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High plasma levels of LDL-cholesterol play a causative role in atherogenesis. The first step in atherogenesis is characterized by oxidation of retained LDL particles in the arterial wall, and scavenger receptors-mediated entry of oxidized-LDL into macrophages, resulting in the accumulation of cholesterol within macrophages. Therefore, cholesterol efflux from macrophages to HDL particles is an essential process in inhibiting the formation of atherosclerotic plaques. Previous in-vitro experiments conducted in our laboratory, revealed that 9-cis β-carotene (9CBC) enhanced the efflux of radioactively labeled cholesterol from macrophages to HDL. However, the mechanism by which 9CBC enhances cholesterol efflux remained obscure. We found, in a controlled in-vitro assay, that treating macrophages both with the Dunaliella bardawil alga derived carotenoid 9CBC, and 22R-hydroxycholesterol (22-HC), two ligands of the nuclear receptors Retinoid X Receptor (RXR) and Liver X Receptor (LXR) respectively, resulted in a synergistically increase in fluorescently-labeled cholesterol efflux. Moreover, concurrent treatment of 9CBC with liarozole, which blocks the metabolism of Retinoic Acid (RA), increased cholesterol efflux from macrophages. This result strengthens our hypothesis that 9CBC activates the RXR nuclear receptor by its conversion to RA. Furthermore, cholesterol efflux was greater in macrophages isolated from alga Dunaliella-fed WT or apoE-/- mice compared to chow diet-fed mice. Our results suggest that simultaneous activation of nuclear receptors affects regulation of cellular cholesterol and may accelerate reverse cholesterol transport, by increasing cholesterol efflux from macrophages and thus, reducing the risk for atherogenesis.

The overall ability of the brain to synthesise neuroactive steroids led us to the identification of compounds that would reproduce aspects of neurosteroid pharmacology. The rate-determining step in neurosteroid biosynthesis is the import of the substrate cholesterol into the mitochondria, where it is metabolised into pregnenolone via the intermediate 22R-hydroxycholesterol. The levels of translocator protein 18-kDa, mediating the import of cholesterol into mitochondria, correlated with increased pregnenolone formation and reduced levels of 22R-hydroxycholesterol in biopsies from Alzheimer’s disease (AD), but not age-matched control, brains. 22R-hydroxycholesterol was shown to protect against b-amyloid (Ab42)-induced neurotoxicity. In search of 22Rhydroxycholesterol stable analogues, we identified the naturally occurring heterospirostenol, (22R,25R)-20a-spirost-5-en-3b-yl hexanoate (caprospinol) and derivatives that protect neuronal cells against Ab1–42 neurotoxicity. The neuroprotective effect of caprospinol is the result of a combination of overlapping properties, including: (i) the ability to bind to Ab42 and reduce plaque formation in the brain in vivo; (ii) interaction with components of the mitochondria respiratory chain resulting in an anti-uncoupling effect; (iii) the capacity to scavenge Ab42 monomers present in mitochondria; and (iv) the property of being a sigma-1 receptor ligand. In vivo, caprospinol crosses the blood–brain barrier, accumulates in the brain, and restores cognitive impairment in a pharmacological rat model of AD. Caprospinol is stable, does not bind to known steroid receptors, is devoid of mutagenic and genotoxic properties, and is devoid of acute toxicity in rodents. The pharmacokinetics and pharmacodynamics of caprospinol were studied, and long-term toxicity studies are under investigation, aiming to develop this compound as a disease-modifying drug for the treatment of AD.

