MINKOWSKI(l) found that after complete extirpation of the pancreas

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1 ON THE PRODUCTION AND UTILISATION OF GLY- COGEN IN NORMAL AND DIABETIC ANIMALS. BY E. W. H. CRUICKSHANK, M.B. (From the Institute of Physiology, University College.) I. On the glycogen of the liver. MINKOWSKI(l) found that after complete extirpation of the pancreas there was a rapid disappearance of glycogen from the liver, so that in animals killed on the second day or later after the operation the liver was practically free from this substance. The administration of glucose to such animals did not give rise to any accumulation of glycogen, the glucose administered passing entirely into the urine. On the other hand, if lavulose were administered there was at first no corresponding rise in the sugar excretion, and the animals killed shortly after the administration were found to have glycogen in their liver. On account of this statement of Minkowski, levulose has been administered in a number of cases of diabetes in man, in the hope that the patient would be able to utilise this sugar although unable to tutilise glucose. In such cases it has been found that the administration of levulose does not at first proportionally increase the output of sugar in the urine. The excretion however seems to be only retarded, since, after two or three days' administration of levulose, an equivalent amount of sugar, either as glucose or laevulose, is excreted in the urine. So far as I am aware the observation of Minkowski on the production of glycogen in the liver of depancreatised animals, as a result of the administration of laevulose, has never been repeated. I have therefore carried out a number of experiments on this subject, since a marked difference in the fate of laevulose and glucose respectively in the organism of diabetic animals would be of fundamental importance to our knowledge and understanding of the carbohydrate metabolism of the body. Methods. The experiments were carried out exclusively on female dogs of medium size, i.e. from 5 to 10 kilos. The depancreatisation was performed by Prof. Starling according to Hddon'sw method. In PH. XLVII. 1

2 2 E. W. H. CR UICKSHANK. this method the only ligature which is applied is that to the main pancreatic duct, the separation of the rest of the pancreas from the surrounding tissues being carried out by means of the fingers, the vessels connecting the pancreas with the surrounding parts being simply torn through. By this means it is possible to effect an absolutely complete extirpation of the pancreas in a very short time, the whole duration of the experiment, including preliminary anaesthetisation, and subsequent bandaging, only occupying about 30 minutes. The skin was sterilised by painting with iodine, and the ordinary aseptic precautions observed. Operating by this method it is really unnecessary to perform the extirpation in two stages. Suppuration, which was so constant a feature in Minkowski's experiments, practicallv never occurred. For this result, in all probability, the rapidity of the operation is responsible, the animal after recovery from the anesthetic probably having one or two hours during which its reparative powers are still normal, so that the processes of repair can set in before the anirnal becomes diabetic. The thoroughness of the operation is probably responsible for the very severe and rapidly fatal course taken by the diabetes which ensues. For the first two or three days after the operation the animal is normal in behaviour. It takes its food well and, apart from the rapid emaciation, presents little deviation from normal. It then becomes somewhat sleepy and stiff in its movements, and finally comatose. In most cases the animals were killed between the second and sixth day. Those kept till the seventh day were practically moribund, and there is no doubt that death would have ensued in most cases by the eighth or ninth day after the operation. The emaciation is conditioned in the first place by a rapid disappearance of fat from the subcutaneous tissues and abdominal depots. This fat disappearance is due to what has been called " fat mobilisation," the fat being carried in large quantities by means of the blood to the liver. From the second to the fifth days therefore there is a pronounced lipremia, and the liver becomes loaded with fat. This condition may last longer in animals which had at the operation a large store of subcuitaneous fat. In animals which are lean to start with, the condition of lipsemia disappears first and later on the liver gets small and loses a great part of its fat, though even in moribund animals the liver is more fatty than normal. A marked fatty infiltration may also affect the heart muscle, especially after four or five days. The animals from which the livers were taken were generally employed for other purposes, i.e. for the estimation of the respiratory quotient of the heart-lung preparation or for the measurement of the

