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Published in final edited form as: Endocrinology. 2006 Dec 14;148(3):961–966. doi: 10.1210/en.2006-0603

Liver-Selective Transgene Rescue of Hypothalamic-Pituitary-Adrenal Axis Dysfunction in 11βHydroxysteroid Dehydrogenase Type 1-Deficient Mice

Janice M Paterson, Megan C Holmes, Christopher J Kenyon, Roderick Carter, John J Mullins 1, Jonathan R Seckl
PMCID: PMC6443039  EMSID: EMS82334  PMID: 17170103

Abstract

11β-Hydroxysteroid dehydrogenase type 1 (11β-HSD1) acts as a reductase in vivo, regenerating active glucocorticoids within cells from circulating inert 11-keto forms, thus amplifying local glucocorticoid action. 11β-HSD1 is predominantly expressed in liver and also adipose tissue and brain. Mice deficient in 11β-HSD1 (11β-HSD1-/-) exhibit adrenal hyperplasia, raised basal corticosterone levels, and increased hypothalamic-pituitary-adrenal (HPA) axis responses to stress. Whereas reduced peripheral glucocorticoid regeneration may explain adrenal hypertrophy and exaggerated stress responses, elevated basal glucocorticoid levels support a role for 11β-HSD1 within the brain in amplifying glucocorticoid feedback. To test this hypothesis, apolipoprotein E-HSD1 mice overexpressing 11β-HSD1 in liver were intercrossed with 11βHSD1-/- mice to determine whether complementation of hepatic 11β-HSD1 can restore adrenal and HPA defects. Transgene-mediated delivery of 11β-HSD1 activity to the liver rescued adrenal hyperplasia and reversed exaggerated HPA stress responses in 11β-HSD1-/- mice. Unexpectedly, elevated nadir plasma corticosterone levels were also restored to control levels. Consistent with this, CYP11B1 mRNA expression in the adrenal cortex of 11β-HSD1-/- mice was increased by 50% but returned to control levels in 11βHSD1-/- mice bearing the apolipoprotein E-HSD1 trans-gene. 11β-HSD1-/- mice have lower plasma glucose levels, but the fall in plasma corticosterone with sucrose supplementation was similar in 11β-HSD1-/- and control mice, suggesting glucose deficiency is not the main mechanism whereby basal corticosterone levels are elevated in the null mice. Thus, regeneration of glucocorticoids by 11β-HSD1 in the liver normalizes all aspects of HPA axis dysregulation in 11β-HSD1-/- mice, without restoration of enzyme activity in key feedback areas of the forebrain. Therefore, hepatic glucocorticoid metabolism influences basal as well as stress-associated functions of the HPA axis.

Introduction

Glucocorticoids (cortisol, corticosterone) are intrinsic to the homeostatic regulation of many physiological processes, notably metabolism and the response to stress. Circulating levels of glucocorticoids are regulated by the hypothalamic-pituitary-adrenal (HPA) axis, which responds to diurnal cues and stressful stimuli. Because both chronic excess and deficiency of glucocorticoids have severe adverse consequences, the HPA axis is under tight homeostatic regulation. This is achieved via negative feedback control by glucocorticoids acting primarily through intracellular receptors in pituitary, hypothalamus, and suprahypothalamic sites, notably the hippocampus (1).

However, cellular glucocorticoid signaling is determined by not only circulating steroid levels and the cellular density of receptors but also 11β-hydroxysteroid dehydrogenases (11β-HSDs), which control intracellular substrate levels (2, 3). 11β-HSDs catalyze interconversion of active 11-hydroxy glucocorticoids (cortisol, corticosterone) and their inert 11keto derivatives (cortisone, 11-dehydrocorticosterone). 11β-HSD type 1 is a predominant 11β-reductase that, in expressing cells, regenerates active glucocorticoids from inert forms, thus amplifying local glucocorticoid action. In contrast, 11βHSD type 2 catalyzes rapid 11β-dehydrogenation, potently inactivating intracellular glucocorticoids and thus protecting intrinsically nonselective mineralocorticoid receptors in the distal nephron from activation by glucocorticoids in vivo. Whereas 11β-HSD2 is barely if at all expressed in the adult rat, mouse, and human forebrain (4 – 6), 11β-HSD1 is highly expressed throughout the brain, including the hippocampus and hypothalamus and also the anterior pituitary (7–9). 11β-HSD1 is also expressed at high levels in the liver and adipose tissues (10, 11).

