Ingemar Björkhem

Brain Cholesterol: Long Secret Life Behind a Barrier

Abstract—Although an immense knowledge has accumulated concerning regulation of cholesterol homeostasis in the body, this does not include the brain, where details are just emerging. Approximately 25% of the total amount of the cholesterol present in humans is localized to this organ, most of it present in myelin. Almost all brain cholesterol is a product of local synthesis, with the blood–brain barrier efficiently protecting it from exchange with lipoprotein cholesterol in the circulation. Thus, there is a highly efficient apolipoprotein-dependent recycling of cholesterol in the brain, with minimal losses to the circulation. Under steady-state conditions, most of the de novo synthesis of cholesterol in the brain appears to be balanced by excretion of the cytochrome P-450–generated oxysterol 24S-hydroxycholesterol. This oxysterol is capable of escaping the recycling mechanism and traversing the blood–brain barrier. Cholesterol levels and cholesterol turnover are affected in neurodegenerating disorders, and the capacity for cholesterol transport and recycling in the brain seems to be of importance for the development of such diseases. The possibility has been discussed that administration of inhibitors of cholesterol synthesis may reduce the prevalence of Alzheimer disease. No firm conclusions can, however, be drawn from the studies presented thus far. In the present review, the most recent advances in our understanding of cholesterol turnover in the brain is discussed.

Hepatic cholesterol metabolism and resistance to dietary cholesterol in LXRβ-deficient mice

The nuclear oxysterol-receptor paralogues LXRalpha and LXRbeta share a high degree of amino acid identity and bind endogenous oxysterol ligands with similar affinities. While LXRalpha has been established as an important regulator of cholesterol catabolism in cholesterol-fed mice, little is known about the function of LXRbeta in vivo. We have generated mouse lines with targeted disruptions of each of these LXR receptors and have compared their responses to dietary cholesterol. Serum and hepatic cholesterol levels and lipoprotein profiles of cholesterol-fed animals revealed no significant differences between LXRbeta(-/-) and wild-type mice. Steady-state mRNA levels of 3-hydroxy-3-methylglutaryl coenzyme A reductase, farnesyl diphosphate synthase, and squalene synthase were increased in LXRbeta(-/-) mice compared with LXRbeta(+/+) mice, when fed standard chow. The mRNA levels for cholesterol 7alpha-hydroxylase, oxysterol 7alpha-hydroxylase, sterol 12alpha-hydroxylase, and sterol 27-hydroxylase, respectively, were comparable in these strains, both on standard and 2% cholesterol chow. Our results indicate that LXRbeta(-/-) mice - in contrast to LXRalpha(-/-) mice - maintain their resistance to dietary cholesterol, despite subtle effects on the expression of genes coding for enzymes involved in lipid metabolism. Thus, our data indicate that LXRbeta has no complete overlapping function compared with LXRalpha in the liver.

The antioxidant butylated hydroxytoluene protects against atherosclerosis.

Rabbits fed a 1% cholesterol diet with or without the antioxidant butylated hydroxytoluene (BHT) developed typical atherosclerotic lesions. The addition of BHT gave higher levels of total cholesterol (+40%), triglycerides (+250%), low density lipoprotein (LDL), and very low density lipoprotein (VLDL) in plasma. Despite the lower plasma lipid levels, the degree of atherosclerosis of the aortic surface was considerably higher in rabbits fed cholesterol than in the group treated with cholesterol and BHT. The mean atherosclerotic involvement was 18.6 +/- 4.4% in the former group and 5.9 +/- 1.7% in the latter group (p = 0.02). In all animals, there was a high correlation between the area of the arterial lesion and cholesterol content (r = 0.96). Serum levels of cholesterol autooxidation products (7-ketocholesterol and cholesterol 5 alpha,6 alpha-epoxide) were lower in the group of rabbits treated with BHT (p less than 0.005). Serum levels of vitamin E were slightly higher in the BHT group. There was no significant difference in the clearance of beta-VLDL between the two treatment groups after using either beta-VLDL from cholesterol-fed animals or beta-VLDL from BHT-fed animals. The results are in accord with the contention that oxidative modification of lipoproteins is important for the development of atherosclerosis and that antioxidants may have a protective effect. At present, however, other explanations cannot be completely excluded, for example, effects of antioxidants on immunologic factors or monocyte adhesion.

Oxysterols. Friends, Foes, or Just Fellow Passengers?

Oxysterols are oxygenated derivatives of cholesterol that are intermediates or even end products in cholesterol excretion pathways. Because of their ability to pass cell membranes and the blood-brain barrier at a faster rate than cholesterol itself, they are also important as transport forms of cholesterol. In addition, oxysterols have been ascribed a number of important roles in connection with cholesterol turnover, atherosclerosis, apoptosis, necrosis, inflammation, immunosuppression, and the development of gallstones. According to current concepts, oxysterols are physiological mediators in connection with a number of cholesterol-induced metabolic effects. However, most of the evidence for this is still indirect, and there is a discrepancy between the documented potent effects of oxysterols under in vitro conditions and the studies demonstrating that they are of physiological importance in vivo. Oxysterol-binding proteins, such as liver X receptor-&agr; (a nuclear receptor), do have a regulatory role in cholesterol turnover, but the physiological ligand of the protein has not yet been defined with certainty. Recently developed genetically engineered mouse models with markedly reduced or increased concentration of some of the oxysterols have exhibited surprisingly small changes in cholesterol turnover and homeostasis. The present review is a critical evaluation of the literature on oxysterols, in particular, the in vivo evidence for a role of oxysterols as physiological regulators of cholesterol homeostasis and as atherogenic factors.

