Improvement of LXR-mediated lipid metabolism in nephrotic model kidney accompanied by suppression of inflammation and fibrosis.

Kidney disease affects millions of people worldwide. Chronic kidney diseases, such as diabetic nephropathy, are often accompanied by nephrotic syndrome, which causes a large amount of protein and lipid to leak out into the urine. Leaked lipids are well known to accumulate in the proximal tubules as lipid droplets. However, the role of lipid metabolism in the kidney has not been thoroughly studied, and the relationship between accumulated lipid and pathological progression is often unknown. In this study, we showed that reducing accumulated lipids by exerting an agonistic effect on Liver X receptor, one of the nuclear receptors known to play an important role in lipid metabolism, suppressed the development of pathological conditions, such as inflammation and fibrosis, in a nephrosis model. Until now, many renal disease treatments have focused on suppressing the inflammatory response. However, it is now clear that even if the direct anti-inflammatory response is weak, the spread of inflammation and fibrosis can be suppressed by reducing accumulated lipids. Our results suggest that reducing abnormal lipid accumulation in the kidney could lead to disease treatment.

Liver X receptor agonists exert antitumor effects against hepatocellular carcinoma via inducing REPS2 expression.

Recent studies show that liver X receptor (LXR) agonists exert significant antitumor effects in a variety of tumor cell lines including hepatocellular carcinoma (HCC). But the molecular mechanisms underlying LXR antitumor activity are not fully understood. In this study we investigated the effect of LXR agonist T0901317 (T317) on HCC development and its relationship with RalA binding protein 1 (RALBP1)-associated EPS domain containing 2 (REPS2)/epidermal growth factor receptor (EGFR) signaling axis. We showed that T317 (0.1-0.5 µM) dose-dependently increased REPS2 expression in normal hepatocytes (BNLCL.2 and LO2) and HCC cells (HepG2 and Huh-7). Using promoter activity assay and chromatin immunoprecipitation (CHIP) assay we demonstrated that T317 enhanced REPS2 expression at the transcriptional level via promoting the binding of LXR protein to the LXR-response element (LXRE) in the REPS2 promoter region. We showed that the inhibitory effect of T317 on the proliferation and migration of HCC cells was closely related to REPS2. Moreover, we revealed that T317 (400 nM) increased expression of REPS2 in HepG2 cells, thus inhibiting epidermal growth factor (EGF)-mediated endocytosis of EGFR as well as the downstream activation of AKT/NF-κB, p38MAPK, and ERK1/2 signaling pathways. Clinical data analysis revealed that REPS2 expression levels were inversely correlated with the development of HCC and reduced REPS2 expression associated with poor prognosis, suggesting that REPS2 might be involved in the development of HCC. In conclusion, this study provides new insights into the potential mechanisms of LXR agonist-inhibited HCC.

Design, synthesis and structure-activity relationship of 4-(1,1,1,3,3,3-hexafluoro-2-hydroxyisoprop-2-yl)phenylsilane derivatives as liver X receptor agonists.

Liver X receptor (LXR) α and LXRß are nuclear receptors playing key roles in lipid metabolism, and LXR ligands are attractive drug candidates for metabolic disorders. Here we report the structural development of 4-(1,1,1,3,3,3-hexafluoro-2-hydroxyprop-2-yl)phenylsilane derivatives as LXR agonists bearing silyl functionalities as the hydrophobic pharmacophore, based on the structure of the known sulfonamide LXR agonist T0901317. Most of the synthesized compounds exhibit agonistic activity toward LXRs, but the LXR subtype-selectivity differs depending upon the substituents on the silicon atom. Among them, tri(n-propyl) derivative 12 shows potent LXR-agonistic activity with moderate α subtype-selectivity, while dimethylphenylsilyl derivative 19 shows modest ß-selectivity. These results indicate that silanes can serve as an alternative to the sulfonamide moiety of LXR agonists, and are promising structural options for the development of novel subtype-selective LXR agonists.

Tritium hydrogen-isotope exchange with electron-poor tertiary benzenesulfonamide moiety; application in late-stage labeling of T0901317.

