On the regulatory role of side-chain hydroxylated oxysterols in the brain. Lessons from CYP27A1 transgenic and Cyp27a1−/− mice

The two oxysterols, 27-hydroxycholesterol (27OH) and 24S-hydroxycholesterol (24OH), are both inhibitors of cholesterol synthesis and activators of the liver X receptor (LXR) in vitro. Their role as physiological regulators under in vivo conditions is controversial, however. In the present work, we utilized a previously described mouse model with overexpressed human sterol 27-hydroxylase (CYP27A1). The levels of 27OH were increased about 12-fold in the brain. The brain levels of HMG-CoA reductase mRNA and HMG-CoA synthase mRNA levels were increased. In accordance with increased cholesterol synthesis, most of the cholesterol precursors were also increased. The level of 24OH, the dominating oxysterol in the brain, was decreased by about 25%, most probably due to increased metabolism by CYP27A1. The LXR target genes were unaffected or slightly changed in a direction opposite to that expected for LXR activation. In the brain of Cyp27^−/− mice, cholesterol synthesis was slightly increased, with increased levels of cholesterol precursors but normal mRNA levels of HMG-CoA reductase and HMG-CoA synthase. The mRNA levels corresponding to LXR target genes were not affected. The results are consistent with the possibility that both 24OH and 27OH are physiological suppressors of cholesterol synthesis in the brain. The results do not support the contention that 27OH is a general activator of LXR target genes in this organ.     

27-hydroxycholesterol is an endogenous ligand for liver X receptor in cholesterol-loaded cells

The nuclear receptors liver X receptor alpha (LXRalpha) (NR1H3) and LXRbeta (NR1H2) are important regulators of genes involved in lipid metabolism, including ABCA1, ABCG1, and sterol regulatory element-binding protein-1c (SREBP-1c). Although it has been demonstrated that oxysterols are LXR ligands, little is known about the identity of the physiological activators of these receptors. Here we confirm earlier studies demonstrating a dose-dependent induction of ABCA1 and ABCG1 in human monocyte-derived macrophages by cholesterol loading. In addition, we show that formation of 27-hydroxycholesterol and cholestenoic acid, products of CYP27 action on cholesterol, is dependent on the dose of cholesterol used to load the cells. Other proposed LXR ligands, including 20(S)-hydroxycholesterol, 22(R)-hydroxycholesterol, and 24(S),25-epoxycholesterol, could not be detected under these conditions. A role for CYP27 in regulation of cholesterol-induced genes was demonstrated by the following findings. 1) Introduction of CYP27 into HEK-293 cells conferred an induction of ABCG1 and SREBP-1c; 2) upon cholesterol loading, CYP27-expressing cells induce these genes to a greater extent than in control cells; 3) in CYP27-deficient human skin fibroblasts, the induction of ABCA1 in response to cholesterol loading was ablated; and 4) in a coactivator association assay, 27-hydroxycholesterol functionally activated LXR. We conclude that 27-hydroxylation of cholesterol is an important pathway for LXR activation in response to cholesterol overload.     

12α-Hydroxylated bile acid induces hepatic steatosis with dysbiosis in rats

There is an increasing need to explore the mechanism of the progression of non-alcoholic fatty liver disease. Steroid metabolism is closely linked to hepatic steatosis and steroids are excreted as bile acids (BAs). Here, we demonstrated that feeding WKAH/HkmSlc inbred rats a diet supplemented with cholic acid (CA) at 0.5 g/kg for 13 weeks induced simple steatosis without obesity. Liver triglyceride and cholesterol levels were increased accompanied by mild elevation of aminotransferase activities. There were no signs of inflammation, insulin resistance, oxidative stress, or fibrosis. CA supplementation increased levels of CA and taurocholic acid (TCA) in enterohepatic circulation and deoxycholic acid (DCA) levels in cecum with an increased ratio of 12α-hydroxylated BAs to non-12α-hydroxylated BAs. Analyses of hepatic gene expression revealed no apparent feedback control of BA and cholesterol biosynthesis. CA feeding induced dysbiosis in cecal microbiota with enrichment of DCA producers, which underlines the increased cecal DCA levels. The mechanism of steatosis was increased expression of Srebp1 (positive regulator of liver lipogenesis) through activation of the liver X receptor by increased oxysterols in the CA-fed rats, especially 4β-hydroxycholesterol (4βOH) formed by upregulated expression of hepatic Cyp3a2, responsible for 4βOH formation. Multiple regression analyses identified portal TCA and cecal DCA as positive predictors for liver 4βOH levels. The possible mechanisms linking these predictors and upregulated expression of Cyp3a2 are discussed. Overall, our observations highlight the role of 12α-hydroxylated BAs in triggering liver lipogenesis and allow us to explore the mechanisms of hepatic steatosis onset, focusing on cholesterol and BA metabolism.     

