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-3 Fatty Acids
School of Anatomy and Human Biology (C.S.W., P.J.M., B.J.W.), The University of Western Australia, Nedlands, Perth, Western Australia 6009, Australia; and School of Medicine and Pharmacology, Royal Perth Hospital Unit (T.A.M., I.B.P.), The University of Western Australia, Perth, Western Australia 6001, Australia
Address all correspondence and requests for reprints to: Brendan J. Waddell, School of Anatomy and Human Biology, The University of Western Australia, Nedlands, Perth, Western Australia 6009, Australia. E-mail: bwaddell{at}anhb.uwa.edu.au.
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Abstract |
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-3 fatty acids attenuates programmed hyperleptinemia and hypertension. Pregnant rats were treated with dexamethasone (Dex) from d 13 to term, and offspring were cross-fostered to mothers on either a standard diet or a diet high in -3 fatty acids and remained on these diets postweaning. Maternal Dex reduced birthweight and delayed the onset of puberty in offspring. Hyperleptinemia (associated with elevated leptin mRNA expression in adipose tissue) and hypertension were evident in offspring by 6 months of age in Dex-exposed animals consuming a standard diet, but these effects were completely blocked by a high -3 diet. These results demonstrate for the first time that manipulation of postnatal diet can limit adverse outcomes of fetal programming, with programmed hyperleptinemia and hypertension prevented by a postnatal diet enriched with -3 fatty acids. This raises the possibility that dietary supplementation with -3 fatty acids may provide a viable therapeutic option for preventing and/or reducing adverse programming outcomes in humans. |
Introduction |
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Poor nutrition and fetal glucocorticoid exposure are two key mediators of fetal programming. Experimental models involving dietary restriction in pregnancy have demonstrated the influence of maternal undernutrition on programming of insulin resistance, obesity, and hypertension (5, 6, 7). Glucocorticoids are important for fetal maturation in late gestation, but overexposure retards fetal growth and delays puberty onset in the rat (8). Additionally, excess glucocorticoid exposure in utero results in offspring with a variety of adverse physiological outcomes, including cognitive impairment (9), insulin resistance (10), hyperleptinemia (11), and hypertension (12). Thus, excessive fetal exposure to glucocorticoids can result in hyperleptinemia either with (13) or without (11) increased adiposity. Treatment of pregnant rats with glucocorticoids also elevates blood pressure of adult offspring (11, 12, 14), as does increased fetal exposure to endogenous maternal glucocorticoids via pharmacological inhibition of the placental glucocorticoid barrier (15, 16).
The interaction between fetal programming and the postnatal environment has been recognized, with evidence that postnatal diet can amplify detrimental programming effects. Thus, hypercaloric postnatal nutrition exacerbates the adverse effects of fetal undernutrition in relation to hyperinsulinemia, hyperleptinemia, hypertension, and obesity (6), and a postnatal high-fat diet fed to offspring of ethanol-treated mothers worsens glucose intolerance (17). Although postnatal pharmacological manipulation (such as treatment with IGF-I) can attenuate adverse programming effects (18, 19, 20), it is not known whether similar beneficial outcomes can be achieved with postnatal dietary manipulation.
The major objective of this study was to determine whether a postnatal diet rich in -3 (n-3) fatty acids can attenuate glucocorticoid-induced fetal programming of hyperleptinemia and hypertension. A diet high in n-3 fatty acids has been shown to lower plasma leptin levels in rats (21), and Mori et al. (22) recently reported that dietary fish, a rich source of n-3 fatty acids, augments the leptin lowering effects of a weight loss program. Furthermore, dietary supplementation with n-3 fatty acids in adults has been shown to protect against heart disease, particularly in relation to hypertension (23), and provides beneficial effects with regard to vascular reactivity, expression of inflammatory markers, and adiposity (24, 25, 26). Our fetal programming model involved administration of dexamethasone (Dex) to pregnant rats from d 13 to term (8). At birth, offspring from treated and control (Con) mothers were cross-fostered to mothers on either a standard semipure diet (containing an n-3 fatty acid profile comparable with normal rat chow) or a semipure diet high in long-chain n-3 fatty acids, and they remained on these diets postweaning.
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Materials and Methods |
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). Preliminary experiments established that Wistar rats consumed similar quantities of the standard and high-n-3 diets over a 10-wk period (data not shown). The semipure diets were manufactured by Specialty Feeds (Glen Forrest, Australia) and were sterilized by 
-irradiation.
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Rats were mated overnight, and the day on which spermatozoa were present in a vaginal smear was designated d 1 of pregnancy. Dex acetate (Sigma Chemical Co., St. Louis, MO) was administered in the drinking water (0.75 µg/ml) from d 13 of pregnancy until birth in those mothers on normal rat chow (28).
