Selective ability of rat 7-Dehydrocholesterol reductase (DHCR7) to act on some 7-Dehydrocholesterol metabolites but not on lumisterol metabolites

Abstract

7-Dehydrocholesterol reductase (DHCR7) catalyses the final step of cholesterol biosynthesis in the Kandutsch-Russel pathway, the reduction of 7-dehydrocholesterol (7DHC) to cholesterol. 7DHC can be acted on by a range of other enzymes including CYP27A1 and CYP11A1, as well as by UVB radiation, producing a number of derivatives including hydroxy-metabolites, some of which retain the C7-C8 double bond and are biologically active. These metabolites include lumisterol (L3) which is a stereoisomer of 7DHC produced in the skin by UVB radiation of 7DHC, as well as vitamin D3. The aim of this study was to test whether these metabolites could act as substrates or inhibitors of DHCR7 in rat liver microsomes. To initially screen the ability of these metabolites to interact with the active site of DHCR7, their ability to inhibit the conversion of ergosterol to brassicasterol was measured. Sterols that significantly inhibited this reaction included 7DHC (as expected), 20S(OH)7DHC, 27(OH)DHC, 8DHC, 20S(OH)L3 and 22(OH)L3 but not 7-dehydropregnenolone (7DHP), 25(OH)7DHC, L3 or vitamin D3 and its hydroxyderivatives. Sterols that inhibited ergosterol reduction were directly tested as substrates for DHCR7. 20S(OH)7DHC, 27(OH)DHC and 7-dehydrodesmosterol were confirmed to be substrates, giving the expected product with the C7-C8 double bond removed. No products were observed from 8DHC or 20S(OH)L3 indicating that these sterols are inhibitors and not substrates of DHCR7. The resistance of lumisterol and 7DHP to reduction by DHCR7 in cells will permit other enzymes to metabolise these sterols to their active forms retaining the C7-C8 double bond, conferring specificity to their biological actions.

Keywords: 7-dehydrocholesterol reductase; 7-dehydropregnenolone; DHCR7; Lumisterol; Vitamin D3.

Fig. 1.

Pathways for the biosynthesis of the major metabolites of 7DHC used on this study. The 7DHC structure is boxed. See text for references.

Fig. 2.

HPLC analysis of microsomal DHCR7 activity towards ergosterol in the absence and presence of 7DHC. Rat microsomes were incubated with 30 μM substrates for 1 h at 37 °C, saponified and extracted 3 times with hexane. Stigmasterol was added prior to saponification as an internal standard. Samples were analysed by reverse phase HPLC at 205 nm on a C18 Alltima column using a methanol-water solvent system as described in the Methods. (A), Incubation with ergosterol in the absence of NADPH (negative control); (B), test incubation with ergosterol; (C), test incubation with ergosterol in the presence of 30 μM 7DHC. Relevant peaks are highlighted with grey bars and numbered for identification from standards run separately.

Fig. 3.

Characteristics of the assay of DHCR7 in microsomes using ergosterol as substrate. (A), Time dependence of DHCR7 activity measured with 30 μM ergosterol and a microsomal protein concentration of 1.0 mg/mL, as in Fig. 2. (B), Linearity of DHCR7 activity with microsomal protein concentration measured with 30 μM ergosterol and an incubation time of 15 min. (C) K_m for NADPH measured with 30 μM ergosterol, 0.5 mg/mL microsomal protein and an incubation time of 10 min.

Fig. 4.

Metabolism of 7DMD by rat liver microsomes. Microsomes were incubated with 30 μM 7DMD for 20 min at 37 °C and products analysed by HPLC as in Fig. 2. In this experiment however, ergosterol (15 nmol) was added as an internal standard at the end of the incubation, prior to extraction. (A), Chromatograms of standards, (B), control reaction lacking NADPH and (C), test reaction with NADPH. Chromatogram traces up to 30 min were similar to those in Fig. 2 and are not shown as no reactants nor products eluted during this period.

Fig. 5.

Metabolism of 20S(OH)7DHC and 27(OH)7DHC by DHCR7 in microsomes. Microsomes were incubated with 60 μM 20S(OH)7DHC (A) or 27(OH)7DHC (B) for 1 h at 37 °C and products analysed by HPLC as in Fig. 2. Each panel shows chromatograms of standard(s), control reaction lacking NADPH and the test reaction with NADPH. Chromatogram traces up to 30 min were similar to those in Fig. 2 and are not shown as no reactants nor products eluted during this period.

Fig. 6.

7DHP and 8DHC are not metabolised by DHCR7 in microsomes. Microsomes were incubated with 60 μM 7-dehydropregnenolone (A) or 8DHC (B) for 1 h at 37 °C and products analysed by HPLC as in Fig. 2. Each panel shows chromatograms of standard(s), control reaction lacking NADPH and the test reaction with NADPH.

Fig. 7.

Lumisterol (L3) and 20S(OH)L3 are not metabolised by DHCR7 in microsomes. Microsomes were incubated with 60 μM L3 (A) or 30 μM 20S(OH) L3 for 1 h at 37 °C and products analysed by HPLC as in Fig. 2. Each panel shows chromatograms of the control reaction lacking NADPH and the test reaction with NADPH. Relevant peaks are highlighted with grey bars and numbered for identification from standards run separately.