Caitlin S. Wyrwoll

11β-hydroxysteroid dehydrogenases and the brain: from zero to hero, a decade of progress.

Graphical abstract Expression and function of 11β-hydroxysteroid dehydrogenase (11β-HSD) type 1 and 2. 11β-HSD1 is widely expressed throughout the adult CNS and is key to HPA axis function and cognitive decline during ageing. Conversely, the major central effects of 11β-HSD2 are seen in development, as expression of 11β-HSD2 is high in fetal tissues including the neonate brain and placenta. Loss of 11β-HSD2 from the fetus and fetally-derived tissues results in a life-long phenotype of anxiety, consistent with developmental programming.

Altered Placental Function of 11β-Hydroxysteroid Dehydrogenase 2 Knockout Mice

Fetal glucocorticoid exposure is a key mechanism proposed to underlie prenatal programming of adult cardiometabolic and neuropsychiatric disorders. Regulation of fetal glucocorticoid exposure is achieved by the placental glucocorticoid barrier, which involves glucocorticoid inactivation within the labyrinth zone of the murine placenta by 11beta-hydroxysteroid dehydrogenase 2 (11beta-HSD2). Thus, the absence of placental 11beta-HSD2 may impact on fetal and placental development. The current study investigated transport of amino acids and glucose, key factors required for fetal growth, and vascular development in placentas from 11beta-HSD2(+/+), (+/-), and (-/-) fetuses derived from 11beta-HSD2(+/-) matings. At embryonic d 15 (E15) (term = E19), 11beta-HSD2(-/-) fetal weight was maintained in comparison to 11beta-HSD2(+/+) fetuses. The maintenance of 11beta-HSD2(-/-) fetal weight occurred despite a reduction in placental weight, suggesting that compensatory changes occur in the placenta to maintain function. However, by E18, 11beta-HSD2(-/-) fetal and placental weights were both reduced. Transport studies revealed up-regulation of placental amino acid transport to 11beta-HSD2(-/-) offspring at E15, coinciding with an increase in the expression of the amino acid transporters. Furthermore, at E18, placental glucose transport to 11beta-HSD2(-/-) offspring was markedly reduced, correlating with lower fetal weight and a decrease in glucose transporter 3 expression. Stereological analyses of the labyrinth zone of the placenta revealed that the reduction in placental weight at E18 was associated with restriction of the normal increase in fetal vessel density over the final third of pregnancy. Our data suggest that restriction of fetal growth in 11beta-HSD2(-/-) mice is mediated, at least in part, via altered placental transport of nutrients and reduction in placental vascularization.

Developmental Programming of Renal Glucocorticoid Sensitivity and the Renin-Angiotensin System

Fetal glucocorticoid excess leads to subsequent adult hypertension, but the mechanisms involved in this developmental programming remain largely unknown. In this study we tested the hypothesis that programmed hypertension in rats is linked to altered renal expression of the glucocorticoid receptor, mineralocorticoid receptor, and 11β-hydroxysteroid dehydrogenase type 2 and components of the intrarenal and adipose renin-angiotensin system. The interactive effects of a postnatal diet enriched in omega-3 fatty acids, which prevents emergence of the hypertensive phenotype, were also examined. Maternal dexamethasone (0.75 μg/mL of drinking water from day 13 to term) markedly increased renal expression of the glucocorticoid receptor in 6-month–old offspring, and this was associated with hypomethylation of the glucocorticoid receptor promoter; renal MR was unaffected. In contrast, maternal dexamethasone reduced renal 11β-hydroxysteroid dehydrogenase type 2 in offspring, but this effect was prevented by a high omega-3 diet. Consistent with these effects, renal Na/K-ATPase-α1 was elevated in offspring of dexamethasone-treated mothers, but only in those raised on the standard diet. Maternal dexamethasone also programmed increased expression of renal and adipose angiotensin-converting enzyme and renal renin, but among these changes, only that of renal angiotensin-converting enzyme was prevented by the omega-3 diet. Our data support the hypothesis that programmed hypertension is mediated, in part, by increased renal glucocorticoid sensitivity, with consequent stimulatory effects on Na/K-ATPase-α1 and intrarenal renin-angiotensin system components. Partial prevention of programmed changes in renal gene expression by postnatal dietary omega-3 fatty acids provides insight into how this intervention prevents hypertension induced by fetal glucocorticoid excess.

Prenatal Excess Glucocorticoid Exposure and Adult Affective Disorders: A Role for Serotonergic and Catecholamine Pathways