Studies on the relationships between 7α-hydroxylation and the ability of 25- and 27-hydroxycholesterol to suppress the activity of HMG-CoA reductase

The metabolism of 25-hydroxycholesterol in different cell types was studied and the role of 7α-hydroxylation for the effect of 25-hydroxycholesterol on the activity of HMG-CoA reductase was determined. Human diploid fibroblasts (HDF) and the human melanoma cell line SK-MEL-2 converted 25-hydroxycholesterol into 7α,25-dihydroxycholesterol and 7α,25-dihydroxy-4-cholesten-3-one while the virus-transformed fibroblast line 90VA-VI, the colon carcinoma cell line WiDr and the breast cancer cell line MDA-231 did not express 7α-hydroxylase activity. The 7α-hydroxylation of 25-hydroxycholesterol in HDF could be stimulated by dexamethasone and cortisol and inhibited by metyrapone. An unidentified, possibly 4-hydroxylated, metabolite was formed by 90VA-VI cells and a polar, probably conjugated, metabolite was formed by WiDr cells. The 7α-hydroxylated metabolites of 25-hydroxycholesterol suppressed the activity of HMG-CoA reductase to a similar extent as 25-hydroxycholesterol in HDF but not in 90VA-VI cells, while the 7α-hydroxylated metabolites of 27-hydroxycholesterol suppressed the activity of HMG-CoA reductase also in 90VA-VI cells. The suppression of HMG-CoA reductase activity by 25- and 27-hydroxycholesterol was decreased or abolished by dehydroepiandrosterone or pregnenolone which have little or no effect on the 7α-hydroxylation. The results indicate that 7α-hydroxylation is not directly involved, positively or negatively, in the action of 25- or 27-hydroxycholesterol as suppressors of HMG-CoA reductase activity.     

Lipid reducing activity of novel cholic acid (CA) analogs: Design,synthesis and preliminary mechanism study

Bile acids, initially discovered as endogenous ligands of farnesoid X receptor (FXR), play a central role in the regulation of triglyceride and cholesterol metabolism and have recently emerged as a privileged structure for interacting with nuclear receptors relevant to a large array of metabolic processes. In this paper, phenoxy containing cholic acid derivatives with excellent drug-likeness have been designed, synthesized, and assayed as agents against cholesterol accumulation in Raw264.7 macrophages. The most active compound 14b reduced total cholesterol accumulation in Raw264.7 cells up to 30.5% at non-toxic 10 μM and dosage-dependently attenuated oxLDL-induced foam cell formation. Western blotting and qPCR results demonstrate that 14b reduced both cholesterol and lipid in Raw264.7 cells through (1) increasing the expression of cholesterol transporters ABCA1 and ABCG1, (2) accelerating ApoA1-mediated cholesterol efflux. Through a cell-based luciferase reporter assay and molecular docking analysis, LXR was identified as the potential target for 14b. Interestingly, unlike conventional LXR agonist, 14b did not increase lipogenesis gene SREBP-1c expression. Overall, these diverse properties disclosed herein highlight the potential of 14b as a promising lead for further development of multifunctional agents in the therapy of cardiovascular disease.     

