11β-Hydroxysteroid dehydrogenase type 1 contributes to the balance between 7-keto- and 7-hydroxy-oxysterols in vivo

Abstract

11β-Hydroxysteroid dehydrogenase 1 (11βHSD1; EC 1.1.1.146) generates active glucocorticoids from inert 11-keto metabolites. However, it can also metabolize alternative substrates, including 7β-hydroxy- and 7-keto-cholesterol (7βOHC, 7KC). This has been demonstrated in vitro but its consequences in vivo are uncertain. We used genetically modified mice to investigate the contribution of 11βHSD1 to the balance of circulating levels of 7KC and 7βOHC in vivo, and dissected in vitro the kinetics of the interactions between oxysterols and glucocorticoids for metabolism by the mouse enzyme. Circulating levels of 7KC and 7βOHC in mice were 91.3±22.3 and 22.6±5.7 nM respectively, increasing to 1240±220 and 406±39 nM in ApoE(-/-) mice receiving atherogenic western diet. Disruption of 11βHSD1 in mice increased (p<0.05) the 7KC/7βOHC ratio in plasma (by 20%) and also in isolated microsomes (2 fold). The 7KC/7βOHC ratio was similarly increased when NADPH generation was restricted by disruption of hexose-6-phosphate dehydrogenase. Reduction and oxidation of 7-oxysterols by murine 11βHSD1 proceeded more slowly and substrate affinity was lower than for glucocorticoids. in vitro 7βOHC was a competitive inhibitor of oxidation of corticosterone (Ki=0.9 μM), whereas 7KC only weakly inhibited reduction of 11-dehydrocorticosterone. However, supplementation of 7-oxysterols in cultured cells, secondary to cholesterol loading, preferentially slowed reduction of glucocorticoids, rather than oxidation. Thus, in mouse, 11βHSD1 influenced the abundance and balance of circulating and tissue levels of 7βOHC and 7KC, promoting reduction of 7KC. In health, 7-oxysterols are unlikely to regulate glucocorticoid metabolism. However, in hyperlipidaemia, 7-oxysterols may inhibit glucocorticoid metabolism and modulate signaling through corticosteroid receptors.

Graphical abstract

Fig. 1

(a) Interconversion of glucocorticoids and 7-oxysterols catalyzed by 11β-hydroxysteroid dehydrogenase type 1 (11βHSD1). The equilibrium of interconversion of inert 11-keto and active 11β-hydroxy forms of glucocorticoids (shown here as 11-dehydrocorticosterone and corticosterone, the principle rodent glucocorticoids) favors predominant reduction. 11βHSD1 can also interconvert 7-keto and 7β-hydroxycholesterol but the favored equilibrium position between the two reactions is not understood. (b)–(e) In Silico modeling of interactions between 7-oxysterols and residues in the active site of murine 11β-hydroxysteroid dehydrogenase 1 (m11βHSD1). 2D Modeling of the active site of m11βHSD1 (retrieved from PDB 1Y5 M) using LigPlot. Hydrogen bond lengths of interactions between (b) 7-ketocholesterol and (c) 7β-hydroxycholesterol and the critical residues of catalytic tetrad are shorter than those for (d) 7α-hydroxycholesterol (7αOHC). (e) 3D modeling of interactions between active site residues Serine 170 (S170) and Tyrosine 183 (Y183) of m11βHSD1 and the 7-oxygenated moieties using PyMOL. Positioning of 7βOHC (pink) or 7KC (yellow) into the active site demonstrated their more favorable orientation over 7αOHC (turquoise), for hydrogen bonding with key amino acids of m11βHSD1 active site.

Fig. 2

7-Oxysterols inhibit the metabolism of glucocorticoids by 11β-hydroxysteroid dehydrogenase 1 (11βHSD1). (a) and (b): The velocities of (a) reduction of 11-dehydrocorticosterone (11-DHC) to corticosterone and (b) oxidation of corticosterone to 11-DHC were quantified following incubation of HEK293 cells (stably transfected to generate murine 11βHSD 1) with a range of concentrations of oxysterols. Non-linear regression was used to assign IC₅₀ values. 7-Ketocholesterol (7KC) only inhibited the reduction of 11-DHC by ∼40%. 7β-Hydroxycholesterol (7βOHC) completely inhibited oxidation of corticosterone; other oxysterols did not have an effect. Data (mean ± SEM) are % control (absence of oxysterol), n = 6 for 7-oxysterols and n = 3 for other oxysterols. OHC = hydroxycholesterol. (c)–(f) 7-Oxysterols inhibited metabolism of glucocorticoids by recombinant and microsomal 11βHSD1; in both cases they inhibited dehydrogenation more potently than reduction. Competitive inhibition of (c) reduction of 11-DHC to corticosterone in the presence of 7KC and (d) oxidation of corticosterone to 11-DHC in the presence of 7βOHC, by recombinant 11βHSD1, demonstrated by Dixon Plots (mean data). Inhibition of (e) reduction of 11-DHC to corticosterone in the presence of 7KC and (f) oxidation of corticosterone to 11-DHC in the presence of 7βOHC by microsomal 11βHSD1. n = 3–7. (g) Supplementation of cholesterol impeded reduction of glucocorticoids by 11βHSD1. The velocity of reduction of 11-DHC (open bars) by 11βHSD1 stably transfected in HEK293 was suppressed when 7-oxysterol levels were supplemented by delivery of a complex of cholesterol and methyl-β-cyclodextrin (1:6). *p < 0.01 compared by 2 way ANOVA with Bonferroni post-test, n = 6.