The multifunctional radioligand [3 H]T0901317 ([3 H]1) has been employed as a powerful autoradiographic tool to target several receptors, such as liver X, farnesoid X, and retinoic acid-related orphan receptor alpha and gamma subtypes at nanomolar concentrations. Although [3 H]1 is commercially available and its synthesis via tritiodebromination has been reported, the market price of this radioligand and the laborious synthesis of corresponding bromo-intermediate potentially preclude its widespread use in biochemical, pharmacological, and pathological studies in research lab settings. We exploit recent reports on hydrogen-isotope exchange (HIE) reactions in tertiary benzenesulfonamides where the sulfonamide represents an ortho-directing group that facilitates CH activation in the presence of homogenous iridium(I) catalysts. Herein, we report a time- and cost-efficient method for the tritium late-stage labeling of compound 1-a remarkably electron-poor substrate owing to the tertiary trifluoroethylsulfonamide moiety. Under a straightforward HIE condition using a commercially available Kerr-type NHC Ir(I) complex, [(cod)Ir (NHC)Cl], the reaction with 1 afforded a specific activity of 10.8 Ci/mmol. Additionally, alternative HIE conditions using the heterogeneous catalyst of Ir-black provided sufficient 0.72 D-enrichment of 1 but unexpectedly failed while repeating with tritium gas.

Inhibition of notch enhances the anti-atherosclerotic effects of LXR agonists while reducing fatty liver development in ApoE-deficient mice.

Liver X receptor (LXR) activation can achieve satisfactory anti-atherosclerotic activity, but can also lead to the development of fatty liver and hypertriglyceridemia. In contrast, Notch inhibition can suppress both atherosclerosis and the hepatic accumulation of lipids. In the present study, we sought to assess whether combining LXR ligand agonists (T317) with Notch receptor inhibitors (DAPT) would lead to enhanced anti-atherosclerotic activity while overcoming the adverse events associated with LXR ligand agonist therapy. The impact of the combined T317 + DAPT therapeutic regimen on atherosclerosis, fatty liver development, and hypertriglyceridemia was assessed using ApoE deficient (ApoE-/-) mice. The results of this analysis suggested that DAPT was able to improve the anti-atherosclerotic activity of T317 without reducing the stability of lesion plaques while simultaneously reducing blood lipids in treated ApoE-/- mice. This combination T317 + DAPT treatment was also linked with a significant upregulation of ABCA1 and the stimulation of reverse cholesterol transport (RCT), as well as with decreases in the levels of intercellular cell adhesion molecule-1 (ICAM-1) and p-p65, and with altered M1/M2 macrophage proportions within atherosclerotic plaques. Importantly, DAPT was also able to reduce T317-mediated lipid accumulation within the liver owing to its ability to reduce SREBP-1 expression while simultaneously increasing that of Pi-AMPKα and PPARα. Together, our results suggest that administering Notch receptor inhibitors to ApoE-/- mice may be an effective means of enhancing the anti-atherosclerotic activity of LXR ligand agonists while simultaneously limiting associated fatty liver and hypertriglyceridemia development in these animals.

Activation of liver X receptor suppresses angiogenesis via induction of ApoD.

Liver X receptors (LXRs) are important sensors and regulators for cholesterol, fatty acid, and glucose. LXRs play essential roles in the development and progression of cardiovascular diseases. We examined the effects of T0901317, a potent LXR agonist, on angiogenesis of human umbilical vein endothelial cells (HUVECs). Treatment with T0901317 inhibited the tube formation and migration of HUVECs and reduced the in vivo angiogenesis, as determined by chorioallantoic membrane assay. T0901317 stimulated gene and protein expression of LXR target gene apolipoprotein D (ApoD). Overexpression of ApoD suppressed the tube formation of HUVECs. ApoD interacted with scavenger receptor class B member 1 (SR-B1), while knockdown of SR-B1 blocked suppressive effects of T0901317 on HUVEC migration. T0901317 treatment or overexpression of ApoD lessened expression of proteins regulating angiogenesis, including phospho-eNOS S1177, phospho-Akt T308, phospho-Akt S473, eNOS, mammalian target of rapamycin, VEGF-A, VEGF-C, IL-8, RhoB, matrix metalloproteinase (MMP)-8, -9, and monocyte chemoattractant protein 1. Our study suggested that activation of LXR interferes with angiogenesis through induction of LXR target gene ApoD, which in turn suppresses PI3K-Akt-eNOS signaling, an essential pathway regulating angiogenesis. ApoD may be a potential therapeutic target for tumor angiogenesis.-Lai, C.-J., Cheng, H.-C., Lin, C.-Y., Huang, S.-H., Chen, T.-H., Chung, C.-J., Chang, C.-H., Wang, H.-D., Chuu, C.-P. Activation of liver X receptor suppresses angiogenesis via induction of ApoD.

