(From the Department of Biochemistry, McGill University, Montreal.)

Transcription

1 385 6I :6I I THE EFFECT OF ANTERIOR PITUITARY EXTRACTS ON ACETONE BODY EXCRETION IN THE RAT. BY PETER T. BLACK, J. B. COLLIP AND D. L. THOMSON. (From the Department of Biochemistry, McGill University, Montreal.) (Received June 26, 1934.) HOFFMANN and ANSELMINO [1931] and MAGISTRIS [1932] have described increased amounts of acetone bodies in the blood of rats after the injection of anterior pituitary extracts; Eitel, Lohr and Loeser [1933] ascribe this phenomenon to a temporary increase in thyroid activity. Burn and Ling [1933] found that the injection of an alkaline extract of bovine anterior lobes greatly increased the acetonuria of female rats receiving a diet of filtered butter; Butts, Cutler and Deuel [1934] obtained similar effects in fasted rats of either sex, and in fasted rats receiving sodium acetoacetate by stomach tube. Anderson and Collip [1934] have found that rats subjected to prolonged treatment with thyreotropic fractions from the anterior pituitary eventually become insensitive, and that there is present in the serum of such animals a substance which inhibits the action of thyreotropic extracts on the metabolic rate. We commenced the present study in order to see if the acetonuric response to pituitary extracts would show similar behaviour, and to prove or disprove the identity of the principle active in this response with the thyreotropic hormone. METHODS. We have confined ourselves, in this work, to a study of the urinary excretion of total acetone bodies, determined by the gravimetric method of van Slyke [1917]. The rats were kept in pairs in Hopkins metabolism cages; the urine was collected separately from the faeces, and preserved with copper sulphate solution. In preliminary studies the filtered butter diet described by B urn and Ling was used throughout the experimental periods. Many of the rats in this colony excrete rather large amounts of acetone on this diet, without other treatment. We have found, however, that rats 40 days of age,

2 386 P. T. BLACK, J. B. COLLIP AND D. L. THOMSON. taken directly from the litter cages, consistently excrete minimal amounts of " acetone " on this diet; with increasing age, an increasing proportion of the animals will display a relatively large acetone excretion, which is still clearly apparent when recalculated in terms of a standard body weight of 100 g., or in terms of the weight of butter consumed. These results, which are summarized in Table I, led us at first to use 40-day animals for TABLE I. Effect of age on acetone excretions total urinary acetone bodies in mg. of acetone on the third day of butter diet. Total acetone, mg. per Total acetone Number 100 g. body weight mg. per g. butter of Age Average, consumed animals days weight g. Average Range Average * * *4 2*7-16*5 4S assay purposes. In later experiments, however, we preferred to study the influence of the pituitary extracts on the acetone body excretion of the fasting animal, thereby assuring ourselves of consistently low control values. All the extracts tested were shown to be free from acetone, sodium acetate, and other simple ketogenic substances. Loss OF SENSITIVITY TO THE KETOGENIC SUBSTANCE. The effect of previous prolonged treatment with active extracts was necessarily studied upon mature animals. A group of eight male rats received, twice daily for 3 months, injections of 0 5 c.c. of the purified thyreotropic extract described by Anderson and Collip [1933]. This extract contains no demonstrable amount of the growth hormone, but is not devoid of gonad-stimulating and adrenotropic potency; each c.c. corresponds to 1 g. of fresh bovine anterior pituitary, and 0 01 c.c. is a. sufficient daily dose to produce thyroid hyperplasia in young guinea-pigs or significant elevation of the basal metabolic rate of hypophysectomized rats. After 3 months' treatment the rats have become quite insensitive to this extract; their thyroid glands are in a resting state, and the metabolic rates are low [Anderson and Collip, 1934]; it will be seen from Table II that they have also lost the capacity to respond to the ketogenie principle of pituitary extracts. The rats at the end of this preliminary treatment were 140 days old; since animals of this age excrete acetone when placed on a high fat diet, this experiment was carried out during a 3-day fasting period. Animals

