(Sherrington School of Physiology, St Thomas's Hospital, London.)

Transcription

1 6I2.352.I7:6II-0I3.85 MATERNAL CONTROL OF THE PLACENTAL GLYCOGEN. BY A. ST G. HUGGETT. (Sherrington School of Physiology, St Thomas's Hospital, London.) CLAUDE BERNARD(1), in 1859, showed that glycogen exists in the placenta of the rabbit. He also demonstrated that it exists in lesser quantity, with a somewhat different distribution in the Ruminants, uterus and trophoblast, and that it appears in the feotal liver in later uterine life. PflI iger (2) showed, however, that traces were to be found in the foetal liver before the middle of pregnancy. In 1908 Lochhead and Cramer (3) proved that glycogen is mainly present in the maternal portion, but is found in the fcetal portion of the placenta in minute traces; it was maximal at the 21st-22nd day of pregnancy in the rabbit. After that date it decreased in quantity and percentage in the placenta, while it steadily increased in the feetal liver. They further showed that the glycogen of the placenta is not increased by feeding with carrots, but that phloridzin injections will lower the glycogen content. They also found an amylolytic ferment in glycerine extracts of the placenta. The object of this paper is to determine what are the factors which control the deposition of glycogen in the placenta of the rabbit at its maximum on the 21st day, and, further, the agencies concerned in its removal from the placenta in the final period of pregnancy. The placental deposit of glycogen may be controlled by influences from the mother or foetus or both. It may be a transition stage for carbohydrate passing through the placenta, or it may be a storehouse for glycogen on behalf of the foetus. In this case we would expect it to be controlled by the fcetal factors. The maternal stimuli determining the liver and muscle glycogen deposits of the mother may modify the deposition in the placenta. Finally, the control of the placental glycogen may be in no way parallel to other glycogen reserves, but be determined by entirely new factors. In support of the view that the control may be maternal in origin is the fact that the glycogen is in those placental tissues which arise

2 MATERNAL CONTROL OF PLACENTAL GLYCOGEN. 361 from the uterus, that is the decidua basalis, and not in the chorionic portion of the placenta (Maximov(6)). In this paper some of the possible maternal influences are investigated and comparison is made between the glycogen of the placenta and the glycogen of the muscles and liver. Methods. The experiments were confined to rabbits, and were performed on the 21st day of pregnancy, at which time it is believed the percentage of glycogen in the whole placenta is maximal. The glycogen was determined in the whole placenta, no separation of the decidual from the chorionic portions being carried out. The carbohydrate in the foetal portion being almost negligible, this result served as an index of the maternal placental glycogen. Glycogen estimations. These were carried out by Pfliiger's method. The placenta or liver were dissolved in their own volume of boiling 60 p.c. potash and were heated in a boiling water bath for 3 hours. The hydrolizate was then cooled and diluted with five or more volumes of water. 25 c.c. of this solution forming an exact aliquot part was transferred to a centrifuge tube of 100 c.c. capacity. The precipitation of the glycogen by the two volumes of alcohol and the washing of the precipitate with alkaline alcohol and 66 p.c. alcohol were carried out entirely in the centrifuge tube. It was redissolved in water, the centrifuge tube being heated in a water bath. It was purified by reprecipitation with alcohol and redissolved in water. This solution was then hydrolized by hydrochloric acid in a water bath for 3 hours and the glucose formed was estimated by the Wood-Berry(4) method. The 66 p.c. alcohol used for washing the glycogen contained sodium chloride to prevent loss by solution in the washings. Simultaneous estimations were performed of the glycogen in the maternal liver, maternal thigh muscles, the whole foetus, but not the foetal liver alone. In all cases, except where specifically stated, the pregnant animal was starved for 24 hours before killing. The normal placental glycogen. The control results on pregnant rabbits killed at the 21st day of pregnancy are given in Table I. In these results the most obvious facts are the relative constancy of the placental figures compared with the hepatic percentages. Mere starvation for 24 hours in a rabbit (that is removal of all food from the cage) is known to have a variable effect on the liver glycogen (5), especially as there is always a quantity of undigested material of varying composition in the animal's stomach. The second point worthy of note is that the percentage of glycogen in the whole foetus at that date is rela-