Synthesis and activity evaluation of a series of cholanamides as modulators of the liver X receptors

The Liver X receptors (LXRs) are members of the nuclear receptor family, that play fundamental roles in cholesterol transport, lipid metabolism and modulation of inflammatory responses. In recent years, the synthetic steroid N,N-dimethyl-3β-hydroxycholenamide (DMHCA) arised as a promising LXR ligand. This compound was able to dissociate certain beneficial LXRs effects from those undesirable ones involved in triglyceride metabolism. Here, we synthetized a series of DMHCA analogues with different modifications in the steroidal nucleus involving the A/B ring fusion, that generate changes in the overall conformation of the steroid. The LXRα and LXRβ activity of these analogues was evaluated by using a luciferase reporter assay in BHK21 cells. Compounds were tested in both the agonist and antagonist modes. Results indicated that the agonist/antagonist profile is dependent on the steroid configuration at the A/B ring junction. Notably, in contrast to DMHCA, the amide derived from lithocholic acid (2) with an A/B cis configuration and its 6,19-epoxy analogue 4 behaved as LXRα selective agonists, while the 2,19-epoxy analogues with an A/B trans configuration were antagonists of both isoforms. The binding mode of the analogues to both LXR isoforms was assessed by using 50?ns molecular dynamics (MD) simulations. Results revealed conformational differences between LXRα- and LXRβ-ligand complexes, mainly in the hydrogen bonding network that involves the C-3 hydroxyl. Overall, these results indicate that the synthetized DMHCA analogues could be interesting candidates for a therapeutic modulation of the LXRs.     

22(R)-hydroxycholesterol and pioglitazone synergistically decrease cholesterol ester via the PPARγ–LXRα–ABCA1 pathway in cholesterosis of the gallbladder

Cholesterosis is a disease of cholesterol metabolism characterized by the presence of excessive lipid droplets in the cytoplasm. These lipid droplets are mainly composed of cholesterol esters derived from free cholesterol. The removal of excess cholesterol from gallbladder epithelial cells (GBECs) is very important for the maintenance of intracellular cholesterol homeostasis and the preservation of gallbladder function. Several lines of evidence have indicated that the activation of either peroxisome proliferator-activated receptor gamma (PPARγ) or liver X receptor α (LXRα) relates to cholesterol efflux. While pioglitazone can regulate the activation of PPARγ, 22(R)-hydroxycholesterol can activate LXRα and is a metabolic intermediate in the biosynthesis of steroid hormones. However, the effect of 22(R)-hydroxycholesterol in combination with pioglitazone on cholesterosis of the gallbladder is unclear. GBECs were treated with pioglitazone, 22(R)-hydroxycholesterol or PPARγ siRNA followed by Western blot analysis for ATP-binding cassette transporter A1 (ABCA1), PPARγ and LXRα. Cholesterol efflux to apoA-I was determined, and Oil Red O staining was performed to monitor variations in lipid levels in treated GBECs. Our data showed that 22(R)-hydroxycholesterol can modestly up-regulate LXRα while simultaneously increasing ABCA1 by 56%. The combination of 22(R)-hydroxycholesterol and pioglitazone resulted in a 3.64-fold increase in ABCA1 expression and a high rate of cholesterol efflux. Oil Red O staining showed an obvious reduction in the lipid droplets associated with cholesterosis in GBECs. In conclusion, the present findings indicate that the anti-lipid deposition action of 22(R)-hydroxycholesterol combined with pioglitazone involves the activation of the PPARγ–LXRα–ABCA1 pathway, increased ABCA1 expression and the efflux of cholesterol from GBECs. Thus, 22(R)-hydroxycholesterol synergistically combined with pioglitazone to produce a remarkable effect on lipid deposition in cholesterosis GBECs.     

The discovery of tertiary-amine LXR agonists with potent cholesterol efflux activity in macrophages

The liver X receptors (LXR) play a key role in cholesterol homeostasis and lipid metabolism. SAR studies around tertiary-amine lead molecule 2, an LXR full agonist, revealed that steric and conformational changes to the acetic acid and propanolamine groups produce dramatic effects on agonist efficacy and potency. The new analogs possess good functional activity, demonstrating the ability to upregulate LXR target genes, as well as promote cholesterol efflux in macrophages.     

