Endocrinology Vol. 142, No. 7 2996-3005
Copyright © 2001 by The Endocrine Society
Lack of Effect of Protein Deprivation-Induced Intrauterine Growth Retardation on Behavior and Corticosterone and Growth Hormone Secretion in Adult Male Rats: A Long-Term Follow-Up Study1
L. A. Nolan,
E. J. Hart,
R. J. Windle,
S. A. Wood,
X. W. Hu,
A. J. Levi,
C. D. Ingram and
A. Levy
University Research Centre for Neuroendocrinology (L.A.N., E.J.H.,
R.J.W., S.A.W., C.D.I., A.L.), University of Bristol, Bristol Royal
Infirmary, Bristol BS2 8HW, United Kingdom; and Department of
Physiology (X.W.H., A.J.L.), Cardiovascular Research Laboratories,
University of Bristol, Bristol BS8 1TD, United Kingdom
Address all correspondence and requests for reprints to: Dr. A. Levy, University Research Centre for Neuroendocrinology, University of Bristol, Bristol Royal Infirmary, Lower Maudlin Street, Bristol BS2 8HW, United Kingdom. E-mail: a.levy{at}bris.ac.uk
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Abstract
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To further define the neuroendocrine consequences of intrauterine
growth retardation (IUGR), we have used a rat model of maternal protein
restriction throughout pregnancy to examine the pattern of
corticosterone and GH secretion under basal conditions and in response
to psychological stress in male offspring at 4, 9, and 18 months of
age. The findings were correlated with studies of behavioral activity.
Despite a consistent reduction in birth weight and failure of catch-up
growth, there were no significant differences in GH secretory profiles
between IUGR and control rats at any age. We were unable to demonstrate
a difference in the number, amplitude, length, or area of
corticosterone secretory pulses between control and IUGR animals;
although again, there was a significant decrease with age. The mean
peak plasma concentration of corticosterone in response to a noise
stress also declined with age but was unaffected by IUGR. There were no
consistent, statistically significant differences in behavioral
responses between normal control and IUGR animals or between groups of
animals at different ages. These results do not, therefore, support the
presence of major functional abnormalities in either GH or
corticosterone secretory responses in adult male rats subjected to
IUGR.
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Introduction
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THE EPIDEMIOLOGICAL ASSOCIATION between
intrauterine growth retardation (IUGR) with catch-up growth after 1 yr
of age, and enhanced lifetime risk of both cardiovascular events and
metabolic problems, such as impaired glucose tolerance, is now well
established (1, 2, 3, 4, 5). Rat models that use maternal
undernutrition or prenatal stress to perturb the intrauterine
environment have been shown to induce IUGR and the hypertension and
persistent metabolic and neuroendocrine changes that are associated
with it (6, 7, 8, 9, 10, 11). Although inadequate maternoplacental
nutrient supply is strongly implicated as the principle cause of
persistent adaptive changes, the nature and extent of specific trophic,
behavioral, and neuroendocrine consequences of IUGR in postnatal life
remain to be fully defined.
Persistent effects on regional expression of hippocampal glucocorticoid
and mineralocorticoid receptors have been identified in the IUGR
offspring of pregnant rats treated with dexamethasone (12)
or subjected to protein restriction (8), and programming
of hypothalamo-pituitary-adrenal (HPA) axis responsiveness through
excessive fetal exposure to maternal glucocorticoids may be one common
factor in the pathway that ultimately leads to adult disease in both
rats and man (6, 13, 14, 15, 16, 17, 18). In some human studies, low birth
weight has been shown to correlate with raised fasting plasma cortisol
levels (14, 16). In animal models, basal corticosterone
concentrations have been found to be unaltered in the progeny of
protein-restricted rats (8) but raised in prenatally
stressed animals (6) and in pups derived from
dexamethasone-treated dams (12). Differences in the timing
and nature of these intrauterine insults may produce different
long-term effects (13); and because the characteristics of
the HPA axis may also be modified by early postnatal events
(19, 20, 21, 22), the nature of programming is clearly extremely
complex.
Because early postnatal growth is GH and insulin-like growth factor 1
(IGF-1)-dependent, the failure of catch-up growth in a subset of IUGR
children and rats has been attributed to alterations in somatotrophic
axis regulation. In some studies, abnormalities in GH secretory
profiles and mean plasma IGF-1 levels have been identified (7, 23, 24). In others, resistance to GH, IGF-1, and/or insulin in
the presence of normal hormone profiles has been implicated
(25, 26, 27).
