11beta-hydroxysteroid dehydrogenase type 1 knockout mice show attenuated glucocorticoid-inducible responses and resist hyperglycemia on obesity or stress

Abstract

Glucocorticoid hormones, acting via nuclear receptors, regulate many metabolic processes, including hepatic gluconeogenesis. It recently has been recognized that intracellular glucocorticoid concentrations are determined not only by plasma hormone levels, but also by intracellular 11beta-hydroxysteroid dehydrogenases (11beta-HSDs), which interconvert active corticosterone (cortisol in humans) and inert 11-dehydrocorticosterone (cortisone in humans). 11beta-HSD type 2, a dehydrogenase, thus excludes glucocorticoids from otherwise nonselective mineralocorticoid receptors in the kidney. Recent data suggest the type 1 isozyme (11beta-HSD-1) may function as an 11beta-reductase, regenerating active glucocorticoids from circulating inert 11-keto forms in specific tissues, notably the liver. To examine the importance of this enzyme isoform in vivo, mice were produced with targeted disruption of the 11beta-HSD-1 gene. These mice were unable to convert inert 11-dehydrocorticosterone to corticosterone in vivo. Despite compensatory adrenal hyperplasia and increased adrenal secretion of corticosterone, on starvation homozygous mutants had attenuated activation of the key hepatic gluconeogenic enzymes glucose-6-phosphatase and phosphoenolpyruvate carboxykinase, presumably, because of relative intrahepatic glucocorticoid deficiency. The 11beta-HSD-1 -/- mice were found to resist hyperglycamia provoked by obesity or stress. Attenuation of hepatic 11beta-HSD-1 may provide a novel approach to the regulation of gluconeogenesis.

Figure 1

Targeted inactivation of 11β-HSD-1 gene. (a) Structure of the targeting vector, the 11β-HSD-1 locus, and the targeted allele. RV, EcoRV; BHI, BamHI. Synthesis of 5′ homology arm, 5′ external probe, and initial screening of colonies was done by PCR with oligos 1, 2, 3, and 4 as shown. (b) Southern blot hybridization analysis of BamHI-digested genomic DNA from neomycin-resistant targeted (+/−) and nontargeted (+/+) ES clones. (c) Northern blot analysis of 11β-HSD-1 expression. Thirty micrograms of total RNA from liver of homozygous (−/−) and heterozygous (+/−) mutants and wild type (+/+) litter mates was hybridized with 11β-HSD-1 and control (U-1) cDNA probes. (d) 11β-HSD activity in the liver homogenates of the same animals expressed as % conversion corticosterone to 11-DHC (n = 4 for −/−, n = 5 for −/+, and n = 5 for +/+).

Figure 2

The effect of dehydrocorticosterone (11-DHC) or vehicle pellets in vivo on plasma corticosterone and thymus weight (Inset) in adrenalectomized 11β-HSD-1 +/+ and −/− mice. The black columns represent data obtained from mice with vehicle pellets, and the hatched columns mice with A pellets. LD, limit of detection. Values are means ± SEM, n = 5–6. ∗, P < 0.05 compared with vehicle control (t test).

Figure 3

(a) Adrenal weights, (b) basal, unstressed, plasma corticosterone levels in vivo, and (c) ACTH-stimulated corticosterone production from adrenal glands in vitro of 11β-HSD-1 +/+ and −/− mice. Open bars and symbols are wild type. Solid bars and symbols are 11β-HSD-1 −/− mice. ACTH (0–10 nmol/liter) was applied to adrenal glands in vitro for 60 min. Results are means ± SEM, n = 8–10. ∗, P < 0.05 compared with +/+ control (t test). Two-way ANOVA showed a significant effect of dose of ACTH (P = 0.02) and mouse genotype (P = 0.002), with no interaction between the two variables (P = 0.47).

Figure 4

Effect of a high-fat diet on body weight and fasting blood glucose levels in 11β-HSD-1 +/+ and −/− mice. Each value indicates mean ± SEM, n = 8–10/group. ∗, P < 0.05 significant to corresponding −/− value.