Litter management
Within 12 h of birth, offspring sex was identified by examination of external genital morphology, and males and females were weighed separately. Within 24 h of birth, all pups from Con and Dex-treated mothers were cross-fostered to a mother on either a standard diet or high-n-3 diet. Cross-fostering resulted in four treatment groups (Con/Standard, Con/High n-3, Dex/Standard, and Dex/High n-3) as shown in Fig. 1. Pups remained with their foster mothers until weaning, at which point male and female offspring were caged separately and remained on their allocated diets (Standard or High n-3).
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Systolic blood pressure (SBP) measurements
SBP was measured by tail-cuff plethysmography in trained animals at 1, 2, 4, and 6 months of age from the same member of each litter in all treatment groups. Rats were restrained in a clear, plastic tube at 39 C, and the cuff was placed on the tail and inflated to 200 mm Hg. The reappearance of a pulse during deflation of the cuff was used to determine SBP. To minimize stress, no animal was restrained for more than 10 min at a time, and a minimum of three clear SBP recordings were taken per animal.
Blood sampling, tissue collections, and dual-energy x-ray absorptiometry (DEXA) measurements
Blood (2 ml) and tissue samples were collected from offspring at 1 and 2 months of age. One male and one female were randomly chosen from each litter, anesthetized with halothane/nitrous oxide, and a blood sample obtained from the dorsal aorta and collected into a heparinized tube. Blood samples were centrifuged at 11,000 rpm for 5 min, and plasma was stored at –20 C until subsequent analysis. Weights for the left epididymal fat pad, left kidney, and the heart were recorded.
At 6 months of age, those animals that had undergone blood pressure measurements were anesthetized with 40 mg/kg Nembutal (Rhone Merieux, Pinkenba, Australia), and body composition was determined by DEXA (GE Lunar Prodigy Series, GE Lunar, Madison, WI) using small animal software (Encore 2004, version 8.50.093, GE Lunar). Animals were then further anesthetized with halothane/nitrous oxide, and blood samples and tissue weights were obtained immediately as described above. Samples of epididymal and retroperitoneal fat pads were snap frozen in liquid nitrogen and stored at –80 C before analysis.
Analysis of serum fatty acids
Serum fatty acids were measured in male and female offspring at 1 and 6 months of age. Serum (200 µl) was extracted with chloroform/methanol (2:1, 2 ml). Fatty acid methyl esters were prepared by treatment of total lipid extracts with 4% H2SO4 in methanol at 90 C for 20 min and analyzed by gas liquid chromatography using a Hewlett-Packard model 5980A gas chromatograph (Hewlett Packard, Rockville, MD). The column was a BPX70 (25 mm x 0.32 mm, 0.25-µm film thickness) (SGE, Ringwood, Victoria, Australia) with a temperature program from 150–210 C at 4 C/min and using N2 as the carrier gas at a split ratio of 30:1. Peaks were identified by comparison with a known standard mixture. Individual fatty acids were calculated as a relative percentage with the evaluated fatty acids set at 100%.
Leptin RIA
Plasma leptin concentrations were measured using a leptin RIA kit (Linco Research, St. Charles, MO). All samples were analyzed in a single assay, for which the intraassay coefficient of variation was 2.6%.
Quantitative RT-PCR analysis of leptin mRNA
Total RNA from epididymal and retroperitoneal fat samples at 6 months of age was extracted using a QIAGEN RNeasy Lipid Tissue Mini Kit (QIAGEN, Clifton Hill, Australia) and then 1 µg reverse transcribed at 55 C for 50 min using Maloney murine leukemia virus RT (Promega, Annandale, Australia). The resultant cDNA was purified using the Ultraclean PCR cleanup kit (MoBio Laboaratories Inc., Solana Beach, CA). The cDNA primers for rat leptin were designed using Primer3 software (http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi), positioned to span introns to distinguish cDNA from genomic DNA and were as follows: sense, 5'-TGA CAC CAA AAC CCT CAT CA-3'; and antisense, 5'-ATG AAG TCC AAA CCG GTG AC-3'. External standards were generated from 10-fold serial dilutions of regular PCR products made in RNase-free water (1–107-fold dilutions). Quantitative PCR was performed in 10-µl reaction volumes using the Rotorgene 3000 system (Corbett Research, Sydney, Australia) with 0.5 U Immolase enzyme (Bioline, Alexandria, Australia) per reaction. Primers were used at a final concentration of 0.5 µM and MgCl2 at 4 mM. The PCR cycling conditions for rat leptin included an initial denaturation at 94 C for 7 min followed by 50 cycles at 94 C for 1 sec, 58 C for 15 sec, and 72 C for 5 sec. Melting curve analysis from 70–99 C showed a single PCR product that was confirmed by gel electrophoresis (data not shown) and sequence analysis of the product. Fluorescence values were analyzed, a standard curve constructed using the Rotorgene software and all samples standardized against the internal control ribosomal L19 (31).