Using a maternal protein restriction model of IUGR, we have recently
shown that IUGR pups exhibit persistent failure of catch-up growth, and
diastolic hypertension and a predisposition to afterload-induced
cardiac arrhythmias in adulthood (28). The aim of the
current study was to investigate the effects of protein
deprivation-induced IUGR, in the rat, on basal corticosterone and GH
secretory profiles and on the behavioral and HPA responses to
psychologically stressful stimuli up to 18 months of age.
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Materials and Methods
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Animal model
Virgin female Wistar rats (225–250 g) were mated with young
stud males. Day zero of pregnancy was defined by the presence of a
vaginal plug, after which dams were randomly assigned to one of two
dietary groups. IUGR was induced in the pups from one group of dams by
allowing free access to a low-protein diet (TD93328; Harlan Teklad
Premier, Purina Mills, St. Louis, MO) for the duration of pregnancy. A
second group of control dams was allowed free access to normal rat
food. Both groups were allowed free access to drinking water and were
maintained under 14-h light, 10-h dark cycles, with lights on at
0500 h. Within 8 h of birth, all female pups were removed
from the litters, and males were cross-fostered onto normal dams
(eight/dam). For the purposes of weighing, pups were briefly handled
daily for the first month after birth and then once per week up to the
age of 6 months. At 21 days old, pups were ear-clipped, weaned, and
housed in cages containing equal numbers of IUGR and control animals.
Further groups of control and IUGR rats were generated at intervals
throughout the study; and experimental procedures were carried out at
4, 9, and 18 months of age. Successive breeding runs ensured that the
effects of small differences in animal housing conditions, over the
duration of the study, would be minimized. All animal procedures were
carried out in accordance with UK Home Office animal welfare
regulations.
Jugular vein cannulation
Cannulation was carried out as described previously (29, 30). Animals were anesthetized using a combination of Hypnorm
(0.32 mg/kg fentanyl citrate and 10 mg/kg fluanisone, im; Janssen Pharmaceuticals, Oxford, UK) and diazepam (2.6
mg/kg ip; Phoenix Pharmaceuticals, Inc., Gloucester, UK).
The right jugular vein was exposed, and a SILASTIC-tipped polythene
cannula (Dow Corning Corp., Midland, MI; od, 0.96 mm; id,
0.58 mm; Portex, Hythe, UK), filled with 10 U/ml heparinized isotonic
saline, was inserted into the vessel until it lay close to the entrance
of the right atrium. The free end of the cannula was exteriorized
through a scalp incision and then tunneled through a protective spring
that was anchored to the parietal bones using two stainless steel
screws and self-curing dental acrylic. After recovery, animals were
moved to individual housing cages, and the end of the spring was
attached to a mechanical swivel that rotated through 360 degrees,
giving the animals maximum freedom of movement. The cannulae were
flushed daily with heparinized saline solution.
Experimental paradigm
Four days after surgery, the cannulae were connected to an
automated sampling system via air-tight swivels, as previously
described (29). The animals were connected to the system
at 1800 h, and sampling was initiated at 0500 h the next
morning. Blood samples (80 µl) were collected every 10 min, for two
periods of 6 h (0500–1100 h and 1700–2300 h), to determine the
basal profiles of corticosterone and GH release. Circulating blood
volume was replaced with an equal volume of heparinized saline at each
sampling point. At 0530 h the following day, sampling was
restarted; and at 0800 h, a white noise generator was activated,
and the animals were exposed to 114 decibels (frequency range,
12–60,000 hertz) for 10 min to test for responses to acute
psychological stress. Sampling continued for a further 3 h, after
which the animals were killed by decapitation.
All blood samples were collected at a 1:4 dilution in heparinized
saline, i.e. a total sample vol of 400 µl. The plasma
fraction was separated by centrifugation and used for the measurement
of corticosterone and GH concentrations. Throughout the sampling
periods, the behavior of the animals was recorded onto videotape
remotely, using video cameras (WV-BP 100, Panasonic, Osaka, Japan), a
sequential camera selection system (Gem Mono Multiplexer, Norbain
Security Ltd., Wokingham, Berkshire, UK), and a high-performance
video cassette recorder unit (HS5424, Mitsubishi Electric
Company, Osaka, Japan). Observations were monitored and saved for later
analysis.
