accompanied by a more rapid flow of secretion. But after administration

Transcription

1 612.34: THE EFFECT OF INJECTED INSULIN ON THE STORAGE OF GLYCOGEN IN THE PANCREAS AND LIVER. By CATHERINE 0. HEBB. From the Department of Physiology, McGill University, Montreal, Canada. (Received for publication 1 1th Augu8t 1937.) IN a previous communication the writer has presented detailed experimental evidence to show that after administration of insulin in the rabbit the reaction of the pancreas to electrical stimulation of the vagal nerves is remarkably altered [Hebb, 1937]. When no insulin has been given, such stimulation invariably produces a marked increase in the discharge of enzymes from the gland, this increase in most cases being accompanied by a more rapid flow of secretion. But after administration of insulin (from 10 to 40 units per kg. body-weight) the glandular nervous mechanism is altered in some way, so that parasympathetic stimulation is attended either by only a very slight augmentation of the secretory activity or, more often, by an inhibition of the secretion. The change thus produced by insulin cannot be said to be due to its having altered the character of the nervous impulses, since it has been shown that insulin similarly reverses the response of the pancreas to physiological doses of acetylcholine (i.e. the amount of acetylcholine which, when given intravenously, exerts on the pancreas a secretory effect equal in magnitude to that produced by weak stimulation of the vagal nerves-for rabbits this dose is approximately mg. per kg.). It was also shown that the inhibitory effect of insulin could not be due to depletion of the secretory products of the acinous cells, since at any time after the administration of insulin intravenous injection of dextrose brings about an immediate restoration of the normal secretory activity with an increased discharge of enzymes. Injection of dextrose not only causes a recovery of the secretion after its inhibition by insulin, but when a quantity of dextrose sufficient to produce hyperglyceemia is injected the response of the pancreas to parasympathetic stimuli is no longer inhibited, the secretory effect of these stimuli being once more normal. The most probable explanation of this inhibitory effect of hypoglyeawmia seems to the writer to be that it depends upon some interference with the normal carbohydrate metabolism of the pancreas.

2 238 Hebb There is some indirect evidence to support this theory. In the first place, the results of Still and his co-workers [cf. Still, Bennet, and Scott, 1933, and Gerard and Still, 1933] indicate that the energy by which the pancreatic secretion is maintained is produced by the oxidation of carbohydrates within the pancreas. Secondly, it has been ascertained by the writer in experiments, the results of which are not yet published, that poisoning with iodoacetic acid produces effects on the pancreatic secretion similar to those produced by administration of massive doses of insulin. In view of these considerations it seems not improbable that inhibition of the pancreatic secretion by means of the injection of insulin may be due to disturbance of the normal utilization of carbohydrates in the pancreas. The experiments described below represent a preliminary step towards determining how precisely insulin does affect the carbohydrate metabolism of the pancreas. An attempt has been made to investigate the effect of the blood-sugar level on the process of glycogenesis in the resting gland and the relation of this process to the carbohydrate metabolism of the body as a whole. METHODS. The experiments were all of the acute type and were performed on dogs, the pancreas in these animals being a relatively large organ, from which numerous tissue samples may be removed without serious impairment of its function and without detriment to the blood supply of the remaining tissue. In the majority of the experiments the anesthetic used was nembutal (Abbott's '844' sterile solution), an initial dose of 0 5 c.c. per kg. bodyweight being given intraperitoneally. Control experiments were carried out, in which the spinal cord was cut (immediately below the medulla) during brief ether anesthesia. Reference will also be made to a small group of experiments in which other anaesthetics were employed, viz. chloralose-0d1 g. per kg. in 1-4 per cent. solutioninjected intravenously, urethane-1-0 g. per kg. in 14 per cent. solution-injected intravenously, or sodium amytal (prepared from the Eli Lilly product, iso-amyl-ethyl barbituric acid)-0-5 c.c. per kg. in 10 per cent. solution-injected intraperitoneally. The animals were kept on a diet of water, meat, purina, oatmeal porridge, and milk. Previous to an experiment the dog was fasted for a period of from 12 to 48 hours, receiving only water during this time. A routine operative procedure was followed. After the anesthetic had taken effect tracheotomy was performed. If desirable, the vagal nerves were isolated in the neck region and cut. A cannula was then inserted in the right carotid artery for the removal of blood samples, and another in a branch of the jugular vein for injection purposes.