3 ANTERIOR PITUITARY AND ACETONURIA. 387 TABLE II. Effect of anterior pituitary extracts on acetone excretion in fasting rats previously treated with thyreotropic hormone over long periods. Total acetone, mg. per 100 g. body weight Rat Weight Extract-A No. g. tested lst day 2nd day 3rd day TGS 1* ,, 1.l ,,t 1.4 1*4 1* ,5, Burn's ,, 1* * ,, O9 0*9 0* ,, 1.4 1*6 1*6 Controls Burn'sj ,,t TGS *3 18.O ,,. 1* In this experiment the rats were studlied individually (not in pairs); each received 1*0 c.c. of extract on each day of fasting. 1-4 received 1 c.c. of the thyreotropic extract ("TGS ") on each day of fasting, while animals 5-7 received similar doses of a crude alkaline extract prepared by the method described by Burn and Ling [1933]. The amount of acetone excreted by these animals was very much less than in controls of the same age, which had not received the preliiary treatment.i EFFECT OF THE ANTI-THYREOTROPIC SUBSTANCER. Fourteen female rats, 33 days old, were treated twice daily for 7 days with 0*5 c.c. of serum from a horse which had been injected daily for 4 months with the thyreotropic extract in amounts increasing from 10 to 100 c.c. This serum has been shown to be capable of inhibiting the effect of the thyreotropic hormone on the basal metabolic rate of hypophysectomized rats [Anderson and ColIip, 1934]. The animals were then fasted for 3 days, during which the acetonuria was followed. On the first of these days they received a further I1-0 c.c. of the serum diluted with 2-0 c.c. of saline; on the second day 1-0 c.c. of thyreotropic hormone ("TGS ") with 2-0 c.c. of saline, and on the thr day 3-0 c.c. of saline. It was found desirable to ensure that a fairly large volume of urine should be excreted. Three groups of animals served as controls for this experiment; the first, of fifteen similar rats, received exactly the same preliminary and experimental treatment as the experimental animals, except that they received serum from a normal, untreated horse. The second group had no preliminary treatment, but were fasted -for 3 days and injected with

4 388 P. T. BLACK, J. B. COLLIP AND D. L. THOMSON. 3 0 c.c. saline on the first and third, and 1.0 c.c. thyreotropic extract and 2x0 c.c. saline on the second, of these days; the third group received saline only. The results are summarized in Table III. The experimental animals TABLE III. Effect of thyreotropic extract on acetone excretion in young fasting rats previously treated with anti-thyreotropic serum. Total acetone, mg. per 100 g. Average body body weight: Number Serum in weight averages and standard deviations of preliminary -,A, animals treatment Initial Final 1st day 2nd day 3rd day 14 Anti-TG ±05 2-2± ±0*4 15 Normal ± ± h None ± ± ±z06 Controls (saline injected) 4 None *4 All animals except the last (control) received -Oc.c. of thyreotropic extract on the second day of fasting. treated with the anti-thyreotropic serum did not respond to the thyreotropic extract, and excreted no more acetone than the negative (salinetreated) controls. The animals treated with normal horse serum excreted far more acetone than these, but less than the positive controls subjected to no preliminary treatment. This latter difference may indicate the presence of an inhibitory substance in normal horse serum, but may be due merely to random variation. A similar phenomenon in experiments with normal horse serum has been encountered in the studies on metabolic rate [Anderson and Collip, 1934]. IDENTITY OF THE KETOGENIC SUBSTANCE. The preceding experiments show that some substance present in an anterior pituitary fraction of high thyreotropic potency is capable of increasing the acetonuria in fasting rats, and that there accumulates in the serum of animals subjected to prolonged treatment with this fraction a substance capable of inhibiting this action. It is not clear, however, whether the acetonuria is a consequence of thyroid stimulation; and if this can be disproved, it still remains possible that the ketogenic activity is a secondary and independent property of the substance which stimulates the thyroid. The reports of other investigators on this point are conflicting; our own investigations lead us to believe that the ketogenic effect is not a consequence of thyroid stimulation, and indeed is not due to the thyreotropic substance but to some other active principle in these 0 extracts.

