On the role of liver X receptors in lipid accumulation in adipocytes

The pivotal role of liver X receptors (LXRs) in the metabolic conversion of cholesterol to bile acids in mice is well established. More recently, the LXRalpha promoter has been shown to be under tight regulation by peroxisome proliferator-activated receptors (PPARs), implying a role for LXRalpha in mediating the interplay between cholesterol and fatty acid metabolism. We have studied the role of LXR in fat cells and demonstrate that LXR is regulated during adipogenesis and augments fat accumulation in mature adipocytes. LXRalpha expression in murine 3T3-L1 adipocytes as well as in human adipocytes was up-regulated in response to PPARgamma agonists. Administration of a PPARgamma agonist to obese Zucker rats also led to increased LXRalpha mRNA expression in adipose tissue in vivo. LXR agonist treatment of differentiating adipocytes led to increased lipid accumulation. An increase of the expression of the LXR target genes, sterol regulatory binding protein-1 and fatty acid synthase, was observed both in vivo and in vitro after treatment with LXR agonists for 24 h. Finally, we demonstrate that fat depots in LXRalpha/beta-deficient mice are smaller than in age-matched wild-type littermates. These findings imply a role for LXR in controlling lipid storage capacity in mature adipocytes and point to an intriguing physiological interplay between LXR and PPARgamma in controlling pathways in lipid handling.     

Tissue-specific autoregulation of the LXRalpha gene facilitates induction of apoE in mouse adipose tissue

The functions of the liver X receptors (LXRs) are not well documented in adipose tissue. We demonstrate here that expression of the LXRalpha gene is highly induced in vivo and in vitro in mouse and human adipocytes in the presence of the synthetic LXR agonist T0901317. This autoregulation is caused by an identified LXR-responsive element motif in the mouse LXRalpha promoter, which is conserved in the human LXRalpha promoter. Using different LXR-deficient mice, we demonstrate that the basal expression level of LXRalpha is increased in LXRbeta(-/-) mice, whereas the basal expression level of LXRbeta is unchanged in LXRalpha(-/-) mice. The two LXRs can compensate for each other in mediating ligand-activated regulation of LXR target genes involved in lipid homeostasis in adipose tissue. Sterol regulatory element binding protein-1 (SREBP-1), ATP binding cassette transporter A1 (ABCA1), ABCG1, as well as apolipoprotein E (apoE) are induced in vivo by T0901317 in wild-type, LXRalpha(-/-) or LXRbeta(-/-) mice but not in LXRalpha(-/-)beta(-/-) mice. Although SREBP-1 and ABCG1 are induced in liver, muscle, and adipose tissue, the apoE, glucose transporter-4 (GLUT4), and LXRalpha genes are specifically induced only in adipose tissue. We suggest that an important aspect of LXRalpha autoregulation in adipose tissue may be to increase the level of LXRalpha over a threshold level necessary to induce the expression of certain target genes.     

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.     

LXRbeta is required for adipocyte growth, glucose homeostasis, and beta cell function

Liver X receptors (LXR) alpha and beta are nuclear oxysterol receptors with established roles in cholesterol, lipid, and carbohydrate metabolism. Although LXRs have been extensively studied in liver and macrophages, the importance for development and metabolism of other tissues and cell types is not as well characterized. We demonstrate here that although LXRalpha and LXRbeta are not required for adipocyte development per se, LXRbeta is required for the increase in adipocyte size that normally occurs with aging and diet-induced obesity. Similar food intake and oxygen consumption in LXRbeta-/- mice suggests that reduced storage of lipid in adipose tissue is not due to altered energy balance. Despite reduced amounts of adipose tissue, LXRbeta-/- mice on a chow diet have insulin sensitivity and levels of adipocyte hormones similar to wild type mice. However, these mice are glucose-intolerant due to impaired glucose-induced insulin secretion. Lipid droplets in pancreatic islets may result from accumulation of cholesterol esters as analysis of islet gene expression reveals that LXRbeta is required for expression of the cholesterol transporters, ABCA1 and ABCG1. Our data establish novel roles for LXRbeta in adipocyte growth, glucose homeostasis, and beta cell function.     

