Phosphorylation of the liver X receptors

The liver X receptors (LXRs) function as nutritional sensors for cholesterol and have important roles in lipid metabolism, glucose homeostasis, and inflammation. We provide the first evidence that LXRs are phosphorylated proteins. Mutational analysis and metabolic labeling indicate LXRalpha is phosphorylated on serine 198 in the hinge region. This is a consensus target for the MAPK family. A phosphorylation-deficient mutant, LXRalpha S198A, remains nuclear and responds to ligands like the wild-type protein. The biological significance of LXR phosphorylation remains to be elucidated but could provide a novel mechanism for the regulation of LXR signaling pathways and cellular metabolism.     

T0901317 is a potent PXR ligand: implications for the biology ascribed to LXR

The liver X receptors (LXRalpha and beta) are nuclear receptors that coordinate carbohydrate and lipid metabolism. Insight into the physiologic roles of the LXRs has been greatly facilitated by the discovery of potent synthetic agonists. Here we show that one of these compounds, T0901317, is also a high-affinity ligand for the xenobiotic receptor pregnane X receptor (PXR). T0901317 binds and activates PXR with the same nanomolar potency with which it stimulates LXR activity. T0901317 induces expression not only of LXR target genes, but also of PXR target genes in cells and animals, including the scavenger receptor CD36, a property not shared by more specific LXR ligands, such as GW3965. Activation of PXR targets may explain why T0901317 induces dramatic liver steatosis, while GW3965 has a milder effect. These results suggest that many of the biological activities heretofore associated with LXR activation may be mediated by PXR, not LXR. Since T0901317 has been widely used in animals to study LXR function, the in vivo effects of this compound ascribed to LXR activation should be re-examined.     

Ligand-binding regulation of LXR/RXR and LXR/PPAR heterodimerizations: SPR technology-based kinetic analysis correlated with molecular dynamics simulation

Liver X receptor (LXR) and peroxisome proliferator-activated receptor (PPAR) are two members of nuclear receptors involved in the nutrient metabolisms of dietary fatty acid and cholesterol. They are found to be of cross-talk function in that LXR regulates fatty acid synthesis and PPAR controls fatty acid degradation. LXRs (LXRalpha and LXRbeta) function by forming obligate heterodimers with the retinoid X receptor (RXR), and subsequently binding to specific DNA response elements within the regulatory regions of their target genes. In this work, the kinetic features concerning LXR/RXR and LXR/PPAR interactions have been fully investigated using surface plasmon resonance (SPR) technology. It is found that LXRs could bind to all the three PPAR subtypes, PPARalpha, PPARgamma and PPARdelta with different binding affinities, and such receptor/receptor interactions could be regulated by ligand binding. Moreover, molecular dynamics (MD) simulations were performed on six typical complex models. The results revealed that ligands may increase the interaction energies between the receptor interfaces of the simulated receptor/receptor complexes. The MD results are in agreement with the SPR data. Further analyses on the MD results indicated that the ligand binding might increase the hydrogen bonds between the interfaces of the receptor/receptor complex.     

Improved metabolic control by depletion of Liver X Receptors in mice

Liver X Receptors (LXRs) coordinate the regulation of lipid and carbohydrate metabolism and insulin signaling. LXR-ligands lower plasma glucose in hyperglycemic rodents and have consequently been proposed as anti-diabetic agents. We investigated the metabolic effects induced by high carbohydrate diet in LXRalpha(-/-)beta(-/-) mice. Irrespective of diets, LXRalpha(-/-)beta(-/-) mice had reduced fatty acid, insulin, and C-peptide plasma levels than wild-type controls, suggesting a lower insulin production. High carbohydrate diet decreased the plasma glucose levels and the homeostasis model assessment (HOMA)-index in LXRalpha(-/-)beta(-/-) mice and increased hepatic triglyceride content and mRNA levels of lipogenic genes in wild-type and LXRalpha(-/-)beta(-/-) mice, proportionally. In wild-type mice high carbohydrate diet was associated with induced expression of LXR (1.5-fold), despite unchanged SREBP-1c expression. LXRalpha(-/-)beta(-/-) mice responded to this diet by induction of SREBP-1c. Our study suggests that in LXRalpha(-/-)beta(-/-) mice, glucose utilization seems to be privileged possibly due to reduced circulating free fatty acid levels.     