Mice homozygous for targeted disruption of the 11β-HSD1 gene (11β-HSD1-/- mice) are viable, fertile, and apparently healthy and show that 11β-HSD1 is the sole 11β-reductase (12). 11β-HSD1-/- mice exhibit improved glucose tolerance, insulin sensitization, and a cardioprotective lipid profile with resistance to diet-induced obesity and its metabolic sequelae (12–14), effects compatible with reduced intracellular glucocorticoid action in liver and adipose tissue. However, 11β-HSD1-/-mice also show increased HPA axis activity and adrenal hyperplasia (12, 15). Adrenocortical hyperplasia might be predicted on the basis that 11β-HSD1-/- mice lack normal regeneration of glucocorticoids in liver and adipose tissue, which, at least in humans, contributes around 40% of active circulating glucocorticoids (16); thus, the null mice require increased adrenal corticosterone production merely to maintain plasma levels, a contention supported by increased adrenal corticosterone secretion to ACTH in vitro (12). However, basal (diurnal nadir) corticosterone and ACTH levels are also elevated in 11β-HSD1-/-mice (15). This suggests attenuated glucocorticoid feedback, a notion supported by blunted suppression of stress responses by exogenous glucocorticoids in 11β-HSD1-/-mice (15). Moreover, failure of neuronal glucocorticoid target genes to display expected changes in expression after stimulation of the HPA axis suggests a requirement for local glucocorticoid regeneration in mediating feedback control. However, the role of peripheral glucocorticoid metabolism in control of the HPA axis remains unexplored.

We recently generated transgenic mice overexpressing 11β-HSD1 in the liver (ApoE-HSD1 mice) (17). ApoE-HSD1 mice exhibit modest insulin resistance and dyslipidemia together with hypertension but not glucose intolerance or obesity. Adrenal weight and basal plasma corticosterone and ACTH levels remain unaltered in this model. We reasoned that aspects of HPA axis dysfunction in 11β-HSD1-/- mice secondary to lack of peripheral regeneration of active corticosterone would be restored, at least in part, by replacement of the enzyme in the liver, whereas components of HPA dysfunction due to lack of 11β-HSD1 in the brain and pituitary would be unaffected. Here we examine this issue by intercross of liver transgenic ApoE-HSD1 mice with 11βHSD1-/- mice.

Materials and Methods

Animals

Adult male mice were studied throughout. C57BL/6J congenic 11βHSD1-/- mice (14) were intercrossed with ApoE-HSD1 mice (line 1065) at F3 C57BL/6J backcross (17) to generate four genotype-defined study groups of age-matched littermates; control nontransgenic (non-TG), ApoE-HSD1 transgenic (TG), 11β-HSD1-/-, and 11β-HSD1-/-/ApoEHSD1 (11β-HSD1-/- xTG) mice with transgenic rescue in the liver alone. Animals bearing the ApoE-HSD1 transgene did so in the hemizygous state (the transgene is integrated at a single chromosomal site distinct to the hsd11b1 gene mutated in 11β-HSD1-/- mice, so each genetic modification segregates as a separate allele). Mice were fed standard chow ad libitum under 12-h light, 12-h dark conditions (lights on 0700, lights off 1900 h) and were housed individually for 4 –5 d before experiments. All experiments were carried out after independent ethical review and were licensed under the provisions of the U.K. Animals (Scientific Procedures) Act, 1986.

Diurnal corticosterone profile

Blood sampled by tail venesection into Microvette CB 300 EDTA tubes (Sarstedt Ltd., Beaumont Leys, Leicester, UK) from individually housed mice was taken within 1 min of disturbing the cage at 0800 and 2000 h to coincide with the expected nadir and peak of circulating levels. Plasma was separated, stored at -20 C and corticosterone assayed by RIA, as previously described (15).