Statistical analysis
All data are expressed as mean ± SEM, with each litter representing an n of one. In all cases, each experimental group had an n of 6–8. Litter size and sex ratio were assessed by one-way ANOVA followed by post hoc least significance difference (LSD) tests (32). All other variables were analyzed by ANOVA (one-, two-, or three-way as appropriate to account for variation due to sex, age, maternal treatment, and diet) followed by post hoc LSD tests. When significant interaction terms were found in these ANOVAs, analyses of subsets of data were made (see Results for specific applications).
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Results |
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Postnatal growth and puberty onset
Male offspring weight varied with both age (P < 0.001) and treatment (P < 0.001), and there was significant interaction between these effects (P < 0.001). Therefore, separate two-way ANOVAs were conducted on weekly weights, and these demonstrated that from birth to 6 months, male offspring of Dex-treated mothers weighed less than their Con counterparts, with no catch-up growth evident (Fig. 2A). Similar results were observed in female offspring of Dex-treated mothers (Fig. 2B). Thus, at 6 months of age body weight was reduced in those animals exposed to Dex in utero, regardless of postnatal diet (reductions of 17 and 10% in males and females, respectively). Although both high-n-3 groups appeared to be consistently heavier throughout the experiment, this trend did not reach statistical significance at any time point.
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) and female (P < 0.05, LSD test) (Table 3) offspring from Dex-treated mothers in comparison with Con offspring, and these effects were not altered by differences in dietary n-3 fatty acids.
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Tissue weights
Epididymal fat pad weight varied with both diet (P < 0.05) and treatment (P < 0.01) at 1 month of age, whereas only diet accounted for variation at subsequent ages (P < 0.001 at 2 months, P < 0.05 at 6 months) (Table 2
). Those groups consuming the high-n-3 diet had decreased epididymal fat pad weight (P < 0.05, LSD test), except at 1 month when it was similar among all groups.
High-n-3 fatty acid consumption resulted in a higher relative kidney weight in males at 2 months of age (21 and 9% heavier in offspring of Con and Dex-treated, respectively; Table 2) and in both males (13 and 17% heavier) and females (13% heavier in both) at 6 months of age (Tables 2 and 3). At 2 months of age, relative heart size was lower in the Con/Standard male offspring compared with all other groups (P < 0.05, LSD test; Table 2), but this difference was no longer evident at 6 months.
Serum fatty acids
In female offspring at 1 month of age, serum concentration of several fatty acids varied markedly with diet. Of particular interest is that in offspring consuming the high-n-3 semipure diet, serum levels of the n-3 fatty acids C20:5, C22:5, and c22:6 were substantially elevated (P < 0.01, LSD test, in all cases) (Table 4). In contrast, animals on the high-n-3 diet had lower levels of the n-6 fatty acids C20:4 and C22:4 (P < 0.05, LSD test, in both cases) (Table 4). Similar patterns of serum fatty acid concentrations were seen in male offspring at 1 month of age and in all offspring at 6 months of age.
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) and female (40% higher, Fig. 3F) (P < 0.05, LSD test) offspring reared on a standard diet. In contrast, offspring raised on a high-n-3 diet showed no evidence of programmed hyperleptinemia by fetal Dex exposure. Additionally, all offspring on the high-n-3 diet had substantially lower plasma leptin than Con animals on a standard diet. Trends for programmed hyperleptinemia were evident in offspring on both standard and high-n-3 diets at earlier ages (Fig. 3, A–D), but this reached statistical significance only in the high-n-3 group at 1 month. The leptin lowering effect of the high-n-3 diet was clearly evident by 2 months of age in female offspring (Fig. 3D; P < 0.05).
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). In addition, high-n-3 diet lowered retroperitoneal fat leptin mRNA expression in offspring of Con mothers. Prenatal Dex also programmed increased leptin mRNA expression in epididymal fat (P < 0.01), but again only when offspring were raised on a standard diet. Interestingly, unlike its effect in retroperitoneal fat, the high-n-3 diet did not lower leptin mRNA expression in the epididymal fat pad of Con animals (Fig. 4B).
