C. N. H. Long

Effect of Epinephrine on Adrenal Cholesterol and Ascorbic Acid.

It has previously been shown that the injection of pure adrenotrophic hormone into rats is followed by a rapid fall in adrenal ascorbic acid, a slower fall in adrenal cholesterol, and an increase in liver glycogen.1,2,3 It has been found (unpublished experiments) that exposure of rats to such conditions as cold, scalds, or hemorrhage is also followed by a depletion of adrenal cholesterol and ascorbic acid. However, since the above stresses are without any effect on the adrenal cholesterol and ascorbic acid of hypophysectomized rats, it is evident that a release of adrenotrophic hormone from the anterior pituitary is a preliminary and necessary step in the adrenal cortical response to an increased need for cortical hormone. The factors, humeral or nervous, that are associated with stress and which lead to an increased secretion of adrenotrophic hormone are not known, but it would appear that one common denominator is an increased degree of activity of the sympathetic nervous system, with a consequent release of epinephrine. We have, therefore, investigated the effect of epinephrine on the cholesterol and ascorbic acid levels of normal and hypophysectomized rats. Methods. Male rats about 200 g in weight of either Sprague-Dawley or Yale strain were used for this investigation. Non-fasted animals were hypophysectomized by the parapharyngeal approach under ether anesthesia. They were used on the third postoperative day after a fast of 18 hours, while food was withheld from normal rats 24 hours before the experiment. A 4-hour course of injections was given subcutaneously to both groups. Every hour each animal received 0.02 mg/100 g of epinephrine in physiological saline. One hour following the last injection the rats were quickly anesthetized with nembutal. The adrenals were removed and weighed on a torsion balance.

Effect of Adrenotropic Hormone on Ascorbic Acid and Cholesterol Content of the Adrenal.

Summary A single dose of pure adrenotropic hormone diminishes both the ascorbic acid and cholesterol content of the adrenals. The level of ascorbic acid is reduced to two-thirds within 20 minutes and one-half of its normal value in one hour. It has again returned to its initial concentration 9 hours after hormone administration. On the other hand the maximum decrease in cholesterol is not reached until 3 hours after injection and its restoration also occurs at a slower rate. It is suggested that the two processes may be related to cortical hormone synthesis in or release from the adrenal gland.

Release of Adrenocorticotrophic Hormone by Direct Application of Epinephrine to Pituitary Grafts.

Summary 1. Homologous grafts of pituitary tissue to the anterior chamber of the eye of the hypophysectomized rat retain the ability to secrete ACTH, both spontaneously and in response to activation by epinephrine. 2. The effect of epinephrine in releasing ACTH may be brought about by its direct application to anterior pituitary tissue.

THE GROWTH AND METABOLIC HORMONES OF THE ANTERIOR PITUITARY

The discussion of the anterior pituitary factors that influence the growth and metabolism of animals is one of unusual complexity. In the first place, it is obviously impossible to distinguish between effects described as LLgrowth promotion” and those that are termed effects on metabolism, since in the last analysis growth in the true sense is dependent on chemical reactions involving not only the synthesis of cellular components but also those by which energy is provided for such synthesis. Furthermore, the term growth has only a general meaning and unless more rigidly defined contributes but little to our understanding of the role played by the entlocrinc glands in its occurrence and continuance. When true growth occurs the cells of an individual organ or those of a hrger part of the animal increase not only in size but also in numbe;. This means that not only is the rate of accumulation of the characteristic cellular constituents (mainly protein, salts, and water) increased but also their absolute quantities in the organism. This continues until the animal has reached adulthood, a t which time the quantity of certain constituents of the body, notably protein and inorganic salts, remains constant although the animal may continue to increase in size and weight by the addition of fat. It is important to remember that, although the quantity of protein remains constant, this constituent of protoplasm is now known to be undergoing constant breakdown and resynthesis. This knowledge has recently been gained by the brilliant work of Schoenheimer and his colleagues, who made use of the heavy isotope of nitrogen, and this information is of the utmost importance not only to our understanding of protein metabolism hut also because i t indicates that those processes by which protein is formed during the period of rapid growth * The experiments reported from the Department of Physiological Chemistry. Yale University Sehool of Medicine, were sup orted by a grant from the Committee on Research in Endocrinology. National Research Council, a n z t h e Fluid Research Fund, Yale University School of Medicine.

