Background
The incidence of obesity and related metabolic disorders (hypertension, insulin resistance and dyslipidemia) have risen dramatically in recent decades, with major implications for cardiovascular disease risk [
1]. There is growing evidence that a range of insults in early life, including in utero, can developmentally program adverse outcomes for the adult phenotype. Most notably, undernutrition, overnutrition and glucocorticoid excess can all program adverse phenotypic outcomes. Accordingly, offspring born small (a proxy for a poor fetal environment) are predisposed to several adult-onset diseases including diabetes and obesity [
2,
3]. We previously demonstrated that offspring of rat mothers treated with dexamethasone during pregnancy were growth retarded at birth and as adults, were hyperleptinemic, hyperinsulinemic and hypertensive [
4]. These offspring also exhibited a pro-inflammatory state, with plasma levels of tumor necrosis factor alpha (TNFα), interleukin 6 (IL6) and IL1β all increased by prenatal dexamethasone [
5]. Moreover, because this pro-inflammatory state and most other adverse programming outcomes were prevented by postnatal dietary omega (n)-3 fatty acid supplementation, we have proposed that inflammation may be a key driver of the overall programmed phenotype [
5]. In particular, adipose tissue inflammation may underlie the programmed pro-inflammatory phenotype, since adipose tissue leptin expression was elevated in offspring that had been exposed prenatally to dexamethasone [
4]. To test this hypothesis we characterised the adipose tissue phenotype and serum fatty acid profiles in adult offspring of dexamethasone-treated mothers. Specifically, we measured adipocyte size (by unbiased stereology) and adipose expression of pro-inflammatory cytokines, peroxisome proliferator-activated receptors, and determinants of glucocorticoid sensitivity (i.e., the glucocorticoid receptor (GR;
Nr3c1) and 11β-hydroxysteroid dehydrogenase (
11bHsd) enzymes). This focus on adipose glucocorticoid sensitivity was considered important given that enhanced glucocorticoid activity in adipocytes appears to be obesogenic (for review see [
6]). Moreover, because the programmed phenotype in our model was largely rescued in offspring raised on a diet enriched with n-3 fatty acids [
4,
5] we also characterised the adipose phenotype and fatty acid profiles of these offspring.
Discussion
Fetal glucocorticoid excess is recognised as a key mechanism involved in the programming of adverse phenotypic outcomes in adult offspring, including hypertension and insulin resistance. The present study showed that prenatal dexamethasone exposure programmed increased adipose inflammation and serum fatty acid levels at 6 months of age, and that consumption of a postnatal diet enriched with n-3 fatty acids alleviated many of these adverse outcomes. Specifically, adipose expression of Tnfa, Il6, Il1b, GR, 11bHsd1 and Ppara and serum fatty acid levels (males only) were all elevated in adult offspring of dexamethasone-treated mothers. Consumption of a diet enriched with n-3 fatty acids from birth corrected the programmed increases in serum fatty acids and adipose expression of Il6 and Il1b. Dietary supplementation with n-3 fatty acids also upregulated expression of Ppard and reduced mean adipocyte size regardless of prenatal treatment.
Disturbances to the normal fetal environment, such as excess glucocorticoid exposure or undernutrition, have been associated with a predisposition for adverse physiological outcomes in adult offspring including type II diabetes, hypertension and obesity in humans [
18,
19] and rats [
4,
5,
20‐
22]. Our developmental programming model involves fetal glucocorticoid excess over the final third of rat pregnancy, which leads to fetal growth restriction [
4,
7,
23] and subsequent development of offspring hypertension, hyperleptinemia [
4], hyperinsulinemia and elevated plasma cytokine levels [
5]. Although percent adiposity appeared unaffected in these programmed offspring [
4], the present study shows that fetal glucocorticoid excess programmed marked changes in the adipose tissue phenotype. Most notably, adipose mRNA expression of the pro-inflammatory cytokines
Tnfa,
Il6 and
Il1b was elevated in male and female offspring of dexamethasone-treated mothers, consistent with our previous report showing that plasma levels of these cytokines were elevated in this same cohort of animals [
5]. Furthermore, although insulin sensitivity was not measured in these animals, analysis of fasting insulin levels indicated that prenatal dexamethasone treatment resulted in hyperinsulinemia [
5]. This suggests that the pro-inflammatory state of adipose tissue may contribute to systemic inflammation and insulin resistance. Moreover, programming of the pro-inflammatory adipose phenotype was largely prevented when offspring were raised on the Hn3 diet, which normalised both
Il6 and
Il1b expression (but interestingly not that of
Tnfa) and circulating insulin levels. This partial correction i.e. normalisation of
Il6 and
Il1b but not
Tnfa, parallels the effects of the Hn3 diet on systemic inflammatory state [
5] and the anti-inflammatory effects of the high n-3 diet are consistent with observations in a range of previous animal and human studies (for review see [
24]). Exactly why programmed
Tnfa expression is not corrected is unclear and requires further investigation.