Restraint stress

Mice were subjected to acute restraint stress in a Perspex tube for 10 min. Blood was sampled by venesection from each animal immediately and 45 min after initiation of restraint. Then 120 min after the start of restraint, mice were decapitated, trunk blood collected into Microvette CB 300 EDTA tubes (Sarstedt) for plasma separation, and the adrenal glands collected and fixed in 10% neutral buffered formalin (left) or frozen for cryostat sectioning (right).

Sucrose supplementation

To test whether carbohydrate supplementation differentially affected HPA axis activity in control and 11β-HSD1-/- mice, animals were given sucrose (1 mol/liter) or vehicle (water) to drink for 14 d. Basal plasma corticosterone levels were measured in samples taken at 0900 h after each treatment (blood sampling was by tail venesection as described above).

Adrenal weights and mRNA quantitation in adrenals by in situ hybridization

Adrenals were carefully trimmed of fat and weighed. Cryostat sections (10 pm) were prepared from the frozen adrenals and serial sections hybridized in situ with antisense riboprobes for the detection of CYP11B1 (11β-hydroxylase) mRNA and sense controls, as previously described (18). In brief, cDNA fragments of mouse CYP11B1 and CYP11B2 mRNAs were generated by PCR from reverse-transcribed adrenal gland total RNA and ligated into pBluescriptII plasmid vector to facilitate generation of sense and antisense cRNA probes from T3 and T7 promoters flanking the insert. The CYP11B1 sequence corresponds to nucleotides 1593–1794 representing the 3'-untranslated region of mouse CYP11B1 cDNA (GenBank accession no. NM_001033229.1) and shows no significant sequence identity with CYP11B2. In situ hybridization was carried out as described (19). After washing to remove nonspecific hybridization, sections were exposed to autoradiographic film (Hyperfilm; Amersham Biosciences, part of GE Healthcare UK Ltd., Little Chalfont, Buckinghamshire, UK) for image analysis (MCID; Imaging Research, part of GE Healthcare UK Ltd.). Specific signal per unit area was recorded for each section. Six sections were analyzed per mouse and the mean value (arbitrary units) for each was multiplied by the weight of the adrenal to obtain a measure representative of the whole adrenal response in vivo for each mouse. The CYP11B1 cRNA probe showed hybridization only to the zona fasciculata-reticularis of control mouse adrenals (supplemental Fig. 1A, published as supplemental data on The Endocrine Society’s Journals Online Web site at http://endo.endojournals.org). As an additional control for specificity, adjacent sections were hybridized with a 237bp cRNA probe corresponding to the 3'-untranslated region of CYP11B2 mRNA equivalent to nucleotides 1563–1790 of a mouse genomic DNA clone containing part of this locus (GenBank accession no. S85260). The CYP11B2 cRNA hybridized only to the zona glomerulosa (supplemental Fig. 1B).

11β-HSD1 in situ hybridization series of 10 pm sagittal cryostat sections through the brains of control, 11β-HSD1-/-, and 11β-HSD1-/- xTG mice were prepared and used for in situ hybridization with an 11β-HSD1 cRNA riboprobe, as described (20). After film autoradiography, sections were dipped in photographic emulsion (NTB2, Kodak, Hertfordshire, UK) for microscopic analysis to precisely locate any signal.

Statistics

Plasma corticosterone data showed inhomogeneity of variance and were log transformed before analysis. Two-way ANOVA was used to compare corticosterone levels between groups with respect to genotype and time. One-way ANOVA was used to examine differences with respect to a single variable (genotype) between groups. Post hoc differences between groups were determined by Fisher’s least significant differences test. P < 0.05 was accepted as the threshold of statistical significance. Data are means ± sem.