3 Effect of Injected Insulin on Storage of Glycogen in Liver 239 The abdomen was opened by means of a short mid-line incision and the main pancreatic duct was cannulated. After the completion of the preliminary operation described above the animal was left undisturbed for a period of 15 to 30 minutes. Then the first samples of arterial blood and of tissue were removed. During the course of the experiment, which was usually continued for 6 hours, samples of tissue were taken at intervals of 2 or 3 hours, while blood samples were withdrawn every hour. The blood sugar was determined according to Somogyi's [1930] second modification of the original Hagedorn-Jensen method. The quantitative estimation of glycogen was carried out according to Good, Kramer, and Somogyi's [1933] modification of the original Pfiuger method. The glycogen determinations were always made in duplicate. In the determination of glycogen changes occurring in a single animal under the conditions of acute experiment it is possible for many errors to arise, some of which have been pointed out by Cori [1931]. Glycogenolysis may occur in the tissue as a result of bleeding, cauterization, or other injuries, while other changes are often brought about by the anaesthetic or by reflex secretion of adrenaline. Moreover, since in the present experiments it was practically impossible to analyse whole organs at once, and since the glycogen concentration was known to vary in different parts of the same organ, it was apparent to the writer that exact knowledge of the total content of glycogen in any one organ or group of organs analysed could not be claimed. Many possible sources of error were eliminated, however, by the performance of numerous control experiments, by making duplicate determinations whenever possible, and by rigid adherence to the same procedure in the taking of tissue samples. Two samples were taken from the pancreas (each approximately 1 g. in weight). Since in any one gland the widest variation in the glycogen concentration was found to be the difference between the concentrations at the splenic end and the duodenal end respectively, one sample was removed from each of these regions. The tissue was held lightly between forceps while the blood-vessels supplying it were ligated, and immediately afterwards it was excised and deposited in caustic potash solution. The time occupied by ligation of the bloodvessels and depositing of the tissue in the potash solution was always between 5 and 8 seconds. Duplicate samples were also taken from the liver, the two pieces being obtained from different lobes. The time required for their removal was always between 4 and 6 seconds. Pieces of liver removed from the same lobe were found to contain almost equal amounts of glycogen, while samples removed simultaneously from different lobes might show a divergence of 5 to 10 per cent. in the concentration of glycogen. In view of this possible divergence, duplicate determinations

4 240 Hebb were made for separate lobes of the liver so that the mean value obtained represented more nearly the true figure for the percentage of glycogen present in the whole liver. The samples removed weighed approximately 1 g. each. Bleeding was checked with a cautery. Tissue for the determination of muscle glycogen was obtained from the muscles of the hind legs. Duplicate determinations were made on tissue obtained from bilateral pairs of muscles. The sample for each determination weighed 1 g. or more. Removal of the tissue was effected within 6 to 9 seconds. Bleeding was prevented by means of ligatures. The differences between individual animals and the errors liable to occur in this type of experiment have prevented the results from being absolutely accurate. However, the conclusions drawn from the data obtained are based on estimations of relative rather than of absolute changes. EXPERIMENTAL RESULTS. A preliminary series of experiments was first carried out with the object of determining the normal range of variation in the concentration of the pancreatic glycogen and also how this would be affected by the different methods of anesthetization mentioned above. This was the more important since no data on the glycogen content of the pancreas could be found in the available literature. As judged by the amount of glycogen present in samples of tissue removed from the pancreas immediately after the short preparatory operation, the concentration of glycogen in the pancreas of the dog varies between 0x10 and 0*30 g. per 100 g. of tissue. Under the various sets of experimental conditions here encountered the lowest concentration recorded was 0'05 g. per cent. and the highest 0 45 By means of control experiments it was found that each one of the different anesthetics employed produced slight but characteristic changes in the concentration of the pancreatic glycogen. For example, when a dog was anesthetized with amytal the glycogen concentration invariably diminished, showing a reduction of from 10 to 20 per cent. in 5 or 6 hours. On the other hand, chloralose anesthesia was always associated with an increase in the glycogen concentration, so that by the end of 6 hours the increase was often as great as 25 per cent. On section of the spinal cord (with brief ether induction), if the concentration of the pancreatic glycogen had been originally less than 0*2 g. per cent., it increased sometimes quite markedly, while if it had at first been higher than 0X2 g. it remained fairly constant for 5 or 6 hours, with -a slight fall perhaps of not more than 5 per cent. in that time. Animals anesthetized with nembutal showed very little loss or gain of pancreatic glycogen, so that the concentration was usually maintained at an even level during 6 hours of anesthesia.