Multiple roles of the nuclear receptors for oxysterols liver X receptor to maintain male fertility

Oxysterol nuclear receptors liver X receptor (LXR)alpha and LXRbeta are known to regulate lipid homeostasis in cells exposed to high amounts of cholesterol and/or fatty acids. In order to elucidate the specific and redundant roles of the LXRs in the testis, we explored the reproductive phenotypes of mice deficient of LXRalpha, LXRbeta, and both, of which only the lxralpha;beta-/- mice are infertile by 5 months of age. We demonstrate that LXRalpha-deficient mice had lower levels of testicular testosterone that correlated with a higher apoptotic rate of the germ cells. LXRbeta-deficient mice showed increased lipid accumulation in the Sertoli cells and a lower proliferation rate of the germ cells. In lxralpha;beta-/- mice, fatty acid metabolism was affected through a decrease of srebp1c and increase in scd1 mRNA expression. The retinoid acid signaling pathway was also altered in lxralpha;beta-/- mice, with a higher accumulation of all-trans retinoid receptor alpha, all-trans retinoid receptor beta, and retinoic aldehyde dehydrogenase-2 mRNA. Combination of these alterations might explain the deleterious phenotype of infertility observed only in lxralpha;beta-/- mice, even though lipid homeostasis seemed to be first altered. Wild-type mice treated with a specific LXR agonist showed an increase of testosterone production involving both LXR isoforms. Altogether, these data identify new roles of each LXR, collaborating to maintain both integrity and functions of the testis.     

Activation of liver X receptors prevents statin-induced death of 3T3-L1 preadipocytes

The biological functions of liver X receptors (LXRs) alpha and beta have primarily been linked to pathways involved in fatty acid and cholesterol homeostasis. Here we report a novel role of LXR activation in protecting cells from statin-induced death. When 3T3-L1 preadipocytes were induced to differentiate by standard isobutylmethylxanthine/dexamethasone/insulin treatment in the presence of statins, they failed to differentiate and underwent massive apoptosis. The simultaneous addition of selective LXR agonists prevented the statin-induced apoptosis. By using mouse embryo fibroblasts from wild-type (LXRalpha+/+/LXRbeta+/+), LXRalpha knock-out mice (LXRalpha(-/-)/LXRbeta+/+), LXRbeta knock-out mice (LXRalpha+/-/LXRbeta(-/-)), and LXR double knock-out mice (LXRalpha(-/-)/LXRbeta(-/-)) as well as 3T3-L1 cells transduced with retroviruses expressing either wild-type LXRalpha or a dominant negative version of LXRalpha, we demonstrate that the response to LXR agonists is LXR-dependent. Interestingly, LXR-mediated rescue of statin-induced apoptosis was not related to up-regulation of genes previously shown to be involved in the antiapoptotic action of LXR. Furthermore, forced expression of Bcl-2 did not prevent statin-induced apoptosis; nor did LXR action depend on protein kinase B, whose activation by insulin was impaired in statin-treated cells. Rather, LXR-dependent rescue of statin-induced apoptosis in 3T3-L1 preadipocytes required NF-kappaB activity, since expression of a dominant negative version of IkappaBalpha prevented LXR agonist-dependent rescue of statin-induced apoptosis. Thus, the results presented in this paper provide novel insight into the action of statins on and LXR-dependent inhibition of apoptosis.     

