help button home button Endocrine Society Endocrinology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology, doi:10.1210/en.2006-0603
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Supplemental Data
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Paterson, J. M.
Right arrow Articles by Seckl, J. R.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Paterson, J. M.
Right arrow Articles by Seckl, J. R.
Endocrinology Vol. 148, No. 3 961-966
Copyright © 2007 by The Endocrine Society

BRIEF COMMUNICATION

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

Janice M. Paterson, Megan C. Holmes, Christopher J. Kenyon, Roderick Carter, John J. Mullins and Jonathan R. Seckl

Molecular Physiology Group (J.M.P., J.J.M.) and Endocrinology Unit (M.C.H., C.J.K., R.C., J.R.S.), Centre for Cardiovascular Science, Queen’s Medical Research Institute, University of Edinburgh, Edinburgh EH16 4TJ, Scotland, United Kingdom

Address all correspondence and requests for reprints to: Professor Jonathan Seckl, Centre for Cardiovascular Science, Endocrinology Unit, Queen’s Medical Research Institute, 47 Little France Crescent, Edinburgh EH16 4TJ, Scotland, United Kingdom. E-mail: j.seckl{at}ed.ac.uk.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
11ß-Hydroxysteroid dehydrogenase type 1 (11ß-HSD1) acts as a reductase in vivo, regenerating active glucocorticoids within cells from circulating inert 11-keto forms, thus amplifying local glucocorticoid action. 11ß-HSD1 is predominantly expressed in liver and also adipose tissue and brain. Mice deficient in 11ß-HSD1 (11ß-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 11ß-HSD1 within the brain in amplifying glucocorticoid feedback. To test this hypothesis, apolipoprotein E-HSD1 mice overexpressing 11ß-HSD1 in liver were intercrossed with 11ß-HSD1–/– mice to determine whether complementation of hepatic 11ß-HSD1 can restore adrenal and HPA defects. Transgene-mediated delivery of 11ß-HSD1 activity to the liver rescued adrenal hyperplasia and reversed exaggerated HPA stress responses in 11ß-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 11ß-HSD1–/– mice was increased by 50% but returned to control levels in 11ß-HSD1–/– mice bearing the apolipoprotein E-HSD1 transgene. 11ß-HSD1–/– mice have lower plasma glucose levels, but the fall in plasma corticosterone with sucrose supplementation was similar in 11ß-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 11ß-HSD1 in the liver normalizes all aspects of HPA axis dysregulation in 11ß-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
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
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 11ß-hydroxysteroid dehydrogenases (11ß-HSDs), which control intracellular substrate levels (2, 3). 11ß-HSDs catalyze interconversion of active 11-hydroxy glucocorticoids (cortisol, corticosterone) and their inert 11-keto derivatives (cortisone, 11-dehydrocorticosterone). 11ß-HSD type 1 is a predominant 11ß-reductase that, in expressing cells, regenerates active glucocorticoids from inert forms, thus amplifying local glucocorticoid action. In contrast, 11ß-HSD type 2 catalyzes rapid 11ß-dehydrogenation, potently inactivating intracellular glucocorticoids and thus protecting intrinsically nonselective mineralocorticoid receptors in the distal nephron from activation by glucocorticoids in vivo. Whereas 11ß-HSD2 is barely if at all expressed in the adult rat, mouse, and human forebrain (4, 5, 6), 11ß-HSD1 is highly expressed throughout the brain, including the hippocampus and hypothalamus and also the anterior pituitary (7, 8, 9). 11ß-HSD1 is also expressed at high levels in the liver and adipose tissues (10, 11).

Mice homozygous for targeted disruption of the 11ß-HSD1 gene (11ß-HSD1–/– mice) are viable, fertile, and apparently healthy and show that 11ß-HSD1 is the sole 11ß-reductase (12). 11ß-HSD1–/– mice exhibit improved glucose tolerance, insulin sensitization, and a cardioprotective lipid profile with resistance to diet-induced obesity and its metabolic sequelae (12, 13, 14), effects compatible with reduced intracellular glucocorticoid action in liver and adipose tissue. However, 11ß-HSD1–/– mice also show increased HPA axis activity and adrenal hyperplasia (12, 15). Adrenocortical hyperplasia might be predicted on the basis that 11ß-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 11ß-HSD1–/– mice (15). This suggests attenuated glucocorticoid feedback, a notion supported by blunted suppression of stress responses by exogenous glucocorticoids in 11ß-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 11ß-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 11ß-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 11ß-HSD1 in the brain and pituitary would be unaffected. Here we examine this issue by intercross of liver transgenic ApoE-HSD1 mice with 11ß-HSD1–/– mice.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Adult male mice were studied throughout. C57BL/6J congenic 11ß-HSD1–/– 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), 11ß-HSD1–/–, and 11ß-HSD1–/–/ApoE-HSD1 (11ß-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 11ß-HSD1–/– mice, so each genetic modification segrates 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 11ß-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 µm) were prepared from the frozen adrenals and serial sections hybridized in situ with antisense riboprobes for the detection of CYP11B1 (11ß-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).

