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. 1996 Jul 26;271(30):18024-31.
doi: 10.1074/jbc.271.30.18024.

Disruption of cholesterol 7alpha-hydroxylase gene in mice. II. Bile acid deficiency is overcome by induction of oxysterol 7alpha-hydroxylase

M Schwarz  1 , E G Lund, K D Setchell, H J Kayden, J E Zerwekh, I Björkhem, J Herz, D W Russell
Affiliations

Disruption of cholesterol 7alpha-hydroxylase gene in mice. II. Bile acid deficiency is overcome by induction of oxysterol 7alpha-hydroxylase

M Schwarz et al. J Biol Chem. .

Abstract

Past experiments and current paradigms of cholesterol homeostasis suggest that cholesterol 7alpha-hydroxylase plays a crucial role in sterol metabolism by controlling the conversion of cholesterol into bile acids. Consistent with this conclusion, we show in the accompanying paper that mice deficient in cholesterol 7alpha-hydroxylase (Cyp7-/- mice) exhibit a complex phenotype consisting of abnormal lipid excretion, skin pathologies, and behavioral irregularities (Ishibashi, S., Schwarz, M., Frykman, P. K. , Herz, J., and Russell, D. W.(1996) J. Biol. Chem. 261, 18017-18023). Aspects of lipid metabolism in the Cyp7-/- mice are characterized here to deduce the physiological basis of this phenotype. Serum lipid, cholesterol, and lipoprotein contents are indistinguishable between wild-type and Cyp7-/- mice. Vitamin D3 and E levels are low to undetectable in knockout animals. Stool fat content is significantly elevated in newborn Cyp7-/- mice and gradually declines to wild-type levels at 28 days of age. Several species of 7alpha-hydroxylated bile acids are detected in the bile and stool of adult Cyp7-/- animals. A hepatic oxysterol 7alpha-hydroxylase enzyme activity that may account for the 7alpha-hydroxylated bile acids is induced between days 21 and 30 in both wild-type and deficient mice. An anomalous oily coat in the Cyp7-/- animals is due to the presence of excess monoglyceride esters in the fur. These data show that 7alpha-hydroxylase and the pathway of bile acid synthesis initiated by this enzyme are essential for proper absorption of dietary lipids and fat-soluble vitamins in newborn mice, but not for the maintenance of serum cholesterol and lipid levels. In older animals, an alternate pathway of bile acid synthesis involving an inducible oxysterol 7alpha-hydroxylase plays a crucial role in lipid and bile acid metabolism.

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Figures

Fig. 1
Fig. 1. Lipoprotein analysis in wild-type and 7α-hydroxylase-deficient mice
Plasma was obtained from one 8-month-old female mouse of the indicated Cyp7 genotype and fractionated on a Superose 6 column. The cholesterol content was determined and plotted as a function of column fraction. The positions at which known lipoproteins eluted from the column are indicated. VLDL, very high density lipoprotein; LDL, low density lipoprotein; HDL, high density lipoprotein.
Fig. 2
Fig. 2. Stool fat content as function of age in wild-type and 7α-hydroxylase-deficient mice
Total lipid content in stool was measured as described under “Experimental Procedures” and plotted as a function of animal age. Values obtained from wild-type (○) and Cyp7−/− mice (▲) are shown. All animals were weaned on day 30 of the experiment.
Fig. 3
Fig. 3. Oxysterol 7α-hydroxylase activity in wild-type and 7α-hydroxylase-deficient mice
Liver microsomes were prepared from mice of the indicated Cyp7 genotype and incubated with [3H]cholest-5-ene-3β,25-diol (25-hydroxycholesterol) in the presence or absence of NADPH. After 15 min at 37 °C, sterols were extracted and analyzed by thin-layer chromatography. An autoradiogram of the experiment is shown.
Fig. 4
Fig. 4. Authentication of oxysterol 7α-hydroxylase product
Cholest-5-ene-3β,7α,25-triol, an anticipated product of the oxysterol 7α-hydroxylase enzyme activity in mouse liver, was chemically synthesized as described under “Experimental Procedures.” In lane 1, an aliquot of this compound was subjected to thin-layer chromatography in a solvent system containing toluene/ethyl acetate (2:3), and the lane was stained with phosphomolybdic acid (PMA). In lane 2, an aliquot of the authentic standard was mixed with the sterol products arising from incubation of mouse liver microsomes with [3H]cholest-5-ene-3β,25-diol (25-hydroxycholesterol), and the mixture was subjected to thin-layer chromatography and staining. In addition to steroids, dithiothreitol and Triton X-100, two components of the enzyme assay, were also stained by phosphomolybdic acid. In lane 3, an autoradiogram of lane 2 is presented. Comparison of the results obtained by autoradiography (lane 3) with those obtained by staining (lanes 1 and 2) reveals comigration of the authentic standard with the radiolabeled product of the enzyme.
Fig. 5
Fig. 5. Product inhibition of oxysterol 7α-hydroxylase activity
Liver microsomes prepared from wild-type mice were incubated with 0.12 μM [3H]cholest-5-ene-3β,25-diol (25-hydroxycholesterol), 1.5 mM NADPH, and the indicated concentrations of hydroxysterols. After a 15-min incubation, the formation of [3H]cholest-5-ene-3β,7α,25-triol was determined by thin-layer chromatography. A, an autoradiogram of the resulting thin-layer chromatogram is shown together with the positions to which substrate and product migrated. B, the results shown in A were quantitated by phosphoimaging and plotted as a standard inhibition curve. Among the three sterols tested, only the product of the oxysterol 7α-hydroxylase enzyme, cholest-5-ene-3β,7α,25-triol, was inhibitory.
Fig. 6
Fig. 6. Ontogeny of oxysterol 7α-hydroxylase expression in mice
Liver microsomes were prepared from wild-type (+/+) or 7α-hydroxylase-deficient mice (−/−) of the indicated ages and assayed for oxysterol 7α-hydroxylase activity. A, shown are autoradiograms of thin-layer chromatography plates. Oxysterol 7α-hydroxylase activity was first detected above background in 3 (day 22) to 4 (day 30)-week-old animals and remained detectable in the livers of older animals. B, data from a more extensive survey of animals of different ages and Cyp7 genotypes were quantitated by phosphoimaging and plotted as oxysterol 7α-hydroxylase enzyme activity versus age in days. Animals were weaned on day 24 in this experiment.
Fig. 7
Fig. 7. Qualitative analysis of fur lipids in wild-type and mutant mice
The fur of nursing females of the indicated Cyp7 genotype was cut from the abdomen or back. Total lipids were extracted with chloroform/methanol (2:1), concentrated by evaporation, and then analyzed by thin-layer chromatography in a solvent system containing toluene/ethyl acetate (7:3). After development, the chromatogram was stained with phosphomolybdic acid and photographed. Squalene and cholesterol standards were chromatographed in adjacent lanes of the plate. The spot in the fur samples that comigrated with the squalene standard contained a mixture of squalene and cholesterol esters. At least three lipids (designated X, Y, and Z) were present at higher levels in the abdomen and back fur sheared from the Cyp7−/− animals.
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