5 Effect of Injected Insulin on Storage of Glycogen in Liver 241 Fasting produced no diminution of the pancreatic glycogen unless it was prolonged for 36 hours. This conclusion was based on the results of 30 experiments in which the length of the fasting period before each experiment had been carefully controlled. The following were the average figures for pancreatic glycogen after different lengths of fast: 12 hours' fasting, 0-19 (8 experiments); 24 hours' fasting, 0-19 (8 experiments); 36 hours' fasting, 0-17 g. per cent. (7 experiments); 48 to 72 hours' fasting, 0-12 (7 experiments). These values were taken only as a general index of the effect of fasting, since the individual variations encountered in different animals were so great that it was difficult to estimate the effect that fasting might produce on a single animal. When these control observations had been completed, experiments were undertaken for the purpose of determining what effect the level of the blood-glucose might exert on the storage of the pancreatic and the hepatic glycogen. It was found that experimental raising of the TABLE I. Three Experiments. Aniesthetic, nembutal. Vagal nerves cut. Amount of dextrose injected Concentration of glycogen, g. per 100 g. pancreas. per kg. body-weight per hour in each experiment.afe Control. 6 hours. Difference. (1) 1 g. (5 c.c. 20 per cent. solution) (2) 2 g. (10 c.c. 20 per cent. solution) (3) 2 g. (10 c.c. 20 per cent. solution) blood-sugar by the intravenous injection of dextrose increased the concentration of glycogen in the pancreas. When dextrose was continuously injected over a period of 6 hours there was a continuous deposition of glycogen in the pancreatic tissue, so that at its final level the concentration was often two or three times greater than at the outset. With the introduction of a sufficient amount of dextrose the rate of deposition was such that the glycogen concentration in some cases attained a level 50 per cent. higher than the highest recorded for uninjected animals. The magnitude of the increase was found to be proportional to the amount of dextrose per kilogram of body-weight injected every hour. As shown in Table I., in one experiment the injection of 1 g. of dextrose per kilogram per hour produced in 6 hours a difference of +0 1 between the initial and the VOL. XXVII., NO

6 242 Hebb final level of the glycogen concentration; in the two other experiments, where twice as much dextrose was administered, the differences between the initial and the final levels were and From these figures it may be seen that, although not strictly quantitative, there was a definite relation between the amount of sugar injected and the amount deposited as glycogen in the pancreas. TABLE II. Experiment 26.-Dog, 9-1 kg. Fasting 24 hours. Section of spinal cord (under ether). Vagal nerves cut. Dextrose only. Experiment 19.-Dog, 857 kg. Fasting 36 hours. Section of spinal cord (under ether). Vagal nerves cut. Dextrose and insulin. 6 hours' continuous intra- Experimental 6 hours' continuous intra- venous injection of 20 per procedure. venous injection of 20 per cent. dextrose solution: total cent. dextrose solution: total dextrose given, 60 g. dextrose given, 20 At beginning of 1st and 4th dextrosegie, 2g. hours 10 units of insulin injected. Total Total Control. After After differ- Control. After After differ- 3 hours. 6 hours. ence in 3 hours. 6 hours. ence in 6 hours. 6 hours. Pancreaticglycogen, Liver glycogen, Blood sugar, m The intravenous administration of dextrose in dogs previously injected with insulin" did not promote the formation of pancreatic glycogen as readily as it did in uninjected controls, as may be seen from Tables II. and III. In the two experiments of Table II. the spinal cord was cut during brief ether anesthesia. In one experiment (No. 26) the administration of 20 g. of dextrose over a period of 6 hours was accompanied by an increase of 0-05 in the concentration of the pancreatic glycogen. In the other experiment (No. 19) the injection of three times as much dextrose in addition to 20 units of insulin produced an increase of only I should like here to express my thanks to the authorities of the Connaught Laboratories, University of Toronto, for kindly supplying me with insulin free of charge.