5 ANTERIOR PITUITARY AND ACETONURIA. This belief is based principally on the fact that increased acetonuria may be obtained with the purified growth hormone fraction [Collip, Selye and Thomson, 1933], which has certainly less than 1 p.c. of the power of the thyreotropic extract to elevate the basal metabolic rate of hypophysectomized rats, and does not produce thyroid hyperplasia. Each c.c. of this extract ("Q 40a") represents 1 g. of fresh bovine anterior lobe; its power to increase body weight in hypophysectomized rats has been reported elsewhere. The thyreotropic fraction (" TGS ") used in this study has no such power, even if administration be prolonged until the metabolic rate is no longer elevated. A definite fractionation of activity has therefore been obtained; the two extracts, however, are of almost equal activity in acetonuria in young fasting rats (Table IV). From these TABLE IV. Effect of separated thyreotropic and growth hormones on acetone excretion in young fasting rats. Total acetone, mg. per 100 g. Dose c.c. Average body body weight: Number per 100 g. weight averages and standard deviations of on 2nd and A,, animals 3rd days Initial Final 1st day 2nd day 3rd day Thyreotropic extract "TGS." ± ± ± ± ± ± ± ± ± ± ± ±0-3 Growth-hormone extract "Q40a." ± ± ± ± ± ± ± ± ± ± ± ± observations it may apparently be concluded that the active principle is not identical with the thyreotropic hormone, nor with the growth hormone. We have recently been able to prepare growth-promoting extracts, by repeated adsorption on calcium phosphate, which no longer increase fasting ketonuria. Further evidence that the thyreotropic hormone is not responsible for this effect has been collected. In the first place, it has been found that the injection of 0-1 mg. thyroxine daily into young rats during a 3-day fasting period does not increase the output of acetone; the highest figure from six animals so treated was only 1-1 mg. per 100 g. It is true that animals which have received a longer preliminary treatment with thyroxine do at times develop a considerable ketonuria when fasted; but the rapid response to thyreotropic extracts apparently cannot be duplicated with

6 390 P. T. BLACK, J. B. COLLIP AND D. L. THOMSON. thyroxine, even in doses which (as in this case) exert a greater influence on the metabolic rate. In the second place, it is possible to show that the effect of thyreotropic or growth-promoting extracts is demonstrable in fasting thyroidectomized rats (Table V), and in earlier experiments we could detect a TABLE V. Effect of anterior pituitary extracts on acetone excretion in fasting thyroidectomized female rats. Dose in c.c. Total acetone, mg. per 100 g. Number on days body weight of Body Extract,, A animals weight tested st day 2nd day 3rd day TGS Q40a Q40a Saline marked response to the injection of pituitary extracts in hypophysectomized-thyroidectomized rats on the butter diet. It would seem, therefore, that the "ketogenic" principle of anterior pituitary extracts is not identical with the thyreotropic hormone, nor with the growth hormone; nor can it be identified with the adrenotropic hormone, which is absent from the active extract " Q40a," and which in purified form [Collip, Anderson and Thomson, 1933] has been found to be inert as far as acetone excretion is concerned, both by ourselves in fasting rats and by Butts, Cutler and Deuel [1934] in rats receiving sodium acetoacetate and injected with the extract prepared in our laboratory. The ketogenic principle is therefore present only as an accidental contaminant in the thyreotropic fractions which we have chiefly used; and if there is present in the serum of horses treated for some time with this fraction a substance capable of inhibiting the ketogenic principle (Table III), this substance is presumably (though not certainly) distinct from the one which accompanies it and which inhibits the action of the thyreotropic hormone upon the basal metabolic rate of hypophysectomized rats [Anderson and Collip, 1934]. SUMMARY. 1. The injection of suitable anterior pituitary extracts strikingly increases acetonuria in rats, either during a fasting period of 3 days, or during the administration of a filtered butter diet. 2. This effect may be obtained with growth-hormone preparations practically free from thyreotropic hormone, or with thyreotropic fractions

7 ANTERIOR PITUITARY AND ACETONURIA. 391 free from growth hormone; it is not obtainable with adrenotropic fractions. 3. The effect may be obtained in thyroidectomized animals, and is not obtainable with moderate doses of thyroxine under similar conditions in normal animals. 4. The effect is not obtainable in rats subjected to prolonged preliminary treatment with thyreotropic extracts containing the ketogenic principle. 5. The serum of a horse subjected to prolonged treatment with the thyreotropic extract inhibits the ketogenic action of this extract in young fasting rats. REFERENCES. Anderson, E. M. and Collip, J. B. (1933). Proc. Soc. exp. Biol., N.Y., 30, 680. Anderson, E. M. and Collip, J. B. (1934). Lancet, i, 76, 784. Burn, J. H. and Ling, H. W. (1933). Quart. J. Pharm. Pharmacol. 6, 31. Butts, J. S., Cutler, C. and Deuel, H. J. (1934). J. biol. Chem. 105, 45. 'Collip, J. B., Anderson, E. M. and Thomson, D. L. (1933). Lancet, ii, 347.,Collip, J. B., Selye, H. and Thomson, D. L. (1933). Proc. Soc. exp. Biol., N.Y., 30, 44 Eitel, H., Lohr, G. and Loeser, A. (1933). Arch. exp. Path. Pharmak. 172, 205. Hoffmann, F. and Anselmino, K. J. (1931). Klin. Wschr. 10, Magistris, H. (1932). Endokrinologie, 11, 172. Van Slyke, D. D. (1917). J. biol. Chem. 32, 455.