3 UTILISATION OF GLYCOGEN. sugar consumption in the heart and lungs. The liver was therefore only removed after the animal had been anasthetised and the heartlung preparation made. It had thus been exposed to the action of the anesthetic and its circulation had been in most cases completely cut off for 15 or 20 minutes before it could be removed from the body. During this time no doubt a certain amount of glycogenolysis might occur, which would tend to reduce the amount of glycogen subsequently found but would not cause the disappearance of more than a small percentage of this substance. The figures for the glycogen content of livers must however be taken as minimal. The estimation of the glycogen was carried out in the following way:-50 grms. of fresh tissue were weighed as accurately as possible, and heated in a Jena flask with 50 c.c. caustic potash containing /0 KOH, for three to four hours in the water bath. The solution was then cooled, diluted with about 100 c.c. of water and pouired into 400 c.c. of 960/0 alcohol, the flask being washed out with a little alcohol. The alcoholic mixture was then allowed to stand over night to ensure complete precipitation of the glycogen. The supernatant fluid was then poured off through a fluted filter, the precipitate washed by decantation several times with 900/0 alcohol, two or three times with absolute alcohol and finally with ether, so that a comparatively lvhite precipitate was obtained. This was dissolved in abouit 200 c.c. of water, carefully neutralised with acetic acid, and 10 c.c. HCl (Sp. Gr. 1-19) were added, the solution being then heated in the water bath for at least three hours. The whole of the glycogen having thus been converted into glucose, the solution was neutralised with NaOH, and filtered into a 250 c.c. flask, the filter washed repeatedly and the volume made up to the mark with distilled water. Of this solutioln 10 to 50 c.c. were taken, according to the amount of sugar found by trial, and the glycogen estimated as glucose by Bertrand's method. In the case of the heart the cavities were washed free from blood and blood clot by rinsing in isotonic salt Eolution. Experimental results. I have arranged the results of my determinations in two tables. TABLE I. Glycogen in the livers of normal dogs fed on the ordinary diet. No. Condition of animal 0/oglycogen in liver Remarks 1 Normal 8*75 Fed on puppy biscuit. 2,, 5-72 Fed on meat and biscuit. 3,, 5.75,., 1-2 3

4 4 E. W. H. CRUICKSHANK. TABLE II. Diabetic animals fed during first day after operation on mitlc and afterwards on meat and pancreas. No. Condition of animal Ol glycogen in liver Remarks 4 Dog, 2 days diabetic *05-*025 In these experiments the liver was 5 Dog, 36 hours,, 266-*288 removed after complete isolation 6 Dog, 2 days,, 025-'037 of the heart had been effected, 7 Cat, 3 days,, 024 usually within half to three-quar- 8 Dog, 3 days,, 040 ters of an hour after commencement of ansesthesia. Note:-Where two figures are given these are the results of duplicate estimations. The diabetic animals were fed during the first day after the operation on milk and after that on meat (horse flesh). In many cases a certain amount of raw ox pancreas was minced up with the meat in order to aid digestion and absorption. It has been often shown that the administration by the mouth of pancreas has no effect on the course of the diabetes itself. It will be seen from Table II that the glycogen very rapidly disappears from the liver after the operation. 36 hours after the operation two estimations gave *266 and '288 o/o. Dogs killed on the second day after the operation varied between *02 and '04 0/5, i.e. there was practically no glycogen present. In Table III I give the results of a number of estimations on animals which had been fed by the stomach tube with glucose or with hevulose together with meat. The two experiments first in the list refer to normal dogs. The diabetic animals were killed between the third and the sixth day after the operation. From Table III it will be seen that in every case there was practically no glycogen in the liver, the amounts found varying between '03 and '18 0/0. It made no difference whether these animals received glucose or laevulose-in neither case was any accumulation of glycogen effected. After all, in view of the condition of the liver, this is what might be expected. It has been shown by Rosenfeld(3) that the fat content of the liver rises in the fasting animal presumably because of the loss of its glycogen store. If carbohydrates are given to a fasting animal, the fat conitent of the liver falls, and if both carbohydrates and fats are given, the liver stores the carbohydrate as glycogen in preference to the fat, so that there is in a sense an antagonism between the glycogen and fat content of the liver. The liver may be loaded with glycogen or with fat but never contains any quantity of both these substances simultaneously. All these diabetic animals developed a typical fatty liver and therefore it would seem difficult, if not impoible, for this