Development of novel silanol-based human pregnane X receptor (PXR) agonists with improved receptor selectivity.

Pregnane X receptor (PXR) is a ligand-dependent transcription factor that is considered to be a potential therapeutic target for multiple diseases. Herein, we report the development and structure-activity relationship studies of a new series of hPXR agonists. Focusing on our recently developed silanol-sulfonamide scaffold, we developed the potent hPXR agonist 28, which shows good selectivity over hLXRα and ß, hFXR, and hRORα and γ. Examination of the structure-activity relationship suggested a possible strategy to manipulate the selectivity. Docking simulation indicated the presence of an additional binding cavity and polar contacts in the ligand-binding pocket of hPXR. This information should be helpful for the future development of more potent and selective hPXR ligands.

Endothelial cellular senescence is inhibited by liver X receptor activation with an additional mechanism for its atheroprotection in diabetes.

Senescence of vascular endothelial cells leads to endothelial dysfunction and contributes to the progression of atherosclerosis. Liver X receptors (LXRs) are nuclear receptors whose activation protects against atherosclerosis by transcriptional regulation of genes important in promoting cholesterol efflux and inhibiting inflammation. Here we found that LXR activation with specific ligands reduced the increase in senescence-associated (SA) ß-gal activity, a senescence marker, and reversed the decrease in telomerase activity, a replicative senescence marker, in human endothelial cells under high glucose. This effect of LXR activation was associated with reduced reactive oxygen species and increased endothelial NO synthase activity. A series of experiments that used siRNAs indicated that LXRß mediates the prevention of endothelial cellular senescence, and that sterol regulatory element binding protein-1, which was up-regulated as a direct LXRß target gene, may act as a brake of endothelial cellular senescence. Although oral administration of the LXR ligand led to severe fatty liver in diabetic rats, concomitant therapy with metformin avoided the development of hepatic steatosis. However, the preventive effect of the LXR ligand on SA ß-gal-stained cells in diabetic aortic endothelium was preserved even if metformin was coadministered. Taken together, our studies demonstrate that an additional mechanism, such as the regulation of endothelial cellular senescence, is related to the antiatherogenic properties of LXRs, and concomitant treatment with metformin may provide a clinically useful therapeutic strategy to alleviate an LXR activation-mediated adverse effects on liver triglyceride metabolism.

A systems biology approach reveals the physiological origin of hepatic steatosis induced by liver X receptor activation.

Liver X receptor (LXR) agonists exert potent antiatherosclerotic actions but simultaneously induce excessive triglyceride (TG) accumulation in the liver. To obtain a detailed insight into the underlying mechanism of hepatic TG accumulation, we used a novel computational modeling approach called analysis of dynamic adaptations in parameter trajectories (ADAPT). We revealed that both input and output fluxes to hepatic TG content are considerably induced on LXR activation and that in the early phase of LXR agonism, hepatic steatosis results from only a minor imbalance between the two. It is generally believed that LXR-induced hepatic steatosis results from increased de novo lipogenesis (DNL). In contrast, ADAPT predicted that the hepatic influx of free fatty acids is the major contributor to hepatic TG accumulation in the early phase of LXR activation. Qualitative validation of this prediction showed a 5-fold increase in the contribution of plasma palmitate to hepatic monounsaturated fatty acids on acute LXR activation, whereas DNL was not yet significantly increased. This study illustrates that complex effects of pharmacological intervention can be translated into distinct patterns of metabolic regulation through state-of-the-art mathematical modeling.

Altered activity profile of a tertiary silanol analog of multi-targeting nuclear receptor modulator T0901317.

