Carbohydrate metabolism in experimental intrauterine growth retardation in rats

Carbohydrate metabolism in experimental intrauterine growth retardation in rats WILLIAM

OH,

MABEL

D.

LUCY

L.

D’AMODIO, YAP,

LEONHARD Torrance, With JOYCE

M.D.” M.D.

M.D. HOHENAUER,

California,

the technical A.

and

assistance GUY,

M.D.

Chicago,

Illinois

of

B.S.

Intrauterine growth retardation was induced in 34 pregnant rats by ligating the uterine artery supplying one uterine horn of a bicornate uterus at 17 days of gestation. The opposite uterine horn was left untouched and served as the control. The fetuses were delivered at 21 days of gestation. One hundred and fifty-eight control and 141 intrauterine growth-retarded fetuses were used in a series of three experiments. The body weight, placenta, liver, heart, and kidney weights were smaller in the intrauterine growth-retarded fetuses. The blood glucose of intrauterine growth-retarded fetuses at birth was lower than in the controls. The liver glycogen contents were lower in the intrauterine growth-retarded fetuses during the last 2 days of pregnancy. Intravenous glucose infusion to the mother 2 hours prior to delivery of the fetuses eflectively raised the fetal blood glucose to a hyperglycemic range and repletes the previously observed lower hepatic glycogen in the intrauterine growth retardation.

KECE N T wo RK s have shown that in human intrauterine growth retardation, or “small-for-date” infants, the postnatal glucose balance is disturbed with a relatively high incidence of hypoglycemia during the first few days of life.le3 Indirect evidences, based on the reduced glycemic response to glucagon-epinephrine administration, attribute the development of hypoglycemia to decreased glycogen reserve.3v 4 Direct evidence for this concept is still lacking.

Therapeutically, the intrauterine growthretarded infants with neonatal hypoglycemia respond adequately to glucose infusion and,/ or steroid treatment. Furthermore, recent work by Rabor and associates4 has shown that early provision of glucose by promptly feeding these infants after birth parenterally may reduce the incidence of hypoglycemia and improve the glucose homeostasis during early neonatal life. In this study we induced experimental intrauterine growth retardation in pregnant rats by unilateral uterine artery ligation. This animal model, originally described by Wigglesworth, permits the evaluation of carbohydrate metabolism in intrauterine growth retardation, with specific reference to (1) fetal hepatic glycogen reserve in utero, (2) fetal blood glucose values at birth, and (3) effects of maternal glucose infusion prior to delivery of the fetus on the fetal

From the Departments of Pediatrics, UCLA School of Medicine, Harbor General Hospital, Torrance, and Michael Reese Medical Center, Chicago. Supported by Grants HD3863-01 and HD4610-01 of the National Institute of Child Health and Human Development. “Address for reprints: William Oh, M.D., Department of Pediatrics, UCLA School of Medicine, Harbor General Hospital, 1000 West Carson Street, Torrance, California 90509. 415

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Oh et al.

Fig. 1. Schematic presentation of the experimental from one experiment. (Modified from Wigglesworth,

glycogen at birth. Material

reserves

and

and

methods

glucose

homeostasis

Female rats obtained from Holtzman Farm (Madison, Wisconsin) were mated overnight and the pregnancy was confirmed by a positive urine sperm test. At 17 days of gestation (duration of rats pregnancy = 21 days), a laparotomy was performed under ether anesthesia with sterile technique. The uterus was exposed. As shown in Fig. 1, the rat uterus is composed of two uterine horns, each with its own uteroovarian blood supply. A silk ligature was applied to the cervical end of the uterine artery. The opposite uterine horn was left untouched and the fetuses located in this horn were used as control. The uterus was returned to the abdominal cavity, the laparotomy wound was closed and the pregnancy was allowed to continue. The fetuses were delivered at 21 days of gestation by cesarean section under either ether inhalation anesthesia or intraperitoneal injections of 100 mg. per kilogram body weight of sodium inactine. The reduction of body and visceral organ weights in the intrauterine growth-retarded fetuses when compared with the control fetuses indicate the successful production of intrauterine