5 TABLE III. UTILISATION OF GLYCOGEN. 5 Animals fed by stomach tube with dextrose or levulose. %.0glycogen No. Condition of animal in liver Remarks 1 Normal dog Fed twice daily with 30 grms. dextrose, the amount given being in excess of that utilised. 2,,,, 575 Fed twice daily with 30 grms. Inevulose for three days. 3 Dog, 3 days diabetic 0504 Fed with 30 grms. dextrose at 10 a.m. and 4 p.m. for three consecutive days. 4,,,,,, 075 Fed, as in previous experiment, with lievulose. These dogs were both sick on the third day, i.e. the day of experiment. 5 Dog, 4 days diabetic *0586 Fed with 25 grms. dextrose twice daily for three days. Liver very fatty and friable. 6,,,,,, 0374 Fed with 25 grms. l2vulose twice daily for three days. These feeds were well retained. 7 Dog, 3 days diabetic.057 Fed with 25 grms. lvulose twice daily, and once on day of experiment. Liver fatty. 8,,,,,, *080 Fed with 25 grms. dextrose as in previous case. 9 Dog, 6 days diabetic *0372 1st day after operation, fed with 40 grms. lsvulose-the solution being taken without the tube. 2nd, 3rd, 4th and 5th days given 40 grms. per diem by means of the tube. Liver was very fatty. 10,,,,,, *051 Fed with 25 grms. levulose twice daily for three days: one day then missed, and fed again on day previous to experiment. Liver fatty. 11 Dog, 4 days diabetic *53 Fed with lievulose. Three feeds of 30 grms. each, with two-hourly intervals. Dog killed instantaneously two hours after last feed. Another lobe contained *18 In Exp. 11 the dog was fed by the stomach tube at 11 a.m., 1 p.m. and 3 p.m., receiving in all 90 grms. of leevulose. About 20 minutes after the second and third feeds the dog vomited a little fluid. It was killed instantaneously by a blow upon the head to avoid the possible source of error due to glycogenolysis occurring previous to the removal of the liver. The stomach and upper part of small intestine were found to contain a considerable amount of fluid, the stomach alone containing a large amount of semi-digested meat which had been given on the previous day. organ to store up glycogen in whatever form the carbohydrate is presented to it. I am at a loss to account for the divergence between my results and those obtained by Minkowski. It should be noted however that the dogs on which my estimations were made suffered from a diabetes which was apparently more rapid in its course and therefore severer than in those recorded by Mli nkowski, and this in spite of the total absence of sepsis in my cases. Is it possible that in Minkowski's cases

6 6 E. W. H. CR UICKSHANK. the extirpation of the pancreas was really not complete, so that the animals could live longer, could survive the loss of their fat even from the liver, and retain some vestige of the power of assimilating carbohydrates, so that the liver was able to build up a certain amount of glycogen from the l]evulose presented to it? I hope to test this possibility later on by investigating the effects of administering laevulose to animals with severe diabetes but in which a minute portion of the pancreas has been left. My estimations thus lend no support to the view that there is any fundamental difference between the fate of laevulose and glucose respectively in the normal or the diabetic animal. II. The glycogen of the heart. I have taken advantage of a number of experiments carried out in this laboratory on the metabolism of the heart-lung preparation (Knowlton and Starling) on normal and diabetic animals to determine the amount of glycogen in the heart under varying conditions. (1) NVormal animals. A certain number of determinations were made on the hearts of animals which died during the preliminary anaesthetisation before the experiment was commenced. The results, which may be taken as typical of the minimal amount present in the normal heart, are given in the following table: TABLE IV. Normal dogs. No. 0/0 glycogen in heart Remarks 1 *631 Died at commencement of experiment Died during isolation of the heart Died after isolation of the heart. 4 *460 Piece of heart taken after its isolation. 5 *630 Ditto Dog fed with 25 grms. dextrose twice daily for three days. 7 *360 Dog fed with leevulose as in preceding case. These isolated heart-lung exps. were continued for 2i hrs. It will be seen that the amounts vary considerably, between *631 and *306, the average amount being *463. Although I am unable to state now the exact condition of nutrition of these animals it does not seem that feeding has the marked influence on the amount of glycogen in the heart that it has on the glycogen content of the liver. Thus in two experiments normal dogs were fed with meat to which lhevulose or dextrose was added. These are the two animals referred to in Table III, in which the livers were found to contain 5 7 /0 of glycogen. The

