Primary human astrocytes produce 24(S),25-epoxycholesterol with implications for brain cholesterol homeostasis

Cholesterol is an essential component of the CNS and its metabolism in the brain has been implicated in various neurodegenerative diseases. The oxysterol produced from cholesterol, 24( S )-hydroxycholesterol, is known to be an important regulator of brain cholesterol homeostasis. In this study, we focussed on another oxysterol, 24( S ),25-epoxycholesterol (24,25EC), which has not been studied before in a neurological context. 24,25EC is unique in that it is synthesized in a shunt in the mevalonate pathway, parallel to cholesterol and utilizing the same enzymes. Considering that all the cholesterol present in the brain is derived from de novo synthesis, we investigated whether or not primary human neurons and astrocytes can produce 24,25EC. We found that astrocytes produced more 24,25EC than neurons under basal conditions, but both cell types had the capacity to synthesize this oxysterol when the enzyme 2,3-oxidosqualene cyclase was partially inhibited. Furthermore, both added 24,25EC and stimulated cellular production of 24,25EC (by partial inhibition of 2,3-oxidosqualene cyclase) modulated expression of key cholesterol-homeostatic genes regulated by the liver X receptor and the sterol regulatory element-binding protein-2. Moreover, we found that 24,25EC synthesized in astrocytes can be taken up by neurons and exert downstream effects on gene regulation. In summary, we have identified 24,25EC as a novel neurosterol which plays a likely role in brain cholesterol homeostasis.     

Vitamin E decreases endogenous cholesterol synthesis and apo-AI-mediated cholesterol secretion in Caco-2 cells

Intestine is the gateway for newly absorbed tocopherols. This organ also plays a crucial role in cholesterol metabolism. Because tocopherols are known to impact cholesterol metabolism in the liver, we hypothesized that tocopherols could also modulate cholesterol metabolism in the intestine. This study aimed to verify this hypothesis and to unveil the mechanisms involved, using Caco-2 cells as a model of the human intestinal cell.Both α- and γ-tocopherol significantly (P<.05) decreased endogenous cholesterol synthesis and apo-AI-mediated cholesterol secretion in Caco-2 cells. Tocopherols down-regulated (P<.05) up to half of the genes involved in the cholesterol synthesis pathway, together with CYP27A1, which is involved in oxysterol production. The activity of this enzyme, as well as the levels of intracellular oxysterols, was significantly diminished by tocopherols. Finally, tocopherols significantly reduced ABCA1 mRNA levels in Caco-2 cells.We conclude that tocopherols impair the endogenous synthesis and apo-AI-mediated secretion of cholesterol in Caco-2 cells. This effect involves a down-regulation of genes involved in the cholesterol synthesis pathway, resulting in down-regulation of CYP27A1 which, in turn, diminishes oxysterol concentrations. The outcome is a decrease of LXR activity, resulting in down-regulation of ABCA1. These data reinforce the effect of α- and γ-tocopherol on cholesterol metabolism via gene expression regulation.     

Oxysterols, cholesterol homeostasis, and Alzheimer disease

Aberrant cholesterol metabolism has been implicated in Alzheimer disease (AD) and other neurological disorders. Oxysterols and other cholesterol oxidation products are effective ligands of liver X activated receptor (LXR) nuclear receptors, major regulators of genes subserving cholesterol homeostasis. LXR receptors act as molecular sensors of cellular cholesterol concentrations and effectors of tissue cholesterol reduction. Following their interaction with oxysterols, activation of LXRs induces the expression of ATP-binding cassette, sub-family A member 1, a pivotal modulator of cholesterol efflux. The relative solubility of oxysterols facilitates lipid flux among brain compartments and egress across the blood-brain barrier. Oxysterol-mediated LXR activation induces local apoE biosynthesis (predominantly in astrocytes) further enhancing cholesterol re-distribution and removal. Activated LXRs invoke additional neuroprotective mechanisms, including induction of genes governing bile acid synthesis (sterol elimination pathway), apolipoprotein elaboration, and amyloid precursor protein processing. The latter translates into attenuated beta-amyloid production that may ameliorate amyloidogenic neurotoxicity in AD brain. Stress-induced up-regulation of the heme-degrading enzyme, heme oxygenase-1 in AD-affected astroglia may impact central lipid homeostasis by promoting the oxidation of cholesterol to a host of oxysterol intermediates. Synthetic oxysterol-mimetic drugs that activate LXR receptors within the CNS may provide novel therapeutics for management of AD and other neurological afflictions characterized by deranged tissue cholesterol homeostasis.     

