RELATIONS BETWEEN INSULIN AND PITUITARY HORMONES IN AMINO ACID METABOLISM

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1 RELATIONS BETWEEN INSULIN AND PITUITARY HORMONES IN AMINO ACID METABOLISM BY WILLIAM D. LOTSPEICH* WITH THE TECHNICAL ASSISTANCE OF JOAN B. SHELTON (From the Department of Physiology, Syracuse University College of Medicine, Syracuse) (Received for publication, December 2, 1949) In a previous communication (1) evidence was presented to support the conclusion that insulin acts to accelerate the rate at which proteins are synthesized from free amino acids. This evidence was based on the fact that there is a correlation between the rates at which the individual amino acids are removed from the blood after insulin and the relative molecular proportions of these same amino acids in a representative body protein such as skeletal muscle. In order to extend these studies and fit them into a more comprehensive scheme, a study was made of the interrelations between insulin and the growth and adrenocorticotropic hormones of the anterior pituitary as they influence the metabolism of the amino acids. The purpose of the present paper is to present the results of these experiments. EXPERIMENTAL Experiments were performed on normal and alloxan-diabetic adult female dogs, maintained on a diet of laboratory chow, supplemented twice a week with horse meat. The animals were fasted 18 to 24 hours before an experiment. After a control sample of blood was taken, either growth hormone (GH) or adrenocorticotropic hormone (ACTH) was injected intraperitoneally. The hormones were dissolved in N sodium hydroxide in 0.9 per cent sodium chloride, and given in a dose of 10 mg. per kilo of body weight. Blood samples, in amounts of 35 ml., were drawn in oxalated syringes at selected intervals thereafter. Neutralized tungstic acid filtrates of these blood samples were prepared immediately and used for the analysis of the amino acids. The amino acids were determined by microbiological assay. The specific methods were the same as those reported previously (1). Only the natural L forms of the amino acids were determined by these methods; therefore all data presented herein are for those forms only. Experimental diabetes mellitus was induced in dogs, previously fasted * Scholar in the Medical Sciences of the John and Mary R. Markle Foundation. Present address, Department of Biochemistry, Oxford University, Oxford, England. 1 The author is indebted to Dr. I. M. Bunding of Armour and Company for the generous supplies of growth and adrenocorticotropic hormones used in this etudy. 221

2 222 HORMONES AND AMINO ACID METABOLISM for 3 days, by the single, rapid, intravenous injection of alloxan monohydrate in a dose that varied from 60 to 75 mg. per kilo of body weight. The dose of 75 mg. was often followed by death in a diabetic-uremic state. Much better preparations were obtained from the use of slightly lower doses. Before use in an experiment these dogs were allowed to recover from the acute effects of the alloxan for a period of at least 2 weeks. By that time the diabetes had become stabilized and the animal was eating well. These dogs were maintained on a mixture of regular and protamine zinc insulin.2 All insulin was withdrawn for a period of at least 48 hours before an experiment. f ~~~~.~~~ - NORMAL -----ALLOXAN DIABETIC I 2 3 I23 I 2 3 I23 I23 HOURS AFTER GROWTH HORMONE FIQ. 1. The changes in blood concentrations of the ten essential amino acids after growth hormone in the normal and the alloxan-diabetic dog. The dose of the hormone was 10 mg. per kilo of body weight; Results Experiments with Growth Hormone Simultaneous changes in the blood concentration of the ten essential amino acids were followed in normal and diabetic dogs for a period of 3 hours after GH injection. The results of one pair of such experiments are presented graphically in Fig. 1. Change in blood concentration is 1 The author is grateful to Eli Lilly and Company for the supplies of insulin used in this study.

3 W. D. LOTSPEICH 223 plotted either above or below the control value. The normal dog had a fasting blood glucose of 75 mg. per cent; the diabetic dog had a fasting blood glucose of 280 mg. per cent. It is striking to note the marked difference in response of the blood amino acids to GH in the normal and diabetic dogs. In the normal dog the blood level of each amino acid was depressed. However, in the diabetic animal this was not the case in all instances. There was either essentially no change at all, a fall of short duration followed by a rise to control levels or above, or, as in the case of isoleucine, leucine, and lysine, a definite rise in blood concentration. Preliminary experiments had established the fact that after this dose of GH t,he fall in blood concentration of all amino acids persisted for at least G IO 8-6- FIG. 2. The relative molecular proportions of the ten essential amino acids in the protein of dog skeletal muscle. hours. Therefore the response of isoleucine, leucine, and lysine was ccrtainly abnormal, and that of threonine and valine as well, because the preliminary fall in their concentrations was of such short duration. There is sufficient evidence to allow the statement that GH causes an acceleration of the rate of protein synthesis in the tissues (2). Therefore if the amino acids that disappear from the blood after GH are going int.0 the reactions of protein synthesis, those that appear in increased quantity in the blood of the diabetic after GH must be the reflection of the reversal of the synthetic reactions, namely protein breakdown. It appears from these data that GH, in the presence of insulin, is an agent of protein synthesis, whereas in the absence of insulin, it is an agent of protein breakdown. In Fig. 2 are presented the relative molecular proportions sf the ten,