7 UTILISA TION OF GL YCOGEN. hearts contained *306 and '360 0/0, figures which agree with the average obtained in the previous series of animals. There is evidence however that the glycogen content of the heart is altered by the nutritional condition of the animal. To determine whether in a normal animal fed solely on butter, the glycogen content of the heart and liver would fall and its place be taken by fat due to the condition of evident lipeemia and loss of glycogen from the lack of carbohydrate supply, a normal dog was taken and fed with fresh butter, about 100grms. being taken on three consecutive days: no other food was given. The dog was killed instantaneously and liver and heart taken at once. Weight of heart grms. Weight of liver ,, Weight of dog kilos. The liver constitutes 3'1 0/0 of body weight. Glycogen content of liver *26 0/0-*33 0/0. Glycogen content of heart '15 0/0. The glycogen of the liver had therefore been almost completely drawn upon for the energy requirements of the body, its place being taken by fat. The heart had also drawn upon its local store of glycogen, reducing it to *15 O/o within three days. While one can definitely state that there is an antagonism between the fat and glycogen content, one cannot as yet attribute the same to the heart muscle because naked eye appearances are deceptive, and such figures would require to be correlated with those of the fat content of both heart and liver. That such an antagonism should exist is highly probable, as there is no reason why the fat, which has migrated to the liver in fatty infiltration, should not overflow to the heart when the glycogen store of that organ and the body generally has been exhausted. On the other hand, there is evidence of well marked glycogenolysis occurring in the heart after death. In two experiments an artificial circulation had been kept up for three hours through the lungs by cannulae placed in the pulmonary artery and in the left auricle, so that the heart was kept warm in the chest but no circulation maintained through -its vessels. In these cases the amount of glycogen in the heart was found to be only '145 0/0 and '016 0/0 respectively. In another case the heart was divided in two parts, one half was treated at once, the other half was kept at about 350C. for three hours. The figures obtained in this experiment were: Heart treated at once /0 Heart kept 3 hours at 350 C. '08 0/0 7

8 8 E. W. H. CRUICKSHA.NK. I next sought to determine whether the glycogen of the heart is used up in the normal functioning of this organ. The following table gives the results of determinations of the glycogen in the heart which had been maintaining a circulation at a normal arterial pressure and with a normal outpuit through an isolated heart-lung preparation for varying times from 1 to 31 hours. TABLE V. No. Duration of exp. 0/0 glycogen Remarks 1 2 hours * hour *352 Heart fibrillated. 3 3i hours ' ,, ,, i,150 Dog received no food for 36 hours previous to experiment. Average '494. It will be seen that the figures obtained are very variable and do not show any marked deviation from the results obtained from the normal heart not subjected to experiment. On the other hand, it must be remembered that in practically all these experiments glucose had been added to the circulating blood, so that the serum of this blood contained from '4 to '6 0/0 glucose, i.e. an amount very considerably in excess of that present under normal circumstances. It is possible therefore that the heart was able to satisfy its carbohydrate needs at the expense of the circulating sugar without drawing on its local store of glycogen. It might in fact utilise some of the excess of the circulating sugar to add to its store. I see no method however by which one may decide this latter point, since it is impossible to take a sample of the heart muscle and analyse it for comparison with the results of the analysis of the heart muscle obtained at the end of the experiment. I thought however that I might decide the question whether the heart could draw upon its local store of glycogen by increasing the metabolic needs of this organ. This can be done by making the heart beat against an abnormally high pressure, i.e. 180 to 200 mm. Hg for three to four hours, and still better by stimulating the heart during this time by repeated doses of adrenalin. It has been shown by Evans that the respiratory exchanges of the heart are doubled or trebled under the influence of adrenalin. Of course to this latter method it might be objected that the adrenalin has a specific action on the glycogen,-to use the language of the Vienna school, that

