Upsala J Med Sci 95: 147-155, 1990 

Perinatal Hypoadrenalism in the Rat does 
not Alter Glucose Tolerance and Insulin 

Secretory Response to Glucose 
Mats G. Karlsson, Ulf J. Eriksson, Ingemar Swenne 

Department of Medical Cell Biology, Uppsala University, Uppsala, Sweden 

ABSTRACT 
Fetal and neonatal hypoadrenalism was induced by treating rat 
mothers with metyrapone from day 12 of pregnancy to day 7 
postnatally. Hypoadrenalism in the neonates was indicated by a 
slight reduction in body weight, adrenal hyperplasia and a 
tendency towards reduced adrenal corticosterone concentration. An 
intraperitoneal glucose tolerance test on the 7-day old neonates 
did not show any disturbancies of glucose disposal or insulin 
secretory response to glucose. The data suggest that adrenal 
corticosteroids are not essential for the perinatal development of 
the B-cell secretory response to glucose. 

INTRODUCTION 
The functional development of the pancreatic B-cell takes place 
in an orderly fashion during fetal and neonatal life. In the rat 
fetus B-cells can be identified morphologically on day 12 of 
gestation (27, 35) but they do not attain functional competence 
until later in development. Glucose-stimulated insulin secretion 
is absent on gestational day 15 (28), can be demonstrated on day 
18 (3, 1 9 ) 1  develops further during the neonatal period (1) but 
does not reach its full capacity until adult life (11). The 
factors governing these developmental changes are not fully 
understood, but glucose itself has been demonstrated to stimulate 
the development of a glucose-sensitive insulin secretory response 
(2, 8).Howeverl it is also of interest to note that serum 
concentrations of growth hormone ( 4 ,  29, 30), thyroid hormones 
(7) and corticosterone (16) all increase dramatically during the 
perinatal period and could potentially modulate B-cell develop- 

147 



ment. Growth hormone increases the insulin output of fetal 
pancreatic islets in vitro but fails to increase the glucose 
sensitivity of the secretory process or islet cell replication 
(32).Fetal decapitation in utero, which minimizes pituitary 
influence on pancreatic development, does not retard the develop- 
ment of insulin secretion (17, 20).Thyroid hormones are similarly 
without effect on B-cell maturation in vitro (32) and perinatal 
hypothyroidism does not affect the development of the insulin 
secretory response to glucose in vivo (31). So far the possible 
role of corticosterone in this process has not been investigated. 

In the present study insulin secretory development is inves- 
tigated in the offspring of rats treated with metyrapone during 
pregnancy and lactation. This substance crosses the placenta and 
diminishes both maternal and fetal corticosterone synthesis (10) 
by inhibition of adrenal hydroxylases ( 5 ,  22), thereby inducing a 
state of hypoadrenalism. 

MATERIALS AND METHODS 
Female Sprague-Dawley rats from a local colony (Biomedicum, 
Uppsala, Sweden) were caged overnight with males and vaginal 
smears were taken on the following morning. Day 0 of pregnancy 
was considered as the day on which a sperm-containing smear was 
found. In order to induce hypoadrenalism in the offspring, the 
rat mothers were treated with metyrapone from day 12 of pregnancy 
to the end of the experiment on day 7 postnatally.Two ml of an 
aqueous solution of metyrapone ( MetopironR; kindly provided by 
Ciba-Geigy, GBteborg, Sweden) was administered with a gastric 
tube as a once daily dose of 50-70 mg/kg body weight. Control 
animals were not treated with gastric intubation. All animals had 
free access to drinking water and pelleted laboratory animal chow 
(R2 pellets; A-lab, Sodertalje, Sweden) throughout the experi- 
ment. Delivery took place spontanously on day 22 of pregnancy in 
both experimental and control group. 

On day 7 postnatally a glucose tolerance test was performed on 
the offspring. The pups were weighed and some removed for sampling 
of basal serum concentrations of glucose and insulin or for 
dissection of pancreas and adrenals. The remaining pups received 
an i.p. injection of 2 g/kg body weight of glucose in a 30% (w/v) 

148 



aqueous solution. The injection site was sealed with vaseline and 
the animals returned to their mothers. At 30, 60 and 120 min 
after the injection animals from each litter were killed by 
decapitation and blood collected from the severed neck vessels. At 
the end of the experiment the mothers were weighed, killed by 
cervical dislocation and blood collected from the cut neck 
vessels. 