We describe a highly sensitive and specific method for the quantification of key regulatory oxysterols in biological samples. This method is based upon a stable isotope dilution technique by liquid chromatography-tandem mass spectrometry (LC-MS/MS). After alkaline hydrolysis of human serum (5 microl) or rat liver microsomes (1 mg protein), oxysterols were extracted, derivatized into picolinyl esters, and analyzed by LC-MS/MS using the electrospray ionization mode. The detection limits of the picolinyl esters of 4beta-hydroxycholesterol, 7alpha-hydroxycholesterol, 22R-hydroxycholesterol, 24S-hydroxycholesterol, 25-hydroxycholesterol, 27-hydroxycholesterol, and 24S,25-epoxycholesterol were 2-10 fg (5-25 amol) on-column (signal-to-noise ratio = 3). Reproducibilities and recoveries of these oxysterols were validated according to one-way layout and polynomial equation, respectively. The variances between sample preparations and between measurements by this method were calculated to be 1.8% to 12.7% and 2.9% to 11.9%, respectively. The recovery experiments were performed using rat liver microsomes spiked with 0.05 ng to 12 ng of oxysterols, and recoveries of the oxysterols ranged from 86.7% to 107.3%, with a mean recovery of 100.6%. This method provides reproducible and reliable results for the quantification of oxysterols in small amounts of biological samples.

22R-hydroxycholesterol, a steroid intermediate in the pathway of pregnenolone formation from cholesterol, was found at lower levels in Alzheimer's disease (AD) hippocampus and frontal cortex tissue specimens compared to age-matched controls. beta-Amyloid (Abeta) peptide has been shown to be neurotoxic and its presence in brain has been linked to AD pathology. 22R-hydroxycholesterol was found to protect, in a dose-dependent manner, against Abeta-induced rat sympathetic nerve pheochromocytoma (PC12) and differentiated human Ntera2/D1 teratocarcinoma (NT2N) neuron cell death. Other steroids tested were either inactive or acted on rodent neurons only. The effect of 22R-hydroxycholesterol was found to be stereospecific because its enantiomer 22S-hydroxycholesterol failed to protect the neurons from Abeta-induced cell death. Moreover, the effect of 22R-hydroxycholesterol was specific for Abeta-induced cell death because it did not protect against glutamate-induced neurotoxicity. The neuroprotective effect of 22R-hydroxycholesterol was seen when using Abeta1-42 but not the Abeta25-35 peptide. To investigate the mechanism of action of 22R-hydroxycholesterol we examined the direct binding of this steroid to Abeta using a novel cholesterol-protein binding blot assay. Using this method the direct specific binding, under native conditions, of 22R-hydroxycholesterol to Abeta1-42 and Abeta17-40, but not Abeta25-35, was observed. These data suggest that 22R-hydroxycholesterol binds to Abeta and the formed 22R-hydroxycholesterol/Abeta complex is not toxic to rodent and human neurons. We propose that 22R-hydroxycholesterol offers a new means of neuroprotection against Abeta toxicity by inactivating the peptide.

Recently, we demonstrated that triiodothyronine (T3) stimulated steroid hormone biosynthesis and steroidogenic acute regulatory (StAR) protein expression in mLTC-1 mouse Leydig tumor cells through the mediation of steroidogenic factor 1 (SF-1). We now report a dual response mechanism of T3 on steroidogenesis and StAR expression, and on LH receptor (LHR) expression and binding in mLTC-1 cells. T3 acutely (8 h), induced a 260% increase in StAR messenger RNA (mRNA) expression over the basal level which was coincident with an increase in progesterone (P) production. In contrast, chronic stimulation with T3 (beyond 8 h), resulted in an attenuation of StAR expression and P production. This attenuation was most likely caused by a decrease in cholesterol delivery to the inner mitochondrial membrane as demonstrated by incubations with the hydrophilic steroid precursors, 22R hydroxycholesterol and pregnenolone, which restored P synthesis. In similar studies, chronic treatment with T3 increased the levels of cytochr...