11ß-HSD1 in situ hybridization series of 10 µm sagittal cryostat sections through the brains of control, 11ß-HSD1–/–, and 11ß-HSD1–/–xTG mice were prepared and used for in situ hybridization with an 11ß-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
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Loss of the diurnal rhythm of corticosterone in 11ß-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. 1Go). 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 11ß-HSD1 in TG mice. As previously reported on another genetic background (MF1/129) (15), basal 0800 h corticosterone levels were significantly increased in 11ß-HSD1–/– mice, compared with non-TG controls (P < 0.05; Fig. 1Go), indicating abnormal HPA regulation. In this study, peak 2000 h corticosterone levels tended to be reduced in 11ß-HSD1–/– mice, suggesting suppression of the normal circadian maximum and rhythmicity. Indeed, whereas all other genotypes showed significant diurnal variation, 11ß-HSD1–/– mice had no significant variation between morning and evening and the day-night change in plasma corticosterone was significantly less in 11ß-HSD1–/– mice than all other genotypes (F = 3.8, P = 0.025). Importantly, transgenic replacement of 11ß-HSD1 solely in 11ß-HSD1–/– liver (11ß-HSD1–/–xTG) restored both basal and peak corticosterone level to non-TG control levels.


Figure 1
View larger version (18K):
[in this window]
[in a new window]

  		FIG. 1. Elevated basal and blunted circadian rhythm of corticosterone in 11ß-HSD1–/– mice is rescued by the apoE-HSD1 transgene. Plasma samples were taken after tail venesection at 0800 h (nadir) and 2000 h (peak) in non-TG controls (black columns), apoE-HSD1-TG mice (gray columns), 11ß-HSD1–/– mice (black-and-white-striped columns) and 11ß-HSD1–/–xTG mice (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 except 11ß-HSD1–/– showed a significant difference between morning and evening, and the diurnal change was significantly less in 11ß-HSD1–/– than all other groups.

 
Hyperplasia of the adrenal gland in 11ß-HSD1–/– mice is rescued by the ApoE-HSD1 transgene
Mean adrenal mass in control non-TG mice and ApoE-HSD1 TG mice did not differ (1.47 ± 0.11 and 1.58 ± 0.07 mg, respectively). 11ß-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 11ß-HSD1–/–xTG mice was significantly lower than 11ß-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 11ß-HSD1 within the liver.

Increased expression of CYP11B1 mRNA in adrenal glands of 11ß-HSD1–/– mice is returned to control level by the ApoE-HSD1 transgene
Within the adrenal cortex, 11ß-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 11ß-HSD1–/– mice are increased, both basally and during recovery from stress (15). It is therefore unsurprising that 11ß-HSD1–/– mice have greater expression of CYP11B1 mRNA (>50% increase, P < 0.001; Fig. 2Go) in their adrenal cortex than control non-TG mice. Whereas liver TG mice have control levels of CYP11B1 mRNA, replacement of 11ß-HSD1 in the liver alone in 11ß-HSD1–/–xTG mice also restores adrenal CYP11B1 levels to control values (Fig. 2Go).


Figure 2
View larger version (39K):
[in this window]
[in a new window]

  		FIG. 2. Increased adrenocortical expression of CYP11b1 mRNA in the adrenal cortex in 11ß-HSD–/– mice is rescued by restoration of 11ß-HSD1 in the liver alone. CYP11ß1 mRNA was measured by in situ hybridization histochemistry in non-TG controls (black columns), apoE-HSD1-TG mice (gray columns), 11ß-HSD1–/– mice (black-and-white-striped columns), and 11ß-HSD1–/–xTG mice (gray-and-black-striped 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 11ß-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. 3Go). 11ß-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 poststress at 120 min. Restoration of 11ß-HSD1 in liver alone in 11ß-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 11ß-HSD1–/– mice. These data suggest that the rise in corticosterone in response to restraint is potently modulated by intrahepatic glucocorticoid regeneration.