We report the design, synthesis, and physicochemical/biological evaluation of novel silanol derivative 6 (sila-T) as a silanol analog of multi-target nuclear receptor modulator T0901317 (5). Compound 6 showed intermediate hydrophobicity between the corresponding alcohol 13 and perfluoroalcohol 5. While 5 exhibited potent activities toward liver X receptor α and ß, farnesoid X receptor, pregnane X receptor (PXR) and retinoic acid receptor-related orphan receptor (ROR)γ, silanol 6 exhibited activity only toward PXR and RORs. Incorporation of silanol instead of perfluoroalcohol is a promising option for developing novel target-selective, biologically active compounds.

Serum CETP status is independently associated with reduction rates in LDL-C in pitavastatin-treated diabetic patients and possible involvement of LXR in its association.

BACKGROUND: Statins decrease cholesteryl ester transfer protein (CETP) levels, which have been positively associated with hepatic lipid content as well as serum low density lipoproteins-cholesterol (LDL-C) levels. However, the relationship between the CETP status and statin-induced reductions in LDL-C levels has not yet been elucidated in detail. We herein examined the influence of the CETP status on the lipid-reducing effects of pitavastatin in hypercholesterolemic patients with type 2 diabetes mellitus as well as the molecular mechanism underlying pitavastatin-induced modifications in CETP levels. METHODS: Fifty-three patients were treated with 2 mg of pitavastatin for 3 months. Serum levels of LDL-C, small dense (sd) LDL-C, and CETP were measured before and after the pitavastatin treatment. The effects of pitavastatin, T0901317, a specific agonist for liver X receptor (LXR) that reflects hepatic cholesterol contents, and LXR silencing on CETP mRNA expression in HepG2 cells were also examined by a real-time PCR assay. RESULTS: The pitavastatin treatment decreased LDL-C, sdLDL-C, and CETP levels by 39, 42, and 23%, respectively. Despite the absence of a significant association between CETP and LDL-C levels at baseline, baseline CETP levels and its percentage change were an independent positive determinant for the changes observed in LDL-C and sdLDL-C levels. The LXR activation with T0901317 (0.5 µM), an in vitro condition analogous to hepatic cholesterol accumulation, increased CETP mRNA levels in HepG2 cells by approximately 220%, while LXR silencing markedly diminished the increased expression of CETP. Pitavastatin (5 µM) decreased basal CETP mRNA levels by 21%, and this was completely reversed by T0901317. CONCLUSION: Baseline CETP levels may predict the lipid-reducing effects of pitavastatin. Pitavastatin-induced CETP reductions may be partially attributed to decreased LXR activity, predictable by the ensuing decline in hepatic cholesterol synthesis. TRIAL REGISTRATION: UMIN Clinical Trials Registry ID UMIN000019020.

Divergent effect of liver X receptor agonists on prostate-specific antigen expression is dependent on androgen receptor in prostate carcinoma cells.

BACKGROUND: Liver X receptor (LXR) isoforms, LXRα and LXRß, have similar protein structures and ligands, but diverse tissue distribution. We used two synthetic, non-steroidal LXR agonists, T0901317 and GW3965, to investigate the effects of LXR agonist modulation on prostate specific antigen (PSA) via the expressions of androgen receptors (AR), LXRα, or LXRß, in prostate carcinoma cells. METHODS: LXRα- or LXRß-knockdown cells were transduced with specific shRNA lentiviral particles. LXRα and LXRß expressions were assessed by immunoblotting and RT-qPCR assays. Cell proliferation was determined by (3) H-thymidine incorporation assays. The effects of LXR agonists and epigallocatechin gallate (EGCG) on PSA expression were determined by ELISA, immunoblotting, or transient gene expression assays. RESULTS: Treatment with either T0901317 or GW3965 significantly attenuated cell proliferation of LNCaP cells. T0901317 treatment suppressed PSA expression while GW3965 treatment enhanced PSA expression. The increase of PSA promoter activity by GW3965 was dependent on the expression of AR. Either LXRα- or LXRß-knockdown did not affect the activation of androgen on PSA gene expression. However, as compared with mock knockdown-LNCaP cells, the LXRα-knockdown but not the LXRß-knockdown attenuated the effects of T0901317 and GW3965 on PSA expressions. The effect of GW3965 on PSA expression was blocked by the addition of EGCG. CONCLUSIONS: Our results indicate that T0901317 and GW3965 have divergent effects on PSA expressions. The effects of LXR agonists on PSA expression are LXRα-dependent and AR-dependent. EGCG blocks the inducing effect of GW3965 on PSA expression.

