Inhibition of cholesterol biosynthesis by Δ22-unsaturated phytosterols via competitive inhibition of sterol Δ24-reductase in mammalian cells

Dietary phytosterols are cholesterol-lowering agents that interfere with the intestinal absorption of cholesterol. In the present study, we have studied their effects on cholesterol biosynthesis in human cells, particularly in the sterol-conversion pathway. For this, both Caco-2 (intestinal mucosa) and HL-60 (promyelocytic) human cell lines were incubated with [(14)C]acetate, and the incorporation of radioactivity into sterols was determined using HPLC and radioactivity detection online. Sterols containing a double bond at C-22 in the side chain (stigmasterol, brassicasterol and ergosterol) dramatically inhibited the activity of sterol Delta(24)-reductase, as indicated by the decrease in radioactivity incorporation into cholesterol and the accumulation of its precursors (mainly desmosterol). Phytosterols with the saturated side chain (beta-sitosterol and campesterol) were inactive in this regard. The inhibition of sterol (24)-reductase was confirmed in rat liver microsomes by using (14)C-labelled desmosterol as the substrate. The (22)-unsaturated phytosterols acted as competitive inhibitors of sterol (24)-reductase, with K(i) values (41.1, 42.7 and 36.8 microM for stigmasterol, brassicasterol and ergosterol respectively) similar to the estimated K(m) for desmosterol (26.3 microM). The sterol 5,22-cholestedien-3beta-ol, an unusual desmosterol isomer that lacks the alkyl groups characteristic of phytosterols, acted as a much stronger inhibitor of (24)-reductase (K(i)=3.34 microM). The usually low intracellular concentrations of the physiological substrates of (24)-reductase explains the strong inhibition of cholesterol biosynthesis that these compounds exert in cells. Given that inhibition of sterol (24)-reductase was achieved at physiologically relevant concentrations, it may represent an additional mechanism for the cholesterol-lowering action of phytosterols, and opens up the possibility of using certain (22)-unsaturated sterols as effective hypocholesterolaemic agents.