3 362 A. ST G. HUGGETT. TABLE I. Percentages of glycogen in rabbit's tissues at 21st day of pregnancy after 24 hours' starvation. Matemnal Whole Maternal Whole liver placenta muscle fcetus *79 3* *58 0*09 2* * * * Average tively high. This includes of course the foetal liver which at the end of the second week of pregnancy is gradually increasing its glycogen stores. It was not separated from the rest of the foetus for separate estimation on account of the practical limitation of the experiments. The muscle glycogen quantities are in accordance with the results for animals starved for 24 hours(7). Carbohydrate feeding. Loclhhead and Cramer found that the ingestion of carrots had no effect on the quantity of glycogen in the placenta. Their results were confirmed and are shown in Table II. TABLE II. Placental glycogen at 21st day. Effect of feeding with carrots. Placenta Rabbit Liver Placenta Ratio Liver Muscle Fcetus (i) 6*0 2*48 0*41 0* (ii) *40 0* (iii) * It is evident that there is no parallel between quantities of glycogen in the placenta and maternal liver or maternal muscles. The same applies to the foetal tissues as a whole. This is well brought out by comparing the percentages of glycogen in the placenta and liver. Percentage Glycogen in Placenta Percentage Glycogen in Liver Normal animal starved for 24 hours 3 10 = 1-84 Carbohydrate fed rabbit =038 The higher the ratio the smaller the percentage of glycogen in the liver relative to the placental percentage, and vice versa. Injections of glucose. Attempts were made to increase glycogen by injections of glucose. These were carried out by intravenous and intraperitoneal injections. Again it was found that the glycogen of the placenta varied much less than the glycogen of the liver.

4 MATERNAL CONTROL OF PLACENTAL GLYCOGEN. 363 TABLE III. Rabbit's glycogen deposits. Effect of glucose injections. Dose of glucose Manner of Liver Placenta Muscle Fcatus grin. injection p.c. p.c. p.c. p.c. 16 Ear vein slowly Intraperitonea The glucose used was a 20 p.c. solution in Ringer's fluid. The action ofinsulin. The effect of insulin was ascertained. The results are given in Table IV. The dose of insulin varied. In each case it was given subcutaneously in solution containing 2 units per c.c. TABLE IV. Action of insulin on glycogen stores in pregnant rabbit. Percentages. Mode of administration Liver Placenta P/L Muscle Fcetus Group A. (i) units per kg. after 24 hours' (ii) starvation (iii) (iv) Group B. 6 units per kg., 3 plus 3 units (v) on the same day ) X2} Group a. 2 units and 3 units per kg. on successive days, 5 units in all with hours' starvation (vi) (vii) ' * Rabbit (i) was killed after 3 hours. Its blood sugar had fallen from 133 mg. to 26 mg. per 100 c.c., but no convulsions had supervened. Rabbit (ii) was killed 4 hours after the injection of insulin. It showed no convulsions. Rabbits (iii) and (iv) were killed 4 hours after insulin and neither showed convulsions. Rabbits (ii), (iii) and (iv) all gave typical sugar depressions following the injection of insulin. Rabbit (v) was killed 2 hours after the second dose of insulin which itself followed the first dose by 2 hours. It never had convulsions. Here we first see signs of the placental glycogen being low. Rabbits (vi) and (vii) both had violent convulsions after the second dose, but none after the first dose on the first day. Rabbit (vi) died in convulsions and the estimation was carried out at once. Rabbit (vii) was killed after convulsions had lasted 40 minutes off and on. Here the same rather low placental glycogen was observed as in rabbit (v), but it is nevertheless high compared with the glycogen in the liver which has almost gone and in the muscle of rabbit (vi) which died in convulsions. In rabbit (vi)

5 364 A. ST G. HUGGETT. incidentally the total feetal glycogen is reduced. No stress, however, is laid on the significance of these foetal figures. It has been stated (Best, Hoet and Marks(8)) that there is an accumulation of glycogen in the liver and muscles when glucose and insulin are given simultaneously. It was therefore of interest to know whether anything similar was observable in the placenta. TABLE V. Effect of insulin and glucose on rabbit's glycogen stores. Percentages. Dose Liver Placenta P/L Muscle Fcetus (i) 2 units insulin per kg. and) 1*36 10 grm. glucose subcutaneous f (ii) 3 units insuin per kg. and grm. glucose intravenous J 3* *20 It seems from Table V that in the liver formation of glycogen is stimulated but nothing similar occurs in the placenta. Adrenaline and glycogen percentage. This is usually considered to have a diminishing effect on the glycogen of the liver (Pollak (9)). The glycogen of the placenta is within the maternal portion and consequently it is of interest to determine the action of adrenaline on the decidual glycogen. TABLE VI. Effect of adrenaline on glycogen percentages in the pregnant rabbit after 24 hours' starvation. Dose Liver Placenta PIL Muscle Foetus (a) Intravenous. (i) 0-01 mg., repeated twice} 0 03 mg. in all f (ii) 010 mg. repeated twice 0 3 mg. in all t 0* (b) Subcutaneous. (iii) 0 3 mg. per kg. (iv) 0-3 mg. per kg. (v) 0 9 mg. in two portions of 0 3 and 0-6 mg. at 1 hour interval Rabbits (i) and (ii) killed 2 hours after the last injection, rabbits (iii), (iv) and (v) killed 31 hours after the last injection. The effect of adrenaline on the placental glycogen is negative, neither increasing nor decreasing it. The effect on the liver is a diminution of glycogen, but on the placenta it has no such effect. With the maternal muscles there is no such definite change as with the liver. The thyroid gland. The thyroid gland increases basal metabolism causing an increase of the nitrogenous excretion and of the respiratory