Combination of mangiferin and T0901317 targeting autophagy promotes cholesterol efflux from macrophage foam cell in atherosclerosis.

BACKGROUND: The synthetic liver X receptor ligand (LXR) T0901317 (T0) has been reported to attenuate atherosclerosis (AS) without hyperglyceridemia due to innovative drug combination or nano-sized drug delivery. Given the key roles of mangiferin (MGF) in lipid metabolism and atherogenesis, it is critical to investigate progression of atherosclerotic lesion after combined treatment of MGF and T0. METHODS: Atherosclerotic plaque formation and hepatic lipid accumulation were compared in Apoe-/- mice among T0 and/or MGF treatment. The in vitro functions of MGF and T0 were analyzed by Oil-red O staining, cholesterol efflux assay, transmission electron microscopy and western blot analyses with or without acetylated low density lipoprotein. RESULTS: The combination therapy are effective regulators for atherosclerotic plaque formation in Apoe-/- mice, due to upregulation of ABCA1 and ABCG1 induced by LXR activation. Subsequently, we identified autophagy promoted by MGF and T0 treatment establishes a positive feedback loop that increases cholesterol efflux, resulted from LXRα activation. Under atherogenic conditions, the autophagy inhibitor CQ abolished the enhancement effect on cholesterol outflow of MGF and T0. Mechanically, MGF and T0 promotes LXRα and mTOR/AMPK signaling cascade in macrophage, and promotes AMPK signaling cascade in hepatocyte, leading to lipid metabolic homeostasis. CONCLUSIONS: Altogether, our findings reveal that MGF and T0 engages in AS therapy without side effects by activating AMPK-dependent autophagy to promote macrophage cholesterol efflux, and MGF might serve as a natural compound to assist T0 in AS via targeting autophagy.

Piperine activates human pregnane X receptor to induce the expression of cytochrome P450 3A4 and multidrug resistance protein 1.

Activation of the pregnane X receptor (PXR) and subsequently its target genes, including those encoding drug transporters and metabolizing enzymes, while playing substantial roles in xenobiotic detoxification, might cause undesired drug-drug interactions. Recently, an increased awareness has been given to dietary components for potential induction of diet-drug interactions through activation of PXR. Here, we studied, whether piperine (PIP), a major component extracted from the widely-used daily spice black pepper, could induce PXR-mediated expression of cytochrome P450 3A4 (CYP3A4) and multidrug resistance protein 1 (MDR1). Our results showed that PIP activated human PXR (hPXR)-mediated CYP3A4 and MDR1 expression in human hepatocytes, intestine cells, and a mouse model; PIP activated hPXR by recruiting its coactivator SRC-1 in both cellular and cell-free systems; PIP bound to the hPXR ligand binding domain in a competitive ligand binding assay in vitro. The dichotomous effects of PIP on induction of CYP3A4 and MDR1 expression observed here and inhibition of their activity reported elsewhere challenges the potential use of PIP as a bioavailability enhancer and suggests that caution should be taken in PIP consumption during drug treatment in patients, particularly those who favor daily pepper spice or rely on certain pepper remedies.

Resveratrol inhibits LXRα-dependent hepatic lipogenesis through novel antioxidant Sestrin2 gene induction.