Rhein Protects against Obesity and Related Metabolic Disorders through Liver X Receptor-Mediated Uncoupling Protein 1 Upregulation in Brown Adipose Tissue

Liver X receptors (LXRs) play important roles in regulating cholesterol homeostasis, and lipid and energy metabolism. Therefore, LXR ligands could be used for the management of metabolic disorders. We evaluated rhein, a natural compound from Rheum palmatum L., as an antagonist for LXRs and investigated its anti-obesity mechanism in high-fat diet-fed mice. Surface plasmon resonance assays were performed to examine the direct binding of rhein to LXRs. LXR target gene expression was assessed in 3T3-L1 adipocytes and HepG2 hepatic cells in vitro. C57BL/6J mice fed a high-fat diet were orally administered with rhein for 4 weeks, and then the expression levels of LXR-related genes were analyzed. Rhein bound directly to LXRs. The expression levels of LXR target genes were suppressed by rhein in 3T3-L1 and HepG2 cells. In white adipose tissue, muscle and liver, rhein reprogrammed the expression of LXR target genes related to adipogenesis and cholesterol metabolism. Rhein activated uncoupling protein 1 (UCP1) expression in brown adipose tissue (BAT) in wild-type mice, but did not affect UCP1 expression in LXR knockout mice. In HIB-1B brown adipocytes, rhein activated the UCP1 gene by antagonizing the repressive effect of LXR on UCP1 expression. This study suggests that rhein may protect against obesity and related metabolic disorders through LXR antagonism and regulation of UCP1 expression in BAT.     

Identification of human low-density lipoprotein receptor as a novel target gene regulated by liver X receptor alpha

Liver X receptor alpha (LXRalpha) is a member of the nuclear receptor superfamily that is activated by oxysterols, and plays a pivotal role in regulating the metabolism, transport and uptake of cholesterol. Here, we demonstrate that LXRalpha also regulates the low-density lipoprotein receptor (LDLR) gene, which mediates the endocytic uptake of LDL cholesterol in the liver. An LXR agonist induced the expression of LDLR in cultured hepatoblastoma cells. Moreover, the LDLR promoter contained an LXR response element that was recognized by LXRalpha/RXRalpha (retinoid X receptor alpha) heterodimers in hepatoblastoma cells. These results suggest a novel pathway whereby LXRalpha might modulate cholesterol metabolism.     

Synthesis, modification, and evaluation of (R)-de-O-methyllasiodiplodin and analogs as nonsteroidal antagonists of mineralocorticoid receptor

Macrolide (R)-de-O-methyllasiodiplodin (1), discovered to be a potent nonsteroidal antagonist of the mineralocorticoid receptor (MR), was synthesized via an efficient method and evaluated for MR antagonistic activity together with its analogs. Among all the tested compounds, compounds 18a, 18b and 18c, exhibited more potent antagonistic activity against MR with IC₅₀ values ranging from 0.58 to 1.11 μM. Generally, it was obviously demonstrated that acetylation at phenolic hydroxyl groups and the ring size in analogs of 1 were very important for MR antagonist activity.     