6 MATERNAL CONTROL OF PLACENTAL GLYCOGEN. 365 exchange. Feeding thyroid to rabbits has resulted in a decrease of the hepatic glycogen. This was shown by Cramer and Krause(lo) in 1913 and has been confirmed frequently since then. The effect on the glycogen of the placenta was tested by giving two rabbits 0 75 grm. of dried thyroid gland (Armour), by the mouth daily for six days. Food was withheld for the last day.. TABLE VII. Effect of 075 grm. dried thyroid gland daily for 6 days on the glycogen in the pregnant rabbit. P.C. loss of Liver Placenta P/L Muscle Fcetus weight * *46 2* These two values for placental glycogen are lower than the normal percentages, but the reduction is not marked and is in no way proportionate to the loss in the hepatic glycogen, as shown by the relatively high P/L ratios of 2-1 and 4-8 compared with the normal average of Phlorrhizin. Lochhead and Cramer examined the action of phlorrhizin on the placental and foetal glycogen deposits. They found it caused a diminution in the weight of the glycogen in the placenta and foetus. This reduction was more marked in the foetus than in the placenta and most pronounced at the 27th day of pregnancy. The glycogen percentage of the placenta was practically unaffected at the 21st day, but the absolute weight of the placenta and foetus was decreased, especially at the 27th day. Consequently phlorrhizin caused a diminution of growth of the placenta and of the foetus, and associated with this there was a diminution in glycogen percentage. This retardation of growth was confined to the foetal tissues, while the glycogen of the placenta is mainly in the maternal decidual portion. With the thyroid-fed rabbits of Table VII the glycogen percentage diminishes at the 21st day in the placenta. This is associated with a 20 p.c. loss in weight in the mother, but there is no diminution in placental weight as compared with the normal. This is shown in Table IX. Tetrahydro-/3-naphthylamine. Adrenaline caused no disappearance of the decidual glycogen, while thyroid given in repeated doses produced some diminution. It was therefore of interest to investigate the effect of tetrahydro-p-naphthylamine (T.H.N.). The action of this substance is marked by a rise of temperature and by restlessness (Fawcett and Hale White, 1897 (1i)). This condition has been described by Cramer (12) as "sympathetic fever" and is marked by a diminution of the liver glycogen.

7 366 A. ST G. HUGGETT. The T.H.N. was dissolved in warm saline and injected subcutaneously. This was given on the 16th to 21st days inclusive, the animal being killed on the 21st day 2 hours after the injection. The results are shown in Table VIII. TABLE VIII. Effect of 6 days' administration of tetrahydro-,-naphthylamine on the glycogen percentages at the 21st day of pregnancy. Temperature Dose rise Liver Placenta P/L Muscle Fcetus 60mg. per day 10 C '67 80mg. per day 1.5 C mg. per day 2 50 C The most outstanding point is that the placental glycogen is markedly reduced as well as the liver glycogen. This reduction in percentage is not accompanied by a reduction in weight of the placenta or foetus, and it is therefore a change peculiar to the glycogen itself. It is more pronounced than that of phlorrhizin in the dosage used by Lochhead and Cramer. It is of interest to correlate the actions of these reagents on the weights of the placenta and fcetus with their effect on the glycogen deposits. These results are tabulated in Table IX. The phlorrhizin is the only reagent that lowers both the foetal weight and the fcetal glycogen percentage at the 21st day. At this stage it has no material effect on the placenta. The T.H.N. markedly lowers the placental glycogen but has no action on the placental weight or the foetus, whether whole weight or glycogen percentage. Thyroid feeding like T.H.N. causes a diminution of placental glycogen percentage but the effect is not so marked. The weight is barely affected while the foetuses are unaffected. TABLE IX. Weights of placenta and foetuses together with their glycogen percentages at the 21st day of pregnancy. Effect of adrenaline, thyroid, T.H.N. and phlorrhizin. Placental Foetus Weight Glycogen Weight Glycogen Treatment grm. p.c. grm. p.c. 1. Normal-average Adrenaline (i) (ii) Thyroid (i) (ii) T.H.N. (i) (ii) (23rd day) (iii) Phlorrhizin (Lochhead) and Cramer) N.B. The third T.H.N. rabbit was 23 days pregnant. This accounts for the high fcetal weight. This compares with Lochhead and Cramer's averages of 4-01 grm. placental weight, 1-98 p.c. placental glycogen, 7.20 grm. fcetal weight with 0.28 p.c. foetal at the 23rd day.en720gi.ftawegtwt0-8pcfealgyon glycogen