Metabolic changes associated with hemorrhage.

The study of the dynamics of the circulatory system necessarily takes first place in the physiology of hemorrhage. I t has led to the dcvelopment of therapeutic measures which, when applied in good time, can bring about recovery in many instances after severe hemorrhage associated with profound shock. The study of metabolism after hemorrhage has as its principal objective the solution of problems raised by the limitations of the present therapeutic methods. This solution has to be based on an understanding of the metabolic changes accompanying, and perhaps responsible for, the so-called “irreversible” state of shock. One such change, the production of vasodepressor material by the anoxic ,/.

The Endocrine Control of the Blood Sugar

The study of diabetes mellitus in man and of experimental diabetes produced by various means in animals has already furnished a wealth of information on the nature of those chemical transformations in living cells that we term metabolism, and upon which all bodily function depends. Let me recall to you a few of these landmarks of our knowledge that have been established by observation and experiment on the diabetic organism. First, the recognition by physicians in several countries that the sweetness of diabetic urine was due to the presence of a sugar, later identified as glucose. Then the detection of beta-hydroxybutyric acid and acetoacetic acid as constituents of diabetic urine, which led in time to an appreciation of the nature of acidosis; the relationship between protein catabolism and glucose formation; and, of course, the recognition that there was an internal secretion of the pancreas provided by the islets of Langerhans.

Effect of Adrenal Cortical Hormone on Carbohydrate Stores of Fasted Hypophysectomized Rats.

Summary The injection of adequate amounts of either adrenal cortical extract or the crystalline compound B of Kendall will not only prevent the depletion of the carbohydrate stores of fasted hypophysectomized rats, but will also restore them after they have been depleted by fasting.

Glycogen Content of the Rat Heart

The cardiac glycogen of albino rats has been determined under a variety of conditions. The figures quoted are for glycogen as glucose in milligrams percent, and unless otherwise stated are for 24-hour fasted animals. Amytal anesthesia was used when securing hearts and muscles. Fifty-two 24-hour fasted animals, used as controls, showed a quite constant cardiac glycogen of 497±56 mg. %; the gastrocnemii of the same animals contained 525 ±48 mg. %. Animals fasted for 48 hours had more glycogen in the hearts (578±81) and less in the gastrocnemii (455±37) than controls. Non-fasted animals showed considerably less cardiac glycogen (341±54) but more glycogen in gastrocnemii (574±74) than controls. It was found also, that 24-hour fasted animals which had been fed sufficient glucose by mouth to provide for maximum absorption for 4 hours, and which were taken at the end of such time, had somewhat less glycogen in the hearts (449±50), although considerably more in the gastrocnemii (690±48) than controls. When, however, insulin injection accompanied such glucose feeding, both hearts and gastrocnemii contained increased glycogen (hearts 703± 212, gastrocnemii 830±183). Epinephrine sufficient to reduce the glycogen of gastrocnemii to 55% of its control value did not alter cardiac glycogen appreciably. The cardiac glycogen after subcutaneous injection of 0.02 mg. of epinephrine per 100 gm. of rat, was (a) in 1/2 hour 475±52, (b) in 3 hours 509±54. One-half hour after large intravenous doses of epinephrine the hearts contained 475±26. Similarly, exercise, both natural and electrically produced, sufficient to reduce the glycogen of gastrocnemii to 70% of its control value did not decrease cardiac glycogen, the values being (a) after natural exercise 546±40, (b) after electrical stimulation 512±19. When, however, electrical stimulation was severe enough to produce cyanosis through interference with respiratory movements the cardiac glycogen was much reduced, being 226±33.