A second key feature of the programmed adipose tissue phenotype was an apparent increase in glucocorticoid sensitivity, a characteristic linked to the aetiology of obesity [
25‐
27]. Thus, adipose expression of both the GR (
Nr3c1) and
11bHsd1 mRNAs were both increased in offspring of dexamethasone-treated mothers. Glucocorticoid activation of the GR is enhanced by local expression of 11βHSD1, which catalyses regeneration of active corticosterone from inert 11-dehydrocorticosterone [
28]. As such, these changes, coupled with increased stress-responsiveness of programmed offspring in this model [
29], are likely to promote GR activation in adipose tissue. These observations extend those of Gnanalingham et al. (2005), who showed that prenatal dexamethasone exposure in a sheep model increased GR and 11βHSD1 levels in perirenal adipose tissue of newborns [
30]. Our data are also consistent with the programmed upregulation of GR expression in kidney [
31] and liver [
32] following prenatal dexamethasone, and in adipose tissue after maternal undernutrition [
22]. Collectively, these studies show that adipose glucocorticoid sensitivity is enhanced by fetal insults (nutritional or excess glucocorticoids).
This outcome might be expected to reduce adipose inflammation, given that glucocorticoids are normally potent anti-inflammatory agents. Indeed, glucocorticoids and pro-inflammatory cytokines are thought to act synergistically to stimulate
11bHsd1 expression in acute inflammation, thereby further enhancing local levels of active glucocorticoid to perhaps limit inflammation (for review see [
6]). But if this acute inflammation is not resolved and chronic inflammation ensues, as in metabolic disease, the anti-inflammatory action of glucocorticoids appears to be lost [
6]. Interestingly, a similar scenario is evident in the late gestation rat placenta, where increased expression of pro-inflammatory cytokines [
33],
GR and
11bHsd1[
23] all occur despite rising levels of maternal and fetal glucocorticoids.
The PPAR transcription factors play key roles in adipocyte differentiation and function, influencing the balance between fat storage (via PPARγ) and utilisation (via PPARδ) (for reviews see [
34,
35]). Although prenatal dexamethasone did not affect adipose expression of either
Pparg or
Ppard, it did program a marginal increase in adipose expression of
Ppara, particularly in female offspring. This effect may have developed as a compensatory response to the heightened adipose inflammatory state, since PPARα activation is known to exert anti-inflammatory effects in other cell types [
36]. Interestingly, these
Ppard responses to diet are different to those observed in the skeletal muscle of the same rats [
5], where elevated
Ppard expression was programmed by prenatal Dex exposure, but was unaffected by diet. This likely represents tissue-specific regulation of
Ppard.
Male offspring of dexamethasone-treated mothers showed elevated serum levels of total fatty acids, an effect evident across all fatty acid groups. While the specific reasons why this effect was limited to male offspring are not known, gender differences have been noted in fatty acid oxidation rates in humans (females lower than males; for review see [
37]). Although previous studies have reported a programmed increase in serum free fatty acid and triacylglycerol levels (by maternal obesity; [
38]), importantly this was observed in offspring that were themselves obese. In contrast, the programmed increase in serum fatty acids in the present study occurred in the absence of increased adiposity, possibly indicative of a greater susceptibility to obesogenic stimuli. It is possible that the programming of elevated serum fatty acids by dexamethasone may be linked, in part, to the elevated levels of pro-inflammatory cytokines [
5], since these cytokines are known to increase lipolysis in both humans and rats [
39,
40]. In this context it is noteworthy that adipose expression of
Il6 was also corrected by the Hn3 diet.
Consumption of the Hn3 diet markedly reduced serum levels of triacylglycerols and cholesterol, but did not affect HDL-C levels, consistent with known effects of n-3 fatty acids on lipid profiles in humans [
41]. This dietary effect may reflect inhibition of hepatic triacylglycerol synthesis and stimulation of beta-oxidation by n-3 fatty acids [
42], effects that are likely to have contributed to the decreased adipocyte size in rats fed the Hn3 diet. Elevated expression of
Ppard was also observed in these smaller adipocytes, consistent with previous studies in rats that consumed n-3 fatty acids [
43]. Activation of PPARδ in adipocytes leads to improved lipid profiles and reduced adiposity [
44] and n-3 fatty acids are known ligands for PPARs [
45]. Decreased adipocyte size has also been associated with increased insulin sensitivity, a reduced incidence of type II diabetes in rats [
46] and humans [
47] as well as reduced expression and release of adipocytokines [
48,
49], suggestive of a less detrimental phenotype associated with the smaller adipocytes. Furthermore, this reduced adipocyte size is consistent with the observed decline in epididymal fat pad weight previously reported for these animals [
4].
Competing interests
The authors declare that there is no competing of interest that could be perceived as prejudicing the impartiality of the research reported.
Authors’ contributions
PJM, CSW, TAM and BJW designed research; CSW, PJM, and ISZ conducted research and analysed data; PJM and BJW wrote the paper and BJW had primary responsibility for final content. All authors read and approved the final manuscript.