4 224 HORMONES AND AMINO ACID METABOLISM essential amino acids in dog skeletal muscle, which represents the largest single protein mass in the body. Leucine, whose molar concentration was the highest, was given a value of 10, and the concentrations of the other amino acids compared to it on the basis of their relative molecular proportions. By comparison of this graph with the curves for blood amino acid of the normal dog in Fig. 1, it is apparent that under the influence of GH there is, in general, a good direct correlation between the relative rates of fall of the individual amino acids in the blood and the relative molecular proportions of those same amino acids in the protein. Thus it appears that each amino acid is removed from blood in amounts necessary to meet its concentration requirements in the proteins being synthesized. This same relation was found to obtain after the administration of insulin to the dog (1). Therefore these data with GH, a known agent of protein synthesis, strengthen the view that insulin is likewise concerned in the synthesis of body protein. Experiments with Adrenocorticotropic Hormone In these experiments the simultaneous change in blood concentrations of three amino acids was followed after the injection of ACTH. Leucine, valine, and histidine were chosen for study because they represent amino acids that are in high, intermediate, and low concentration, respectively, in the protein of skeletal muscle. This is seen by reference to Fig. 2. In the first type of experiment ACTH was given after a control sample, and the blood concentrations of the three amino acids were followed for a period of 6 hours thereafter. In Fig. 3 are shown the results of such an experiment. It is evident that there was a marked elevation in the blood concentration of all three amino acids. Furthermore there was a direct correlation between the rate of elevation of blood level of each amino acid and the relative proportions of the amino acids in the protein. Leutine, in highest relative proportion in the protein, showed the highest rate of elevation in the blood. Valine, in intermediate concentration in the protein, showed an intermediate rate of elevation in the blood. Histidine, in relatively low concentration in the protein, showed the lowest rate of elevation after ACTH. In the second type of experiment the dog was first given an intravenous injection of regular insulin in a dose of 2 units per kilo of body weight. At the end of an hour ACTH was given and the blood amino acids followed until the end of the 3rd hour. In Fig. 4 are shown the results of such an experiment. Change in blood concentration, below the control value, is plotted against time in hours. It is seen that the insulin caused a marked fall in the blood level of all three amino acids during the 1st hour. The administration of ACTH then caused a temporary reversal of

5 W. D. LOTSPEICH 225 the insulin effect, as is seen by the elevation of the blood levels of the amino acids after the ACTH. In this experiment both the rate of fall in concentration of blood amino acid after insulin and the degree of the reversal of the insulin effect with ACTH showed a direct correlation with the relative molecular proportion of each amino acid in the protein. Thus LEUCINE VALINE TIME IN HOURS FIG. 3. The effect of ACTH on the blood concentration of leucine, valine, and histidine in the normal dog. The dose of ACTH was 10 mg. per kilo of body weight. leucine, in high concentration in the protein, showed both the steepest fall aft,er insulin and the greatest rise after ACTH. Valine, in intermediate concentration in the protein, showed both an intermediate rate of fall after insulin and rate of rise after ACTH. Histidine, in relatively low concentration in the protein, showed both the lowest rate of fall after insulin and rate of rise after ACTH. This proportionality appears in still another instance. Reference to

6 226 HORMONES AND AMINO ACID METABOLISM Fig. 1 will show that, in the diabetic, after GH the greatest elevation in blood concentration was in those amino acids (leucine, isoleucine, and lysine) which are in highest concentration in protein, and that the least VALINE I I 2 3 TIME IN HOURS LEUCINE FIG. 4. The effect of ACTH on the blood amino acid response to insulin. The dose of insulin was 2 units per kilo, and that of ACTH 10 mg. per kilo of body weight. change in blood concentration was in those amino acids (histidine, methionine, and tryptophan) which are in lowest concentration in protein. Thus if fall in the concentration of blood amino acid after GH and insulin in the normal connotes an accelerated rate of protein synthesis, and rise