Results

Loss of the diurnal rhythm of corticosterone in 11β-HSD1-/ mice is restored by the ApoE-HSD1 transgene

Two-way ANOVA showed significant effects of time and an interaction with genotype (F = 3.8, P = 0.025); at 0800 h, there were significant effects of genotype (F = 4.7, P = 0.009), and at 2000 h, there were no significant differences by genotype. In control non-TG mice, plasma corticosterone levels exhibited the expected 0800 h nadir and significantly higher 2000 h peak levels (P < 0.01). The low morning basal corticosterone levels (30 nmol/liter) are consistent with stress-free sampling at this time (Fig. 1). Morning and evening plasma corticosterone levels were similar in ApoE-HSD1 TG and non-TG mice, indicating normal HPA axis regulation despite 5-fold liver overexpression of 11β-HSD1 in TG mice. As previously reported on another genetic background (MF1/129) (15), basal 0800 h corticosterone levels were significantly increased in 11β-HSD1-/- mice, compared with non-TG controls (P < 0.05; Fig. 1), indicating abnormal HPA regulation. In this study, peak 2000 h corticosterone levels tended to be reduced in 11β-HSD1-/- mice, suggesting suppression of the normal circadian maximum and rhythmicity. Indeed, whereas all other genotypes showed significant diurnal variation, 11β-HSD1-/- mice had no significant variation between morning and evening and the day-night change in plasma corticosterone was significantly less in 11β-HSD1-/- mice than all other genotypes (F = 3.8, P = 0.025). Importantly, transgenic replacement of 11β-HSD1 solely in 11β-HSD1-/-liver (11β-HSD1-/- xTG) restored both basal and peak corticosterone level to non-TG control levels.

Fig. 1.

Fig. 1

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Elevated basal and blunted circadian rhythm of corticosterone in 11β-HSD1-/- mice is rescued by the apoE-HSD1 transgene. Plasma samples were taken after tail venesection at 0800h(nadir) and2000h (peak)in non-TG controls (black columns),apoE-HSD1-TGmice (gray columns),11β-HSD1-/-mice (black-and-white-striped columns) and 11β-HSD1-/-x TGmice (gray-and-black-striped columns).Values are mean±SEM,n=6-10/group. *,P<0.05, compared with other genotypes at the same time. All genotypes except11β-HSD1-/-showed a significant difference between morning and evening, and the diurnal change was significantly less in 11β-HSD1-/-than all other groups.

Hyperplasia of the adrenal gland in 11β-HSD1-/- mice is rescued by the ApoE-HSD1 transgene

Mean adrenal mass in control non-TG mice and ApoEHSD1 TG mice did not differ (1.47 ± 0.11 and 1.58 ± 0.07 mg, respectively). 11β-HSD1-/- mice had significantly heavier adrenals than non-TG controls (2.00 ± 0.17 mg, P = 0.02), as previously found on another genetic background (12). Adrenal mass in 11β-HSD1-/- xTG mice was significantly lower than 11β-HSD1-/- mice (P < 0.05) and was similar to control non-TG values (1.25 ± 0.12 mg, P = 0.004), indicating reversal of adrenal hyperplasia by restoration of 11β-HSD1 within the liver.

Increased expression of CYP11B1 mRNA in adrenal glands of 11β-HSD1-/- mice is returned to control level by the ApoE-HSD1 transgene

Within the adrenal cortex, 11β-hydroxylase (CYP11B1) performs the final step in the synthesis of corticosterone. CYP11B1 expression is enriched in the zona fasciculata and reticularis and is regulated, in part, by circulating ACTH. ACTH levels in 11β-HSD1-/- mice are increased, both basally and during recovery from stress (15). It is therefore unsurprising that 11β-HSD1-/- mice have greater expression of CYP11B1 mRNA (>50% increase, P < 0.001; Fig. 2) in their adrenal cortex than control non-TG mice. Whereas liver TG mice have control levels of CYP11B1 mRNA, replacement of 11β-HSD1 in the liver alone in 11β-HSD1-/- xTG mice also restores adrenal CYP11B1 levels to control values (Fig. 2).

Fig. 2.

Fig. 2

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Increased adrenocortical expression of CYP11b1 mRNA in the adrenal cortex in 11β-HSD-/- mice is rescued by restoration of 11β-HSD1 in the liver alone. CYP11β1 mRNA was measured by in situ hybridization histochemistry in non-TG controls (black columns), apoE-HSD1-TG mice (gray columns), 11β-HSD1-/- mice (black-andwhite-striped columns), and 11β-HSD1-/- xTG mice (gray-and-blackstriped columns). Values represent mean OD measurements over the adrenal cortex of five sections/mouse with four to six mice per genotype ± SEM. ***, P < 0.001, compared with all other groups.