Disruption of Cholesterol 7α-Hydroxylase Gene in Mice II. BILE ACID DEFICIENCY IS OVERCOME BY INDUCTION OF OXYSTEROL 7α-HYDROXYLASE

Past experiments and current paradigms of cholesterol homeostasis suggest that cholesterol 7α-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 7α-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 7α-hydroxylated bile acids are detected in the bile and stool of adult Cyp7−/− animals. A hepatic oxysterol 7α-hydroxylase enzyme activity that may account for the 7α-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 7α-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 7α-hydroxylase plays a crucial role in lipid and bile acid metabolism.

Effects of Cholesteryl Ester Transfer Protein Inhibition on High-Density Lipoprotein Subspecies, Apolipoprotein A-I Metabolism, and Fecal Sterol Excretion

Objective—Pharmacological inhibition of the cholesteryl ester transfer protein (CETP) in humans increases high-density lipoprotein (HDL) cholesterol (HDL-C) levels; however, its effects on apolipoprotein A-I (apoA-I) containing HDL subspecies, apoA-I turnover, and markers of reverse cholesterol transport are unknown. The present study was designed to address these issues. Methods and Results—Nineteen subjects, 9 of whom were taking 20 mg of atorvastatin for hypercholesterolemia, received placebo for 4 weeks, followed by the CETP inhibitor torcetrapib (120 mg QD) for 4 weeks. In 6 subjects from the nonatorvastatin cohort, the everyday regimen was followed by a 4-week period of torcetrapib (120 mg BID). At the end of each phase, subjects underwent a primed-constant infusion of (5,5,5-2H3)-l-leucine to determine the kinetics of HDL apoA-I. The lipid data in this study have been reported previously. Relative to placebo, 120 mg daily torcetrapib increased the amount of apoA-I in &agr;1-migrating HDL in the atorvastatin (136%; P<0.001) and nonatorvastatin (153%; P<0.01) cohorts, whereas an increase of 382% (P<0.01) was observed in the 120 mg twice daily group. HDL apoA-I pool size increased by 8±15% in the atorvastatin cohort (P=0.16) and by 16±7% (P<0.0001) and 34±8% (P<0.0001) in the nonatorvastatin 120 mg QD and BID cohorts, respectively. These changes were attributable to reductions in HDL apoA-I fractional catabolic rate (FCR), with torcetrapib reducing HDL apoA-I FCR by 7% (P=0.10) in the atorvastatin cohort, by 8% (P<0.001) in the nonatorvastatin 120 mg QD cohort, and by 21% (P<0.01) in the nonatorvastatin 120 mg BID cohort. Torcetrapib did not affect HDL apoA-I production rate. In addition, torcetrapib did not significantly change serum markers of cholesterol or bile acid synthesis or fecal sterol excretion. Conclusions—These data indicate that partial inhibition of CETP via torcetrapib in patients with low HDL-C: (1) normalizes apoA-I levels within &agr;1-migrating HDL, (2) increases plasma concentrations of HDL apoA-I by delaying apoA-I catabolism, and (3) does not significantly influence fecal sterol excretion.

Lipoprotein Oxidation and Progression of Carotid Atherosclerosis

BACKGROUND Epidemiological studies and animal experiments have provided evidence supporting the role of lipid peroxidation in atherogenesis and cardiovascular diseases. Direct evidence linking lipid oxidation to atherosclerotic progression in humans, however, has been lacking. We investigated the association of lipid oxidation products with the progression of early carotid atherosclerosis in hypercholesterolemic men from eastern Finland. METHODS AND RESULTS Twenty subjects with a fast progression and 20 with no progression of carotid atherosclerosis in 3 years were selected from > 400 participants in the Kuopio Atherosclerosis Prevention Study. Progression of carotid atherosclerosis was assessed by high-resolution B-mode ultrasonography. Serum 7 beta-hydroxycholesterol, a major oxidation product of cholesterol in membranes and lipoproteins, and seven other cholesterol oxidation products were measured by isotope dilution-mass spectrometry, lipid hydroperoxides in LDL fluorometrically as thiobarbituric acid-reactive substances (TBARS) and oxidation susceptibility of LDL and VLDL kinetically. High concentrations of serum 7 beta-hydroxycholesterol (beta = 47, P = .0005), cigarette smoking (beta = .35, P = .0167), and LDL TBARS (beta = .23, P = .0862) and an increased oxidation susceptibility of VLDL + LDL (beta = .22 P = .1114) were the strongest predictors of a 3-year increase in carotid wall thickness of more than 30 variables tested in step-up least-squares regression models. A 10-variable model explained 60% of the atherosclerotic progression. In a multivariate logistic model, the risk of experiencing a fast progression increased by 80% (P = .013) per unit (microgram/L) of 7 beta-hydroxycholesterol. CONCLUSIONS The findings of this study provide further evidence to support an association between lipid oxidation and atherogenesis in humans.