Liver X receptor-α (LXRα), a member of the nuclear receptor superfamily of ligand-activated transcription factors, regulates de novo fatty acid synthesis that leads to stimulate hepatic steatosis. Although, resveratrol has beneficial effects on metabolic disease, it is not known whether resveratrol affects LXRα-dependent lipogenic gene expression. This study investigated the effect of resveratrol in LXRα-mediated lipogenesis and the underlying molecular mechanism. Resveratrol inhibited the ability of LXRα to activate sterol regulatory element binding protein-1c (SREBP-1c) and thereby inhibited target gene expression in hepatocytes. Moreover, resveratrol decreased LXRα-RXRα DNA binding activity and LXRE-luciferase transactivation. Resveratrol is known to activate Sirtuin 1 (Sirt1) and AMP-activated protein kinase (AMPK), although its precise mechanism of action remains controversial. We found that the ability of resveratrol to repress T0901317-induced SREBP-1c expression was not dependent on AMPK and Sirt1. It is well established that hepatic steatosis is associated with antioxidant and redox signaling. Our data showing that expression of Sestrin2 (Sesn2), which is a novel antioxidant gene, was significantly down-regulated in the livers of high-fat diet-fed mice. Moreover, resveratrol up-regulated Sesn2 expression, but not Sesn1 and Sesn3. Sesn2 overexpression repressed LXRα-activated SREBP-1c expression and LXRE-luciferase activity. Finally, Sesn2 knockdown using siRNA abolished the effect of resveratrol in LXRα-induced FAS luciferase gene transactivation. We conclude that resveratrol affects Sesn2 gene induction and contributes to the inhibition of LXRα-mediated hepatic lipogenesis.

Structure-based design of substituted hexafluoroisopropanol-arylsulfonamides as modulators of RORc.

The structure-activity relationships of T0901317 analogs were explored as RORc inverse agonists using the principles of property- and structure-based drug design. An X-ray co-crystal structure of T0901317 and RORc was obtained and provided molecular insight into why T0901317 functioned as an inverse agonist of RORc; whereas, the same ligand functioned as an agonist of FXR, LXR, and PXR. The structural data was also used to design inhibitors with improved RORc biochemical and cellular activities. The improved inhibitors possessed enhanced selectivity profiles (rationalized using the X-ray crystallographic data) against other nuclear receptors.

Nanodisc delivery of liver X receptor agonist for the treatment of diabetic nephropathy.

Dyslipidemia is recognized to be an important contributor to the progression of diabetic nephropathy (DN), leading to lipoprotein dysregulation, excessive mesangium expansion as well as inflammation in the glomeruli. Thus, dual targeting of abnormal cholesterol metabolism and inflammatory responses of mesangial cells represents an alternative approach for DN treatment. Herein, we sought to develop a renal-targeting therapeutic strategy for diabetic nephropathy by modifying synthetic high-density lipoprotein (sHDL) nanodiscs with a kidney targeting ligand (KT peptide) and encapsulating a liver X receptor (LXR) agonist in the modified sHDL. LXR agonists delivered by sHDL can facilitate the removal of excessive lipids from mesangial cells, ameliorate inflammation and restore normal renal function. Overall, our data suggests that our optimized KT-targeted sHDL/TO nanodiscs (KT-sHDL/TO) generate potent therapeutic efficacy not only by more efficient cholesterol efflux, but also by suppressing mesangial cell proliferation. Most importantly, in a DN murine model, KT-sHDL/TO ameliorated dyslipidemia and inflammation superior to blank sHDL and non-targeting sHDL/TO formulations, showing promise for future clinical translation in DN treatment.

LXRß Activation Inhibits the Proliferation of Small-cell Lung Cancer Cells by Depleting Cellular Cholesterol.

BACKGROUND/AIM: Liver X receptors (LXRs) are nuclear receptors with various functions, including the regulation of cholesterol metabolism, glucose homeostasis, and inflammation. We previously reported that LXR activation inhibits the growth of oral cancer cells by inducing cellular cholesterol efflux and that LXRß is expressed mainly in small-cell lung cancer (SCLC) tissues. SCLC is one of the most aggressive cancers, and identifying an effective therapeutic target molecule is desirable. Therefore, we investigated whether LXRß could be an effective target molecule for SCLC treatment through in vitro experiments. MATERIALS AND METHODS: We evaluated the influence of treatment with the LXR agonist T0901317 on cell proliferation and apoptosis in SCLC cell lines using cell viability, BrdU-ELISA, FACS, and western blot analyses. Moreover, the mechanism by which T0901317 inhibits SCLC cell proliferation was elucidated using qRT-PCR, western blot, a cholesterol quantification assay, and a genome editing technique. RESULTS: We showed that cultivated SCLC cells expressed LXRß and that an LXR agonist inhibited the proliferation of SCLC cells without toxicity to normal cells. Furthermore, the antitumoral effect of an LXR agonist on SCLC cells was attributed to the induction of ABCA1 by LXRß activation, resulting in an increase in cellular cholesterol efflux via ABCA1. CONCLUSION: The activation of LXRß up-regulates ABCA1 expression, causing cholesterol depletion in cancer cells. This mechanism could be a novel target strategy for SCLC.