Elevated plus maze
The elevated plus maze consisted of a central 10 x 10 cm
platform (14.5 x 14.5 cm for the 9- and 18-month groups) with a
pair of horizontal open-arm runways, 10 cm wide and 50 cm long
(14.5 x 70 cm for the 9- and 18-month groups), at 90 degrees to a
pair of closed-arm platforms of similar proportions, bounded on either
side by 12-cm-high walls, the whole standing 72 cm above floor level.
For the larger animals in the 9- and 18-month groups, the width of the
platforms was increased to 14.5 cm, and the closed-arm wall height to
14 cm. Rats were placed onto the central platform of the maze, facing a
closed-arm at the start of the test, and monitored remotely by video
camera for 10 min. The apparatus was thoroughly cleaned with ethanol
between tests. Parameters retrospectively scored were: 1) number of
open- and closed-arm entries, where an entry was defined as all four
paws passing the threshold of an arm; 2) time spent in open arms; and
3) time before first entering an open arm (latency period).
Open field
The open field consisted of a white square arena (120 x
120 cm) divided into 16 equal squares (30 x 30 cm) by blue lines.
The inner area consisted of the 4 central squares and was surrounded by
the outer area of 12 squares adjacent to the walls of the arena. Rats
were placed in the center of the arena and monitored remotely by video
camera for 10 min. The number of inner and outer segment crossings was
scored retrospectively, together with the total length of time spent in
the inner area of the apparatus, and the time before the animals
returned to the inner area after they initially moved to the outer rim
at the start of the test. A crossing was defined as all 4 paws passing
over 1 of the blue lines on the floor of the arena.
Behavioral responses to noise stress
Total time spent engaged in any type of activity, the time spent
self-grooming, and the number of rearings (transitions from standing on
all four paws, to the two back paws, not the total duration spent on
two paws) made by each animal were retrospectively scored for 10 min
before the application of noise stress, 10 min during noise stress, and
for the 10 min immediately after stress.
Hormone assays
Total plasma corticosterone concentrations were measured
directly in 50 µl diluted plasma samples by RIA using a citrate
buffer at pH 3.0 to denature the binding globulin, antiserum kindly
supplied by Prof. G. Makara (Institute of Experimental Medicine,
Budapest, Hungary) and [125I]-corticosterone
(ICN Biomedicals, Inc., Irvine, CA) with a specific
activity of 2–3 mCi/µg (30). The assay had a limit of
detection of 5 ± 1 ng/ml, and intra- and interassay coefficients
of variation were measured at 12.4% and 16.0%, respectively.
Plasma GH was determined by RIA using reagents kindly supplied by Dr.
A. F. Parlow, NIDDK-NIH (Torrance, CA). Purified rat GH
(NIDDK-rGH-1–5; AFP-3190B) was iodinated with
125I using the hloramine-T method. Recombinant GH
(rGH) reference preparation (NIDDK-rGH-RP-2) was stored at a
concentration of 32 ng/ml, and standards were prepared by doubling
dilutions in human serum. Monkey antirat GH antiserum (NIDDK-rGH-S-5)
was used at a final dilution of 1:120,000. Antibody-bound fractions
were separated using a final concentration of 12% polyethyleneglycol.
Samples were measured in duplicate, and the averaged results were
expressed (in nanograms per ml) in terms of the NIDDK standard RP-2.
The intraassay coefficient of variation ranged between 10.3 and 13.2%,
and the limit of detection was 0.05 ng/ml.
Statistics
Instat (GraphPad Software, Inc., San Diego, CA) was
used to perform statistical calculations. The pulsatility of
corticosterone and GH data from serial plasma samples were analyzed
using the PC PULSAR program, version 2 (31). Peak
frequency, amplitude, length, and area, and mean hormone concentrations
were measured. The following G values were employed: G1 = 5,
G2 = 3.5, G3 = 2.5, G4 = 1.5, and G5 = 1.2,
together with a peak splitting parameter of 2 (SD units).
Differences between multiple groups were evaluated using one-way ANOVA
followed by post hoc Tukey-Kramer multiple-comparison tests.
P < 0.05 was considered statistically significant.