Figure 3
View larger version (22K):
[in this window]
[in a new window]

  		FIG. 3. Elevated basal and poststress corticosterone levels in 11ß-HSD1–/– 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), 11ß-HSD1–/– mice (black-and-white-striped columns), and 11ß-HSD1–/–xTG mice (gray-and-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 11ß-HSD1 mice
11ß-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 11ß-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 11ß-HSD1–/– mice (F = 16.6, P < 0.001), but the fall in corticosterone levels did not differ by genotype (Fig. 4Go).


Figure 4
View larger version (29K):
[in this window]
[in a new window]

  		FIG. 4. Sucrose ingestion leads to similar falls in plasma corticosterone in 11ß-HSD1–/– and control mice. Plasma samples were taken by tail venesection at 0900 h in non-TG controls (black columns) and 11ß-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-11ß-HSD1 transgene in the forebrain is unlikely to explain HPA axis rescue of 11ß-HSD1–/– mice
A possible explanation of the apparent rescue of HPA axis function in 11ß-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 11ß-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. 5AGo). 11ß-HSD1 mRNA was not detected in the brains of 11ß-HSD1–/– mice (Fig. 5BGo). 11ß-HSD1–/–xTG had no expression of 11ß-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. 5CGo). 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.


Figure 5
View larger version (49K):
[in this window]
[in a new window]

  		FIG. 5. In situ hybridization for 11ß-HSD1 mRNA in sagittal brain sections from control (A), 11ß-HSD1–/– (B), and 11ß-HSD1–/–/apoE-HSD1-TG mice (C).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The key finding in this paper is that restoration of 11ß-HSD1 in the liver in 11ß-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 poststress corticosterone responses, it was unexpected for basal corticosterone levels.

We previously found on a different strain background (MF1/129) that 11ß-HSD1–/– mice have adrenal hypertrophy and elevated basal and poststress 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 11ß-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, 11ß-HSD1–/– mice show elevated basal and a diminished diurnal rise in corticosterone levels. The basis for the interstrain difference in peak corticosterone levels is uncertain. Given the full reversal of 11ß-HSD1–/– HPA axis abnormalities with restoration of 11ß-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 11ß-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 11ß-HSD1–/– 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, 11ß-HSD1 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 11ß-HSD1–/–xTG mice. Moreover, CYP11B1 was also normalized, supporting the notion that lack of liver glucocorticoid regeneration by 11ß-HSD1 is the main cause of adrenal hypertrophy and corticosteroid hypersynthesis in 11ß-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 11ß-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 poststress HPA hyperresponsivity seen in 11ß-HSD1–/– mice.

However, elevated basal corticosterone and, in this case, reduced evening rise of corticosterone levels in 11ß-HSD1–/– mice were also reversed by rescue of the enzyme in the liver. It has previously been suggested that elevated basal corticosterone levels in 11ß-HSD1–/– mice reflect blunted feedback (15). This now seems unlikely because replacement of 11ß-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). 11ß-HSD1–/– mice have reduced glucose and lipid levels after stress or high-fat feeding (12, 13, 14), which might reduce such metabolic suppression of the HPA axis. However, voluntary sucrose ingestion, whereas effectively reducing corticosterone in both 11ß-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 11ß-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 11ß-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 11ß-HSD1–/– mice is elevated corticosteroid binding globulin, but this does not occur in this line (15)

It is interesting that increased expression of 11ß-HSD1 in the liver in nontransgenic mice had no discernable effect on HPA function. The liver has high 11ß-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 11ß-HSD1 in the liver of 11ß-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 11ß-hydroxylase expression.


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


   Footnotes
 
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.

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.

First Published Online December 14, 2006

Abbreviations: HPA, Hypothalamic-pituitary-adrenal; 11ß-HSD, 11ß-hydroxysteroid dehydrogenase; TG, transgenic.

Received May 9, 2006.

