeletal muscle in rigor when glycogen depletion has occurred [Hoet and Marks, 1926]. 31 FiG. 2 (Expt. 571).-A similar experiment in which the rise of venous pressure was accompanied by missed ventricular beats. Adrenaline began at 3.35 p.m. Aortic pressure, 110 mm. Hg. Output, c.c./min. Glycogen of heart, per cent. Time =minutes. TABLE I. ADRENALINE-DRIVEN HEART-LUNG PREPARATIONS. (Heart failure impending.) Adrenaline period. Expt. No. Duration, min. Mean rate of dose, mg. /hr. Blood during a] nd at end, mg./100 c.c. Glucose. Lactate. Heart glycogen, g./100 g ( / / / / After 20 minutes adrenaline. 2 After 62 minutes adrenaline. 3 Expt. continued for 32 minutes after stopping adrenaline.

6 32 Bogue, Evans, and Gregory The results of four experiments in which failure of the heart, which almost until the end had been in good condition, was imminent at or shortly after the end of the adrenaline period, are given in Table I. It is seen that in all cases the glycogen was below 0.1 per cent., the average being 0*034 per cent. Blood sugar, where estimated, had also fallen to a low level. ADRENALINE ADMINISTRATION-" RECOVERY" PERIOD. In subsequent experiments we were careful to discontinue the administration of adrenaline immediately on the onset of a definite rise in venous pressure, which was usually in about two hours from TABLE II.-ADRENALINE-DRIVEN HEART-LUNG PREPARATIONS. (No glucose or lactate added during recovery period.) Blood sugar and blood lactate. Adrena- Mraten adrenal period. experiment. Recovery No. pierid adrena- Reciover Glycogen, line, in.. -mm g EEE min..~ mg./hr. % 0 0 c -..4!:~s In 573 blood sample taken 22 minutes before end of adrenaline period. In 575 and 577 blood changing. Blood changing. the start. At the same time the output and arterial resistance were reduced, each to about one-half the previous values, so that the work of the heart was reduced to about one-fourth, i.e. from about 60 to about 15 kg.-m./hr., and the preparation was allowed to run on, without any additions to the blood, for two to three hours. The heart rate after discontinuance of the adrenaline declined, at first rapidly and then slowly, reaching about the normal level in from 40 to 60 minutes, so that some adrenaline action was presumably prolonged well beyond the stated periods. The results of these experiments are given in Table II. It is probable that in these experiments the heart glycogen at the end of the "adrenaline period" proper had not fallen to such a low level as in the first series, and it was probably because of this that the hearts were able to continue beating reasonably well throughout the second or "recovery" period. Further, the blood analyses show that

7 The Source of the Heart Glycogen 33 blood sugar and lactate were still present and that there was usage of both blood sugar and lactate during the second period in four of the experiments, while in three of them (573, 575, and 577) fresh blood, containing glucose and lactate, was added to replace loss during the second period, so that glucose and lactate utilisations were certainly greater than would appear from the analyses. These three experiments, with heart glycogens of 0-173, 038, and 0-59 per cent. respectively, suggest that when fresh blood is available regeneration of glycogen may occur rather rapidly. In the other three experiments, where blood sugar and lactate were lower, with glycogens of 04105, 0 043, and 0-14 (mean, 0096 per cent.) there seems to have been little if any glycogen recovery, and it seems safe to conclude that when blood glucose and lactate are sufficiently low no appreciable restoration of the glycogen removed under the influence of adrenaline has taken place in two hours. Hence it would seem unlikely that there can be any rapid local glyconeogenesis taking place from any constituents of the heart itself. ADDITION OF LACTATE. In the next series of experiments the heart was driven as before with adrenaline and hard work, until impending cardiac weakness began TABLE III.-ADRENALINE-DRIVEN HEART-LUNG PREPARATIONS. (Lactic acid added during recovery period.) Blood sugar and blood lactate. Adrena. Rate of adrenaline period. experiment. Re- Lactic No. line adrena- covery acid Glycogen,. period, line,. c. period, added, g./100 g. m_. mg./hr. m. m i g. ~~1b 0 1~~-' d (578) after first addition of lactate. to show itself by a rise of venous pressure. Then the work was reduced to one-fourth and sodium lactate (1.5 to 3-0 g. lactic acid) added slowly at intervals. After a further period of about two hours the experiment was terminated. The results are given in Table III. and they clearly show that there has been no increase in heart glycogen above that presumably present at the end of the adrenaline period. There was certainly some little usage of sugar, but the result has evidently not been VOL. XXVII., NO