Ligand specificity and evolution of liver X receptors

Liver X receptors (LXRs) are key regulators of lipid and cholesterol metabolism in mammals. Little is known, however, about the function and evolution of LXRs in non-mammalian species. The present study reports the cloning of LXRs from African clawed frog (Xenopus laevis), Western clawed frog (Xenopus tropicalis), and zebrafish (Danio rerio), and their functional characterization and comparison with human and mouse LXRs. Additionally, an ortholog of LXR in the chordate invertebrate Ciona intestinalis was cloned and functionally characterized. Ligand specificities of the frog and zebrafish LXRs were very similar to LXRalpha and LXRbeta from human and mouse. All vertebrate LXRs studied were activated robustly by the synthetic ligands T-0901317 and GW3965 and by a variety of oxysterols. In contrast, Ciona LXR was not activated by T-0901317 or GW3965 but was activated by a limited number of oxysterols, as well as some androstane and pregnane steroids. Pharmacophore analysis, homology modeling, and docking studies of Ciona LXR predict a receptor with a more restricted ligand-binding pocket and less intrinsic disorder in the ligand-binding domain compared to vertebrate LXRs. The results suggest that LXRs have a long evolutionary history, with vertebrate LXRs diverging from invertebrate LXRs in ligand specificity.     

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.     

Constitutive activation of LXR in macrophages regulates metabolic and inflammatory gene expression: identification of ARL7 as a direct target

Ligand activation of liver X receptors (LXRs) has been shown to impact both lipid metabolism and inflammation. One complicating factor in studies utilizing synthetic LXR agonists is the potential for pharmacologic and receptor-independent effects. Here, we describe an LXR gain-of-function system that does not depend on the addition of exogenous ligand. We generated transgenic mice expressing a constitutively active VP16-LXRα protein from the aP2 promoter. These mice exhibit increased LXR signaling selectively in adipose and macrophages. Analysis of gene expression in primary macrophages derived from two independent VP16-LXRα transgenic lines confirmed the ability of LXR to drive expression of genes involved in cholesterol efflux and fatty acid synthesis. Moreover, VP16-LXRα expression also suppressed the induction of inflammatory genes by lipopolysaccharide to a comparable degree as synthetic agonist. We further utilized VP16-LXRα-expressing macrophages to identify and validate new targets for LXRs, including the gene encoding ADP-ribosylation factor-like 7 (ARL7). ARL7 has previously been shown to transport cholesterol to the membrane for ABCA1-associated removal and thus may be integral to the LXR-dependent efflux pathway. We show that the ARL7 promoter contains a functional LXRE and can be transactivated by LXRs in a sequence-specific manner, indicating that ARL7 is a direct target of LXR. These findings provide further support for an important role of LXRs in the coordinated regulation of lipid metabolic and inflammatory gene programs in macrophages.     

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.     

Regulation of the aldo-keto reductase gene akr1b7 by the nuclear oxysterol receptor LXRalpha (liver X receptor-alpha) in the mouse intestine: putative role of LXRs in lipid detoxification processes

Liver X receptors (LXRs) regulate the expression of a number of genes involved in cholesterol and lipid metabolism after activation by their cognate oxysterol ligands. AKR1-B7 (aldo-keto reductase 1-B7) is expressed in LXR target tissues such as intestine, and because of its known role in detoxifying lipid peroxides, we investigated whether the AKR1-B7 detoxification pathway was regulated by LXRs. Here we show that synthetic LXR agonists increase the accumulation of AKR1-B7 mRNA and protein levels in mouse intestine in wild-type but not lxr(-/-) mice. Regulation of akr1b7 by retinoic X receptor/LXR heterodimers is dependent on three response elements in the proximal murine akr1b7 promoter. Two of these cis-acting elements are specific for regulation by the LXRalpha isoform. In addition, in duodenum of wild-type mice fed a synthetic LXR agonist, we observed an LXR-dependent decrease in lipid peroxidation. Our results demonstrate that akr1b7 is a direct target of LXRs throughout the small intestine, and that LXR activation plays a protective role by decreasing the deleterious effects of lipid peroxides in duodenum. Taken together, these data suggest a new role for LXRs in lipid detoxification.