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.     

Liver X receptor signaling pathways in cardiovascular disease

The liver X receptors alpha and beta (LXRalpha and LXRbeta) are members of the nuclear receptor family of proteins that are critical for the control of lipid homeostasis in vertebrates. The endogenous activators of these receptors are oxysterols and intermediates in the cholesterol biosynthetic pathway. LXRs serve as cholesterol sensors that regulate the expression of multiple genes involved in the efflux, transport, and excretion of cholesterol. Recent studies have outlined the importance of LXR signaling pathways in the development of metabolic disorders such as hyperlipidemia and atherosclerosis. Synthetic LXR agonists inhibit the development of atherosclerosis in murine models, an effect that is likely to result from the modulation of both metabolic and inflammatory gene expression. These observations identify the LXR pathway as a potential target for therapeutic intervention in human cardiovascular disease.     

24(S),25-epoxycholesterol: a messenger for cholesterol homeostasis

The oxysterol 24(S),25-epoxycholesterol is made in a shunt in the cholesterol biosynthetic pathway in all cholesterogenic cells. Evidence is emerging that endogenous 24(S),25-epoxycholesterol can work at several levels to control acute cholesterol homeostasis. For instance, this oxysterol suppresses activation of the master regulators of cholesterol homeostasis, the sterol regulatory element binding proteins. Indeed, 24(S),25-epoxycholesterol appears to serve as a measure of cholesterol synthesis and to protect against surges in the production of this potentially cytotoxic molecule. In addition, endogenous 24(S),25-epoxycholesterol is a natural ligand for the liver X receptors which induce expression of cholesterol efflux-related genes. Levels of endogenous 24(S),25-epoxycholesterol can be artificially elevated by partially inhibiting the step after the start of the shunt, catalysed by oxidosqualene cyclase. The idea of manipulating a self-governing pathway for the production of a physiological regulator, that can enhance cholesterol removal and decrease uptake and synthesis, is attractive and warrants further evaluation.     

Side chain oxygenated cholesterol regulates cellular cholesterol homeostasis through direct sterol-membrane interactions

Side chain oxysterols exert cholesterol homeostatic effects by suppression of sterol regulatory element-binding protein maturation and promoting degradation of hydroxymethylglutaryl-CoA reductase. To examine whether oxysterol-membrane interactions contribute to the regulation of cellular cholesterol homeostasis, we synthesized the enantiomer of 25-hydroxycholesterol. Using this unique oxysterol probe, we provide evidence that oxysterol regulation of cholesterol homeostatic responses is not mediated by enantiospecific oxysterol-protein interactions. We show that side chain oxysterols, but not steroid ring-modified oxysterols, exhibit membrane expansion behavior in phospholipid monolayers and bilayers in vitro. This behavior is non-enantiospecific and is abrogated by increasing the saturation of phospholipid acyl chain constituents. Moreover, we extend these findings into cultured cells by showing that exposure to saturated fatty acids at concentrations that lead to endoplasmic reticulum membrane phospholipid remodeling inhibits oxysterol activity. These studies implicate oxysterol-membrane interactions in acute regulation of sterol homeostatic responses and provide new insights into the mechanism through which oxysterols regulate cellular cholesterol balance.     