Accepted for publication December 6, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. De Kloet ER 2004 Hormones and the stressed brain. Ann NY Acad Sci 1018:1–15[CrossRef][Medline]
  2. Stewart PM, Krozowski ZS 1999 11ß-Hydroxysteroid dehydrogenase. Vitam Horm 57:249–324[Medline]
  3. Seckl JR, Walker BR 2001 Minireview: 11ß-hydroxysteroid dehydrogenase type 1—a tissue-specific amplifier of glucocorticoid action. Endocrinology 142:1371–1376[Abstract/Free Full Text]
  4. Robson AC, Leckie CM, Seckl JR, Holmes MC 1998 11ß-Hydroxysteroid dehydrogenase type 2 in the postnatal and adult rat brain. Mol Brain Res 61:1–10[Medline]
  5. Roland BL, Li KXZ, Funder JW 1995 Hybridization histochemical localization of 11ß-hydroxysteroid dehydrogenase type 2 in rat brain. Endocrinol 136:4697–4700[Abstract]
  6. Sandeep TC, Yau JL, MacLullich AM, Noble J, Deary IJ, Walker BR, Seckl JR 2004 11ß-Hydroxysteroid dehydrogenase inhibition improves cognitive function in healthy elderly men and type 2 diabetics. Proc Natl Acad Sci USA 101:6734–6739[Abstract/Free Full Text]
  7. Lakshmi V, Sakai RR, McEwen BS, Monder C 1991 Regional distribution of 11ß-hydroxysteroid dehydrogenase in rat brain. Endocrinology 128:1741–1748[Abstract/Free Full Text]
  8. Moisan M-P, Seckl JR, Edwards CRW 1990 11ß-Hydroxysteroid dehydrogenase bioactivity and messenger RNA expression in rat forebrain: localization in hypothalamus, hippocampus and cortex. Endocrinology 127:1450–1455[Abstract/Free Full Text]
  9. Moisan M-P, Seckl JR, Brett LP, Monder C, Agarwal AK, White PC, Edwards CRW 1990 11ß-Hydroxysteroid dehydrogenase messenger ribonucleic acid expression, bioactivity and immunoreactivity in rat cerebellum. J Neuroendocrinol 2:853–858[CrossRef][Medline]
  10. Lakshmi V, Monder C 1988 Purification and characterisation of the corticosteroid 11ß-dehydrogenase component of the rat liver 11ß-hydroxysteroid dehydrogenase complex. Endocrinology 123:2390–2398[Abstract/Free Full Text]
  11. Bujalska IJ, Kumar S, Stewart PM 1997 Does central obesity reflect "Cushing’s disease of the omentum"? Lancet 349:1210–1213[CrossRef][Medline]
  12. Kotelevtsev Y, Holmes MC, Burchell A, Houston PM, Schmoll D, Jamieson P, Best R, Brown R, Edwards CR, Seckl JR, Mullins JJ 1997 11ß-Hydroxysteroid dehydrogenase type 1 knockout mice show attenuated glucocorticoid-inducible responses and resist hyperglycemia on obesity or stress. Proc Natl Acad Sci USA 94:14924–14929[Abstract/Free Full Text]
  13. Morton NM, Holmes MC, Fievet C, Staels B, Tailleux A, Mullins JJ, Seckl JR 2001 Improved lipid and lipoprotein profile, hepatic insulin sensitivity, and glucose tolerance in 11ß-hydroxysteroid dehydrogenase type 1 null mice. J Biol Chem 276:41293–41300[Abstract/Free Full Text]
  14. Morton NM, Paterson JM, Masuzaki H, Holmes MC, Staels B, Fievet C, Walker BR, Flier JS, Mullins JJ, Seckl JR 2004 Novel adipose tissue-mediated resistance to diet-induced visceral obesity in 11ß-hydroxysteroid dehydrogenase type 1-deficient mice. Diabetes 53:931–938[Abstract/Free Full Text]
  15. Harris HJ, Kotelevtsev Y, Mullins JJ, Seckl JR, Holmes MC 2001 Intracellular regeneration of glucocorticoids by 11ß-hydroxysteroid dehydrogenase (11ß-HSD)-1 plays a key role in regulation of the hypothalamic-pituitary-adrenal axis: analysis of 11ß-HSD-1-deficient mice. Endocrinology 142:114–120[Abstract/Free Full Text]
  16. Andrew R, Westerbacka J, Wahren J, Yki-Jarvinen H, Walker BR 2005 The contribution of visceral adipose tissue to splanchnic cortisol production in healthy humans. Diabetes 54:1364–1370[Abstract/Free Full Text]
  17. Paterson JM, Morton NM, Fievet C, Kenyon CJ, Holmes MC, Staels B, Seckl JR, Mullins JJ 2004 Metabolic syndrome without obesity: hepatic overexpression of 11ß-hydroxysteroid dehydrogenase type 1 in transgenic mice. Proc Natl Acad Sci USA 101:7088–7093[Abstract/Free Full Text]