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Results
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The effects of maternal protein deprivation on male pup birth
weight and subsequent growth are shown in Fig. 1
. Mean birth weight was reduced from
6.7 ± 0.8 g to 3.9 ± 0.5 g (P <
0.001). The 12% weight difference between IUGR and control animals at
12 weeks (384.1 ± 4.47 g vs. 337.9 ±
4.2 g) and 13% difference between the two groups at 18 months
(679.0 ± 18.7 g vs. 588.8 ± 17.5 g)
revealed the failure of complete catch-up growth over this time. The
similarity of postnatal growth rate, week to week, is further
illustrated in Fig. 1B.
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Figure 1. The effects of IUGR on postnatal body weight. A,
Differences in mean body weight (± SE) between IUGR rats
and normal controls, up to 78 weeks of age; B, rate of change in body
weight with time. The similarity in rate of body weight change, over
time, seen in B is consistent with failure of postnatal catch-up growth
in IUGR animals.
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Basal corticosterone profiles
Plasma corticosterone levels were measured in samples taken every
10 min, over two 6-h periods, from 0500–1100 h and 1700–2300 h. For
each animal, the individual plasma corticosterone values obtained were
averaged over 1-h periods to prevent the confounding effects of the
different phases of secretory pulses found in individual rats at each
time point, when averaging data (30).
Approximately 1 h after the start of the sampling period on the
first morning of sampling, but not on subsequent days, a relatively
consistent surge of corticosterone secretion occurred that returned to
baseline levels within 2 h. Because of this, analysis of the
effects of IUGR and age on diurnal variation in basal corticosterone
secretion was made by comparing data collected between 0800–1100 h and
2000–2300 h (Fig. 2).
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Figure 2. Diurnal differences in hourly averaged
corticosterone secretion in control and IUGR rats at 4, 9, and 18
months of age (n = 10–13). *, Significant to
P < 0.05; ***, significant to
P < 0.001 (compared with the equivalent values at
9 and 18 months of age).
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As expected, the mean evening plasma corticosterone levels were
elevated, compared with those measured in the morning, although this
only reached statistical significance in the 4-month IUGR group. There
were no significant differences in basal corticosterone secretion
between IUGR animals and normal controls at any individual time point
or within any age group. However, between 4 and 9 months of age,
evening plasma corticosterone levels fell significantly in both IUGR
and normal control groups (Fig. 2
and Table 1). At 4 months of age, the mean plasma
corticosterone concentration, measured over the entire 6-h evening
sampling period, was not significantly different between normal control
and IUGR rats (127 ± 16.7 vs. 139 ± 26.3 ng/ml;
n = 13; Table 1). With increasing age, mean evening corticosterone
levels in normal rats fell significantly, to 35.6 ± 7.5 ng/ml by
9 months (P < 0.01; n = 11) and to 20.5 ±
8.0 ng/ml by 18 months of age (P < 0.001; n =
13), and similar decreases were observed in age-matched IUGR groups
(Table 1). The differences between 9 and 18 months of age did not reach
statistical significance.
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Table 1. The characteristics of the pulsatile pattern of
corticosterone release measured between 1700 and 2300 h in normal
control and IUGR rats at different ages
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The pulsatile characteristics of corticosterone secretion were examined
for the 6-h period from 1700–2300 h (Table 1) when pulse amplitude is
greatest (30). No significant differences were found in
the number, amplitude, length, or area of the peaks of corticosterone
secretion, between normal control and IUGR animals at any age, although
values for all parameters, except peak length, significantly decreased
with age (Table 1). In normal control animals, the frequency of
corticosterone peaks detected using this algorithm fell from an average
of 1.3 detectable pulses/h at the age of 4 months, to only 0.18/h at 18
months.
Effect of noise stress on HPA activity
The application of a 10-min noise stress, beginning at 0800
h, elicited a rapid increase in mean corticosteroid release in both
normal control and IUGR rats, which peaked 20–30 min after the onset
of noise and returned to basal levels between 40 and 50 min after the
cessation of noise (Fig. 3). With the
exception of a small, insignificant increase in the amplitude of the
secretory response made by IUGR animals in the 9-month-old group, there
were no discernible differences in the characteristics of the
corticosterone response to noise stress between normal control and IUGR
rats in any one age group (Fig. 3).
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Figure 3. The effect of noise stress (114 decibels x
10 min, commencing at 0800 h; hatched bar) on
plasma corticosterone concentration in IUGR rats and normal controls at
4, 9, and 18 months of age. Means ± SE are shown;
n = 10–13.