model. The animal weights were taken J. S.: J. Path. Bact. 88: I, 1964.5

growth retardation by Wigglesworth technique. Three separate experiments are reported in this communication. The number of pregnant rats, fetuses and type of anesthesia are Iisted in Table I. In the initial experiment, body and visceral organ weights of 50 fetuses derived from 6 experimental pregnant rats are listed in Table II. In the subsequent experiments, the Wigglesworth model was considered valid if the body and liver weights were significantly lower in the experimental fetuses. To determine the changes of fetal liver glycogen levels during the last 5 days of gestation, 11 pregnant rats and their fetuses were studied, Fourteen fetuses were delivered at 17 days of gestation. In the remaining 9 pregnant rats, Wigglesworth technique was performed on the seventeenth day, and the fetuses were delivered by cesarean section at 18, 19, 20, and 21 days of gestation. The fetuses were killed immediately after delivery by decapitation; bIood samples of those fetuses who were delivered at 21 days of gestation were collected for glucose analysis. The livers were removed and dropped into a thermos containing liquid nitrogen, within 10 seconds. A known amount of liver tissue was homogenized. The glycogen was

Volume Number

108

Experimental

intrauterine

rats and fetuses in various

experiments

growth

retardation

417

3

Table I. Number

of pregnant

__-_

No. No. of mothers

Experiment Body and organ weights Blood glucose and liver glycogen Infusion study Control Saline Glucose -.Total

Table II. Birth growth-retarded

-...-

of fetuses Intrauterine growth-retarded

Control

Anesthesra

6

25

25

Ether

11

48

37

Ether

5 5 7

26 23 36

22 25 32

Sodiun I Inactine

34

158

141

weight and visceral organ (IUGR) rat fetuses

weight

of control

versus

.--..

-.-_---

intrauterine - .--

Intrauterine growthretarded

Controls

.

Birth weight (grams) Placenta (mg. ) Liver (mg.) Heart (mg.) Kidney (mg.) “No.

of fetuses.

Mean

5.76 890 309 60 21

+ 2 + 3 +-

0.07 30 25 2.2 1.4

(25)” (25) (25) (13) (15)

Corporation,

+ + t 5 +

0.16 35 14 3 2.8

(25) (25) (25) (20) (13)

P < < < < <

0.001 0.05 0.001 0.05 0.001

-

5 S.E.M.

precipitated with 60 per cent ethyl alcohol, and subsequently hydrolyzed by sulfuric acid.G The glycogen contents were measured and expressed as mg. of glucose per gram wet liver weight. In the third experiment, involving 17 experimental pregnant rats with 79 intrauterine growth-retarded and 85 control fetuses, the effects of glucose infusion to the mother prior to birth on the fetal glucose balance and hepatic glycogen reserve were evaluated, Under peritoneal inactine anesthesia, 2 mg. per kilogram per minute of glucose (10 per cent solution) was infused continuously by an infusion pump * following a prime dose of 1 Gm. per kilogram body weight, through the external jugular vein of experimental pregnant rats with 36 control and 32 intrauterine growth-retarded rat fetuses. Blood samples were obtained from the warmed maternal tail veins at the designated intervaIs during the infusion period. As controls, 22 intrauterine growth-retarded and 26 control *Halter

4.08 763 178 41 13

Mt.

Laurel,

New

Jersey.

fetuses from 5 pregnant rats at tern.4 were used. In addition, 5 term pregnant rats were infused with 5 ml. of normal saline solution over a lJ/1 hour period. Twenty three control and 25 intrauterine growth--retarded rat fetuses were obtained from the saline infused group. Maternal blood glucose values were determined at the same interval as the glucose infused group. At the end of the 90 minute saline or glucose infusion, the fetuses were delivered by cesarean sectioi7 and killed immediately after birth by dccapitation. Blood samples for glucose and liver glycogen were measured as described under the second experiment. Blood glucose was analyzed by the glucose osidase metIlod.7 Results The body, placenta, liver, heart, a:~1 kidney weights of intrauterine growth-rcttarded fetuses were significantly lower than the controls (Table II) . Fig. 2 shows that the liver glycogfm content of both intrauterine growth-retarded and control fetuses were similarly lo\\ on thr

418

Oh et al.