8 34 Bogue, Evans, and Gregory appreciably influenced by it. Although the actual quantity of lactate consumed was not determined, this must have been considerable, since a simple calculation will show that the amounts of lactate which had been added from time to time would at the end of the recovery period have given lactate concentrations of the order of 200 to 400 mg./100 c.c. if there had been no utilisation of that substance. Evidently the lactic acid loss, which must, in Expt. 588 for instance, have been at the rate of at least 400 mg./100 g./hr., cannot be accounted for as due to its conversion into glycogen. ADDITION OF GLUCOSE. A third series of experiments, in which quantities of 2 to 3 g. of glucose were similarly added during the post-adrenaline period was carried out in a similar way to the preceding* ones, and the results of seven such experiments are given in Table IV. TABLE IV.-ADRENALINE-DRIVEN HEART-LUNG PREPARATIONS. (Glucose added during the recovery period.) - - Blood sugar and blood lactate. Adrena- Adrena- adrenaline period. experiment. Re- Glul N.line line covery adeglycogen, No. period, rate, c; period, g.j100 g. mm. mg./hr. m 0n30 0 m. g o*0.0cb * *0 0* * * *0 0* *38 Four units insulin at commencement of recovery and two units 90 minutes later. With the exception of Expt. 583, where recovery was only for 47 minutes, these all showed return of at least half of the cardiac glycogen in two or three hours, and rough calculations from the blood sugar levels at the end of the two periods of the experiment show that glucose had been utilised in considerable amount. Taken together with the preceding series of experiments, we believe that these results leave no reasonable doubt as to the formation of heart glycogen from blood glucose. Addition of insulin during recovery did not apparently accelerate glycogen formation (Expt. 593). Attention may here be drawn to the remarkable difference between the effects of addition of lactate and addition of glucose on the efficiency

9 The Source of the Heart Glycogen of the heart previously exhausted by adrenaline. Fig. 3 shows that, as judged by the venous pressure, addition of glucose effected no improvement, whereas addition of lactate, provided it be made slowly, considerably improves the condition of the heart, as was shown for the normal heart by Bogue, Evans, and Hsu [1935]. 35 HOURS FIG. 3.-Showing the venous pressures in three experiments with the heart-lung preparation. In each case adrenaline (0.3 mg./hr.) was given for two hours, after which it was stopped and the mechanical work of the hearts reduced. In Expt. 575 no addition was made after the adrenaline period; in Expt. 579 lactate was added where shown; and in Expt. 585 glucose was added. Note the lower pressure after lactate addition. ADDITION OF BOTH LACTATE AND GLUCOSE. In some of the experiments, after driving the heart for two hours with adrenaline and heavy work, both glucose and lactate were added TABLE V.-ADRENALINE-DRIVEN HEART-LUNG PREPARATIONS. (Glucose and lactate added in recovery period.) Blood sugar and lactate. No. adrenaline period. c5 D6 C; c).40 ce 4 by) E experiment. C E c; ^ cq.0 Q - 0_ 4.~ -.--J period, min. g. Adrena- Adrenaline line period, rate, min. mg./hr. Glucose, Lactate, g. Gly. cogen, g.iloog Glucose and lactate added together, at intervals. 2 Glucose added first, then lactate. 3 Glucose first, then lactate. Eleven units insulin added during recovery. 4 After first addition of glucose and lactate. 5 Four units insulin added at 37 minutes of recovery period.