  18. Masuzaki H, Yamamoto H, Kenyon CJ, Elmquist JK, Morton NM, Paterson JM, Shinyama H, Sharp MG, Fleming S, Mullins JJ, Seckl JR, Flier JS 2003 Transgenic amplification of glucocorticoid action in adipose tissue causes high blood pressure in mice. J Clin Invest 112:83–90[CrossRef][Medline]
  19. Holmes MC, French KL, Seckl JR 1997 Dysregulation of diurnal rhythms of serotonin 5-HT2C and corticosteroid receptor gene expression in the hippocampus with food restriction and glucocorticoids. J Neurosci 17:4056–4065[Abstract/Free Full Text]
  20. Rajan V, Chapman KE, Lyons V, Jamieson P, Mullins JJ, Edwards CRW, Seckl JR 1995 Cloning sequencing and tissue-distribution of mouse 11ß-hydroxysteroid dehydrogenase-1 cDNA. J Steroid Biochem Mol Biol 52:141–147[CrossRef][Medline]
  21. Moisan M-P, Seckl JR, Edwards CRW 1990 11ß-Hydroxysteroid dehydrogenase bioactivity and messenger RNA expression in rat forebrain: localization in hypothalamus, hippocampus and cortex. Endocrinology 127:1450–1455[Abstract/Free Full Text]
  22. Yau JLW, Noble J, Kenyon CJ, Hibberd C, Kotelevtsev Y, Mullins JJ, Seckl JR 2001 Lack of tissue glucocorticoid reactivation in 11ß-hydroxysteroid dehydrogenase type 1 knockout mice ameliorates age-related learning impairments. Proc Natl Acad Sci USA 98:4716–4721[Abstract/Free Full Text]
  23. Agarwal AK, Monder C, Eckstein B, White PC 1989 Cloning and expression of rat cDNA encoding corticosteroid 11 dehydrogenase. J Biol Chem 264:18939–18943[Abstract/Free Full Text]
  24. Laugero KD, Bell ME, Bhatnagar S, Soriano L, Dallman MF 2001 Sucrose ingestion normalizes central expression of corticotropin-releasing-factor messenger ribonucleic acid and energy balance in adrenalectomized rats: a glucocorticoid-metabolic-brain axis? Endocrinology 142:2796–2804[Abstract/Free Full Text]
  25. Dallman MF, Akana SF, Laugero KD, Gomez F, Manalo S, Bell ME, Bhatnagar S 2003 A spoonful of sugar: feedback signals of energy stores and corticosterone regulate responses to chronic stress. Physiol Behav 79:3–12[CrossRef][Medline]
  26. la Fleur SE, Manalo SL, Roy M, Houshyar H, Dallman MF 2005 Hepatic vagotomy alters limbic and hypothalamic neuropeptide responses to insulin-dependent diabetes and voluntary lard ingestion. Eur J Neurosci 21:2733–2742[CrossRef][Medline]
  27. Xu Q, Bernardo A, Walker D, Kanegawa T, Mahley RW, Huang YD 2006 Profile and regulation of apolipoprotein E (ApoE) expression in the CNS in mice with targeting of green fluorescent protein gene to the ApoE locus. J Neurosci 26:4985–4994[Abstract/Free Full Text]
  28. Grehan S, Tse E, Taylor J 2001 Two distal downstream enhancers direct expression of the human apolipoprotein E gene to astrocytes in the brain. J Neurosci 21:812–822[Abstract/Free Full Text]
  29. Allan C, Taylor S, Taylor J 1997 Two hepatic enhancers, HCR. 1 and HCR. 2, coordinate the liver expression of the entire human apolipoprotein E/C-I/C-IV/C-II gene cluster. J Biol Chem 272:29113–29119[Abstract/Free Full Text]
  30. Frederic F, Chautard T, Brochard R, Chianale C, Wollman E, Oliver C, DelhayeBouchaud N, Mariani J 1997 Enhanced endocrine response to novel environment stress and endotoxin in Lurcher mutant mice. Neuroendocrinology 66:341–347[Medline]
  31. Hilber P, Lorivel T, Delarue C, Caston J 2004 Stress and anxious-related behaviors in Lurcher mutant mice. Brain Res 1003:108–112[CrossRef][Medline]



This article has been cited by other articles:


Home page
 J EndocrinolHome page
T. Dickmeis
Glucocorticoids and the circadian clock
J. Endocrinol., January 1, 2009; 200(1): 3 - 22.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Supplemental Data
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Paterson, J. M.
Right arrow Articles by Seckl, J. R.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Paterson, J. M.
Right arrow Articles by Seckl, J. R.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology Endocrine Reviews J. Clin. End. & Metab.
Molecular Endocrinology Recent Prog. Horm. Res. All Endocrine Journals