Exaggerated corticosterone responses to acute restraint stress in 11β-HSD1-/- mice is reversed by the ApoE-HSD1 transgene

All groups of mice examined exhibited an increase in circulating corticosterone after exposure to 10 min restraint stress. In control non-TG mice, peak plasma corticosterone at 10 min after stress had declined but had not returned to baseline values at 120 min. ApoE-HSD1 TG mice showed a similar stress response to controls (Fig. 3). 11β-HSD1-/mice showed both elevated basal (0800 h) and peak 10-min stress-induced corticosterone levels (50% higher than non-TG controls, P = 0.02). Thereafter corticosterone levels fell to control post-stress at 120 min. Restoration of 11β-HSD1 in liver alone in 11β-HSD1-/- xTG mice reduced basal and peak (10 min) stress corticosterone levels to non-TG control levels (P = 0.02), suggesting rescue of the rapid elevation of corticosterone in response to stress seen in 11β-HSD1-/mice. These data suggest that thee in corticosterone in response to restraint is potently modulated by intrahepatic glucocorticoid regeneration.

Fig. 3.

Fig. 3

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Elevated basal and poststress corticosterone levels in 11βHSD1-/- mice is normalized by replacement of the enzyme in the liver alone. Plasma samples were taken by tail venesection at 0 (0800 h), 10, and 120 min after restraint stress in non-TG controls (black columns), apoE-HSD1-TG mice (gray columns), 11β-HSD1-/- mice (black-and-white-striped columns), and 11β-HSD1-/- xTG mice (grayand-black-striped columns). Values are mean plasma corticosterone levels per genotype ± SEM;n = 6. *, P < 0.05, compared with other genotypes at that time point.

Caloric supplementation does not differentially alter plasma corticosterone in 11β-HSD1 mice

11β-HSD1-/- mice have lower plasma glucose levels after fasting, stress, or high-fat feeding, although not under basal conditions. To determine whether mild energy deficiency might explain altered HPA axis function in the null mice, control and 11β-HSD1-/- mice were given a high-sucrose supplement via the drinking water. Both genotypes consumed the same amount of sucrose (not shown). Plasma glucose levels were identical on chow diet in the genotypes and were unaltered by sucrose supplementation. Sucrose caused a fall in plasma corticosterone in both control and 11β-HSD1-/- mice (F = 16.6, P < 0.001), but the fall in corticosterone levels did not differ by genotype (Fig. 4).

Fig. 4.

Fig. 4

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Sucrose ingestion leads to similar falls in plasma corticosterone in 11β-HSD1-/-and control mice. Plasma samples were taken by tail venesection at 0900 h in non-TG controls (black columns) and 11β-HSD1-/- mice (black-and-white-striped columns). Values represent the mean difference in plasma corticosterone levels in mice having access to sucrose, compared with those just having water; means ± SEM;n = 12/group.

Expression of the ApoE-11β-HSD1 transgene in the forebrain is unlikely to explain HPA axis rescue of 11β-HSD1-/- mice

A possible explanation of the apparent rescue of HPA axis function in 11β-HSD1-/- mice by the ApoE-HSD1 transgene is ectopic expression in finely localized central nervous system sites responsible for HPA axis control. In situ hybridization showed high expression of 11β-HSD1 mRNA in hippocampus, cortex, cerebellum, and other brain regions in control mice, paralleling previous reports in the rat (7, 9, 21) and human (6) (Fig. 5A). 11β-HSD1 mRNA was not detected in the brains of 11β-HSD1-/- mice (Fig. 5B). 11β-HSD1-/- xTG had no expression of 11β-HSD1 mRNA in the forebrain and specifically none in hypothalamus, hippocampus, or cingulate cortex, areas important in glucocorticoid feedback or the choroid plexus in which the enzyme might alter activation of glucocorticoids entering cerebrospinal fluid. Interestingly, expression of the transgene was detected in a subfield of the cerebellum (Fig. 5C). Microscopic analysis showed this to be localized specifically to the Purkinje cell/Bergman glial layer (data not shown). The cerebellum is not known to alter basal HPA axis function. Moreover, no abnormalities in cerebellar structure or function were apparent in mice bearing the apoE-HSD1 transgene.