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At 4 months of age, the mean peak plasma concentration in response to
the noise stress in normal control animals was 420 ± 25 ng/ml
(n = 12). By 9 months, this value had fallen significantly, to
131 ± 34 ng/ml (n = 10; P < 0.001, compared
with the 4-month group); and by 18 months, it had decreased further to
66 ± 16 ng/ml (n = 13; not significant, compared with
9-month group). A similar decline was measured in IUGR animals (Fig. 3). There was no effect of age on either the timing of the peak or on
the rate of the subsequent rapid decline to prestress levels.
Behavioral response to noise stress
All groups of animals showed an increase in total activity during
the period of noise stress and in the period immediately after
cessation of the noise stimulus (Fig. 4, A–C). This increase in total activity was significant in all groups in
the period after noise cessation and during the noise stress in the
4-month-old group (Fig. 4A), but it did not quite reach statistical
significance in the period during the noise stress in the 9- or
18-month-old IUGR animals or in the 18-month-old normal control
animals. The mean number of rearings made by the rats, recorded as a
measure of exploratory behavior, was significantly higher in all groups
of animals during the period of noise stress, compared with prestressed
animals (Fig. 4, D–F). Rearing behavior, unlike total activity, began
to decline immediately after cessation of noise. Self-grooming was
recorded as a measure of displacement activity and was increased in all
groups in the immediate poststress period, compared with prestress
levels (Fig. 4, G–I). The large increase in grooming activity
accounted for the further increment in total activity recorded in the
immediate poststress period. There were no statistically significant
differences in the behavior patterns observed between normal control
and IUGR animals, or between groups of animals at different ages,
during any individual time period.
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Figure 4. The effect of IUGR on behavioral responses to
noise stress. Each panel contains 3 pairs of columns that represent
activity during the 10 min preceding white noise stress, during the
stress, and for the 10 min after cessation of noise. Normal controls
are represented by the filled columns, and IUGR animals
by the open columns. Means ± SE are
shown (n = 11–16). *, P < 0.05; **,
P < 0.01; ***, P < 0.001
(compared with pre-noise values in each group). A, B, and C show the
time engaged in total activity during each time period. D, E, and F
show the number of rearings made in each time period (recorded as a
measure of exploratory behavior). G, H, and I show time spent engaged
in self-grooming (a measure of displacement activity).
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Basal GH profiles
Examples of GH profiles measured in plasma samples obtained
between 1700 and 2300 h, in normal control and IUGR rats of
different ages, are shown in Fig. 5; and
the pulsatile characteristics of GH secretion, over the same time
period, are summarized in Table 2. There
was a wide variation in the GH profiles measured in all groups of
animals studied, which accounted for the lack of statistically
significant differences in individual GH secretion parameters between
control and IUGR animals or between groups of animals at different
ages. Though the number and duration of peaks remained relatively
constant, there was a strong trend toward declining peak amplitude,
with increasing age, leading to an overall reduction in mean GH levels
by 9 months of age. The pulsatile characteristics of GH profiles
obtained between 0500 and 1100 h were also similar to those
obtained in the evening (data not shown). In 4-month-old IUGR animals,
there was a small reduction in mean GH secretion during both morning
and evening sampling periods (Fig. 6),
compared with normal control animals of the same age. At 9 and 18
months of age, the mean levels of GH secretion were more comparable
between groups.
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Figure 5. Examples of GH secretory profiles measured in
plasma samples obtained between 1700 and 2300 h in normal control
and IUGR rats at 4, 9, and 18 months of age.
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Table 2. The characteristics of the pulsatile pattern of GH
release measured between 1700 and 2300 h in normal control and
IUGR rats at different ages
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Figure 6. Mean GH secretion (± SE) in IUGR rats
during both the morning and evening 6-h sampling periods, compared with
normal control animals of the same age (n = 6–11).
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Anxiety-related behavioral responses
At 4 months old, IUGR animals made significantly more entries into
the closed arms of the plus maze than normal control rats (Fig. 7a), indicating an increased level of
active behavior, although this difference was reduced by 9 months of
age and completely absent by 18 months of age. In this test, IUGR
animals of 4 and 9 months of age also had a tendency to display
behavioral patterns associated with reduced anxiety, including a
decreased time before first entering the open arms of the apparatus
(Fig. 7b), an increased number of open-arm entries (Fig. 7c), and an
increased time spent in the open arms (Fig. 7d). However, none of the
differences in these parameters reached statistical significance. In
the open field, the behavioral patterns observed did not differ either
between IUGR and normal control rats or between age groups in the
activity (Fig. 7e), anxiety (Fig. 7, f and g), or exploratory (Fig. 7h)
parameters measured.
