Biomarkers of hypothalamic-pituitary-adrenal axis activity in mice lacking 11β-HSD1 and H6PDH

Abstract

Glucocorticoid concentrations are a balance between production under the negative feedback control and diurnal rhythm of the hypothalamic-pituitary-adrenal (HPA) axis and peripheral metabolism, for example by the enzyme 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1), which catalyses the reduction of inactive cortisone (11-dehydrocorticosterone (11-DHC) in mice) to cortisol (corticosterone in mice). Reductase activity is conferred upon 11β-HSD1 by hexose-6-phosphate dehydrogenase (H6PDH). 11β-HSD1 is implicated in the development of obesity, and selective 11β-HSD1 inhibitors are currently under development. We sought to address the concern regarding potential up-regulation of the HPA axis associated with inhibition of 11β-HSD1. We assessed biomarkers for allele combinations of 11β-HSD1 and H6PDH derived from double heterozygous mouse crosses. H6PDH knock out (KO) adrenals were 69% larger than WT while 11β-HSD1 KO and double KO (DKO) adrenals were ~30% larger than WT - indicative of increased HPA axis drive in KO animals. ACTH-stimulated circulating corticosterone concentrations were 2.2-fold higher in H6PDH KO animals and ~1.5-fold higher in 11β-HSD1 KO and DKO animals compared with WT, proportional to the observed adrenal hypertrophy. KO of H6PDH resulted in a substantial increase in urinary DHC metabolites in males (65%) and females (61%). KO of 11β-HSD1 alone or in combination with H6PDH led to significant increases (36 and 42% respectively) in urinary DHC metabolites in females only. Intermediate 11β-HSD1/H6PDH heterozygotes maintained a normal HPA axis. Urinary steroid metabolite profile by gas chromatography/mass spectrometry as a biomarker assay may be beneficial in assaying HPA axis status clinically in cases of congenital and acquired 11β-HSD1/H6PDH deficiency.

Figure 1

(A) Urinary steroid metabolite profiles of WT vs 11β-HSD1 KO/DKO mice. Urine samples were collected from WT, 11β-HSD1 KO and DKO mice of both sexes using filter paper as in Materials and Methods section. Urinary steroids were subsequently extracted and analysed by GC/MS. Urinary steroid metabolites are shown as percentage 11-DHC metabolites as a proportion of total GC. **P<0·01 (n>4/group) between WT and KO of same sex; ^##P<0·01 (n>4/group) between sexes of same genotype; *P<0·05 (n>4/group) between WT and KO of same sex. (B) Urinary steroid metabolite profiles of WT vs H6PDH KO mice. Urine samples were collected from WT and H6PDH KO mice of both sexes on a daily basis using filter paper and were analysed as in (A). **P<0·01 (n>4/group).

Figure 2

Urinary steroid metabolite profiles of WT vs 11β-HSD1 HET/H6PDH HET mice. Urine samples were collected from WT and 11β-HSD1/H6PDH HET mice of both sexes on a daily basis using filter paper and were analysed as in (Fig. 1A).

Figure 3

Corticosterone levels in ACTH-stimulated and -unstimulated male animals. Mice were either left unstimulated or stimulated with ACTH at 1 unit (10 μg)/100 g. 11β-HSD1 KO, H6PDH KO and DKO animals display elevated levels of circulating corticosterone relative to WT levels, which is indicative of enhanced HPA axis drive. However, the mean differences between groups were not found to be statistically significant (n>4/group).

Figure 4

(A) Adrenal weights of male WT vs HSD1 KO/H6PDH KO/DKO animals. Mice were killed and adrenals were dissected and weighed. **P<0·01 (n>4/group) between WT and KOs. ^##P<0·01 (n>4/group) between H6PDH KO and all other genotypes. (B) Adrenal weights of male WT vs 11β-HSD1 HET/H6PDH HET animals. Mice were killed and adrenals were dissected and weighed as in (A). **P<0·01 (n>4/group) between HSD1 KO/H6PDH HET and HSD1 HET/H6PDH KO vs all other genotypes. ^##P<0·01 (n>4/group) between HSD1 HET/H6PDH KO vs all other genotypes.