The retina is the sensory organ in the back of the eye which absorbs and converts light to electrochemical impulses transferred to the brain. Herein, we studied how retinal environment affects enzyme-mediated cholesterol removal. We focused on two mitochondrial cytochrome P450 enzymes, CYPs 27A1 and 11A1, which catalyze the first steps in metabolism of cholesterol in the retina and other tissues. Phospholipids (PL) from mitochondria of bovine neural retina, retinal pigment epithelium, liver and adrenal cortex were isolated and compared for the effect on kinetic properties of purified recombinant CYPs in the reconstituted system in vitro. The four studied tissues were also evaluated for the mitochondrial PL and cholesterol content and levels of CYPs 27A1, 11A1 and their redox partners. The data obtained were used for modeling the retinal environment in the in vitro enzyme assays in which we detected the P450 metabolites, 22R-hydroxycholesterol and 5-cholestenoic acid, unexpectedly found by us in the retina in our previous studies. The effect of the by-product of the visual cycle pyridinium bis-retinoid A2E on kinetics of CYP27A1-mediated cholesterol metabolism was also investigated. The results provide insight into the retina’s regulation of the enzyme-mediated cholesterol removal.

The incorporation of 22R-hydroxycholesterol [(22R)-5-cholestene-3 beta,22-diol] into the bovine erythrocyte membranes remarkably enhanced the degradation of sphingomyelin in erythrocyte membranes by the action of sphingomyelinase from Bacillus cereus, causing much faster hemolysis of erythrocytes. The stimulative effect of 22R-hydroxycholesterol on the breakdown of sphingomyelin was maximal in the presence of Mg2+. On the other hand, in spite of the presence of 22R-hydroxycholesterol, the breakdown of sphingomyelin was inhibited by increasing concentrations of Ca2+. Also, the incorporation of 22R-hydroxycholesterol into the erythrocyte membranes facilitated the specific adsorption of the enzyme onto the surface of the erythrocyte membranes. The specific adsorption of sphingomyelinase amounted to 20-40% of the total activity in the presence of Mg2+ and the absence of divalent metal ions. In the presence of Ca2+, the incorporation of 22R-hydroxycholesterol enhanced the enzyme adsorption, exceeding more than 90% of the total activity. Therefore, the incorporation of 22R-hydroxycholesterol into bovine erythrocyte membranes remarkably accelerates the breakdown of sphingomyelin in the presence of Mg2+, and the specific adsorption of sphingomyelinase onto erythrocytes in the presence of Ca2+.

Recently, we have shown that 22 R -hydroxycholesterol, a steroid intermediate in the pathway of pregnenolone formation from cholesterol, is present at lower levels in Alzheimer’s disease (AD) hippocampus and frontal cortex tissue specimens than in age-matched controls, and that this substance protects against cell death induced by amyloid β-peptide in both rat sympathetic nerve pheochromocytoma (PC12) and differentiated human Ntera2/D1 teratocarcinoma neurons. Herein we report that 22 R -hydroxycholesterol inhibits the proliferation of human Ntera2/D1 teratocarcinoma precursor cells (NT2) and induces these cells to differentiate into “neuron-like” or “astrocyte-like” cells. 22 R -Hydroxycholesterol-induced differentiation of NT2 cells is associated with increases in the expression of neurofilament protein NF200, the cytoskeletal proteins microtubule-associated protein type II (MAP2) a and MAP2b, glial fibrillary acidic protein (GFAP) and glial cell line-derived neurotrophic factor receptor-alpha 2 (GFRα2). These effects of 22 R -hydroxycholesterol are considered to be stereospecific because its enantiomer 22 S -hydroxycholesterol and other steroids failed to induce differentiation of NT2 cells. 22 R -Hydroxycholesterol was found to lack specific binding for numerous receptors, including all steroid receptors tested. However, using a cholesterol protein binding blot assay we demonstrated the presence of a 22 R -hydroxycholesterol-binding protein in NT2 cells distinct from the human oxysterol receptors liver X receptor LXRα and β.