9 UTILISATION OF GLYCOGEN. 9 it " mobilises " the glycogen of the heart muscle. If this were its sole effect the administration of adrenalin should increase the amount of sugar in circulation, whereas results obtained in this laboratory, which will be published shortly, have shown that the administration of adrenalin increases very largely the rate of disappearance of sugar from the blood in the heart-lung preparation. We may take it therefore that any action of adrenalin on the glycogen store of the heart will be in all probability occasioned by the increased functional capacity and excitatory activity of the muscle, induced by this drug. The results of these experiments are given in the following table: No A. Normal hearts worked by adrenalin. Duration of exp. 3 hours 4i, 4,, ,, &bove. No i, 1 hour 3 hours 0/0 glycogen *134 *269 *063 *056 TABLE VI. Amount of adrenalin administered C02 and adrenalin alternately. Increased blood-pressure only. 1 c.c. 1 : 10,000 adrenalin every 15 mins. 1st hour 1 c.c. 1 :10,000 adrenalin every 15'. 2nd,I I rd,, lc.c. 1:5,000,.. 4th,, 1c.c. 1:2,500,. Pulse beats per min. *053 Ditto. *020 1st hour 1 c.c. 1: 10,000 adrenalin every 15'. 2nd hour 1 c.c. 1: 5,000,... 3rd & 4th hrs. 1.c. 1: 2,500,... Pulse beats per min. '030 Ditto. *170 Cat. Died of asphyxia Dog. Heart kept at 240 beats per min. by frequent administration of 1:10,000 adrnln. B. Normal hearts worked by adrenalin, while blood sugar is maintained at normal or Duration of exp. 4i hours 3i,, 3 4i,, s/0 glycogen Amount of adrenalin and sugar added '134 C02 and adrenalin. 10 c.c. 10 %/0 glucose soln. '03 1st hour 1 c.c. 1 :10,000 adrenalin every 15'. 2nd,, Cc.. 1: 5,000,. 3rd,,,,,,, 10 c.c. 10 0/0 glucose soln. '13 1st hour 1 c.c. 1: 10,000 adrenalin every 15'. 2nd,... to.... 3rd & 4th hrs. 1 c.c. 1: 5,000,... At commencement of exp. 10 c.c. 10 0/0 solution of glucose added; after two hours 10 c.c. again added; after 3 hours 5 c.c. 10 0/0 glu. cose added.

10 10 E. W. H. CRUICKSHIANK. It will be seen that increased demands made on the heart muscle by prolonged hard work lead to a great diminution of the glycogen store, and that the exaggerated work of the heart induced by repeated doses of adrenalin causes an almost complet.e disappearance of the glycogen from the heart; and this occurs even when the percentage of sugar in the circulating blood is maintained at or above normal by the repeated addition of glucose to the blood in the course of the experiment. We are therefore justified in looking upon the glycogen in the heart as a local store, wbich can be drawn upon to furnish energy to the contracting muscle whenever the sugar in the blood becomes deficient, or when the requirements of the muscle are raised to an abnormal extent even in the presence of the ordinary amount of sugar in the circulating blood. Whether an increased amount of sugar in the blood leads to increased formation of glycogen in the heart, my experiments are not sufficient to decide. III. The diabetic heart. Estimations of glycogen in the hearts of diabetic animals are said to have shown that these hearts contain glycogen in somewhat larger quantities than those of normal animals. I have been unable to find the authority for this statement. Cammidge4) writes: " If the animal is killed a few days after the pancreas has been removed, the glycogen normally present in the liver and muscles is seen to have disappeared, or only to be present in traces, no matter whether the animal has been starved or liberally fed. It is usually found, however, in excessive quantities in some situations, such as the epithelium of Henle's tubes, the heart muscle, and the leucocytes, where it does not normally occur in abundance." I give here a table of experiments on the bearts of diabetic animals at various dates after the extirpation of the pancreas, the hearts having in most cases been worked in a heart-lung preparation for times varying from 1 to 4 hours. Additional glucose had almost always been added to the blood, makina the percentage about 0-6. In the first two, when the hearts contained over *9 0/0 glycogen, the animals died during the induction of anesthesia. In nearly all the cases however the amount of glycogen was high, the average being *706 0/0 as against *478 0/G obtained in the experiments on normal hearts. We may say then that the diabetic heart as a rule contains an increased amount of glycogen, showing that there is no lack of power to store up this substance in the heart, whatever may be the case in the liver. In