fusion (95 t 10 mg. per cent, No. = 5 1. The reason for this was not apparent. Fig. 4 shows the liver glycogen contrnt of intrauterine growth retardation vs. control fetuses at term in relation to maternal hyperglycemia resulting from glucose infusion. The significantly lower liver glyco,yen content in the intrauterine growth-retarded fetuses was repleted to normal levels in the glucose infused group. As expected, the blood glucose values of the fetuses in the glucose infused group were 2yz times higher than the noninfused and saline infused groups (Fig. 5) . The previously observed difference in blood glucose between intrauterine growth-retarded (lower) and control fetuses without the maternal ,giucose infusion, were also abolished. GESTATION,

Fig. 2. Liver growth-retarded S.E.M. The control versus at 20 and 21

Comment

DAYS

glycogen of normal and intrauterine rat fetuses. Values are means t differences between the values of intrauterine growth-retarded fetuses days are significant (P < 0.001).

seventeenth, eighteenth, and nineteenth days of gestation (range of 2.2 to 7.0 mg. per gram wet weight). On the last 2 days of gestation, the liver glycogen content of intrauterine growth retardation was significantly lower than the control fetuses (9.5 2 1.4 and 30.5 + 1.2 vs. 21.0 f 3.2 and 59.0 + 8.0 mg. per gram wet weight on the twentieth and twenty-first days of gestation, respectively). The lower liver glycogen in intrauterine growth-retarded fetuses at term was associated with a lower blood glucose value. The blood glucose of intrauterine growth-retarded fetuses was 65 k 9 mg. per cent in contrast to the control of 90 + 6 mg. per cent (P < 0.001). In the glucose infusion experiment, the maternal blood glucose values of the glucose infused group ranged from 180 to 2 10 mg. per cent from 15 to 120 minutes after the glucose administration. The saline infused mother had blood glucose values of 63 to 75 mg. per cent (Fig. 3). The latter appeared to be lower than those without in-

The data on body weight, and visceral organ weights were similar to those obtained by Wigglesworth and by Blanc.8 These data confirmed the production of intrauterine fetal growth failure by reducing the blood supply to the maternal portion of the placenta, hence artificially producing placental circulatory insufficiency. A similar model has been produced in pregnant sheep in which fetal growth retardation was observed following single umbilical arterial ligation in the beginning of the third trimester of pregnancy.” It seems reasonable to assume that the reduction of fetal body weight as well as the visceral organ weights is a good index for the intrauterine growth retardation, attributable to the experimental procedure producing uteroplacental circulatory compromise. We have reported the body compositional changes of the rat intrauterine growth retardation model e1sewhere.l” We observed a reduction of carcass dry fat free solid, protein, and fat in intrauterine growthretarded fetuses along. with a reduction of liver glycogen contents reported in this communication. Hence, it seems apparent that in

intrauterine

trauterine

growth substrate

retardation

deprivation

volves all three major nutrients sustain normal fetal growth.

the probably

necessary

inin-

to

Experimental

intrauterine

growth

retardation

419

mg./lOOml. 300

250

-

200

-

150

f

-

Fetuses delIvered

100

-

50

infusbon

o-

(controls)

PWinfusIon

I5

30 MINUTES

Fig.

3. Maternal

mq. /q.

100 90

blood

glucose

levels

45

60

AFTER

75

GLUCOSE

following

90

105

120

INFUSION

intravenous

glucose

infusion

to the

mother;.

wet wt.

r

7 SALINE I NFUSION

NO INFUSION

GLUCOSE INFUSION

00 70 60 50 40

IUGR

30 20 IO CIL

Fig. 4. Effects of maternal intrauterine growth-retarded of observations.

and rat

fetal hyperglycemia fetuses. The numbers

Fetal glycogen synthesis in utero has been studied previously in different species. In rat, Villee’r has shown that during the first three quarters of pregnancy, placenta is the main organ for glycogenesis. During the last quar-

on the within

hepatic glycogen the bars indicate

ter of pregnancy, the major synthesis is shifted to the glycogen storage increased during the last 2 days of ther, ShelleyJz-’ R has shown

contents of the number

site of glycogen liver, where the six- to sevenfold pregnancy. Furthat the hepatic

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October J. Obstet.