Fetal glucocorticoid exposure is a key mechanism proposed to underlie prenatal ‘programming’ of adult affective behaviours such as depression and anxiety. Indeed, the glucocorticoid metabolising enzyme 11β-hydroxysteroid dehydrogenase type 2 (11β-HSD2), which is highly expressed in the placenta and the developing fetus, acts as a protective barrier from the high maternal glucocorticoids which may alter developmental trajectories. The programmed changes resulting from maternal stress or bypass or from the inhibition of 11β-HSD2 are frequently associated with alterations in the hypothalamic-pituitary-adrenal (HPA) axis. Hence, circulating glucocorticoid levels are increased either basally or in response to stress accompanied by CNS region-specific modulations in the expression of both corticosteroid receptors (mineralocorticoid and glucocorticoid receptors). Furthermore, early-life glucocorticoid exposure also affects serotonergic and catecholamine pathways within the brain, with changes in both associated neurotransmitters and receptors. Indeed, global removal of 11β-HSD2, an enzyme that inactivates glucocorticoids, increases anxiety- and depressive-like behaviour in mice; however, in this case the phenotype is not accompanied by overt perturbation in the HPA axis but, intriguingly, alterations in serotonergic and catecholamine pathways are maintained in this programming model. This review addresses one of the potential adverse effects of glucocorticoid overexposure in utero, i.e. increased incidence of affective behaviours, and the mechanisms underlying these behaviours including alteration of the HPA axis and serotonergic and catecholamine pathways.

Directional secretion and transport of leptin and expression of leptin receptor isoforms in human placental BeWo cells.

Placental leptin secretion has important implications for maternal adaptation to pregnancy, fetal growth and development, and local autocrine/paracrine actions within trophoblast. In this study we used a cell culture insert model to examine directional secretion of leptin from the basal and apical surfaces of human choriocarcinoma BeWo cells, and to assess the effects of dexamethasone and syncytialization. Additionally, the effects of dexamethasone on transcellular passage of leptin across BeWo monolayers, and on expression of the leptin receptor isoforms Ob-Rs and Ob-RL were examined. Leptin was secreted into both the basal and apical chambers and was stimulated by dexamethasone. Treatment of BeWo cells with forskolin induced syncytialization and loss of monolayer integrity, but resulted in a marked increase in total leptin secretion, an effect further enhanced by co-treatment with dexamethasone. Bidirectional transfer of 125I-leptin between the apical and basal chambers of BeWo cell cultures was low but indicative of specific transcellular passage of leptin; transfer was unaffected by dexamethasone. Treatment of BeWo cells with forskolin increased Ob-Rs mRNA expression, whilst Ob-RL mRNA expression increased in response to forskolin only in the presence of dexamethasone. In conclusion, our data show that leptin is secreted from both the apical and basal surfaces of BeWo placental cells and is increased by both syncytialization and glucocorticoids. Moreover, transport of exogenous leptin occurred in both the apical to basal and reverse directions, suggesting the potential for maternal-fetal exchange of leptin across the human placenta.

Maternal vitamin D deficiency alters fetal brain development in the BALB/c mouse.

Prenatal exposure to vitamin D is thought to be critical for optimal fetal neurodevelopment, yet vitamin D deficiency is apparent in a growing proportion of pregnant women. The aim of this study was to determine whether a mouse model of vitamin D-deficiency alters fetal neurodevelopment. Female BALB/c mice were placed on either a vitamin D control (2,195 IU/kg) or deficient (0 IU/kg) diet for 5 weeks prior to and during pregnancy. Fetal brains were collected at embryonic day (E) 14.5 or E17.5 for morphological and gene expression analysis. Vitamin D deficiency during pregnancy reduced fetal crown-rump length and head size. Moreover, lateral ventricle volume was reduced in vitamin D-deficient foetuses. Expression of neurotrophin genes brain-derived neurotrophic factor (Bdnf) and transforming growth factor-β1 (Tgf-β1) was altered, with Bdnf reduced at E14.5 and increased at E17.5 following vitamin D deficiency. Brain expression of forkhead box protein P2 (Foxp2), a gene known to be important in human speech and language, was also altered. Importantly, Foxp2 immunoreactive cells in the developing cortex were reduced in vitamin D-deficient female foetuses. At E17.5, brain tyrosine hydroxylase (TH) gene expression was reduced in females, as was TH protein localization (to identify dopamine neurons) in the substantia nigra of vitamin D-deficient female foetuses. Overall, we show that prenatal vitamin D-deficiency leads to alterations in fetal mouse brain morphology and genes related to neuronal survival, speech and language development, and dopamine synthesis. Vitamin D appears to play an important role in mouse neurodevelopment.

Developmental programming of adult hyperinsulinemia, increased proinflammatory cytokine production, and altered skeletal muscle expression of SLC2A4 (GLUT4) and uncoupling protein 3

Fetal glucocorticoid excess programs detrimental effects in the adult phenotype including hyperleptinemia and aberrant glycemic control. In this study, we determined the interactive effects of maternal dexamethasone (Dex) treatment and postnatal dietary omega-3 (n-3) fatty acids on adult proinflammatory cytokine production and skeletal muscle expression of genes central to glucose handling and fatty acid metabolism. Dex acetate was administered to pregnant rats (0.75 microg/ml drinking water) from day 13 to term. Offspring of treated and control mothers were cross-fostered to mothers on either a standard (Std) or high n-3 (Hn3) diet, and remained on these diets postweaning. Adult offspring exposed to Dex in utero exhibited fasting hyperinsulinemia when raised on the Std diet but not when raised on the Hn3 diet. Dex also programmed increased plasma tumour necrosis factor alpha and interleukin 1 beta (IL-1 beta), but the increase in IL-1 beta was also prevented by the Hn3 diet. In skeletal muscle, expression of insulin regulated Slc2a4 (formerly known as GLUT4) was elevated (up to 15-fold) after Dex in utero, and this resulted in elevated intracellular, but not membrane-associated, SLC2A4 protein. Fetal glucocorticoid excess also reduced adult skeletal muscle Ucp3 expression in all offspring, whereas skeletal muscle expression of both Ppard and Ppargc1a were increased in females but not males. In conclusion, our data show that fetal glucocorticoid excess programs adult hyperinsulinemia and increased proinflammatory cytokine production. Related changes in the skeletal muscle Slc2a4, Ucp3, and Ppard indicate that fetal glucocorticoid excess disturbs adult glucose/fatty acid transport and metabolism.

