Evidence that 24- and 27-hydroxylation are not involved in the cholesterol-induced down-regulation of hydroxymethylglutaryl-CoA reductase in mouse liver.
Several authors have suggested that 27-hydroxycholesterol may be an important physiological regulator of cholesterol homeostasis. In the present study we investigated the possibility that 24- or 27-hydroxylation of cholesterol is of importance for the down-regulation of hydroxymethylglutaryl (HMG)-CoA reductase in mouse liver induced by dietary cholesterol. Using an accurate method based on isotope dilution-mass spectrometry with deuterated internal standards, we were able to detect significant levels of both 24- and 27-hydroxycholesterol in liver homogenates from normal mice. Feeding cholesterol, 2% for 4 days, increased the levels by 80 and 30%, respectively. No significant hepatic levels of 25-hydroxycholesterol could be demonstrated in untreated mice, and the level of this steroid in cholesterol-treated mice was just above the detection limit. Mouse liver mitochondria were able to catalyze 24- as well as 27-hydroxylation, but not 25-hydroxylation of cholesterol. There was no such conversion in liver microsomes. When using 24-2H2- or 23,23,24,24,25-2H5-labeled cholesterol as substrate a kinetic isotope effect of about 4.5 was observed for the mitochondrial 24-hydroxylation. When using 26,26,26,27,27,27-2H6-labeled cholesterol as substrate a kinetic isotope effect of about 2.5 was observed for the 27-hydroxylation. Use of those deuterium-labeled cholesterol species thus allowed a specific suppression of the rate of 24- and 27-hydroxylation. Feeding mice with 0.05% unlabeled pure cholesterol in the diet for 24 h inhibited the hepatic HMG-CoA reductase activity by about 50%. The same degree of suppression was obtained after feeding with 23,23,24,24,25-2H5- and 26,26,26,27,27,27-2H6-labeled cholesterol. Were mitochondrial 24- and 27-hydroxylation of importance, one would expect reduced suppression of HMG-CoA reductase when feeding deuterated cholesterol, due to the isotope effects. As this was not the case, it is concluded that neither 24-hydroxylation nor 27-hydroxylation are critical for the cholesterol-induced down-regulation of HMG-CoA reductase in mouse liver.
Lund
E
E
Department of Clinical Chemistry, Karolinska Institutet, Huddinge Hospital, Sweden.
Breuer
O
O
Björkhem
I
I
D000818:Animals; D002784:Cholesterol / Q000302:isolation & purification / Q000378:metabolism* / Q000494:pharmacology*; D002851:Chromatography, High Pressure Liquid; D003903:Deuterium; D006903:Hydroxymethylglutaryl CoA Reductases / Q000378:metabolism*; D007553:Isotope Labeling / Q000379:methods; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008297:Male; D013058:Mass Spectrometry; D051379:Mice; D008810:Mice, Inbred C57BL; D008862:Microsomes, Liver / Q000201:enzymology; D008930:Mitochondria, Liver / Q000201:enzymology; D011865:Radioisotope Dilution Technique; D013261:Sterols / Q000302:isolation & purification / Q000378:metabolism*
D013261:Sterols; D002784:Cholesterol; D003903:Deuterium; D006903:Hydroxymethylglutaryl CoA Reductases