THE INFLUENCE OF INSULIN ON GLYCOGEN DISTRIBU- TION IN MARINE FISHES BY R. W. ROOT, F. G. HALL, AND I. E. GRAY (From the Zoological Laboratory of Duke University, Durham, North Carolina) (Received for publication, January 19, 1931) INTRODUCTION The problem of the mechanism of insulin action has received considerable attention in the past few years. Explanations have been sought in several ways, particularly through the study of the effect of insulin on blood sugar, glycogen distribution, the respiratory quotient, and, more recently, on the intermediary metabolism of the carbohydrates. A few investigators have also been concerned with the influence of insulin on protein, fat, and mineral metabolism. An excellent review of literature up to 1926 has been given by Macleod (1). The more recent work has been summarized by the same author in a series of lectures delivered before the London Hospital (2). Cori and Cori, Macleod and his coworkers, and Lesser and collaborators have made many significant contributions to the specific problem of the influence of insulin on glycogen distribution. Their work has been restricted almost entirely to mammals. We find this same thing to be true of the contributions of other workers. The data obtained have been more or less conflicting, and we believe that it can be safely stated that we have not yet arrived at a point where final conclusions can be drawn. The whole insulin problem is complicated by the fact that the physiological response of animals to insulin seems to differ according to whether they are diabetic, starved, or are absorbing carbohydrates. There seems, also, to be an additional factor, that of the general metabolism of the experimental animals. These, and perhaps other complications, are no doubt responsible in a large measure for the several conflicting theories of the action of insulin. Goldblatt classifies the prevalent theories in a recent paper (3). 27
28 Insulin and Glycogen Distribution In view of the more or less conflicting results that have been obtained in mammals, it was thought advisable to attempt a study on some cold-blooded animal, in which the general metabolic activity is considerably slower. Accordingly fishes were chosen and a series of experiments on them conducted. We feel that the lower metabolism of fishes makes them more favorable for this study than mammals in that changes due to the action of insulin take place more slowly and the details can be more easily detected. The work was carried out during the summer of 1929 at the United States Fisheries Laboratory at Woods Hole, Massachusetts. We are especially grateful to Dr. 0. E. Sette for placing the facilities of the laboratory at our disposal. Procedure In a previous paper, Gray and Hall (4) have shown that insulin shock may be easily produced in species of active fishes. However in sluggish forms there is little external evidence of the action of insulin as convulsions. It was also shown that in fishes a much larger dosage is necessary to produce convulsions and lowering of blood sugar concentration than is required for mammals. Since the scup, Stenotomus chrysops, L., was used in these previous studies, and much information concerning its reaction to insulin had already been obtained, this fish was chosen as the experimental animal. The fishes were either taken from commercial traps or caught by hook and line and brought to the laboratory, where they were kept in hatching boxes for about 24 hours before using. These boxes were supplied with an abundance of running sea water to prevent any asphyxial conditions. The practice of keeping the fishes in these boxes for some time before use is necessary to insure more constant physiological conditions and less individual variation (5). The fishes were divided into two groups and an attempt was made to have animals of about the same weight in each group. The average weight of the animals was about 250 gm. One group was used as a control. At a set time 10 units of insulin (Lilly) were injected intraperitoneally into each of the animals in the experimental group. The control animals were handled in two ways: they were either injected with a volume of physiological saline equal to the volume of insulin, or they were
Root, Hall, and Gray 29 left alone until killed for blood sugar and glycogen determinations. The reasons for the injection of saline will be brought out later. After recorded intervals, following the injection with insulin, one fish from the experimental and one from the control group were used for blood sugar and glycogen determinations. The fishes were quickly removed from the hatching boxes and blood from the severed caudal artery collected in oxalated tubes. The spinal cord was then severed just back of the brain to prevent muscular movements, and the body cavity opened. The liver was removed in toto by grasping it with forceps at its point of attachment and immediately dropped into a tared flask containing the proper amount of hot 60 per cent KOH. After this a strip of muscle was taken from one side of the animal and dropped into another tared flask containing hot KOH. The entire operation consumed less than 2 minutes. The weight of the liver and muscle was determined by reweighing the tared flasks on precision balances. The weight of the entire fish was determined by weighing the remains and adding to the result the weight of liver, muscle, and blood removed. The blood sugar was determined according to the Folin modification of the Folin-Wu method (6); the glycogen according to the well known method of Pfhiger (7). Instead of the glycogen being read directly with a polariscope it was inverted to glucose with 2.2 per cent HCl and the glucose determined by the Hagedorn- Jensen method (8). The results were expressed in terms of glycogen by using the formula, glycogen = glucose X 0.927. Animals were bled and analyses were made on the lst, 2nd, 3rd, 4th, 5th, 6th, Sth, loth, 12th, and 14th hours after the injection of insulin. The data obtained from the analyses of several repeated series were then graphed to show the relation of the blood sugar, muscle, and liver glycogen in the insulinized animals to the same constituents in uninsulinized controls. Results Fig. 1 summarizes the results obtained in this study. The data from the insulinized fishes are expressed in percentage variation from normal concentrations found in the controls. These figures are plotted against time after the fishes were given insulin. Each curve is a composite of results obtained from 32 individuals. The average normal concentration of the liver glycogen was found to
30 Insulin and Glycogen Distribution be 33.07 mg. per gm. of liver, of muscle glycogen 0.883 mg. per gm. of muscle, of blood sugar 46 mg. per 100 cc. of blood. In the insulinized fishes the blood sugar concentration is above normal Time. \n houis after in jectlon FIG. 1. The influence of insulin on carbohydrate distribution in the scup for a period of 3 hours after the insulin injection. This period of hyperglycemia is followed by prolonged hypoglycemia, the blood sugar falling to a very low level. The liver glycogen decreases in