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, A, C, and E), SBP varied significantly with diet (P < 0.001) and age (P < 0.001), and there was significant interaction between diet and treatment (P < 0.01). In male offspring at 1 month of age, SBP was similar among groups (Fig. 5A), but by 2 months (Fig. 5C), there was a significant diet effect (P < 0.001) and an interaction between diet and treatment (P < 0.05). Thus, prenatal Dex exposure programmed higher SBP (P < 0.05, LSD test) in those offspring maintained on a standard diet. In contrast, inclusion of high n-3 in the postnatal diet alleviated this hypertension. SBP in the Dex/High n-3 was as low as the Con/Standard animals at the same age. This overall pattern of SBP among groups was maintained at 4 (data not shown) and 6 (Fig. 5E) months of age. Specifically, SBP at 6 months was elevated due to Dex exposure in utero (P < 0.05), but again this effect was eliminated by the high-n-3 fatty acid postnatal diet. Additionally, all offspring on the high-n-3 diet had lower blood pressure at 2, 4, and 6 months of age than corresponding Con animals on the standard diet.
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, B, D, and F), SBP varied significantly due to diet (P < 0.001) and age (P < 0.001), although interestingly, unlike the male offspring, Dex-induced hypertension was not evident at early time points. At 6 months of age, variation in SBP among groups (Fig. 5F) was comparable with that in male offspring, with an elevation due to prenatal Dex exposure and suppression of this effect by the high-n-3 fatty acid diet (P < 0.05, LSD test). All female offspring on the high-n-3 diet had lower blood pressure than Con animals on a standard diet. |
Discussion |
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Glucocorticoid exposure in utero markedly elevated plasma leptin in postnatal life but supplementation of the postnatal diet with n-3 fatty acids attenuated this hyperleptinemia in both sexes at 6 months of age. Consistent with these findings, a similar pattern of leptin mRNA expression was evident in the retroperitoneal fat pad. Although plasma leptin and leptin mRNA in adipose tissue were also reduced by a high-n-3 diet in Con offspring, this effect was less marked than in offspring of Dex-treated mothers. Accordingly, there was a significant interaction between treatment and dietary effects, indicating that the leptin response to diet was dependent on prenatal treatment. The elevation of leptin due to glucocorticoid-exposure in utero is likely brought about by the influence of glucocorticoids on adipocyte differentiation (33). It would appear that fetal glucocorticoid exposure leads to programmed changes in the adipocyte phenotype such that leptin mRNA and thus leptin production per unit weight of fat are increased. Importantly, these programmed effects on the adipocyte phenotype occurred without any increase in total fat content, as evidenced by epididymal fat pad weights and percentage body fat composition by DEXA analysis. In contrast, plasma leptin and adipose leptin mRNA were both markedly reduced by n-3 fatty acids, and this effect was evident as early as 1 month of age. Similar effects of n-3 fatty acids have been reported previously for adult rats (21). It was also of particular interest that n-3 fatty acids not only reduced leptin mRNA expression in fat depots but also potently reduced the amount of epididymal fat present at both 2 and 6 months of age. A similar effect of n-3 fatty acids on adiposity was recently reported (24).
The observation that hypertension never developed in those offspring maintained on a high-n-3 diet suggests that the postnatal treatment completely suppressed the effects of excess glucocorticoid exposure in utero. As was evident with respect to leptin, the beneficial effects of a high-n-3 postnatal diet on blood pressure were also present in Con offspring. Again, however, there was a highly significant interaction between diet and prenatal treatment, showing that the blood pressure response to diet was dependent on prenatal treatment. These observations are consistent with several mechanisms implicated in the fetal programming of hypertension and with the known effects of n-3 fatty acids on these same mechanisms in adults. Specifically, vascular reactivity is known to be adversely affected in programming models (34, 35), whereas dietary n-3 fatty acids provide beneficial effects in this regard (for review, see Ref.36). Similarly, although the renin-angiotensin system is disturbed by fetal programming (37, 38), the beneficial effects of n-3 fatty acids on blood pressure appear to be mediated, in part, via effects on angiotensin II formation (39).
Our observation that hypertension and hyperleptinemia were both evident by 6 months of age in both sexes is consistent with the well-recognized link between obesity and cardiovascular disease. Leptin is thought to be particularly critical in this link because leptin administration reverses the decreased blood pressure of leptin-deficient ob/ob mice (40). The recent finding that dietary n-3 fatty acids enhance the leptin-lowering effect of a weight reduction program may also account for the substantial fall in blood pressure in the same individuals (22). There is also evidence that programming of hypertension by fetal glucocorticoids involves impaired nephrogenesis (41, 42), and so it is likely that offspring in our model had a reduced nephron number at birth. Nephrogenesis continues postnatally to d 8 in the rat (43), and so it is possible that postnatal n-3 fatty acids could result in compensatory nephrogenesis in this short period, although there is no evidence for this to date. In this context, it is of interest that relative kidney weight was elevated in offspring maintained on the high-n-3 diet, but further histological studies are needed to ascertain what specific structural differences account for this change in weight.