Dihydroxycholesterol and pregnenolone were clearly detected on HPLC when 22R-hydroxycholesterol was incubated with a reconstituted P450scc system containing equimolar amounts of P450scc and adrenodoxin. The dihydroxycholesterol, which has been accepted to be an intermediate in the conversion of 22R-hydroxycholesterol to pregnenolone, accumulated when adrenodoxin was at a subsaturating level with respect to P450scc. The formation of the intermediate increased with increasing pH in the range of 7.2 to 8.1, and the ratio of the intermediate to the product, pregnenolone, increased with increasing pH. When the binding of P450scc to adrenodoxin was weakened by elevation of the ionic strength, the formation of the intermediate relative to the product increased. The apparent Km for dihydroxycholesterol at a subsaturating level of adrenodoxin was about 7 microM, in contrast to 4 microM at a saturating level of adrenodoxin, implying that the affinity of dihydroxycholesterol is lower at a subsaturating level of adrenodoxin than at a saturating one. These results suggest that a subsaturating level of adrenodoxin weakened the binding of dihydroxycholesterol to P450scc and thus the intermediate, dihydroxycholesterol, was released. An intermediate other than dihydroxycholesterol, obtained when cholesterol was used as the substrate, was identified as 22R-hydroxycholesterol by HPLC and mass spectroscopic analysis. The intermediate obtained when 22R-hydroxycholesterol was used as the substrate was identified as 20R,22R-dihydroxycholesterol by HPLC, mass, and 1H-NMR spectroscopic analyses. These results provide direct evidence that cholesterol is metabolized to pregnenolone by way of 22R-hydroxycholesterol and 20R,22R-dihydroxycholesterol by P45 scc.

Using tritiated internal standards, crystalline 22R-hydroxycholesterol and 20α,22R-dihydroxycholesterol have been isolated for the first time from bovine adrenal glands. The identity of the sterols was established by comparison of their melting points and infrared spectra with those of authentic standards. The tissue concentration of the sterols, as determined by both direct weighing of crystalline samples and the double-isotope derivative technique, indicated the presence of 1.5 mg/kg of 22R-hydroxycholesterol and 2.15 mg/kg of 20α,22R-dihydroxycholesterol. The presence of neither the monosulfate nor the disulfate of 22R-hydroxycholesterol could be established.

The effects of thyroidectomy on pregnenolone and progesterone biosynthesis were investigated in rat adrenal cortex mitochondria and microsomes. The sequential hydroxylations of cholesterol, and the latter's side-chain cleavage, which constitute the limiting step in steroidogenesis, were studied by measuring oxygen consumption rates in the presence of cholesterol derivatives. The addition of (20R)-hydroxycholesterol, (22R)-hydroxycholesterol or (20R,22R)-dihydroxycholesterol stimulated theses rates, but addition of cholesterol or (22S)-hydroxycholesterol had little effect. Thyroidectomy significantly reduced oxygen consumption rates in the presence of the sterols by about 30%. Oxygen uptake was small in the presence of respiratory inhibitors; the addition of sterols raised this uptake but subsequent thyroidectomy did not change it. Rat adrenal cortex mitochondria and microsomes converted pregnenolone into progesterone through 3αbol dehydrogenase/ Δ 4−5 isomerase, in two different successive steps. Values for enzyme activities were 0.18, 0.26 and 0.81 nmol progesterone/min/mg protein for the overall complex, the 3βol dehydrogenase and the Δ 4−5 isomerase respectively. All enzyme activities were unchanged by thyroidectomy. Similar results were obtained for corresponding microsomal activities whose values were in the same range. For both microsomes and mitochondria, the dehydrogenase reaction was the limiting step in the enzyme reaction leading to progesterone formation from pregnenolone. The limiting step in corticosteroidogenesis leading to prenenolone formation by an NADPH-dependent step was slowed down by thyroidectomy, probably because the reaction that transfers energy from NADH to NADP was inhibited. The enzyme complex leading to progesterone which involves NAD + as cofactor, was unchanged by thyroidectomy. Thyroid hormones may therefore affect the availability of the energy mechanisms connected with the proton motive force, since thyroidectomy reduces both the phosphorylative oxidation and energy dependent hydroxylation reactions involved in steroidogenesis.