Root, Hall, and Gray concentration from the start, and continues to decrease until only about 5 per cent of the normal amount remains in the liver. The muscle glycogen, on the other hand, rises gradually upon injection of insulin, and reaches the peak of its concentration between the 4th and 5th hours after the injection; there then follows a very decisive drop in concentration. The sudden drop occurs at a time when convulsive symptoms are noted in the animals. The lowest level of concentration is found at the 10th hour after insulin administration; from that point on to the 14th hour the concentration increases again until it is approximately normal. DISCUSSION The results indicate that insulin effects a marked change in the distribution of glycogen in the scup. Concomitant with the drop in liver glycogen and blood sugar there is a rise in muscle glycogen, indicating that insulin may bring about an excessive storage of muscle glycogen at the expense of liver glycogen and blood sugar. It is impossible to say that all the carbohydrate thrown out of the liver and blood is accounted for by storage in the muscles. A considerable part of it may have been oxidized, or have been taken care of in some other manner. The hyperglycemia observed following immediately after the insulin injections is of interest. It was thought that this might be a result of the act of injecting the fishes. As a control some of the fishes were injected with a volume of physiological saline equal to the volume of insulin given the experimental animals. The results were not entirely negative. A slight hyperglycemia followed in the control animals, but it was never as marked as in the experimental animals. It appeared that the insulin did have a definite hyperglycemic effect, We suggest the following interpretation of this situation. The injected insulin stimulates liver glycogenolysis. Immediately following administration of insulin, on account of the high initial concentration of liver glycogen, a large quantity of sugar is supplied to the blood. For a time more sugar is acquired by the blood than is removed by the tissues through oxidative and other processes. However, as the glycogen concentration of the liver gradually decreases, less and less sugar is supplied by the liver, until finally a stage is reached where the supply no longer exceeds the demand and hypoglycemia sets in.
32 Insulin and Glycogen Distribution Collens and Murlin (9) working with dogs found that portal injection of insulin caused a marked hyperglycemia within 5 minutes after the injection. The hyperglycemia lasted for only a short time and was followed by the usual hypoglycemia. They attributed the temporary hyperglycemia to rapid initial glycogenolysis in the liver. The concentration of muscle glycogen appears to be a sensitive indicator of the convulsive stage in insulinized fishes. It will be noted from Fig. 1 that muscle glycogen concentration gradually increased for about 6 hours after insulin injection and then suddenly dropped. This drop occurred at a time when convulsive symptoms were noted in the fishes. At that time they would swim with great rapidity, going in extremely haphazard fashion, and striking the sides of the tank. This period of rapid propulsion was followed by a quiescent period, with the fishes resting bellies upward. At the 10th hour the fishes were in the most pronounced stage of convulsions, and the lowest muscle glycogen concentrations were then recorded. At the 12th and 14th hours the fishes showed only slight convulsions and higher muscle glycogen concentrations. At the same time however, blood sugar and liver glycogen do not increase in concentration, in fact the blood sugar concentration drops even lower than it was at the 10th hour. Thus it is difficult to say whether fishes at the 12th and 14th hours were past the critical stage of insulin convulsion, or were merely less susceptible to the insulin than fishes at the 10th hour. One might think that there would be a rise in blood sugar if the fishes were recovering from the effects of the insulin. If our interpretation rested only on external evidence and the determination of the muscle glycogen concentration, we might say that the fishes were recovering from insulin shock; but the blood sugar concentration does not indicate such a situation. On the other hand, perhaps, in cases of recovery from insulin shock, muscle glycogen recovers before liver glycogen and blood sugar. If this were true our interpretation would seem consistent. Cori and Cori (lo), who have worked extensively on rats, came to the conclusion that there is a. cycle of the glucose molecule in the body. They think that insulin is of significance in that it accelerates the cycle in the direction of blood glucose to muscle glycogen. Acceleration in this direction leads to hypoglycemia,
Root, Hall, and Gray 33 and, secondarily, to a depletion of the glycogen stores of the liver. Barbour et al. (ll), working with the standard white rat, concluded that large doses of insulin injected into fed rats have an inhibitory effect on glycogen formation in the liver, at the same time increasing glycogen in the muscles, the total gained by the muscles being of about the same magnitude as that lost in the liver. Smaller doses, while having the same effect on the liver glycogen, produce no demonstrable change in muscle glycogen. In fasted rats they found that insulin always caused a decrease in both liver and muscle glycogen, but before there was any demonstrable recovery in blood sugar the liver glycogen concentration returned to, or about, the general level. Choi (12) found that muscle glycogen increased when glucose and insulin were injected together. Markowitz et al. (13), working with the dog, found that a rise in muscle glycogen could not be demonstrated in dogs with excised liver and pancreas. If insulin was injected, or if the liver alone was excised the muscle glycogen increased. We feel that Markowitz and his coworkers have performed a critical experiment, in that they demonstrate, in a seemingly irrefragible manner, the ability of insulin to bring about the storage of glycogen in the muscles. We cite the previous experiments for the purpose of illustrating the general agreement of the results we have obtained with fishes with those obtained with mammals. Our results also agree with von Issekutz and Vegh (14), and with Takuwa (15), who worked with turtles. They found a decrease in liver glycogen after the injection of insulin. Conclusions derived from studies on the action of insulin have been very conflicting. Some authors have obtained results that appear to be quite contradictory to those obtained by others. Obviously, our results cannot be in agreement with all. Rather than review all of the contradictory interpretations of insulin action the reader is referred to Macleod (1, 2). Especial mention, however, should be made of a paper by Goldblatt (16). This author worked with young rabbits and obtained results that led him to conclude that the action of insulin is to lock glycogen in the liver. We did not find evidence of such a phenomenon in the scup. Consequently, we are somewhat skeptical of Goldblatt s interpretation, not only on account of our inability to confirm his conclusion, but also because of evidence presented by other investigators.