Targeting macrophage liver X receptors by hydrogel-encapsulated T0901317 reduces atherosclerosis without effect on hepatic lipogenesis.

BACKGROUND AND PURPOSE: Targeting macrophage but not hepatocyte liver X receptors (LXRs) can reduce atherosclerosis without effect on hepatic lipogenesis. In this study, we encapsulated LXR ligands with D-Nap-GFFY to form a nanofibre hydrogel (D-Nap-GFFY-T0901317 or GFFY-T0901317) and determined its effect on atherosclerosis, hepatic lipogenesis and the underlying mechanisms involved. EXPERIMENTAL APPROACH: D-Nap-GFFY-T0901317 was subcutaneously injected to proatherogenic diet-fed apoE-deficient (Apoe-/- ) mice, followed by determination of the development of atherosclerosis, liver steatosis and the involved mechanisms, with comparison of T0901317 oral administration. KEY RESULTS: Subcutaneous injection of D-Nap-GFFY-T0901317 to Apoe-/- mice inhibited atherosclerosis at a comparable level as T0901317 oral administration without effect on hepatic lipogenesis. More importantly, D-Nap-GFFY-T0901317 regressed the advanced lesions. In arterial wall, D-Nap-GFFY-T0901317 reduced macrophage/foam cells, necrotic cores and calcification and increased collagen content. It activated expression of ABCA1/G1 and smooth muscle α-actin, while inhibiting expression of intracellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1). D-Nap-GFFY-T0901317 also reduced serum pro-inflammatory cytokines and facilitated Kupffer cell M2 polarization. Mechanistically, D-Nap-GFFY-T0901317 was selectively taken up by macrophages but not hepatocytes, resulting in activation of macrophage ABCA1/G1 expression, while having no effect on lipogenic genes in hepatocytes. Moreover, the selective uptake of D-Nap-GFFY-T0901317 by macrophages was mainly completed in a scavenger receptor class A-dependent manner. CONCLUSION AND IMPLICATIONS: Our study demonstrates that D-Nap-GFFY-T0901317 reduces atherosclerosis without effect on hepatic lipogenesis by targeting macrophage LXRs selectively, indicating its potential application for atherosclerosis treatment.

Pharmacokinetics of T0901317 in mouse serum and tissues using a validated UFLC-IT-TOF/MS method.

T0901317, a liver X receptors (LXRs) agonist with high-affinity, is widely used to explore the functions of LXRs in various diseases such as atherosclerosis and Alzheimer's disease. However, there is currently little information available about the pharmacokinetics (PK) behavior of T0901317. Here we established a novel ultrafast liquid chromatography-high resolution mass spectrometry method to quantify the concentration of T0901317 in serum, liver, and brain. The chromatographic separation was attained on a C18 (2.1 × 100 mm, 1.8 µm) column using acetonitrile and 0.1 % of formic acid in water as mobile phase operated in gradient elution mode. The mass detection was carried out using negative ions m/z 479.9809 and 322.0882 for T0901317 and internal standard, respectively. The proposed method was fully validated according to the FDA guidelines, and it generally provides good results in terms of linearity (r2 > 0.99), precision (RSD < 18 % and 12 % for LLOQ and other QC levels, respectively), accuracy (between 92.30 % and 108.16 %), and matrix effect (between 86.56 % and 113.81 %). We then for the first time determined and computed the PK parameters of T0901317 in mouse after intraperitoneal administration of a 20 mg/kg dosage. The peak times (Tmax) in serum, liver, and brain were 1.5, 1.5, and 4 h, respectively, while the half-lives (t1/2) were 4.9, 3.3, and 4.5 h, respectively. Taken together, our results provide a significant choice to study the PK property of T0901317, from which the design of the dosing and sampling protocols of LXRs receptor-antagonist experiments employing T0901317 can also benefit.