7 Effect of Injected Insulin on Storage of Glycogen in Liver 243 The results of the two experiments shown in Table III. confirmed the above observations. In these cases the animals were anesthetized with nembutal and each received a total of 100 g. of dextrose by continuous injection during 6 hours. In one experiment (No. 30) no insulin wras administered to the animal, and at the end of 6 hours the total difference in the concentration of the pancreatic glycogen was In the other experiment (No. 33) a dose of TABLE III. Experimslent 30. Dog, 8-2 kg. Fasting 24 hours. Nembutal, 5 c.c. Vagal nerves cut. Dextrose only. Experinm ent 33. Dog, 6 kg. Fasting 36 hours. Nembutal, 3 c.c. Vagal nerves cut. Dextrose and insulin. Experimental procedure. 6 hours' continuous intravenous injection of 40 per cent. dextrose solution: total dextrose given, 100 g. 6 hours' continuous intravenous injection of 40 per cent. dextrose solution: total dextrose given, 100 g. At the beginning of 1st hour insulin injected (40 units intramuscularly and 40 units intravenously). I Total Total After After difference in After After Control. differ-i 3 hours. 6 hours. ence in 6 hours. Control. 3 hours. 6 hours. 6 hours.' Pancieatic glycogen, 0-18 Liver glycogen, Bloodl sugar, le 1* * units of insulin waas injected before the removal of the first tissue samples, and in this case, although actually a larger amount of dextrose per kilogram was given, the total difference in the glycogen concentration was only per cent. It will be observed in Tables II. and III. that changes in the liver glycogen are also recorded. With the administration of dextrose alone there was a distinct increase of glycogen in the liver (Experiments 26 and 30), while the administration of dextrose and insulin in one case increased the hepatic glycogen (Experiment 33) and in the other diminished it (Experiment 19). The observation that injection of dextrose increased the deposition of glycogen in the liver was confirmed il several other experiments. However, as the results of Experiments

8 244 Hebb 19 and 33 indicate, the effects of the injection of sugar in insulinized animals were more variable. The changes observed in the -liver glycogen after the injection of insulin in a number of experiments may be summarized as follows: When no dextrose was administered a considerable loss of glycogen occurred (one experiment); if there was moderate hypoglyceemia in spite of the addition of dextrose, some loss of glycogen still occurred (three experiments); and when sufficient sugar was injected to produce marked hyperglycaomia there was a considerable increase in the deposition of glycogen (three experiments). Under the last-mentioned of these conditions it was occasionally found that the rise in the concentration of glycogen in the liver was greater than that produced by administration of dextrose alone. Thus insulin caused glycogenolysis in the liver only when there was an insufficiency of glucose in the blood, and so the occurrence of glycogenolysis after insulin injection is a secondary effect due to hypoglycemia. In these experiments the point of attack of the injected insulin appeared to be primarily extra-hepatic, and by analogy with the experiments of Cori [1931] and others it seemed probable that the chief effect of insulin injection was to increase the uptake of glucose (i.e. for glycogen formation and oxidation) by the muscles (cf. Experiments 34 and 35, Table IV.). Thus when there was not sufficient glucose in the blood to meet the requirements of the muscles, the liver glycogen was drawn upon for the formation of extra glucose. Conversely, when sufficient glucose was added to the blood to supply the needs of the muscles, the liver was not depleted of its glycogen, and, provided there was an excess of glucose, the organ readily increased its store of glycogen. The diminished ability of the insulinized animal to store glycogen in the pancreas may have been due to similar causes. It may be supposed that the insulin normally secreted by the pancreas aids the storage of glycogen in that organ as well as in the muscles, but that, when injected, it affects the muscles primarily, so that the increased consumption of glucose by them diminishes the amount available for the pancreas. In the production of insulin the pancreas would, of course, benefit from its own secretion, so that when such a secretion occurred, as, for instance, after the injection of dextrose, glycogen would be readily formed both in the pancreatic gland and in the muscles. Furthermore, there is the additional possibility that, during its secretion, some of the insulin might be diffused directly from the islets of Langerhans into the acinous tissue of the pancreas, with the result that the gland as a whole would be affected by a discharge of insulin more quickly than other organs. Some confirmation of this hypothesis was obtained from further experiments. In terms of the percentage increase over the original concentrations of glycogen it was found that the injection of dextrose