1, 1970 Gynec.

250 NO INFUSION

SALINE INFUSION

GLUCOSE INFUSION

175Iso125

Control

Fig. 5. Fetal blood glucose level of intrauterine growth-retarded infused group in contrast to controls and saline infused group. indicate the number of observations.

glycogen level fell rapidly during the first 3 hours of extrauterine life. These finding were later confirmed by Dawkins.14 In our study, the normal fetuses also showed an increment in hepatic glycogen reserve during the last 2 days of pregnancy. During this time, the intrauterine growth-retarded fetuses stored half as much liver glycogen as the controls. Glycogen is an important reserve for optimal glucose homeostasis immediately after birth. A reduction of its reserve could conceivably account for the observed lower blood glucose values in the intrauterine growth-retarded group. While it is not desirable to make comparison between the results of animal experiments with those of human clinical observations, it is of interest that in human intrauterine growth retardation, there are indirect evidences of decreased hepatic glycogen reserve1 accounting for the frequent occurrence of neonatal hypoglycemia in intrauterine uterine growth retardation.1-3 The mechanism for the reduced glycogen content in the liver is not obvious. With uterine artery ligation and its consequence

rat fetuses in the glucoseNumbers within the bars

of uteroplacental circulatory insufficiency, the placentofetal blood flow is reduced. Hence, the amount of substrate presented to the fetus will be reduced. The reduction of glucose supply may result in the reduction of final glycogen reserve in the liver at the time of birth. The above hypothesis was supported by the glucose infusion experiment. In this instance, the blood glucose concentrations of the fetuses were elevated to two to three times the normal. Hence, the apparent deficit in glucose supply to the intrauterine growth-retarded fetuses, due to reduced placentofetal blood flow, was compensated by the increase in substrate concentration ; hence the difference in blood glucose values and liver glycogen content at birth were not observed. The additional implication of the glucose infusion results should be mentioned. Studies in the low birth weight infants have shown that early provision of nutrients by early oral or intravenous feeding in the postnatal period is beneficial for glucose homeostasis.15-16 Such practice would also improve

Experimental

glucose balance in intrauterine malnourished infants. Therefore, one can speculate that, if intrauterine growth retardation is predicted during labor, intravenous glucose infusion to

intrauterine

growth

retardation

421

the mother in labor may improve the infant’s carbohydrate reserve even before birth and during the first hours after birth.

REFERENCES

1.

2. 3. 4.

5. 6.

7. 8.

Cornblath, M., Wybregt, S. H., Baens, G. S., and Klein, R. I.: Pediatrics 33: 388, 1964. Haworth, J. C., and McRae, K. N.: J. Lancet 87: 41, 1967. Raivio, K. O., and Hall, N.: Acta Paediat. (Stockholm) 57: 517, 1968. Rabor, I. F., Oh, W., Wu, P. Y. K., Metcoff, J., Vaughn, M. A., and Gabler, M.: Pediatrics 42: 261, 1968. Wigglesworth, J. S.: J. Path. Bact. 88: 1, 1964. Bergmeyer, H. U., editor: Methods of Enzymatic Analysis, New York, 1963, Academic Press, Inc., p. 59. Marks, V.: Clin. Chem. Acta 4: 395, 1959. Blanc, W. A.: Pediat. Res. 1: 218, 1967.

9. 10. 11. 12. 13. 14. 15.

16.

Emmanouilides, G. C., Townsend, D. E., and Bauer, R. A.: Pediatrics 42: 919, 1968. Hohenauer, L., and Oh, W.: J. Nutrition 99: 23, 1969. Villee, C.: J. Appl. Physiol. 5: 437, 1953. Shelley, H. J.: Brit. Med. Bull. 17: 137, 1961. Shelley, H. J.: Brit. Med. J. 1: 273. 1964. Dawkins, M. J. R.: Brit. Med. Bull. 22: 27, 1961. Wu, P. Y. K., Teilman, P., Gablcr, M.. Vaughn, M., and Metcoff, J. Pediatrics 39: 733, 1967. Beard, A. G., Panos, C. T., Marasijgan, B. V., Eminians, J., Kennedy, H. F., and Lamb. J.: J. Pediat. 68: 329, 1966.