Trans geometric isomers of EPA decrease LXRalpha-induced cellular triacylglycerol via suppression of SREBP-1c and PGC-1beta

Dietary mono- or di-trans fatty acids with chain lengths of 18-22 increase the risk of cardiovascular diseases because they increase LDL cholesterol and decrease HDL cholesterol in the plasma. However, the effects of trans isomers of PUFAs on lipid metabolism remain unknown. Dietary PUFAs, especially eicosapentaenoic acid (EPA) in marine oils, improve serum lipid profiles by suppressing liver X receptor alpha (LXRalpha) activity in the liver. In this study, we compared the effects of trans geometric isomers of eicosapentaenoic acid (TEPA) on triacylglycerol synthesis induced by a synthetic LXRalpha agonist (T0901317) with the effects of EPA in HepG2 cells. TEPA significantly decreased the amount of cellular triacylglycerol and the expression of mRNAs encoding fatty acid synthase, stearoyl-CoA desaturase-1, and glycerol-3-phosphate acyltransferase induced by T0901317 compared with EPA. However, there was no significant difference between the suppressive effect of TEPA or EPA on the expression of sterol-regulatory element binding protein-1c (SREBP-1c) induced by T0901317. We found that TEPA, but not EPA, decreased the mRNA expression of peroxisome proliferator-activated receptor gamma coactivator 1beta (PGC-1beta), which is a coactivator of both LXRalpha and SREBP-1. These results suggest that the hypolipidemic effect of TEPA can be attributed to a decrease not only in SREBP-1 but also in PGC-1beta expression.     

Probing electron transfer reactions between two azurins from Alcaligenes xylosoxidans GIFU 1051 with optically active Ru complexes as molecular recognition probes: Importance of the 43rd residue

Electron transfer reactions between optically-active Ru^II/III complexes incorporating (S)-/(R)-amino acids, and the two azurins, azurin-1 (az-1Cu) and azurin-2 (az-2Cu) isolated from Alcaligenes xylosoxidans GIFU 1051, have been studied to probe molecular recognition sites on the two azurins. The Ru^II/III complexes are K[Ru^II(L)(bpy)] and [Ru^III(L)(bpy)], and have a tripodal ligand (L) derived from the (S)-/(R)-amino acids, which are in turn exchanged for other functional substituent groups, such as (S)-/(R)-phenylalanine, -leucine, -valine, -alanine, and -glutamic acid (L = (S)-/(R)-BCMPA, -BCMLE, -BCMVA, -BCMAL, and -BCMGA). In the oxidation reaction of az-1Cu^I promoted by the Ru^III complexes, the kinetic parameters exhibited enantio- and stereo-selectivities, while the same reaction of az-2Cu^I was less enantio- and stereo-selective. These differences suggest that the processes of formation of the activated states are different for the two azurins. On the other hand, such a difference has not been observed for az-1 and az-2 with respect to the reduction reactions promoted by both azurins Cu^II by the Ru^II complexes within the experimental error. This suggests that the neutrality of the Ru complexes is important for precise molecular recognition of azurins. His117 has been proposed as the electron transfer site. The local structures in the vicinity of the His117 side chain in the two azurins, are essentially identical with the exception of the 43rd residue, Val43 and Ala43 for az-1 and az-2, respectively. Electron transfer reactions between Ru^III complexes and a mutant azurin, V43A-az-1, were also carried out. Interestingly, the activation parameters estimated were very similar to those of az-2, indicating that the 43rd residue acts as the electron transfer site in azurins and provides rationalization for the different mechanisms of az-1 and az-2 in redox reactions.     

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.     

Molecular determinants of the interactions between SRC-1 and LXR/RXR heterodimers

Liver X receptor (LXR)/retinoid X receptor (RXR) heterodimers have been shown to perform critical functions in cholesterol and lipid metabolism. Here, we have conducted a comparative analysis of the contributions of LXR and RXR binding to steroid receptor coactivator-1 (SRC-1), which contains three copies of the NR box. We demonstrated that the coactivator-binding surface of LXR, but not that of RXR, is critically important for physical and functional interactions with SRC-1, thereby confirming that RXR functions as an allosteric activator of SRC-1-LXR interaction. Notably, we identified NR box-2 and -3 as the essential binding targets for the SRC-1-induced stimulation of LXR transactivity, and observed the competitive in vitro binding of NR box-2 and -3 to LXR.