Adrenalectomized-Depancreatized Dogs

We have previously reported1 that removal of the adrenal glands from the cat ameliorates the effects of pancreatic diabetes to an extent comparable to that obtained by hypophysectomy and, furthermore, that the removal of the adrenal cortex is responsible for this effect of adrenalectomy. Houssay and Biasotti2 have recently reported a similar effect of adrenalectomy upon pancreatic diabetes in the toad. Since the dog has been extensively used in the study of experimental diabetes and since several workers have reported that adrenalectomy does not modify an experimental diabetes3,4,5 in this species, it was of some interest to prepare long surviving animals in which all adrenal and pancreatic tissue had been removed. This was accomplished by first removing one adrenal and at a later date, the pancreas. The animals were then maintained by use of protamine insulin until in good health, when the second adrenal was removed and the insulin discontinued. During the remainder of their lives, they received a diet of meat and raw pancreas and, in addition, from 4 to 8 gm. of NaCl daily. They also received 5-10 cc. of cortical extract (Upjohn) daily. Analyses of the blood at the end of the experiment indicated that no gross disturbances in the electrolyte balance had occurred. In consequence, it is assumed that the alterations in the character of the diabetes were not to be attributed to a distorted water and salt metabolism. Table I shows that the glucose, nitrogen and acetone excretion of the 5 dogs studied was strikingly reduced compared to the quantities reported by Chaikoff6 in fasting depancreatized dogs from which insulin had been withdrawn. While we have no comparable figures for the survival of depancreatized dogs, it would appear that as in the cat, the length of life of the adrenalectomized-depancreatized dog is also appreciably extended. None of our dogs relapsed into acidosis or coma but slowly became free from glycosuria as their weight declined. This occurred even if the food intake remained constant. Hypoglycemic episodes relieved by glucose also were observed in one animal. In 2 animals the ketonuria for a short period was greater than we have observed in adrenalectomized-depancreatized cats (40-70 mg. a kilo a day) but this appears to be a species difference as hypophysectomized-depancreatized dogs exhibit a greater ketonuria than do similarly operated cats. It is therefore concluded that adrenalectomy produces the same modification of pancreatic diabetes in the dog as has already been observed in the cat.

Preparation of Pituitary Adrenotropic Hormone.

This is a preliminary description of a relatively simple procedure devised for the isolation of purified and highly active preparations of adrenotropic hormone from whole hog pituitary glands. The extraction and fractionation procedures consist of the following steps: (1) Extraction and isolation of a fraction designated as crude prolactin by the procedure originally developed by Lyons 1 and adopted by White, Bonsnes and Long 2 as the first step for the subsequent preparation of crystalline prolactin. This “crude prolactin” contains adrenotropic hormone activity. (2) Solution of the crude prolactin preparation in 2% concentration at pH 9.0 and removal of precipitates which form by careful adjustment of pH to 8.0, 6.6 and 5.4. (3) Supernatant obtained in Step 2, after removal of pH 5.4 insoluble material (prolactin), is freed from traces of prolactin by the addition of 7 ml of saturated (NH4)2SO4 to each 100 ml of solution. The precipitate is separated and discarded. (4) Acetone is added to the supernatant to a concentration of 80%. The adrenotropic fraction precipitates. (5) This precipitate is dissolved in water (volume used being two-thirds of that employed in Step 2) and the solution mixed with one-half its volume of concentrated NH4OH. The ammoniacal solution is allowed to stand at room temperature for 7 hours. (6) Acetone is then added to above solution to a concentration of 90%. (7) The precipitate is dissolved in one-third volume of water used in Step 2, and dialyzed to remove salt. (8) The adrenotropic fraction is precipitated by lowering of pH to 4.7. The precipitate may be dried by washing 3 times with acetone at the centrifuge and finally drying in vacuo over concentrated sulfuric acid. The yield of dry, purified adrenotropic fraction has been of the order of 500 mg from each 1000 g of whole hog pituitary glands.