:6I2.352.I2:6I observations of Ahlgren (3), who found that pituitary extract increases

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1 :6I2.352.I2:6I THE ACTION OF EXTRACT OF PITUITARY ON THE BLOOD SUGAR AFTER PANCREATECTOMY. BY C. G. IMRIE. (Department of Physiology, The University, Sheffield.) THE experiments about to be described were undertaken in order to observe the action of pituitary extract on the blood sugar in an animal deprived of its pancreas and maintained in a healthy condition on an appropriate diet and adequate doses of insulin. Under such conditions the amount of insulin and glycogen in the tissues could be more or less controlled and the influence of these factors observed. The investigation was suggested by the observations that pituitary extract' diminishes the hyperglycbemia in diabetes mellitus (1) and causes a fall in the blood sugar of eviscerated cats receiving glucose injected intravenously at a uniform rate (2). These effects aze not understood. Clark (2) refers to the observations of Ahlgren (3), who found that pituitary extract increases the oxidation processes in muscle tissue and suggests this as a possible explanation of the fall in the blood sugar produced by the extract in his eviscerated preparations. It was thought that some information regarding the nature of these responses might be furnished by a study of the effects of pituitary extract on the hyperglycsemia of depancreatized animals. Such preparations would also provide an opportunity of investigating the source of the additional glucose in "pituitrin" hyperglycwsmia. Clark's observations suggest that it comes from the glycogen of the liver. They do not support the view of Moehlig and Ainslie(4) that the extra glucose arises from the glycogen in the muscles. Since MacLeo d and his co-workers(5) were unable to detect any significant difference in the amount of glycogen present in the skeletal muscles of depancreatized animals before and after insulin, but observed an increase in the liver, it was thought that by withholding insulin for varying periods of time and depleting the liver of its glycogen, additional evidence regarding the source of the glucose in " pituitrin " hyperglyceemia might be obtained. 1 The pituitary extract employed was the preparation issued by Park, Davis and Co., under the name "pituitrin."

2 PITUITARY ON BLOOD SUGAR. Methods. A fox terrier bitch, weight 14 lb., was depancreatized under ether ansesthesia. For the first 48 hours water, cane sugar and insulin were given. It was then placed on the following diet: meat 400 grm., raw pancreas 100 grm. and cane sugar 100 grm. (MacLeod(5)). This was divided into two feeds given morning and evening. Fourteen units of insulin were injected twice daily after the sugar had been taken. During the first few days the animal ate only part of the diet so that the dose of insulin was less, but by the end of a week the full diet was being consumed. Under this regime the dog survived for 10 months in an excellent state of health, the body weight was maintained and the skin remained in good condition. In all the experiments blood was obtained from the ear at halfhourly intervals and the sugar estimated by the method of Hagedorn and Jensen(6). The observations were made in the afternoons, and no food or insulin was given on the mornings of such days except when stated otherwise. During the experiments the animal was kept in a box devised for the purpose and curtained off in a corner of the laboratory. This procedure was found to be desirable as at times emotional stimulation appeared to influence the shape of the blood sugar curves. Experiments. Blood sugar curves after the injection of 1 c.c. of "pituitrin " were first obtained on the intact animal before the pancreas was removed. Three of these are set out in Table I. The fasting level of the blood sugar is about 0*095 p.c. and the response to "pituitrin" is similar to that previously observed for this and other animals. In the third series the return of the blood sugar to normal is delayed, but in this instance the animal had received food 5 hours before. TABLE I. Blood sugar after 1 c.c. of "pituitrin" in normal dog, intervals of 30 minutes between determinations. A. (p.c.) B. (p.c.) C. (p.c.) 0* * 0.097* 0107* * * * *100 * "Pituitrin" was given immediately after this sample of blood was withdrawn. 265 Observations onq the depancreatized animal were begun a fortnight after the operation. By, this time the wound had healed, the original weight was regained and the dog accustomed to its diet andc injections

3 266 C. G. IMRIE. of insulin. The blood sugar was determined over periods of 2 to 3 hours under different conditions as regards food and insulin. In one instance food and insulin were given in the morning and observations begun 5 hours later, in others no morning ration of food or insulin was given, and in others, again, isulin was- withheld for periods varying from 20 to 95 hours before the examinations were made. During the longer periods of insulin deprivation the animal lost weight and on one occasion became quite ill. Several days intervened, therefore, between such observations to enable the former state of nutrition to be regained. The figures obtained on four occasions are given in Table II. TABLE II. No food in No food in Food and No food or morning, no morning, no insulin at insulin in insulin for insulin for 9.30 a.m. the morning 44 hours 70 hours (p.c.) (p.c.) (p.c.) (p.c.) It is apparent that wide variations in the blood sugar occurred when insulin had been withheld for 44 hours or less; but after 70 hours of insulin deprivation the blood sugar remained constant for 2 hours, though at a lower level. If glycogen were present in the liver in those instances when insulin had not been withheld longer than 44 hours it is likely that variations in the blood sugar were associated with the liberation of glucose from it, for the animal was restless, excitable and barking at times and in a state where emotional stimulation must have been a factor. If no glycogen were present in the liver after 70 hours of insulin deprivation the emotional state of the animal should not have influenced the blood sugar. It did not do so in the instance referred to or on eight other occasions which will be mentioned later. The injection of 0-3 c.c. of adrenaline (1 in 1000) did not produce a rise in the blood sugar when insulin had not been given for 70 hours, indicating the absence or low concentration of glycogen in the liver. When adrenaline was given after insulin had been withheld for only 20 hours a rise occurred. The figures are given in Table III. The action of pituitary extract was next observed after varying periods of insulin deprivation. One c.c. of "pituitrin" was injected on each occasion. The results are set out in Table IV.