Fig. 5.

Fig. 5

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Insitu hybridization for 11β-HSD1mRNA in sagittal brain sections from control (A),11β-HSD1-/-(B),and11β-HSD1-/-/apoE-HSD1-TGmice(C).

Discussion

The key finding in this paper is that restoration of 11βHSD1 in the liver in 11β-HSD1-/- mice normalizes all HPA axis parameters tested. The data imply that liver glucocorticoid metabolism affects HPA axis function. Whereas this might have been anticipated for adrenal mass and hence post-stress corticosterone responses, it was unexpected for basal corticosterone levels.

We previously found on a different strain background (MF1/129) that 11β-HSD1-/- mice have adrenal hypertrophy and elevated basal and post-stress corticosterone levels (15), data confirmed here on a novel genetic background [mainly C57BL/6J (96%) with the remainder CBA/C3H]. Previously we found that peak corticosterone levels were unaltered by 11β-HSD1-/- on the MF1/129 background (15) or inbred on the 129 background (22), although the diurnal rhythm was abnormal with a premature and prolonged peak. Here on the C57BL/6J/CBA/C3H background, 11βHSD1-/- mice show elevated basal and a diminished diurnal rise in corticosterone levels. The basis for the inter-strain difference in peak corticosterone levels is uncertain. Given the full reversal of 11β-HSD1-/- HPA axis abnormalities with restoration of 11β-HSD1 in the liver alone, it may be that peripheral metabolic differences underlie a blunted drive at the diurnal peak and/or exaggerated feedback in the null mice. Nonetheless, the 11β-HSD1-/- gives broadly the same abnormal HPA axis phenotype in young adult mice in this and our previous studies (15, 22). Whether this remains true for other strain backgrounds remains to be determined.

We had anticipated that increased adrenal mass in 11βHSD1-/- mice would be largely due to their requirement for increased adrenal glucocorticoid synthesis because of the loss of peripheral glucocorticoid regeneration. In humans, splanchnic regeneration of cortisol contributes to around 40% of total adrenal production (16). In mice and rats, 11βHSD1 shows highest expression in liver (20, 23); its loss is expected to necessitate increased adrenal production of corticosterone. Thus, restoration of liver enzyme activity is expected to reduce the requirement for adrenal hypertrophy, an outcome clearly shown in the rescued 11β-HSD1-/- xTG mice. Moreover, CYP11B1 was also normalized, supporting the notion that lack of liver glucocorticoid regeneration by 11β-HSD1 is the main cause of adrenal hypertrophy and corticosteroid hypersynthesis in 11β-HSD1-/- mice. Analogously, it might be argued that a hypertrophied adrenal cortex will produce more corticosterone when stimulated by ACTH/stress. This is borne out by the previously documented exaggerated response of 11β-HSD1-/- mouse adrenals to ACTH in vitro (12). Thus, the normalization of adrenal size by replacement of the enzyme in liver accords with the concept that adrenal hypertrophy is the key player in the post-stress HPA hyper-responsivity seen in 11β-HSD1-/- mice.