11 UTILISATION OF GLYCOGEN. 11 No. Duration of diabetes 1 3 days 2 5 it ,, is,, Conditions Isolated heart lung,...,... TABLE VII.,,,,9 2 hrs. Isolated heart lung 4 hrs. Rs qo ep Resp. quot. exp. 4 1&,, Artificial heart, isolated lung Isolated heart lung?9 1, Resp. quot. exp. Isolated heart lung Duiratinn Of exp. /eivcnen in heart Remarks - *96 Died at commencement of anesthesia. - *925 Died during isolation of the heart. 02 L l1 Ddt m Ad 1-g ne77 21i 'L0 *89 *631 Died at commencement 3,, * *600 * of experiment. Killed for blood. Hormone given during exp.=j of dog's pancreas Heart failed after much stress, very fatty. I hr- -*805 3 Is 3,, Average for unfed diabetic hearts= -706 grns. 0/ Heart rather fatty. the case of the heart it seems that there is no such marked antagonism between the fat content and the glycogen content, as has been described for the liver. Thus the hearts obtained from Exps. 13 and 16 were both extremely fat, having a greyish white appearance, yet they contained -26 o/0 and -365 /o glycogen respectively, i.e. rather less than half the amount found in hearts which were to all appearance normal. The excessive amount of glycogen as compared with normal dogs might be ascribed either to the chronic hyperglycaemia present in the diabetic animals or to a lack of power on the part of the heart muscle to convert the glycogen into sugar or to utilise it for its metabolic needs. A certain amount of support is lent to the former of these two suggestions by the result obtained by feeding diabetic animals with laevulose or glucose. Although in the following table in which this feeding was carried out, the amounts of glycogen in the heart muscle vary considerably, the two highest figures I have ever obtained, namely, 1-60/o and 1P30/0 of glycogen, occur in this series of experiments. That the increased amount of glycogen in these hearts is not due to a failure of power to convert it into sugar is shown by the following

12 12 No. Time after op. 1 5 days 2 5-,, 3 4 3,, 3 Pt 5 3 pi 6 7 4,, 4,, 8 6 it 9 3,, It 3 it 12 3,, 13 4,, E. W. H. CRUICKSHANK. Nature of exp. Conditions Isol. heart lung,,i I,, Resp. quot. Isol. heart lung TABLE VIII. Duration e/9 glycogen of exp. m heart Nutriment, meat, U. 2 hrs. *644 Dog given 100 grms. glucose but was sick afterwards. 2,, * grms. livulose vomited 20 mine. after feed. 2,, * grms. dextrose. 2,, * grms. levulose. Dog sick and miserable. 2,, * c.c. 10 e/0 glucose added to blood. 4,, * grms. dextrose twice daily for 3 days. 4,, grms. livulose twice daily for 3 days. 5,, grms. nlvulose twice daily for 3 days. 5,, * grms. livulose twice daily for 2 days, and 30 grms. on day of experiment. 4 *724 I Fed with dextrose as above. 2i it *695 Glucose 30 grms. twice daily for 3 days. i 19,, grms. glucose: 3 days. *78 90 grms. livulose in 1 day. results obtained from a diabetic heart, in which half the heart was treated immediately on removal from the body, while the other half was kept at 350 C. for two or three hours. The post-mortem disappearance of glycogen thus seems to occur as rapidly in the diabetic heart as in the normal heart. Heart treated at once *795 0/0 Heat kept at for 3 hours *130 o/0 On the other hand the experiments of Knowlton and Starling and of MacLean and Smedley(s) seemed to indicate that the heart muscle in diabetic animals loses partially or completely its power of utilising glucose and presumably therefore glycogen, and this conclusion would seem to be borne out by the high percentage of glycogen found in the heart muscle even after experiments lasting as long as four and a half hours. But if glycogen can be formed by the diabetic heart from the sugar of the circulating blood, we cannot decide the question whether it can be used up unless we push the functional activity of the heart to the limit of its capacity. In the diabetic heart therefore, as in the normal heart, a certain number of experiments were carried out in which the heart was made to beat for three or four hours against a very