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Figure 7. Effect of IUGR on anxietyrelated
behavioral responses. In each panel, pairs of columns refer to 4-month,
9-month, and 18-month groups. Normal controls are represented by the
filled columns, and IUGR animals by the open
columns. Means ± SE are shown (n =
7–13). **, P < 0.01, compared with normal
controls of the same age. Raised plus maze results: a, entries into the
closed arms of the plus maze (increases in proportion to overall
activity); b, time before first entry into the open arms of the
apparatus (increases in proportion to anxiety); c, entries into the
open arms of the plus maze (increases in inverse proportion to
anxiety); d, time spent in open arms of the apparatus (increases in
inverse proportion to anxiety). Open field results: e, outer perimeter
crossings (increases in proportion to overall activity); f, time before
the rat ventures away from the perimeter back into the inner area
(increases in proportion to anxiety); g, inner, central area crossings
(increases in inverse proportion to anxiety); h, number of rearings (a
measure of exploratory behavior).
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Discussion
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In our hands, IUGR induced by maternal protein deprivation
throughout pregnancy results in reduced somatic growth and an increase
in predisposition to afterload-induced cardiac arrythmias and in
diastolic hypertension in the adult offspring (28).
Despite the greater-than-10% difference in body weight between IUGR
animals and controls that persisted throughout the study, we were
unable to demonstrate any consistent, statistically significant
differences in basal GH or corticosterone secretory patterns between
the two groups. In terms of corticosterone secretion, the magnitude of
the response to acute psychological stress diminishes with increasing
age but is unaffected by the intrauterine insult used in these studies.
Although 4-month-old IUGR rats are more active than their normal
counterparts, by 9 and 18 months of age these differences are
abolished. IUGR rats at all ages are not significantly more or less
anxious than normal control animals.
Abnormalities in GH levels adversely affect both myocardial growth and
function, and it has been suggested that GH deficiency has more severe
effects if it is present during early heart development (32, 33). The major indication that a period of abnormal GH axis
regulation occurs in the IUGR animals is the persistent failure in
postnatal catch-up growth. Failure of catch-up growth was also observed
in a recent study of 18-month-old rats with IUGR, which was induced by
third trimester bilateral uterine artery ligation (27). In
both models of IUGR (protein restriction and arterial ligation), rates
of body weight gain after birth and GH profiles at 3–4 months of age
were essentially similar between IUGR animals and normal controls.
Furthermore, studies in younger animals have confirmed normal GH
levels, but identified reduced IGF-1 concentrations at 8 and 22 days of
age, which had returned to normal by 63 days (34). In a
further study, IGF-1 levels were found to be reduced only up to
postnatal day 9 in IUGR rats subjected to severe maternal
undernutrition (7). Although administration of excess GH
or IGF-1 promotes postnatal catch-up growth in rats subjected to
maternal protein deprivation (35), this does not
necessarily indicate that this is the primary dysfunction. In contrast
to the lack of effect of IUGR, if protein deprivation is delayed until
post weaning, GH profiles are adversely affected, IGF-1 levels are
normal, and catch-up growth does not occur by the age of 12 weeks
(36). These data suggest a role for IGF-1 and/or GH in
mediating catch-up growth but that a return simply to normal levels may
not be sufficient, on its own, to restore normal adult body weight. The
exact nature and timing of the early insults that result in growth
retardation are unclear, but there is little direct evidence from the
present study that GH axis anomalies persist into adulthood. These data
do not rule out the possibility that cardiac abnormalities induced by
IUGR (28) may be associated with altered tissue
sensitivity to GH axis components, a mechanism suggested as a cause of
growth failure in a subgroup of IUGR children (25, 26).
Altered programming of the HPA axis is an alternative hypothesis that
links fetal experience with adult disease, particularly because
exposure of the fetus to an excess of glucocorticoids has been shown to
be associated with retarded postnatal growth, and hypertension
and hyperglycemia in adulthood (13). The fact that
maternal protein restriction selectively attenuates levels of placental
11ß-hydroxysteroid dehydrogenase type 2 (9) suggests one
mechanism by which the fetus may be exposed to the programming effect
of glucocorticoids in utero.