Conditional Deletion of Hsd11b2 in the Brain Causes Salt Appetite and Hypertension

Background— The hypertensive syndrome of Apparent Mineralocorticoid Excess is caused by loss-of-function mutations in the gene encoding 11&bgr;-hydroxysteroid dehydrogenase type 2 (11&bgr;HSD2), allowing inappropriate activation of the mineralocorticoid receptor by endogenous glucocorticoid. Hypertension is attributed to sodium retention in the distal nephron, but 11&bgr;HSD2 is also expressed in the brain. However, the central contribution to Apparent Mineralocorticoid Excess and other hypertensive states is often overlooked and is unresolved. We therefore used a Cre-Lox strategy to generate 11&bgr;HSD2 brain-specific knockout (Hsd11b2.BKO) mice, measuring blood pressure and salt appetite in adults. Methods and Results— Basal blood pressure, electrolytes, and circulating corticosteroids were unaffected in Hsd11b2.BKO mice. When offered saline to drink, Hsd11b2.BKO mice consumed 3 times more sodium than controls and became hypertensive. Salt appetite was inhibited by spironolactone. Control mice fed the same daily sodium intake remained normotensive, showing the intrinsic salt resistance of the background strain. Dexamethasone suppressed endogenous glucocorticoid and abolished the salt-induced blood pressure differential between genotypes. Salt sensitivity in Hsd11b2.BKO mice was not caused by impaired renal sodium excretion or volume expansion; pressor responses to phenylephrine were enhanced and baroreflexes impaired in these animals. Conclusions— Reduced 11&bgr;HSD2 activity in the brain does not intrinsically cause hypertension, but it promotes a hunger for salt and a transition from salt resistance to salt sensitivity. Our data suggest that 11&bgr;HSD2-positive neurons integrate salt appetite and the blood pressure response to dietary sodium through a mineralocorticoid receptor–dependent pathway. Therefore, central mineralocorticoid receptor antagonism could increase compliance to low-sodium regimens and help blood pressure management in cardiovascular disease.

Developmental programming of adult adrenal structure and steroidogenesis: effects of fetal glucocorticoid excess and postnatal dietary omega-3 fatty acids

Fetal glucocorticoid excess programs a range of detrimental outcomes in the adult phenotype, at least some of which may be due to altered adult adrenocortical function. In this study, we determined the effects of maternal dexamethasone treatment on offspring adrenal morphology and function, as well as the interactive effects of postnatal dietary omega-3 (n-3) fatty acids. This postnatal dietary intervention has been shown to alleviate many of the programming outcomes in this model, but whether this is via the effects on adrenal function is unknown. Dexamethasone acetate was administered to pregnant rats (0.75 microg/ml drinking water) from day 13 to term. Cross-fostered offspring were raised on either a standard or high-n-3 diet. Adrenal weight (relative to body weight) at 6 months of age was unaffected by prenatal dexamethasone, regardless of postnatal diet, and stereological analysis showed no effect of dexamethasone on the volumes of adrenal components (zona glomerulosa, zona fasciculata/reticularis or adrenal medulla). Expression of key steroidogenic genes (Cyp11a1 and Star) was unaffected by either prenatal dexamethasone or postnatal diet. In contrast, adrenal expression of Mc2r mRNA, which encodes the ACTH receptor, was higher in offspring of dexamethasone-treated mothers, an effect partially attenuated by the Hn3 diet. Moreover, stress-induced levels of plasma and urinary corticosterone and urinary aldosterone were elevated in offspring of dexamethasone-treated mothers, indicative of enhanced adrenal responsiveness. In conclusion, this study shows that prenatal exposure to dexamethasone does not increase basal adrenocortical activity but does result in a more stress-responsive adrenal phenotype, possibly via increased Mc2r expression.

Altered placental methyl donor transport in the dexamethasone programmed rat

There is increasing evidence for a role for epigenetic modifications in early life programming effects. Altered placental methyl donor transport may impact on the establishment of epigenetic marks in the fetus. This study investigated the effects of prenatal glucocorticoid overexposure on placental methyl donor transport. Glucocorticoids increased folate but decreased choline transport and reduced fetal plasma methionine levels. There was no change in global DNA methylation in fetal liver. These data suggest prenatal glucocorticoid overexposure causes complex alterations in the placental transport of key methyl donors which may have important implications for maternal diet and nutrient supplementation in pregnancy.