13 UTILISATION OF GLYCOGEN. high arterial resistance, corresponding to an arterial pressure of 180 to 200 mm. Hg, and other experiments in which the cardiac activity was stimulated to its utmost by repeated doses of adrenalin. The results are given in the following table: Days TABLE IX. Diabetic hearts worked. Duration No. after op. Nature of exp. of exp. l/0 glycogen Remarks 1 5 Isol. heart lung 4 hrs. '1 1 c.c. 1: 10,000 adrenalin every 15 mins. 2 2,, 4,, 02 lst hour 1 c.c. 1:10,000 adrenalin every 15 mins. 2nd hr. 1 c. 1: 5,000 ditto. 3rd&4thhrs. lc.c. 1:2,500do. 3 3,, 3,, c.c. 1:10,000 adrenalin every 15 mins. for 2 hrs., then 1 c.c. of 1: 5,000 during last hour. 10 c.c. 10 /o glucose at commencement. 10 c.c. 10 O/l glu. cose after 2 hrs. It will be seen that when the heart is stimulated in this way the results obtained are the same as with a normal heart. Glycogen under these conditions disappears almost completely from the heart muiscle. Since the sugar also disappears from the blood we must ascribe the disappearance to the actual consumption of the glycogen and not simply to its hydrolysis, and we must conclude that the diabetic heart, like the normal heart, can under appropriate circumstances use its stored up glycogen for the pturposes of its metabolism. Whether the power of the diabetic heart to use its glycogen is quantitatively inferior to that of the normal heart my experinments are not sufficient to decide. It is certain however that there is no fuindamental difference in this respect between the normal and the diabetic heart. CONCLUSIONS. 1. The observations of other workers are confirmed to the effect that there is a rapid and almost complete disappearance of glycogen from the liver after extirpation of the pancreas, the liver becoming loaded with fat. 2. Iam unable to confirm Minkowski'sstatementthatadministration of lovulose to diabetic animals leads to storage of glycogen (provided that the extirpation of the pancreas is complete). 3. The heart muscle contains normally a variable quantity of glycogen, amounting on the averaae to about 0-5e/0. 13

14 14 E. W. H. CRUICKSHANK. 4. This amount may be somewhat increased by copious carbohydrate food, and may be diminished by a diet devoid of protein and carbohydrate. 5. Under conditions of increased activity, the heart may uise up nearly the whole of its stored glycogen in a few hours. 6. In pancreatic diabetes the heart muscle contains considerably more glycogen than in normal dogs. 7. As in the normal heart, practically the whole of this glycogen may be used up during activity of the heart, especially if this be evoke(d by adrenalin. 8. Both the normal and the diabetic heart contain after death a glycogenolytic ferment, so that glycogen rapidly disappears if the heart be kept warm after cessation of the circulation. I wish to express my thanks to Prof. Starling for much valuable advice given during the couirse of this work. The cost of this investigation has been defrayed by a grant from the Government Grant Committee of the Royal Society. REFERENCES. (1) Minkowski. Arch. f. exper. Path. u. Pharm. xxvi. S ; xxxi. S (2) Hedon. Compt. Rend. du VIlle Congres intern. de Physiol. 1911, x. pp (3) Rosenfeld. Ergeb. der Physiol. 2. i. S (4) Cammidge. Glycosuria and Allied Conditions, p (5) MacLean and Smedley. This Journal, XLV. p