An increased and prolonged corticosterone secretory response to stress,
together with a decrease in hippocampal glucocorticoid receptors and a
decreased efficiency of glucocorticoid feedback, has been observed in
adult rats after prenatal stress (20, 21, 37, 38). The
effects of prenatal stress or maternal protein deprivation on basal
corticosterone levels are conflicting with both increased and unchanged
levels being reported (8, 37). The data presented in this
study showed no differences in either basal or stress-induced
corticosterone responses or in diurnal rhythms between IUGR and normal
control animals at any individual time-point, and the data are in
agreement with those reported previously for IUGR offspring of
protein-deprived dams (8). Serum cortisol levels and
secretory rhythms have also been studied in cohorts of IUGR children
and found not to correlate with size at birth (39),
although an association between birth size and both glucocorticoid
metabolite levels (40) and fasting plasma cortisol
concentrations (14, 16, 17) have been described.
In contrast to studies carried out in prenatally stressed rats, a study
in IUGR rats exposed to maternal protein deprivation also reported an
increase in hippocampal glucocorticoid receptor binding, a result which
might suggest an increase in glucocorticoid feedback sensitivity
(8). Glucocorticoid receptor levels were not directly
addressed in the present study, but there were no effects of IUGR on
either the timing of the corticosteroid peak after exposure to a noise
stress, or in the subsequent rapid return to prestress levels at any
age studied.
It should be noted that the effects of intrauterine insults on
programming of the HPA axis may be modified by early postnatal
experience. For example, daily handling immediately after birth is
associated with a decreased corticosterone response to stress in later
life, and early adoption can completely reverse the effects of prenatal
stress on all HPA axis parameters (19, 20, 41). These
observations may be relevant to the data reported here, because all
pups were crossfostered at birth and were handled daily for the
first month of life for the purpose of weighing. It thus remains
possible that any corticosterone secretory effects programmed in
utero by maternal protein deficiency were subsequently masked by
the consequences of early postnatal events.
Because it has been shown that behavioral responses in anxiety-related
tests (such as the elevated plus maze, and the open field) correlate
with corticosterone secretory response and are also subject to
modification by exposure to prenatal stress and postnatal handling
(38, 42), behavioral activity was examined in this study.
In view of the lack of effect on stress-induced HPA activity, IUGR rats
were not found to be any more or less anxious than their normal
counterparts in these tests. In addition, behavioral responses made
before, during, and immediately after a 10-min exposure to noise stress
were not significantly different between IUGR and normal control groups
in terms of total activity, self-directed grooming, or exploratory
rearings.
The most marked changes in HPA activity, seen in the present study,
concern age-related changes. Age-related differences in evening plasma
corticosterone concentration and in the amplitude of the corticosterone
response to a novel psychological noise stress were observed, with the
lowest responses being made by 18-month-old animals. The decline in
evening corticosterone levels resulted in the flattening of the diurnal
rhythm of secretion in the two older groups of animals. A commonly held
view is that basal corticosterone level increases in aged rats
(43, 44), although some studies have shown that basal
corticosterone levels either remain unchanged (45) or are
reduced (46). At 18 months, the rats in the present study
should perhaps be regarded as mature adults rather than aged or
senescent animals, as in another study no differences in basal
corticosterone secretion were observed until 24 months
(22).
In conclusion, we have shown that the male IUGR offspring of dams
subjected to protein malnutrition have similar basal GH and
corticosterone profiles as those of their normal counterparts at 4, 9,
and 18 months of age. We have demonstrated an age-related, but not an
IUGR-related, decrease in corticosterone response to a psychological
stress and have shown that the behavioral response to anxiety is not
influenced by IUGR induced by maternal protein restriction. Despite the
fact that IUGR-induced changes in these parameters may have been
ameliorated by early postnatal events, in our hands IUGR animals
generated using the same model have significant cardiac abnormalities
and hypertension that persist throughout adult life (28).
Thus, it can be concluded that the cardiovascular and growth effects
seen in this model do not arise from dysregulation of either the HPA or
somatotroph axis, at least at the levels of corticosterone and
GH secretion seen here.
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Footnotes
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1 This study was supported by the British Heart Foundation and The
Wellcome Trust. 
Received January 22, 2001.
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Abstract
Introduction