8 MATERNAL CONTROL OF PLACENTAL GLYCOGEN. 367 The next point to note is that the increase in metabolism due to T.H.N. is attributed to sympathetic stimulation. But adrenaline injections did not cause the same diminution in placental glycogen as T.H.N. There was, however, this difference that the adrenaline rabbits had injections only on one day, whereas the T.H.N. rabbits were injected on the five days previous to killing. One of the adrenaline rabbits does show, however, a placental glycogen of 2-43 p.c. which is somewhat low for the placenta. Another small point of interest is the relatively high percentage of glycogen in the foetus in these adrenaline rabbits. Without stressing the point it is worth noting that while most observers are agreed that adrenaline causes a diminution in the glycogen of the placenta, some workers, notably Drummond and Paton(13) and more recently Kuriyama(14), state that repeated injections of adrenaline cause an increase in hepatic glycogen. The explanation of these discrepancies has not so far been advanced, though it appears to bear some relation to the initial glycogen percentage of the liver and the degree of protein katabolism. The effects of these reagents were controlled by using a series of normal pregnant rabbits at the 21st day of cyesis. This type of control is open to obvious criticism. It would seem better to control these experiments by using the foetuses of one horn of the uterus as control against those in the second horn. The objection to this is that the necessary anaesthetic might cause glycogenolysis. However, an attempt was made using amytal as the anaesthetic. The amytal was injected either intraperitoneally or subcutaneously, and 1-3 hours later a small quantity of ether given for induction. This was found to be necessary in all cases of amytal anaesthesia-up to 80 mg. per kg. The abdomen was opened, the foetuses removed with their placenta from one horn and, the uterus retracting, the horn was returned to the abdomen. There was no post-partum haemorrhage from the opened horn of the uterus. The abdominal wound was clipped and the drug for the experiment administered. The abdomen was opened 3 hours later and the other foetuses, placentae, liver and muscle tissue removed and the glycogen estimated. The results are given in Table X. In these cases rabbits are included which had proceeded beyond the 21st day of pregnancy. This, however, does not vitiate the experiment as an illustration of the use of one horn of the uterus as a control against the remaining horn. From these results therefore the following facts emerge: (i) That the liver glycogen percentages are much below normal even in the first rabbit which had had no hormones injected. These low result v

9 368 A. ST G. HUGGETT. TABLE X. Amytal ansesthesia. Laparotomy and unilateral Caesarean section. Effect of operation, anaesthetic, insulin and adrenaline on the glycogen percentages of the pregnant rabbit. Percentages of glycogen Placentae Fcetuses Maternal Operation and Differ- Differhormone Before After ence Before After ence Muscle Liver 1 Anesthetic and operation only th day 2. Insulin 3 units per kg th day 3. Insulin 3 units per kg st day 4. Adrenaline Normals 28th day Lochhead andr Cramer may in the rabbits that had hormone be partly due to the hormone, but the ansesthetic and operative processes have undoubtedly lowered the quantity of glycogen present in the liver before the injection of any hormone. (ii) This method of control is of no value with the placental glycogen because the initial values are markedly subnormal, showing that the anesthetic and operative processes are factors removing the glycogen from the placenta. One reason for variation in these cases is that the placenta sometimes retracts off the uterus and develops a blood clot in between it and the uterine mucosa. In rabbits the placenta is deciduate, so the retraction and separation occur through the uterine mucosa. In those cases where abortion occurred or obvious placental separation had existed at the opening of the second horn, the fcetus and placenta were excluded from the glycogen estimations. Included, however, are all those cases in which there was no obvious and definite separation or subplacental hsemorrhage. Therefore some cases of constricted circulation to the placenta might have been included. These results on the use of amytal agree with those of Hines, Lees e and Baker (15) showing that amytal is an unsuitable aneesthetic for glycogen formation experiments.