10 36 Bogue, Evans, and Gregory during the subsequent period of low work without adrenaline addition. It was thought that this procedure might perhaps give better recoveries of glycogen than were found when glucose alone was added, but as shown by the results given in Table V. this was not the case. For some cause, on which we are unable to throw any light, the recoveries of glycogen, though definite, were smaller than those obtained when only glucose was added, and it seems as though the utilisation (and probable combustion) of lactate for some reason hinders the laying down of glycogen. Addition of insulin in Expts. 594 and 600 gave slightly higher glycogen, but this may have been due to the adrenaline periods being shorter, though with more rapid addition, than was usual. (The actual duration of the adrenaline period is, however, of less importance than the behaviour of the venous pressure. We have usually found that when the venous pressure rises the glycogen has reached a low level.) INFLUENCE OF Low SUGAR AND HIGH LACTATE, OR OF HIGH SUGAR AND Low LACTATE, ON THE GLYCOGEN EXHAUSTION. Since it appeared that lactate, though used in large amounts, is not readily convertible into glycogen by the heart, it seemed possible that TABLE VI. HIGH LACTATE THROUGHOUT ADRENALINE PERIOD. Period Blood during and at Adrena- mean end of experiment. Lactate Recovery Heart E~xpt. line rate of added, period, glycogen, No. duration, dose, Glucose, Lactate, g. min. g./100 g. min. mg./hr. mg./100 c.c. mg./100 c.c /70 86/ A /12 63/ B /0 41/ A /18 55/ B /6 59/ Early failure with hoemopericardium. 2 Heart dilated and appeared near to failure. might nevertheless be able, if present in the blood in high concentra- it tion, to exert a glycogen-sparing action when the heart was driven hard by adrenaline and heavy work. A preliminary experiment, in which the adrenaline period was only short, seemed to encourage this belief (Expt. 601, Table VI.). In order to make the experiments more clear-cut it was thought desirable in some to keep the blood sugar as low as possible, and the experiments were therefore carried out as follows: Two heart-lung experiments were made consecutively on the same day; to the blood of the first preparation adrenaline was added by slow infusion in the

11 The Source of the Heart Glycogen usual way, and lactate was also added, at first rather quickly and then more slowly, at a rate comparable with its anticipated usage. After two hours, or earlier if a rise of venous pressure suggested impending deterioration, the experiment was terminated, the heart taken for glycogen, and the blood, now nearly sugar-free, was used for a second similar experiment, followed by a recovery period. The results are given in Table VI. and make it clear that the presence of lactate does not appreciably hinder the loss of glycogen which results from adrenaline administration. Expt. 601, already referred to, was evidently too brief to enable conclusions to be drawn. A similar series of experiments was then made in which glucose was added in such amounts as to give a hyperglycaomia throughout the adrenaline period. These experiments were carried out with the heartoxygenator instead of with the heart-lung preparation, in order to reduce the lactate formed by glycolysis and to which the lungs, if present, would contribute considerably. The results shown in Table VII. gave no convincing proof of glycogen-sparing action. 37 TABLE VIJ.-HIGH GLUCOSE THROUGHOUT ADRENALINE PERIOD. (Heart oxygenator preparation.) Adrena- Period Blood during and at Expt. line end of mean experiment. Glucose Heart NoE. No. duration, rate, ratea Glucose, Lactate, added, g. glycogen, g./100 g. mm Mg-/hr. mg./100 c.c. mg./100 c.c /53 24/ *39 131/55 21/ * /259 47/ DIscusSION. The results are, we think, sufficiently clear-cut to prove that the continued administration of adrenaline may rapidly deplete the heart of glycogen; moreover, deterioration and failure of the heart rapidly sets in when the glycogen reaches a low level, and it would seem that cardiac muscle is incapable of functioning in the absence of glycogen. Whether the lowering of glycogen under the action of adrenaline is merely due to the increased number of contractions, with correspondingly shortened duration of diastole, to which the high rate of mechanical work in the present experiments may have contributed, or whether it is a specific action of the adrenaline comparable to that on skeletal muscle [Cori and Cori, 1928] is a point which must be left for further investigation.