Insulin and Glycogen Distribution In conclusion we would like to suggest a possible explanation to account for the conflicting evidence as to the action of insulin which seems to prevail at the present time. Practically all of the work concerned with the mechanism of insulin action has been carried on with homeothermic animals and scarcely any with poikilothermic animals. The metabolism of a homeothermic animal is much more rapid than that of a poikilothermic animal. Changes go on so rapidly in mammals that some of them may be missed entirely. As proof of the more rapid action of insulin in the homeotherms, we point out that l$ units of insulin per kilo of body weight injected into a rabbit will bring on convulsions in about 5 hours, while the fishes used by us were receiving about 40 units of insulin per kilo of body weight and yet convulsions did not occur until 8 to 10 hours after they were injected. We feel that, due to the slower metabolism, a poikilotherm is more favorable to employ in the study of insulin action. The results are brought to us in the form of a slow motion picture, and the details of the action can be studied with greater facility. SUMMARY 1. Massive injections of insulin (about 40 units per kilo of body weight) elicit a marked change in glycogen distribution in normal fasting scup. 2. Following the injection glycogen is thrown out of the liver, and after a transient hyperglycemia, pronounced hypoglycemia occurs. Concomitant with the drop in liver glycogen concentration and blood sugar concentration, there is a rise in muscle glycogen until convulsive symptoms appear in the animals. The concentration of muscle glycogen then falls rapidly. 3. It appears that insulin causes an increase in storage of muscle glycogen at the expense of liver glycogen and blood sugar. BIBLIOGRAPHY 1. Macleod, J. J. R., Carbohydrate metabolism and insulin, London and New York (1926). 2. Macleod, J. J. R., Lancet, 2, 1 (1929). 3. Goldblatt, M. W., B&hem. J., 23, 243 (1929). 4. Gray, I. E., and Hall, F. G., Bid. Bull., 68, 217 (1930). 5. Hall, F. G., Gray, I. E., and Lepkovsky, S., J. Bid. Chem., 67, 549 (1926).
Root, Hall, and Gray 35 6. Folin, O., J. Biol. Chem., 82,83 (1929). 7. Pfliiger, E., Arch. ges. Physiol., 129, 362 (1909). 8. Hagedorn, H. C., and Jensen, B. N., Biochem. Z., 136, 46 (1923). 9. Collens, W. S., and Murlin, J. R., Proc. Sot. Exp. Biol. and Med., 26, 485 (1929). 10. Cori, C. F., and Cori, G. T., J. Biol. Chem., 81, 389 (1929). 11. Barbour, A. D., Chaikoff, I. L., Macleod, J. J. R., and Orr, M.D., Am. J. Physiol., 80,243 (1927). 12. Choi, Y. O., Am. J. Physiol., 83,406 (1927). 13. Markowitz, J., Mann, F. C., and Bollman, J. L., Am. J. Physiol., 87, 566 (1929). 14. von Issekutz, B., and Vegh, F., Biochem. Z., 192,383 (1928). 15. Takuwa, M., Mitt. med. Akad. Kioto, 3, pt. 4 (1929). 16. Goldblatt, M. W., Biochem. J., 23, 83 (1929).
THE INFLUENCE OF INSULIN ON GLYCOGEN DISTRIBUTION IN MARINE FISHES R. W. Root, F. G. Hall and I. E. Gray J. Biol. Chem. 1931, 91:27-35. Access the most updated version of this article at http://www.jbc.org/content/91/1/27.citation Alerts: When this article is cited When a correction for this article is posted Click here to choose from all of JBC's e-mail alerts This article cites 0 references, 0 of which can be accessed free at http://www.jbc.org/content/91/1/27.citation.full.html #ref-list-1