10 MATERNAL CONTROL OF PLACENTAL GLYCOGEN. 369 DISCUSSION. It is evident from these results that the glycogen of the placenta varies in a different manner from that of the liver. It is much more stable. Factors which lower or raise the blood sugar have no effect upon it. The onset of insulin convulsions alone does not lower it to any marked degree, but repeated doses of insulin on successive days cause a definite decrease. It is more resistant under insulin than the muscle glycogen. On the other hand, those measures which might be expected to raise the glycogen stores and which are effective in this way with the liver deposits are quite ineffective with the placental glycogen. Feeding with carbohydrate, injection of glucose alone or with insulin have no such effect in the case of the placental glycogen. It still preserves its constancy. Thyroid, tetrahydro-,b-naphthylamine and to a lesser degree adrenaline are reagents which are marked by increases in metabolism, as shown by the temperature of the body, nitrogen excretion and oxygen consumption. The effect of the first two on the placental glycogen was to cause a definite decrease. With adrenaline this was barely noticeable. With thyroid there was a slight fall to figures of 145 and 2-23 p.c., and at the same time there was the marked loss in maternal weight of 20 p.c. In the case of the tetrahydronaphthylamine the fall in the placental glycogen was most pronounced, 0-8 p.c. being the average percentage. In none of these cases was there any evidence of a fall in the foetal glycogen reserves or of the foetal weights. Their action has consequently been purely or mainly on the maternal metabolism. As far therefore as the maternal stimuli are concerned the placental glycogen is very insusceptible to changes and is uninfluenced by ordinary factors of maternal metabolism. But with extreme increase in the endogeneous metabolism or gross demands on the carbohydrate stores, such as repeated insulin injections produce, the glycogen of the placenta slowly disappears, though never entirely under the conditions of the experiment. Animals dying in insulin convulsions contain glycogen in their placentae, suggesting that even if it is mobilized in extreme carbohydrate want, it is usually mobilized slowly. The only condition in which it was decreased at all rapidly was where ether and amytal were used as anaesthetics and COsesarean section performed. In this period of 2 to 3 hours there was an appreciable fall of the placental glycogen. The exact cause of this fall cannot be stated on the evidence available.

11 370 A. ST G. HUGGETT. SUMMARY. 1. The placental glycogen is remarkably constant in percentage relative to the glycogen of the liver and muscles of the mother. 2. It is unaffected by carbohydrate feeding and by glucose injections, whether alone or with insulin. 3. Depletion of the glycogen of the maternal liver is unaccompanied by a fall or rise in the placental glycogen. 4. Repeated doses of insulin on successive days cause a slight fall. 5. Ether and amytal cause a decrease in placental glycogen 6. Excessive continued increase in metabolism as in the pyrexia due to tetrahydro-,b-naphthylamine or in thyroid feeding over several days causes a decrease in the glycogen percentage, but this is not proportional to the decrease in the glycogen in the liver or muscle of the mother. 7. The glycogen of the maternal portion of the placenta appears to be a reserve for the foetus which the mother cannot normally draw upon except in extreme cases of disordered metabolism over a prolonged period. 8. These results apply to the rabbit at the 21st day of pregnancy, when the placental glycogen is maximal, but not necessarily to the full term rabbit when it has markedly diminished. In conclusion, I wish to express my thanks to Dr W. Cramer for suggesting the use of tetrahydro-,b-naphthylamine and for giving me some from his stock. I wish further to thank the Beit Memorial Trustees, since it was during the tenure of a Beit Fellowship that this work was initiated. The expenses have been defrayed by a grant from the Government Grant Committee of the Royal Society.

12 MATERNAL CONTROL OF PLACENTAL GLYCOGEN. 371 REFERENCES. 1. B ernard. Journ. Physiol. de 1'Homme, 2. p Pfliiger. Pfluger's Arch p Lochhead and Cramer. Proc. Roy. Soc. B, 80. p Wood and Berry. Proc. Camb. Phil. Soc. 12. Pt ii. p (Described by Mellanby and Woolley. This Journ. 49. p ) 5. Macleod. Carbohydrate Metabolism and Insulin, p London, Maximov. Arch. f. Mikroskop. Anat. 51. p Macleod. Carbo. Metab. and Insulin, p Best, Hoet and Marks. Proc. Roy. Soc. B, 100. p Pollak. Arch. f. exp. Path. u. Pharm. 61. p Cramer and Krause. Proc. Roy. Soc. B, 86. p Fawcett and Hale White. This Joum. 21. p Cramer. Fever, Heat Regulation, Climate and the Thyro-adrenal Apparatus, p. 58. London, Drummond and Noel Paton. This Journ. 31. p Kuriyama. Journ. Biol. Chem. 34. p Hines, Leese and Baker. Proc. Soc. Exp. Biol. and Med. 25. p