Although hypertension was clearly evident in both male and female offspring, the onset occurred at 2 months of age in males but not until 6 months in females. The presence of programmed hypertension in both sexes is consistent with several previous reports (11, 12, 14), although the present work has identified sex differences in its onset. Interestingly, Ortiz et al. (42) found that males but not females were susceptible to glucocorticoid-programmed hypertension, whereas O’Regan et al. (38) reported the opposite effect (i.e. females but not males affected). Clearly, further studies are required to ascertain whether these inconsistencies relate to the differences in timing and dose of maternal Dex treatment used in the various models.
The phenomenon of catch-up growth, whereby postnatal growth rate is enhanced to compensate for low birthweight, has been proposed to be as much of a risk factor for adult-onset diseases as intrauterine growth restriction (44). In our model, complete catch-up growth did not occur, yet adverse effects on blood pressure and leptin were still observed. Indeed, other studies have also noted adverse programming effects in the absence of catch-up growth (11, 45). It is also of interest that fetal glucocorticoid exposure did not result in increased percentage body fat or epididymal fat pad weight in adult offspring. Previously, Dahlgren et al. (13) showed that female offspring exposed to interleukin-6 prenatally and consuming normal rat chow (4.7% fat) had hyperleptinemia in association with increased parametrial and retroperitoneal fat pad weights.
Increased fetal glucocorticoid exposure substantially delayed puberty onset in both males and females, similar to our previous report (8). Interestingly, the high-n-3 diet did not influence the programmed delay in puberty onset, even though rats on the high-n-3 diet had markedly reduced plasma leptin. Although leptin is thought to provide a permissive signal for the onset of puberty (46, 47), the latter appears to be dependent on a range of developmental cues, including increased hypothalamic expression of the leptin receptor (48).
In conclusion, our data clearly indicate that a postnatal diet rich in n-3 fatty acids prevents programmed hyperleptinemia and hypertension. This raises the possibility that dietary supplementation with n-3 fatty acids may provide a viable therapeutic option for preventing and/or reducing adverse programming outcomes in humans.
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Acknowledgments |
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Footnotes |
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First Published Online October 6, 2005
Abbreviations: Con, Control; Dex, dexamethasone; LSD, least significance difference; SBP, systolic blood pressure.
Received June 21, 2005.
Accepted for publication September 23, 2005.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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3 fatty acids, blood lipids and cardiovascular risk reduction. Curr Opin Lipidol 12:11–17[CrossRef][Medline]3 fatty acids. In: Whelton PK, He J, Louis GT, eds. Lifestyle modification for the prevention and treatment of hypertension. New York: Marcel Dekker Inc.; 275–300This article has been cited by other articles:
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  P. D. Gluckman, M. A. Hanson, C. Cooper, and K. L. Thornburg Effect of In Utero and Early-Life Conditions on Adult Health and Disease N. Engl. J. Med., July 3, 2008; 359(1): 61 - 73. [Full Text] [PDF]
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 C. S. Wyrwoll, P. J. Mark, and B. J. Waddell Developmental Programming of Renal Glucocorticoid Sensitivity and the Renin-Angiotensin System Hypertension, September 1, 2007; 50(3): 579 - 584. [Abstract] [Full Text] [PDF]
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 N. Sharma, I. C. Okere, M. K. Duda, D. J. Chess, K. M. O'Shea, and W. C. Stanley Potential impact of carbohydrate and fat intake on pathological left ventricular hypertrophy Cardiovasc Res, January 15, 2007; 73(2): 257 - 268. [Abstract] [Full Text] [PDF]
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 D.-M. Alexe, G. Syridou, and E. Th. Petridou Determinants of Early Life Leptin Levels and Later Life Degenerative Outcomes Clin. Med. Res., December 1, 2006; 4(4): 326 - 335. [Abstract] [Full Text] [PDF]
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, Con/Std; 
, Dex/Std; 
, Con/Hn3; 
, Dex/Hn3). Insets show postnatal growth in the first 4 postnatal weeks. Values are the mean + SEM (n = 7–8 per group). Data were analyzed by three-way ANOVA with repeated measures, followed by post hoc LSD tests. Dex-exposed offspring were lighter at all time points compared with Con offspring (P < 0.05, LSD test).