9 Effect of Injected Insulin on Storage of Glycogen in Liver 245 alone resulted in a slightly greater deposition of glycogen in the pancreas than in the musculature of the legs; but when the animals were first injected with insulin-40 units intravenously and 10 units in the musculature of each leg-the administration of sugar scarcely affected the pancreatic glycogen, while it brought about a much greater deposition of glycogen in the muscles than when insulin had not been given. In Table IV. the protocols of Experiments 34 and 35 are shown. TABLE IV. Experiment 34.-Dog, 4-8 kg. Fasting 24 hours. Nembutal, 2.2 c.c. Vagal nerves intact. Experiment 35.-Dog, 2-6 kg. Fasting 18 hours. Nembutal, 2.4 c.c. Vagal nerves intact. Dextrose only. Dextrose and insulin. Experimental procedure. 3 hours' continuous intravenous injection of 20 per cent. dextrose solution: total dextrose given, 20 g. 3 hours' continuous intravenous injection of 20 per cent. dextrose solution: total dextrose given, 20 g. At the beginning of 1st hour insulin injected (40 units intramuscularly and 40 units intravenously). Control. After 3 hours. Total difference in 3 hours. Percentage increase. Control. After 3 hours. Total difference in 3 hours. Percentage increase. Pancreatic glycogen, Liver glycogen, Muscle glycogen, Blood sugar, m In Experiment 34 it may be seen that the injection of 20 g. of dextrose in 3 hours increased the concentration of glycogen by 31 per cent. in the pancreas, by 218 per cent. in the liver, and by 26 per cent. in the musculature. In Experiment 35, after the injection of insulin 3 hours of sugar injection did not increase the glycogen of the pancreas, nor to any extent that of the liver, but it did increase the muscle glycogen by 79 per cent. (The muscular tissue for the glycogen determinations was taken from different muscles of the legs from those injected with insulin.) The observation that administration of insulin promotes a more

10 246 Hebb rapid deposition of glycogen in the muscles and diminishes the rate of glycogen formation in the pancreas was confirmed in several other experiments. In all of these it was found that injection of dextrose alone increased the glycogen concentration both of the muscles and of the pancreas; but, when dextrose was injected into an animal to which insulin had previously been administered, the formation of glycogen in the muscles was proportionately much greater, while in the pancreas it was proportionately much less. Moreover, in two further experiments it was observed that administration of insulin without dextrose slightly increased the muscle glycogen, while not affecting the pancreatic. In these cases the decrease of glycogen in the liver was more than sufficient to account for its increase in the muscles. Therefore it may be concluded that the main cause of the depression of glycogenesis in the pancreas is the increased uptake of glucose by the muscles. DISCUSSION. The experimental work which is reviewed above constitutes the first step in an investigation of the carbohydrate metabolism of the pancreas. Its chief object has been to determine what conditions affect the formation and storage of glycogen by the pancreas and what relation these processes bear to the carbohydrate metabolism of the body as a whole. From this preliminary study two important conclusions may be drawn. In the first place, it appears that the rate of pancreatic glycogenesis depends upon the amount of glucose available to the gland from the blood-stream. Thus, when the level of the blood-sugar is. normal, the concentration of glycogen in the pancreas remains constant for a fairly long time (up to 36 hours), while experimental raising of the blood-sugar, by the injection of dextrose, increases the glycogen concentration of the pancreas at a rate which is directly proportional to the amount of dextrose given. Secondly, it is shown that the extraneous addition of insulin retards the formation of glycogen in the pancreas, mainly by diverting a larger proportion of the available glucose to the muscles. This hypothesis that injected insulin affects glycogen formation in the pancreas only indirectly, by causing hypoglyceemia to intervene, is in harmony with the observation that insulin, when injected without administration of dextrose, does not bring about any change in the concentration of the pancreatic glycogen. In other words, injected insulin does not diminish the glycogen already formed by the pancreas-it only prevents, wholly or partially, the formation of new glycogen by that organ. In view of previous evidence [Hebb, 1937] that insulin administration in the rabbit inhibits the response of the pancreas to secretory