7 W. D. LOTSPEICH 227 in blood amino acid concentration after ACTH in the normal and GH in the diabetic connotes an accelerated rate of protein breakdown, it would seem that the rate at which each amino acid is taken into the protein synthetic reaction, or liberated from the protein during its breakdown, is dependent on the concentration of the amino acid in the tissue proteins. DISCUSSION Mirsky (3) has studied changes in protein metabolism by following the rate of accumulation of blood urea in the nephrectomized dog. Using an anterior pituitary extract rich in growth properties, he showed that, with the pancreas intact, the extract caused an acceleration of the rate of protein synthesis. With the pancreas removed the same extract caused an acceleration of the rate of protein breakdown. From this Mirsky concluded that the growth hormone of the anterior pituitary requires insulin for its protein synthetic function. Certainly the experiments with purified GH, presented in this paper, are in agreement with this view. Young (4) has shown that the prolonged administration of anterior pituitary extract will cause typical diabetes mellitus in adult dogs and rats. Sufficient evidence has now accumulated to allow the conclusion that both GH and ACTH, which are present in such extracts, are diabetogenic. Milman and Russell (5) have observed that when purified GH is given to either mildly or severely diabetic rats there is a significant rise in blood glucose concentration. The same sort of phenomenon has been presented in this paper in the case of the blood amino acids in the alloxan-diabetic dog. The diabetogenic effect of GH is further illustrated by the clinical course of the alloxan-diabetic dog following GH administration. It invariably causes the death of the dog within 12 to 48 hours in a state of diabetic acidosis. The time of survival is directly related to the severity of the diabetes at the time of GH administration. It is tempting to speculate that the well stabilized alloxan-diabetic dog is analogous to the animal that has been partially depancreatized. Both have a certain amount of functional islet tissue left. The injection of GH then brings about either the destruction or the effective dysfunction of this remaining tissue, and thus there ensues an intensification of the diabetes. The mechanism whereby GH is diabetogenic is not certain. Young (4) tends toward the view that it exhausts the islets by forcing them to secrete beyond their capacity. Anderson and Long (6) favor a completely opposite view. In a series of brilliant experiments they made use of an ingenious device for studying the elaboration of insulin by the isolated perfused pancreas. They showed quite conclusively that insulin is normally secreted in response to a high level of glucose in the perfusing blood. If GH is also present in the perfusate, the normal release of insulin is in-

8 228 HORMOKES AND AMINO ACID METABOLISM hibited. Thus, whether by exhaustion of the islet cells or by the effective inhibition of release of insulin by the islets, GH negates the normal function of insulin and is therefore diabetogenic. Conn and his coworkers (7) have been able to produce a temporary diabetes mellitus in human subjects by the repeated injection of ACTH. The mechanism of action of ACTH in this instance is probably an antagonism of insulin at its peripheral biochemical site of action, rather than an inhibition of its liberation from the islets, as in the case of GH. This view is borne out by the experiments with ACTH presented above. The elevation of blood amino acids after ACTH is a typically diabetic response. The effect of the ACTH is to antagonize insulin, as is seen in the second experiment, in which the amino acid response of an exogenous dose of insulin was reversed by ACTH working through its target gland, the adrenal cortex. Thus the evidence at hand indicates that it is inaccurate to refer to the diabetogenic hormone of the anterior pituitary. Certainly we see that at least two hormones of that organ have a diabetogenic function. Although the mechanism of diabetogenic action is different in each case, the end-result is the same. Perhaps other hormones of the pituitary will be shown to have a diabetogenic effect. SUMMARY Changes in the blood concentration of a number of individual amino acids have been studied in normal and alloxan-diabetic dogs after the administration of purified growth and adrenocorticotropic hormones. The following observations have been made. 1. In the normal dog, with pancreas intact, GH promotes the synthesis of tissue protein from circulating amino acids. In the diabetic dog, on the other hand, GH promotes the catabolism of tissue proteins. 2. The fact that GH in the diabetic causes an intensification of the diabetes lends support to the conclusion that GH is a diabetogenic factor. 3. The diabetogenic action of pituitary ACTH has been demonstrated in the case of the individual amino acids, and the mechanism of its diabetogenic action discussed. 4. Similarities in the response of the blood amino acids after GH and insulin have been shown to lend support to the view that insulin, as well as GH, causes an acceleration of the rate of synthesis of tissue proteins from free amino acids. 5. Evidence has been presented to show that both the rate of disappearance of the amino acids from the blood during protein synthesis and their accumulation in the blood during protein breakdown are directly related to the concentration of each amino acid in tissue proteins.

9 W. D. LOTSPEICH 229 BIBLIOQRAPHX 1. Lotspeich, W. D., J. Biol. Chem., 179, 175 (1949). 2. Li, C. H., and Evans, H. M., in Recent progress in hormone research; Proceedings of the Laurentian Hormone Conference, New York, S (1948). 3. Mirsky, I. A., Endocrinology, 26, 52 (1939). 4. Young, F. G., Biochem. J., 39, 515 (1945). 5. Milman, A. E., and Russell, J. A., Federation Proc., 8, 111 (1949). 0. Anderson, E., and Long, J. A., Endocrinology, 40,98 (1947). 7. Corm, J. W., Louis, L. H., and Wheeler, C. E., J. Lab. and Chin. Med., SS, 651 (1948).

10 RELATIONS BETWEEN INSULIN AND PITUITARY HORMONES IN AMINO ACID METABOLISM William D. Lotspeich and With the technical assistance of Joan B. Shelton J. Biol. Chem. 1950, 185: Access the most updated version of this article at Alerts: When this article is cited When a correction for this article is posted Click here to choose from all of JBC's alerts This article cites 0 references, 0 of which can be accessed free at tml#ref-list-1