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Endocrinology Vol. 142, No. 7 2996-3005
Copyright © 2001 by The Endocrine Society

ARTICLES

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


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
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.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
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.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
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.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
The effects of maternal protein deprivation on male pup birth weight and subsequent growth are shown in Fig. 1Go. 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. 1BGo.



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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.

 
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. 2Go).



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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).

 
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. 2Go and Table 1Go). 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 1Go). 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 1Go). 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

 
The pulsatile characteristics of corticosterone secretion were examined for the 6-h period from 1700–2300 h (Table 1Go) 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 1Go). 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. 3Go). 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. 3Go).



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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.

 
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. 3Go). 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. 4Go, 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. 4AGo), 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. 4Go, 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. 4Go, 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).

 
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. 5Go; and the pulsatile characteristics of GH secretion, over the same time period, are summarized in Table 2Go. 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. 6Go), 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).

 
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. 7aGo), 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. 7bGo), an increased number of open-arm entries (Fig. 7cGo), and an increased time spent in the open arms (Fig. 7dGo). 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. 7eGo), anxiety (Fig. 7Go, f and g), or exploratory (Fig. 7hGo) 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).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
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.


   Footnotes
 
1 This study was supported by the British Heart Foundation and The Wellcome Trust. Back

Received January 22, 2001.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Barker DJ 1992 The fetal origins of adult hypertension. J Hypertens Suppl 10:S39–S44
  2. Barker DJP, Osmond C, Simmonds SJ, Wield GA 1993 The relation of small head circumference and thinness at birth to death from cardiovascular disease in adult life. BMJ 306:422–426
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