However, elevated basal corticosterone and, in this case, reduced evening rise of corticosterone levels in 11βHSD1-/- mice were also reversed by rescue of the enzyme in the liver. It has previously been suggested that elevated basal corticosterone levels in 11β-HSD1-/- mice reflect blunted feedback (15). This now seems unlikely because replacement of 11β-HSD1 in the liver restores corticosterone to control levels. There are several possible explanations of the findings. First, liver glucocorticoid metabolism and its effects on fuel metabolism may affect HPA axis function directly. In support of this contention, consumption of sucrose suppresses HPA axis hyperactivity in adrenalectomized rats (24) and mice (Carter, R., J. R. Seckl, and M. C. Holmes, unpublished data), suggesting metabolic fuels directly inhibit HPA axis drive (25). 11β-HSD1-/- mice have reduced glucose and lipid levels after stress or high-fat feeding (12–14), which might reduce such metabolic suppression of the HPA axis. However, voluntary sucrose ingestion, whereas effectively reducing corticosterone in both 11β-HSD1-/- and control mice, did so equally, suggesting that lack at least of carbohydrate is not responsible for the HPA axis abnormalities. A second, related, possibility is that liver glucocorticoid metabolism and its metabolic consequences signal indirectly to the HPA axis via, for instance, the hepatic vagal innervation. There is a documented hepatic-vagal input to the hypothalamus, sensitive to metabolic fuel status and known to influence HPA axis function (26). Whether this pathway is pertinent to our results is putatively testable by vagotomy, although this procedure is challenging in mice. A further finding supporting the idea that HPA function is sensitive to liver metabolism of glucocorticoids is the restoration of the normal circadian rhythm (trough to peak) corticosterone variation in the 11β-HSD1-/- xTG. Reduced adrenal mass in the liver rescue mice would be anticipated, if anything, to reduce rather than increase peak corticosterone levels. Whereas the mechanism of restoration of full circadian variation of glucocorticoid levels remains unclear, its normalization is in keeping with a key role for liver glucocorticoid-sensitive processes in HPA axis control at both peak and trough.

Another explanation for the findings would be ectopic expression of the 11β-HSD1 transgene in central nervous system sites of HPA axis feedback. Indeed, apolipoprotein E may be expressed in hippocampal neurons after injury, although not under basal circumstances (27). Brain expression of apolipoprotein E involves specific enhancer sequences (28) distinct from those driving hepatic expression (29) and used in our transgene. We found no expression of the transgene in any forebrain region, notably none involved in HPA axis feedback. However, transgene expression was detected in Purkinje cell/Bergman glial layer of the cerebellum. There is no evidence to date that the cerebellum plays a role in HPA axis control. Indeed, Lurcher mutant mice, despite massive postnatal loss of Purkinje cells and subsequent widespread degeneration of the cerebellar cortex and its main connections, have normal basal corticosterone levels (30, 31). It is therefore unlikely that Purkinje layer transgene expression alters HPA function. Another potential explanation of altered total corticosterone levels in 11β-HSD1-/- mice is elevated corticosteroid binding globulin, but this does not occur in this line (15)

It is interesting that increased expression of 11β-HSD1 in the liver in nontransgenic mice had no discernable effect on HPA function. The liver has high 11β-HSD1 levels, but the transgene does have a metabolic phenotype with increased triglyceride levels and modest insulin resistance, although unaltered glucose homeostasis (17). This implies that either the model is too subtle to affect metabolic fuel actions on the HPA axis, that increased fuel availability has no effect on the normal HPA axis (although this is not supported by the actions of sucrose ingestion to reduce basal plasma corticosterone levels in intact mice), and/or that glucose is the key player controlling HPA function in these models rather than insulin resistance/sensitivity or lipid levels (which are altered in ApoE-HSD1 mice). In conclusion, replacement of 11β-HSD1 in the liver of 11β-HSD1-/- mice is sufficient to normalize HPA axis parameters such as circadian rhythmicity, basal and stress plasma corticosterone levels, and key adrenal markers such as adrenal size and 11β-hydroxylase expression.

Supplementary Material

Suppl Fig

Acknowledgments

We thank Morag Meikle and Lynne Ramage for excellent technical assistance.

This work was supported by a Wellcome Trust Programme (to J.R.S. and J.J.M.) and a Wellcome Trust Project Grant (to M.C.H., J.M.P., J.J.M., and J.R.S.). J.J.M. is a Wellcome Trust Principal Fellow. J.M.P. is a Research Councils of the United Kingdom Fellow.

Abbreviations

HPA

Hypothalamic-pituitary-adrenal

11β-HSD

11β-hydroxysteroid dehydrogenase

TG

transgenic

Footnotes

Disclosures: J.M.P., M.C.H., C.J.K., R.C., and J.J.M. have nothing to declare. J.R.S. consults for Incyte, Vitae Pharmaceuticals, Merck, and IPSEN and is an inventor on European Union WO9707789.

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