Structured summary

MINT-7986678, MINT-7986639, MINT-7986700, MINT-7986720, MINT-7986736, MINT-7986760, MINT-7986787: LXR (uniprotkb:Q13133) physically interacts (MI:0915) with SRC1 (uniprotkb:Q15788) and RXR (uniprotkb:P19793) by pull down (MI:0096)MINT-7986596, MINT-7986621: SRC1 (uniprotkb:Q15788) physically interacts (MI:0915) with LXR (uniprotkb:Q13133) by pull down (MI:0096)MINT-7986555, MINT-7986575: LXR (uniprotkb:Q13133) physically interacts (MI:0915) with SRC1 (uniprotkb:Q15788) by two hybrid (MI:0018)MINT-7986808, MINT-7986907, MINT-7986890: SRC1 (uniprotkb:Q15788) binds (MI:0407) to LXR (uniprotkb:Q13133) by pull down (MI:0096)MINT-7986822, MINT-7986848, MINT-7986865: SRC1 (uniprotkb:Q15788) binds (MI:0407) to RXR (uniprotkb:P19793) by pull down (MI:0096)     

Oxysterol represses high-affinity IgE receptor-stimulated mast cell activation in Liver X receptor-dependent and -independent manners

Oxysterols activating liver X receptors (LXRs) repress expression of pro-inflammatory genes and have anti-inflammatory effects. Here, we show for the first time that bone marrow-derived murine mast cells (BMMCs) predominantly express LXRβ. 25-hydroxycholesterol, a representative LXR activating oxysterol, suppressed IL-6 production and degranulation response in BMMCs following engagement of high-affinity IgE receptor (FcεRI). Interestingly, 25-hydroxycholesterol reduced cell-surface FcεRI expression by inhibiting assembly of FcεRIα and FcεRIβ. We demonstrate that LXR activation was involved in the suppression of IL-6 production in BMMCs, but that reduced FcεRI expression and degranulation response was mediated in an LXR-independent manner.     

X-Ray Structures of the LXRα LBD in Its Homodimeric Form and Implications for Heterodimer Signaling

Liver X receptors (LXRs) are nuclear receptors that are central regulators of cholesterol homeostasis, and synthetic LXR agonists have shown promise as promoters of reverse cholesterol transport and anti-inflammatory agents. Here, we present three X-ray structures of three different agonists bound to the ligand binding domain of LXRα. These compounds are GW3965, F₃methylAA, and a benzisoxazole urea, and we show that these diverse chemical scaffolds address common structural themes, leading to high binding affinity for LXR. Our structures show the LXRα ligand binding domain in its homodimeric form, an arrangement previously thought to be stereochemically difficult. A comparison with existing structures of the LXRβ homodimer and LXRα:RXR (retinoid X receptor) heterodimers explains differences in dimer affinity and leads us to propose a model for allosteric activation in nuclear receptor dimers, in which an unactivated RXR partner provides an inhibitory tail wrap to the cofactor binding pocket of LXR.     

Enhancement of androgen receptor expression induced by (R)-methanandamide in prostate LNCaP cells

It has been recently shown that cannabinoids may regulate the growth of many cell types. In the present work we examined the effect of the anandamide analogue (R)-methanandamide (MET) on androgen-dependent prostate LNCaP cell growth. We found that 0.1 microM MET had a mitogenic effect measured by [(3)H]thymidine incorporation into DNA. The effect exerted by MET was blocked by the cannabinoid receptor antagonists SR141716 (SR1) and SR144528 (SR2) as well as by the phosphoinositide 3-kinase (PI3K) inhibitor LY294002, suggesting an involvement of cannabinoid receptors and the PI3K pathway in the mechanism of MET action. MET treatment of LNCaP cells also induced an up-regulation of androgen receptor expression that was blocked by the two cannabinoid receptor antagonists SR1 and SR2. These results show for the first time that cannabinoids may modify androgen receptor expression in an androgen-dependent cell line and by this mechanism could regulate prostate cell growth.