Enzymatic reduction of oxysterols impairs LXR signaling in cultured cells and the livers of mice

Liver X receptors (LXRs) are nuclear receptors that play crucial roles in lipid metabolism in vivo and are activated by oxysterol ligands in vitro. The identity of the ligand that activates LXRs in vivo is uncertain. Here we provide two lines of evidence that oxysterols are LXR ligands in vitro and in vivo. First, overexpression of an oxysterol catabolic enzyme, cholesterol sulfotransferase, inactivates LXR signaling in several cultured mammalian cell lines but does not alter receptor response to the nonsterol agonist T0901317. Adenovirus-mediated expression of the enzyme in mice prevents dietary induction of hepatic LXR target genes by cholesterol but not by T0901317. Second, triple-knockout mice deficient in the biosynthesis of three oxysterol ligands of LXRs, 24S-hydroxycholesterol, 25-hydroxycholesterol, and 27-hydroxycholesterol, respond to dietary T0901317 by inducing LXR target genes in liver but show impaired responses to dietary cholesterol. We conclude that oxysterols are in vivo ligands for LXR.     

Implications of cerebrovascular ATP-binding cassette transporter G1 (ABCG1) and apolipoprotein M in cholesterol transport at the blood-brain barrier

Impaired cholesterol/lipoprotein metabolism is linked to neurodegenerative diseases such as Alzheimer's disease (AD). Cerebral cholesterol homeostasis is maintained by the highly efficient blood-brain barrier (BBB) and flux of the oxysterols 24(S)-hydroxycholesterol and 27-hydroxycholesterol, potent liver-X-receptor (LXR) activators. HDL and their apolipoproteins are crucial for cerebral lipid transfer, and loss of ATP binding cassette transporters (ABC)G1 and G4 results in toxic accumulation of oxysterols in the brain. The HDL-associated apolipoprotein (apo)M is positively correlated with pre-β HDL formation in plasma; its presence and function in the brain was thus far unknown. Using an in vitro model of the BBB, we examined expression, regulation, and functions of ABCG1, ABCG4, and apoM in primary porcine brain capillary endothelial cells (pBCEC). RT Q-PCR analyses and immunoblotting revealed that in addition to ABCA1 and scavenger receptor, class B, type I (SR-BI), pBCEC express high levels of ABCG1, which was up-regulated by LXR activation. Immunofluorescent staining, site-specific biotinylation and immunoprecipitation revealed that ABCG1 is localized both to early and late endosomes and on apical and basolateral plasma membranes. Using siRNA interference to silence ABCG1 (by 50%) reduced HDL-mediated [³H]-cholesterol efflux (by 50%) but did not reduce [³H]-24(S)-hydroxycholesterol efflux. In addition to apoA-I, pBCEC express and secrete apoM mainly to the basolateral (brain) compartment. HDL enhanced expression and secretion of apoM by pBCEC, apoM-enriched HDL promoted cellular cholesterol efflux more efficiently than apoM-free HDL, while apoM-silencing diminished cellular cholesterol release. We suggest that ABCG1 and apoM are centrally involved in regulation of cholesterol metabolism/turnover at the BBB.     

LXR/RXR activation enhances basolateral efflux of cholesterol in CaCo-2 cells

Regulation of gene expression of ATP-binding cassette transporter (ABC)A1 and ABCG1 by liver X receptor/retinoid X receptor (LXR/RXR) ligands was investigated in the human intestinal cell line CaCo-2. Neither the RXR ligand, 9-cis retinoic acid, nor the natural LXR ligand 22-hydroxycholesterol alone altered ABCA1 mRNA levels. When added together, ABCA1 and ABCG1 mRNA levels were increased 3- and 7-fold, respectively. T0901317, a synthetic non-sterol LXR agonist, increased ABCA1 and ABCG1 gene expression 11- and 6-fold, respectively. ABCA1 mass was increased by LXR/RXR activation. T0901317 or 9-cis retinoic acid and 22-hydroxycholesterol increased cholesterol efflux from basolateral but not apical membranes. Cholesterol efflux was increased by the LXR/RXR ligands to apolipoprotein (apo)A-I or HDL but not to taurocholate/phosphatidylcholine micelles. Actinomycin D prevented the increase in ABCA1 and ABCG1 mRNA levels and the increase in cholesterol efflux induced by the ligands. Glyburide, an inhibitor of ABCA1 activity, attenuated the increase in basolateral cholesterol efflux induced by T0901317. LXR/RXR activation decreased the esterification and secretion of cholesterol esters derived from plasma membranes. Thus, in CaCo-2 cells, LXR/RXR activation increases gene expression of ABCA1 and ABCG1 and the basolateral efflux of cholesterol, suggesting that ABCA1 plays an important role in intestinal HDL production and cholesterol absorption.     