Regulation of cholesterol homeostasis by the liver X receptors in the central nervous system

The nuclear oxysterol receptors liver X receptor-alpha [LXRalpha (NR1H3)] and LXRbeta (NR1H2) coordinately regulate genes involved in cholesterol homeostasis. Although both LXR subtypes are expressed in the brain, their roles in this tissue remain largely unexplored. In this report, we show that LXR agonists have marked effects on gene expression in murine brain tissue both in vitro and in vivo. In primary astrocyte cultures, LXR agonists regulated several established LXR target genes, including ATP binding cassette transporter A1, and enhanced cholesterol efflux. In contrast, little or no effect on gene expression or cholesterol efflux was detected in primary neuronal cultures. Treatment of mice with a selective LXR agonist resulted in the induction of several LXR target genes related to cholesterol homeostasis in the cerebellum and hippocampus. These data provide the first evidence that the LXRs regulate cholesterol homeostasis in the central nervous system. Because dysregulation of cholesterol balance is implicated in central nervous system diseases such as Alzheimer's and Niemann-Pick disease, pharmacological manipulation of the LXRs may prove beneficial in the treatment of these disorders.     

Oxysterols: From cholesterol metabolites to key mediators

Oxysterols are cholesterol metabolites that can be produced through enzymatic or radical processes. They constitute a large family of lipids (i.e. the oxysterome) involved in a plethora of physiological processes. They can act through GPCR (e.g. EBI2, SMO, CXCR2), nuclear receptors (LXR, ROR, ERα) and through transporters or regulatory proteins. Their physiological effects encompass cholesterol, lipid and glucose homeostasis. Additionally, they were shown to be involved in other processes such as immune regulatory functions and brain homeostasis. First studied as precursors of bile acids, they quickly emerged as interesting lipid mediators. Their levels are greatly altered in several pathologies and some oxysterols (e.g. 4β-hydroxycholesterol or 7α-hydroxycholestenone) are used as biomarkers of specific pathologies. In this review, we discuss the complex metabolism and molecular targets (including binding properties) of these bioactive lipids in human and mice. We also discuss the genetic mouse models currently available to interrogate their effects in pathophysiological settings. We also summarize the levels of oxysterols reported in two key organs in oxysterol metabolism (liver and brain), plasma and cerebrospinal fluid. Finally, we consider future opportunities and directions in the oxysterol field in order to gain a better insight and understanding of the complex oxysterol system.     

The phospholipid transfer protein gene is a liver X receptor target expressed by macrophages in atherosclerotic lesions

The liver X receptors (LXRs) are members of the nuclear receptor superfamily that are activated by oxysterols. In response to ligand binding, LXRs regulate a variety of genes involved in the catabolism, transport, and uptake of cholesterol and its metabolites. Here we demonstrate that LXRs also regulate plasma lipoprotein metabolism through control of the phospholipid transfer protein (PLTP) gene. LXR ligands induce the expression of PLTP in cultured HepG2 cells and mouse liver in vivo in a coordinate manner with known LXR target genes. Moreover, plasma phospholipid transfer activity is increased in mice treated with the synthetic LXR ligand GW3965. Unexpectedly, PLTP expression was also highly inducible by LXR in macrophages, a cell type not previously recognized to express this enzyme. The ability of synthetic and oxysterol ligands to regulate PLTP mRNA in macrophages and liver is lost in animals lacking both LXRalpha and LXRbeta, confirming the critical role of these receptors. We further demonstrate that the PLTP promoter contains a high-affinity LXR response element that is bound by LXR/RXR heterodimers in vitro and is activated by LXR/RXR in transient-transfection studies. Finally, immunohistochemistry studies reveal that PLTP is highly expressed by macrophages within human atherosclerotic lesions, suggesting a potential role for this enzyme in lipid-loaded macrophages. These studies outline a novel pathway whereby LXR and its ligands may modulate lipoprotein metabolism.