Blood samples were allowed to clot for one hour at + 4 O  before 
centrifugation and separation of serum. Serum samples were stored 
at -20° until analyzed. Serum glucose was determined with a 
glucose oxidase technique using a Beckman Glucose Analyzer 2 
(Beckman, Fullerton, USA) and insulin was assayed radioimmunologi- 
cally (12). Corticosterone was assayed with a commercially 
available radioimmunoassay (Radioimmunoassay Ltd, Cardiff, UK). 

Pancreatic glands of neonates, dissected out immediately after 
decapitation, were weighed and sonicated in 500 ~1 distilled 
water. After sonication 200 p l  of each sample was added to 2 ml 
acid ethanol (70% ethanol with 1.5 ml 1 M HC1/100 ml). The samples 
were thouroughly mixed and incubated overnight at +4O, centrifuged 
and the supernatants stored at -20° until assayed for insulin. 

Adrenal glands of neonates, dissected out immediately after 
decapitation , were weighed and sonicated in 500 pl distilled 
water. Aliquots were removed for determination of their DNA 
content (14, 21). The remaining samples were diluted with equal 
volumes of absolute ethanol and stored at -20° until assayed for 
corticosterone. 

Results are given as means f S.E.M. and statistical analysis 
was performed using Student's two-tailed t-test for independent 
observations. 

RESULTS 
Metyrapone-treated pregnant rats had an apparently normal 
pregnancy and delivery occurred at the same gestational age as in 
the control rats. Litter size was not affected, nor was there any 
difference in maternal body weight on day 7 postnatally (Table 1). 
The basal serum glucose and insulin concentrations of metyrapone- 
treated rat mothers were not different from those of control 
mothers. However, the serum corticosterone concentration was 

149 



Table 1. Effects of metyrapone treatment during pregnancy and 
lactation in the rat. 

Controls Metyrapone- 
treated 

Maternal 
body weight (9) 
serum glucose (mM) 
serum insulin (pg/l) 
serum corticosterone (nM) 

litter size (n) 
body weight (9) 
serum insulin (pg/l) 
adrenal wet weight (mg) 
adrenal wet weight/DNA 

corticosterone/adrenal 
wet weight (pmol/mg) 
insulin/pancreatic wet 
weight (ng/mg) 

Neonatal 

( m g h  1 

303 f 6 (8) 
6.5 f 0.3 (8) 

0.41 f 0.04 (8) 
5.4 t 2.4 (8) 

11.0 f 1.0 (8) 
14.9 f 0.2 (88) 
0.40 f 0.04 (27) 
0.81 t 0.08 (25) 

39 f 3 (25) 

57 f 10 (25) 

111 f 7 (27) 

316 f 8 (11) 
6.5 f 0.3 (8) 

0.43 t 0.08 (7) 
1.3 f 0.2 (8)* 

10.2 f 0.2 (11) 
14.4 f 0.2 (104)* 
0.29 f 0.01 (24)*** 
1.32 f 0.12 (24)*** 

52 1: 10 (22)*** 

37 f 6 (23) 

68 f 11 (25)** 

Table 1. Pregnant and lactating rats were administered metyra- 
pone, 50-70 mg/kg body weight, by gastric intubation from day 12 
of gestation to day 7 postnatally. On day 7 postnatally both 
mother and pups were killed and the adrenal and pancreatic glands 
and serum were collected for assay of hormones. Values are given 
as means f S.E.M. for the number of rats (n) indicated. Sig- 
nificance of difference between metyrapone-treated rats and their 
offspring and control animals: * p<0.05; * *  p<O.Ol; *** p<O.OOl. 

lowered to 24% of the value of the control mothers. 
The litters of metyrapone-treated mothers suckled well and upon 

examination their gastrointestinal tracts were filled with milk, 
indicating that metyrapone-treatment of the mothers probably did 
not interfere with their ability to feed the offspring.Neverthe- 
less, at 7 days of age their neonates had a slightly reduced body 
weight (Table 1). 

Adrenal wet weight was increased in the neonates of metyrapone- 
treated mothers (Table 1). The ratio between adrenal wet weight 
and DNA content was also increased, indicating a cellular 
hypertrophy. There was also a tendency towards a reduced adrenal 
concentration of corticosterone in the offspring of metyrapone- 
treated mothers, but this did not reach statistical significance. 