12 38 Bogue, Evans, and Gregory It is also clear that, after depletion by adrenaline, heart glycogen can rapidly be formed from glucose but not from lactate, though under the action of adrenaline it cannot be formed, or is broken down more rapidly than formed, even from glucose. It appears that the lactate, which is used in such large amount, is not converted into glycogen, but is probably combusted. This probability raises the question whether in the course of its breakdown glycogen passed through the stage of lactic acid. That it can do so under anaerobic conditions is probable, since large amounts of lactic acid are then given off from the heart muscle. If glycogen was also convertible into lactic acid under aerobic conditions this might forthwith be oxidised, together with lactate taken in from the blood-stream; but in that case we might reasonably expect that the process might be reversible when the lactate concentration was high, so that glycogen would be formed, or at least that a high concentration of lactate in the blood would somewhat delay the removal of glycogen by the action of adrenaline. That the latter is not the case does not negative the suggestion, however, because it has been shown that a high concentration of glucose is also unable to check the fall of glycogen under adrenaline. Yet, as previously shown [Bogue et al., 1935], the utilisations of both lactate and glucose are augmented by adrenaline. It has been shown by Evans, Grande, and Hsu [1935 a] that high blood lactate increases lactate usage and diminishes sugar usage, but that the reverse is not so generally true, since high blood sugar has only a small effect on the lactate usage, though increasing the sugar usage. It might be argued from this that when the lactate was high there was less uptake of sugar for conversion into glycogen, and this might be the reason why we obtained smaller increases of glycogen when both lactate and glucose were added than when glucose alone was given. CONCLUSIONS. 1. By continuous administration of adrenaline (together with high mechanical work) the glycogen content of the dog's heart is rapidly lowered. When it has all been exhausted failure suddenly occurs. 2. When the administration of adrenaline is stopped short of failure, and when the glucose and lactate of the blood have also been reduced to low levels, there is no recovery of glycogen; glyconeogenesis therefore does not occur, or is relatively slow. 3. Addition of lactate after glycogen depletion by adrenaline, if the blood glucose is low, leads to no recovery of glycogen in two hours. 4. Addition of glucose after adrenaline depletion and with low blood lactate results in considerable restoration of heart glycogen in two hours. The heart glycogen is therefore formed from glucose. 5. Addition of both glucose and lactate to adrenaline-depleted

13 The Source of the Heart Glycogen hearts leads to a smaller glycogen formation than is obtained by addition of glucose alone. This is perhaps correlated with the fact that high lactate depresses the sugar usage of the heart. 6. Addition of either glucose or lactate, so as to give large concentrations of either in the blood prior to the addition of the adrenaline does not hinder the glycogen depletion caused by adrenaline. The costs of the investigation were in part defrayed out of a grant from the Thomas Smythe Hughes Fund by the University of London to R. A. G., and of a grant from the Government Grants Committee of the Royal Society to J. Y. B. The authors wish to express their thanks for these two grants. 39 REFERENCES. BOGUE, J. Y., EVANS, C. L., GRANDE, F., and Hsu, F. Y. (1935). Quart. J. exp. Physiol. 25, 213. BOGUE, J. Y., EVANS, C. L., and Hsu, F. Y. (1935). J. Physiol. 84, 55 P. BOGUE, J. Y., and GREGORY, R. A. (1937). Unpublished. Shown at Physiol. Soc. meeting, 13th March BURN, J. H., and DALE, H. H. (1924). J. Physiol. 59, 164. CHANG, I. (1937). Quart. J. exp. Physiol. 26, 285. CORI, C. F., and CoRI, GERTY T. (1928). J. biol. Chem. 79, 309. EVANS, C. L., GRANDE, F., and Hsu, F. Y. (1934). Quart. J. exp. Physiol. 24, 283. EVANS, C. L., GRANDE, F., and Hsu, F. Y. (1935 a). Ibid. 24, 347. EVANS, C. L., GRANDE, F., Hsu, F. Y., LEE, D. H. K., and MULDER, A. G. (1935 b). Ibid. 24, 365. HOET, J. P., and MARKS, H. P. (1926). Proc. Roy. Soc., B, 100, 72. McGINTY, D. A., and MILLER, A. I. (1933). Amer. J. Physiol. 103, 712. VISSCHER, M. B., and MULDER, A. G. (1930). Ibid. 94, 630.