Induction of intestinal ATP-binding cassette transporters by a phytosterol-derived liver X receptor agonist

The nuclear receptors liver X receptor (LXR) alpha and LXRbeta serve as oxysterol receptors and regulate the expression of genes involved in lipid metabolism. LXR activation induces the expression of ATP-binding cassette (ABC) transporters, such as ABCG5 and ABCG8, which inhibit intestinal absorption of cholesterol and phytosterols. Although several synthetic LXR agonists have been generated, these compounds have limited clinical application, because they cause hypertriglycemia by inducing the expression of lipogenic genes in the liver. We synthesized derivatives of phytosterols and found some of them to act as LXR agonists. Among them, YT-32 [(22E)-ergost-22-ene-1alpha,3beta-diol], which is related to ergosterol and brassicasterol, is the most potent LXR agonist. YT-32 directly bound to LXRalpha and LXRbeta and induced the interaction of LXRalpha with cofactors, such as steroid receptor coactivator-1, as effectively as the natural ligands, 22(R)-hydroxycholesterol and 24(S),25-epoxycholesterol. Although the nonsteroidal synthetic LXR agonist T0901317 induced the expression of intestinal ABC transporters and liver lipogenic genes, oral administration of YT-32 selectively activated intestinal ABC transporters in mice. Unlike T0901317 treatment, YT-32 inhibited intestinal cholesterol absorption without increasing plasma triglyceride levels. The phytosterol-derived LXR agonist YT-32 might selectively modulate intestinal cholesterol metabolism.     

Phospholipid Transfer Protein Is Expressed in Cerebrovascular Endothelial Cells and Involved in High Density Lipoprotein Biogenesis and Remodeling at the Blood-Brain Barrier

Phospholipid transfer protein (PLTP) is a key protein involved in biogenesis and remodeling of plasma HDL. Several neuroprotective properties have been ascribed to HDL. We reported earlier that liver X receptor (LXR) activation promotes cellular cholesterol efflux and formation of HDL-like particles in an established in vitro model of the blood-brain barrier (BBB) consisting of primary porcine brain capillary endothelial cells (pBCEC). Here, we report PLTP synthesis, regulation, and its key role in HDL metabolism at the BBB. We demonstrate that PLTP is highly expressed and secreted by pBCEC. In a polarized in vitro model mimicking the BBB, pBCEC secreted phospholipid-transfer active PLTP preferentially to the basolateral (“brain parenchymal”) compartment. PLTP expression levels and phospholipid transfer activity were enhanced (up to 2.5-fold) by LXR activation using 24(S)-hydroxycholesterol (a cerebral cholesterol metabolite) or TO901317 (a synthetic LXR agonist). TO901317 administration elevated PLTP activity in BCEC from C57/BL6 mice. Preincubation of HDL₃ with human plasma-derived active PLTP resulted in the formation of smaller and larger HDL particles and enhanced the capacity of the generated HDL particles to remove cholesterol from pBCEC by up to 3-fold. Pre-β-HDL, detected by two-dimensional crossed immunoelectrophoresis, was generated from HDL₃ in pBCEC-derived supernatants, and their generation was markedly enhanced (1.9-fold) upon LXR activation. Furthermore, RNA interference-mediated PLTP silencing (up to 75%) reduced both apoA-I-dependent (67%) and HDL₃-dependent (30%) cholesterol efflux from pBCEC. Based on these findings, we propose that PLTP is actively involved in lipid transfer, cholesterol efflux, HDL genesis, and remodeling at the BBB.     