When an i.p. glucose tolerance test was performed on the 7-day 
old neonates the glucose tolerance was similar in the offspring of 
metyrapone-treated mothers and controls (Fig 1). The basal serum 
insulin concentration was lower in the offspring of metyrapone- 

150 



1 

I I I 1 

0 30 6 0  9 0  1 2 0  
time ( min ) 

15 1 
4- Controls 

5 1  
o f  I I I I 

0 30 6 0  9 0  1 2 0  
time ( min ) 

Fig 1. Effect of metyrapone treatment of rats during pregnancy and 
lactation on the glucose tolerance and insulin secretory response 
to glucose in the offspring. Rat mothers were treated with 
metyrapone (50-70 mg/kg body weight/day) from day 12 of gestation 
to day 7 postnatally when an intraperitoneal glucose tolerance 
test (2 g glucose/kg body weight) was performed on the offspring 
and serum concentrations of glucose (lower panel) and insulin 
(upper panel) measured. Values are given as means +S.E.M. for 7-11 
animals. Significance of difference between the offspring of 
metyrapone-treated animals and the offspring of control animals: * 
p<O. 05. 

treated mothers, but the insulin secretory response to glucose 
during the test was similar in both groups (Fig 1). 

The pancreatic insulin concentration in the neonates of 
metyrapone-treated mothers was only two thirds of that of the 

151 



control group (Table 1). 

DISCUSSION 
Treatment with metyrapone during pregnancy and lactation induces a 
state of hypoadrenalism in both mother and offspring (9, 10, 26). 
In the present study this was confirmed by the decreased serum 
concentration of corticosterone in the mothers. Despite the 
hypoadrenalism the rat mothers thrived, did not lose weight and 
carried their offspring to term without losses. The neonates 
appeared healthy although signs of hypoadrenalism such as adrenal 
hyperplasia and a tendency towards decreased adrenal cortico- 
sterone concentration could be demonstrated. Serum concentrations 
of corticosterone in the offspring (data not shown) showed 
variations in both the experimental and control group. This was 
considered a stress response due to handling and changes in 
environmental temperature during the experiment and would not 
reflect a failure of metyrapone to inhibit fetal adrenal function. 
Indeed, in previous studies of metyrapone-induced fetal hypo- 
adrenalism a decrease of serum corticosterone concentration was 
not observed despite adrenal hypertrophy and diminished adrenal 
corticosterone content (26). 

The peak values of the glucose tolerance test in the neonates 
were higher than in adult rats of the same strain (6) and the 
insulin secretory response to the glucose peaks lower than the 
corresponding response in adults (33). These observations confirm 
the immaturity of the insulin secretory response to glucose in 7- 
day-old rats (11) which as a consequence dispose of glucose less 
efficiently than adults. The insulin secretory response to glucose 
in vivo is similar to the response in vitro in the offspring (11). 
The presently demonstrated lack of difference between the 
offspring of metyrapone-treated rats and the control animals would 
therefore suggest the adrenal corticosteroids are not essential 
for the perinatal development of a glucose-sensitive insulin 
secretion. 

Chronic hypoadrenalism in the adult rat diminishes insulin 
secretion (25, 34) whereas administration of glucocorticoids to 
normal adult animals increases insulin output by stimulation of B- 
cell growth (13, 18, 24) and insulin secretion (23, 24). The 

152 



present finding of lowered serum and pancreatic insulin con- 
centrations could be interpreted to indicate a hypoplasia of the 
islet organ in the neonates with hypoadrenalism. The total 
capacity for insulin secretion would thus be diminished although 
the glucose sensitivity of the B-cell has been reported to remain 
intact (15). Further experiments, including the morphometric 
analysis of the endocrine pancreas of the neonates, would be 
needed to clarify this hypothesis. 

ACKNOWLEDGEMENTS 
The skilful technical assistance of Gina Eriksson and Parri 
Wentzel is gratefully acknowledged. This work was supported by the 
Swedish Diabetes Association, the Nordic Insulin Fund, the 
Expressen Prenatal Research Foundation, the Ernfors Family Fund, 
the Juvenile Diabetes Foundation (grant 185544) and the Swedish 
Medical Research Council (grants 12X-109, 12X-7475, and 12P- 
6947). 

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Adress for correspondance: 

Dr Ingemar Swenne 
Dept of Paediatrics 
Akademiska Sjukhuset 
S-751 85 Uppsala 
SWEDEN 

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