Markedly reduced bile acid synthesis but maintained levels of cholesterol and vitamin D metabolites in mice with disrupted sterol 27-hydroxylase gene.

Sterol 27-hydroxylase is important for the degradation of the steroid side chain in conversion of cholesterol into bile acids and has been ascribed a regulatory role in cholesterol homeostasis. Its deficiency causes the autosomal recessive disease cerebrotendinous xanthomatosis (CTX), characterized by progressive dementia, xanthomatosis, and accelerated atherosclerosis. Mice with a disrupted cyp27(cyp27 −/−) had normal plasma levels of cholesterol, retinol, tocopherol, and 1,25-dihydroxyvitamin D. Excretion of fecal bile acids was decreased (<20% of normal), and formation of bile acids from tritium-labeled 7α-hydroxycholesterol was less than 15% of normal. Compensatory up-regulation of hepatic cholesterol 7α-hydroxylase and hydroxymethylglutaryl-CoA reductase (9- and 2–3-fold increases in mRNA levels, respectively) was found. No CTX-related pathological abnormalities were observed. In CTX, there is an increased formation of 25-hydroxylated bile alcohols and cholestanol. In bile and feces of thecyp27 −/− mice only traces of bile alcohols were found, and there was no cholestanol accumulation. It is evident that sterol 27-hydroxylase is more important for bile acid synthesis in mice than in humans. The results do not support the contention that 27-hydroxylated steroids are critical for maintenance of cholesterol homeostasis or levels of vitamin D metabolites in the circulation.

IMPORTANCE OF A NOVEL OXIDATIVE MECHANISM FOR ELIMINATION OF BRAIN CHOLESTEROL : TURNOVER OF CHOLESTEROL AND 24(S)-HYDROXYCHOLESTEROL IN RAT BRAIN AS MEASURED WITH 18O2 TECHNIQUES IN VIVO AND IN VITRO

The brain is the most cholesterol-rich organ in the body. Brain cholesterol is characterized by a very low turnover with very little exchange with lipoproteins in the circulation. Very recently we showed that there is a continuous age-dependent flux of 24(S)-hydroxycholesterol from the human brain into the circulation (Lütjohann, D., Breuer, O., Ahlborg, G., Nennesmo, I., Sidén, Å., Diczfalusy, U., and Björkhem, I. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 9799–9804). Here we measured the rate of synthesis of cholesterol as well as the conversion of cholesterol into 24(S)-hydroxycholesterol in rat brain in vivo with use of an18O2 inhalation technique and mass isotopomer distribution analysis. Cholesterol synthesis was found to correspond to 0.03 ± 0.01% of the pool per h. Conversion of cholesterol into 24(S)-hydroxycholesterol was of a similar magnitude, about 0.02% of the pool per h. Brain microsomes converted endogenous cholesterol into 24(S)-hydroxycholesterol at a similar rate when incubated in the presence of NADPH. When incubated with whole homogenate and subcellular fractions of rat brain, there was no significant conversion of tritium-labeled 24-hydroxycholesterol into more polar products. Plasma from 18O2-exposed rats contained 24(S)-hydroxycholesterol with an enrichment of 18O similar to that in 24(S)-hydroxycholesterol in the brain. The results suggest that the present 24(S)-hydroxylase mediated mechanism is most important for elimination of cholesterol from the brain of rats. There is a slow conversion of brain cholesterol into 24(S)-hydroxycholesterol with a rapid turnover of the small pool of the latter oxysterol due to leakage to the circulation (half-life of brain 24(S)-hydroxycholesterol is about 0.5 days as compared with 2–4 months for brain cholesterol). It is evident that the 24(S)-hydroxylation greatly facilitates transfer of cholesterol over the blood-brain barrier and that this hydroxylation may be critical for cholesterol homeostasis in the brain.

Crossing the barrier: oxysterols as cholesterol transporters and metabolic modulators in the brain

A normal brain function requires constant levels of cholesterol, and the need for constancy seems to be higher here than in any other organ. Nature has met this need by isolation of brain cholesterol by a highly efficient blood–brain barrier. As a low synthesis of cholesterol is present in the brain, a mechanism for compensatory elimination is required. A decade ago we made the unexpected finding that the favoured mechanism for this involves conversion into 24S‐hydroxycholesterol, followed by diffusion over the blood–brain barrier. Recent studies by us and others on this new pathway have given new insights into the mechanisms by which cholesterol homeostasis is maintained in the brain. We recently demonstrated a flux of another oxygenated product of cholesterol, 27‐hydroxycholesterol, in the opposite direction. The latter flux may be important for neurodegeneration, and may be the link between hypercholesterolaemia and Alzheimers disease. An overview of the above studies is presented and the possibility that the cholesterol 24S‐hydroxylase in the brain may be important for memory and learning and that it may be a new drug target is discussed.