OSBP-related protein 2 is a sterol receptor on lipid droplets that regulates the metabolism of neutral lipids

Oxysterol binding protein-related protein 2 (ORP2) is a member of the oxysterol binding protein family, previously shown to bind 25-hydroxycholesterol and implicated in cellular cholesterol metabolism. We show here that ORP2 also binds 22(R)-hydroxycholesterol [22(R)OHC], 7-ketocholesterol, and cholesterol, with 22(R)OHC being the highest affinity ligand of ORP2 (K_d 1.4 × 10⁻⁸ M). We report the localization of ORP2 on cytoplasmic lipid droplets (LDs) and its function in neutral lipid metabolism using the human A431 cell line as a model. The ORP2 LD association depends on sterol binding: Treatment with 5 μM 22(R)OHC inhibits the LD association, while a mutant defective in sterol binding is constitutively LD bound. Silencing of ORP2 using RNA interference slows down cellular triglyceride hydrolysis. Furthermore, ORP2 silencing increases the amount of [¹⁴C]cholesteryl esters but only under conditions in which lipogenesis and LD formation are enhanced by treatment with oleic acid. The results identify ORP2 as a sterol receptor present on LD and provide evidence for its role in the regulation of neutral lipid metabolism, possibly as a factor that integrates the cellular metabolism of triglycerides with that of cholesterol.     

LXR antagonists induce ABCD2 expression

X-linked adrenoleukodystrophy (X-ALD) is a rare neurodegenerative disorder characterized by the accumulation of very-long-chain fatty acids resulting from a β-oxidation defect. Oxidative stress and inflammation are also key components of the pathogenesis. X-ALD is caused by mutations in the ABCD1 gene, which encodes for a peroxisomal half ABC transporter predicted to participate in the entry of VLCFA-CoA into the peroxisome, the unique site of their β-oxidation. Two homologous peroxisomal ABC transporters, ABCD2 and ABCD3 have been proven to compensate for ABCD1 deficiency when overexpressed. Pharmacological induction of these target genes could therefore represent an alternative therapy for X-ALD patients. Since LXR activation was shown to repress ABCD2 expression, we investigated the effects of LXR antagonists in different cell lines. Cells were treated with GSK(17) (a LXR antagonist recently discovered from the GlaxoSmithKline compound collection), 22(S)-hydroxycholesterol (22S-HC, another LXR antagonist) and 22R-HC (an endogenous LXR agonist). We observed up-regulation of ABCD2, ABCD3 and CTNNB1 (the gene encoding for β-catenin, which was recently demonstrated to induce ABCD2 expression) in human HepG2 hepatoma cells and in X-ALD skin fibroblasts treated with LXR antagonists. Interestingly, induction in X-ALD fibroblasts was concomitant with a decrease in oxidative stress. Rats treated with 22S-HC showed hepatic induction of the 3 genes of interest. In human, we show by multiple tissue expression array that expression of ABCD2 appears to be inversely correlated with NR1H3 (LXRα) expression. Altogether, antagonists of LXR that are currently developed in the context of dyslipidemia may find another indication with X-ALD.     

Cholesterol,Oxysterol, Triglyceride,and Coenzyme Q Homeostasis in ALS. Evidence against the Hypothesis That Elevated 27-Hydroxycholesterol Is a Pathogenic Factor

High plasma levels of cholesterol have been suggested to be neuroprotective for the degenerative disease amyotrophic lateral sclerosis (ALS) and to be associated with increased survival time. The gene encoding cholesterol 27-hydroxylase, CYP27A1, was recently identified as a susceptibility gene for sporadic ALS. A product of this enzyme is 27-hydroxycholesterol. We investigated plasma samples from 52 ALS patients and 40 control subjects (spouses) regarding cholesterol homeostasis, lipid profiles, and coenzyme Q. Eleven of the patients carried mutations in C9orf72 and seven in SOD1. Plasma levels of 27-hydroxycholesterol were significantly lower in male patients with ALS than in controls. It was not possible to link the reduced levels to any specific mutation, and there was no significant correlation between 27-hydroxycholesterol and survival. With normalization for diet using the spouses, a correlation was found between survival and total cholesterol, very low density lipoprotein cholesterol, low density lipoprotein cholesterol, and coenzyme Q. We conclude that cholesterol, 24S-hydroxycholesterol, 25-hydroxycholesterol, 27-hydroxycholesterol and lipid profiles in plasma are of limited prognostic value in individual ALS patients.