4 PITUITARY ON BLOOD SUGAR. 267 TABLE III. No insulin No insulin No insulin H9emoglobin for 20 hours for 70 hours for 70 hours determinations (p.c.) (p.c.) (p.c.) (p.c.) * * 0.177* * Adrenaline was given immediately after this sample was withdrawn. TABLE IV. No insulin given for: 20 hours 44 hours 70 hours 70 hours 70 hours Hb 94 hours (p.c.) (p.c.) (p.c.) (p.c.) (p.c.) (p.c.) (p.c.) * * 0.172* * 0.168* * * 1 c.c. "pituitrin" given immediately after this sample was withdrawn. As these figures show, " pituitrin " produced a rise in the blood sugar when insulin had been withheld for 20 and 44 hours. Since considerable variations occurred spontaneously under similar conditions but with no "pituitrin," these results cannot be stressed. In those instances where no insulin had been given for 70 hours or more, no rise followed the injection of "pituitrin," the blood sugar remained remarkably constant except for a slight fall which appeared 14 to 2 hours later. This fall is comparable in time and degree to that which sometimes occurs in normal animals and man. No fall similar to that observed in eviscerated cats and in diabetes mellitus was obtained. On one occasion h.emoglobin determinations by the Gower-Haldane method were made in order to ascertain whether dilution of the blood could account for the late fall in the blood sugar. No evidence for this was obtained. Similar determination of the hasmoglobin after adrenaline (Table III) in one instance showed a small increase in the concentration of the blood half an hour after the adrenaline was given. This change was not reflected in the blood sugar. It appears, therefore, that in depancreatized animals "pituitrin" causes a rise in the blood sugar so long as there is glycogen present in

5 268 C. G. I3IRIE. the liver. When insulin is withheld for about 70 hours the hyperglycaemic effects of "pituitrin" are no longer apparent. Collip(7) found that if large amounts of sugar were given with inulin, the liver contain ed 20 p.c. or more of glycogen. Liberal amounts of sugar were given to this dog, the liver presumably contained much glycogen and it required about 3 days without insulin to remove it. Since the muscles still contained glycogen, these results confirm the view that the glycogen of the liver is the source of the extra glucose in the hyperglycaemia caused by extract of pituitary. Note. The dog lived for 10 months after the operation. It appeared to be in excellent health except during the longer periods of insulin deprivation, when it lost weight and became less lively. During the last month of its life it refused to take the raw pancreas. At the end of the month it became acutely ill, refused all food and died in 2 days. MacLeod (5) found that without raw pancreas in the diet his animals did not as a rule live longer than 6 weeks and died with symptoms of hepatic insufficiency, and at autopsy a remarkable degree of necrosis and fatty change was found in the liver. The liver in this animal showed the changes described by MacLeod, and in addition the kidneys presented extensive fatty changes most marked in the convoluted tubules but quite abundant in the straight and collecting ones as well. No pancreatic tissue was found. SUMMARY. The action of pituitary extract on the blood sugar of a depancreatized animal maintained in a healthy state on an appropriate diet and adequate doses of insulin has been studied. If insulin is withheld for 44 hours or less before injecting the extract of pituitary, a rise in the blood sugar is produced as in the intact animal. If insulin is withheld for 70 hours or more, the rise does not occur. A slight fall appears about 1J to 2 hours after the injection. Under similar conditions adrenaline too fails to inereme the concentration of the blood sugar. Since the muscle glycogen is not materially altered under the conditions of the experiment, these results support the vew that the extra glucose in the hyperglyetsmia produced by pituitary extract is derived from the glycogen in the liver. The expenses of this research have been defrayed by a grant from the Medical Research Council.

6 PITUITARY ON BLOOD SUGAR. 269 REFERENCES. 1. Tingle and Imrie. This Journ. 62. Proceedings, p. ii Clark. Ibid. 64. p Ahlgren. Skand. Arch. f. Physiol. 47. Supp. p Moehlig and Ainslie. Journ. Am. Med. Assoc. 84. p MacLeod. Carbohydrate Metabolism and Insulin. (Longmans, Green and Co.) Hagedorn and Jensen. Biochem. Zeit p Collip (cf. Banting, Best, Collip and MacLeod). Trans. Roy. Soc. Canada, Sec. v PH. LXVII. 19