11 Effect of Injected Insulin on Storage of Glycogen in Liver 247 stimuli, the next point to be considered is whether this effect may be attributed to a disturbance of the normal carbohydrate metabolism of the gland. Recent researches on the metabolism of the digestive glands have shown that there is a striking similarity between the metabolic changes occurring in muscles during their contraction and the metabolic changes that occur in the digestive glands when they are actively secreting. With respect to the salivary glands, it has been demonstrated by Hober and Ferrari [1933] and by others that the energy for the maintenance of secretion is ultimately obtained through the oxidation of carbohydrates; and Northup [1935] has estimated the rates of glycogenolysis and of lactic acid and phosphoric acid production in the submaxillary gland during secretion. Concerning the metabolic exchanges of the pancreas there is as yet little knowledge. However, Still, Bennet, and Scott [1933] have studied the respiratory metabolism of this gland, and they are of opinion that the process is maintained at the expense of carbohydrate oxidation. If this is true, and there seems to be so far no serious reason for disputing it, one may then assume that, e.g., in the rabbit, where pancreatic secretion is continuous, the pancreas must be continually utilizing carbohydrates and its supply of carbohydrates must be constantly renewed. However, as has been shown above, the administration of insulin in large doses retards glycogenesis in the pancreas; therefore such administration would soon result in a serious depletion of the pancreatic glycogen and consequently in reduced secretory activity of the cells. The hypothesis which has just been advanced to explain the inhibitory effect of insulin on pancreatic secretion is only a tentative one. These studies, which are a necessary preliminary step in the investigation of the problem, are being continued, and it is hoped that in a short time it will be possible to publish results of a more definite nature. SUMMARY. 1. In dogs, anaesthetized with amytal, nembutal, or chloralose, and in dogs whose spinal cord has been sectioned, the concentration of glycogen in the pancreas varies between 0410 g. and 0 30 g. per 100 g. of gland. 2. Fasting does not diminish the concentration of the pancreatic glycogen unless prolonged for 36 hours or more. 3. Continuous intravenous administration of dextrose increases the concentration of the pancreatic glycogen at a rate which bears a definite relation to the amount of dextrose injected per kilogram body-weight per hour. 4. The administration of insulin retards glycogenesis in the pancreas. 5. The injection of dextrose alone readily increases the storage of hepatic glycogen.

12 248 Effect of Injected Insulin on Storage of Glycogen in Liver 6. The injection of dextrose along with insulin will increase the glycogen in the liver only if sufficient sugar is administered to prevent hypoglycaemia. 7. Since the administration of insulin has been found to increase the deposition of glycogen in the muscles, it is concluded that injected insulin has primarily a peripheral point of attack, and that it retards glycogenesis in the pancreas by increasing the uptake of glucose by the muscles. The expenses of this research were defrayed by a grant obtained from the Banting Research Foundation, whose assistance is gratefully acknowledged. The writer wishes to express her sincere appreciation of the helpful advice and encouragement which she has received from Dr. B. P. Babkin, under whose direction the investigation was carried out. She is indebted to Miss J. F. Oswald for help in the preparation of this manuscript for press. REFERENCES. CORI, C. F. (1931). Physiol. Rev. 11, 143. GERARD, R. W., and STILL, E. U. (1933). Amer. J. Physiol. 108, 232. GOOD, C. A., KRAMER, H., and SOMOGYI, M. (1933). J. Biol. Chem. 100, 485. HEBB, C. 0. (1937). Quart. J. exp. Physiol. 26, 339. HOBER, R., and FERRARI, R. (1933). Klin. Wschr. 12, 433. NORTHUP, D. (1935). Amer. J. Physiol. 114, 46. SOMOGYI, M. (1930). J. Biol. Chem. 86, 655. STILL, E. U., BENNET, A. L., and SCOTT, V. B. (1933). Amer. J. Physiol. 106, 509.