Atherosclerosis: lessons from LXR and the intestine

doi:10.1016/j.tem.2012.10.004

Trends in Endocrinology & Metabolism

Volume 24, Issue 3, March 2013, Pages 120-128

https://doi.org/10.1016/j.tem.2012.10.004 Get rights and content

Modulation of the cholesterol-sensing liver X receptors (LXRs) and their downstream targets has emerged as promising therapeutic avenues in atherosclerosis. The intestine is important for its unique capabilities to act as a gatekeeper for cholesterol absorption and to participate in the process of cholesterol elimination in the feces and reverse cholesterol transport (RCT). Pharmacological and genetic intestine-specific LXR activation have been shown to protect against atherosclerosis. In this review we discuss the LXR-targeted molecular players in the enterocytes as well as the intestine-driven pathways contributing to cholesterol homeostasis with therapeutic potential as targets in the prevention and treatment of atherosclerosis..

Section snippets

Atherosclerosis, LXR, and the intestine: what is the link?

Cholesterol is an essential component of cellular membranes and a precursor for bile acid, steroid hormone, and vitamin D synthesis. However, cholesterol levels must be tightly regulated because excess cholesterol can promote the development of atherosclerotic cardiovascular disease (CVD). Epidemiological studies have shown that low-density lipoprotein (LDL) and high-density lipoprotein (HDL) cholesterol levels are directly and inversely correlated, respectively, with clinical events resulting

The key molecular players in the intestinal absorptive cells

In the following paragraphs we review the key players in the intestinal cholesterol trafficking as well as the role of LXR in the transcriptional regulation of these intestinal lipid transporters (Figure 1).

Intestinal cholesterol absorption

Molecules involved in intestinal cholesterol absorption represent attractive targets for cholesterol-lowering drugs due to their impact upon whole-body cholesterol homeostasis. In fact, even a modest reduction in cholesterol absorption in animal models is antiatherogenic and also appears to enhance RCT [90]. The Schering-Plough Research Institute successfully identified the NPC1L1-targeted drug ezetimibe as a potent and specific inhibitor of intestinal cholesterol absorption, and this is now

Feasibility and challenges of developing intestine specific LXR target-therapies

Treatment of hypercholesterolemia for atherosclerosis prevention has been focused for many years on LDL cholesterol-lowering drugs such as statins and ezetimibe. However, alternative strategies are currently actively pursued, particularly HDL-targeted approaches. Although niacin [97] and cholesteryl ester transfer protein (CEPT) inhibitors 114, 115 will provide the earliest wave of critical clinical data for HDL-directed strategies, a myriad of novel drugs whose development stems from attempts

Concluding remarks

The intestine is an attractive target for reducing cardiovascular risk because it contains numerous transporters and NRs that modulate cholesterol homeostasis. In this regard, the major current hurdle, the LXR-coordinated upregulation of hepatic de novo lipogenesis and cholesterol loss, has been (partially) resolved by several studies showing that local activation of LXRs is sufficient to beneficially impact upon the treatment of atherosclerosis. Intestinal drug targeting is thus a promising

Acknowledgments

A.M. is funded by Italian Association for Cancer Research (AIRC, Milan, Italy, IG 10416), Italian Ministry of Health and Education (Finanziamenti per la Ricerca di Base IDEAS RBID08C9N7), Italian Ministry of Health (Young Researchers Grant 2008, GR-2008-1143546), the European Commission 7th Framework Programme FP7/2007–2013 under Grant Agreement No. 202272 (LipidomicNet), Cariplo Foundation Milan, University of Bari, Italy (ORBA 08WEZJ, 07X7Q1, 06BXVC, IDEA GRBA0802SJ). B.B. is a fellow of the

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The activation of liver X receptors in Madin-Darby bovine kidney cells and mice restricts infection by bovine viral diarrhea virus
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Bovine viral diarrhea virus (BVDV) is prevalent worldwide and is an important pathogen that represents a serious threat to the development of the cattle industry by causing significant economic losses. Liver X receptors (LXRs) are members of the nuclear receptor superfamily and have become attractive therapeutic targets for cardiovascular disease. In the present study, we found that LXRs in both Madin-Darby bovine kidney (MDBK) cells and mice were associated with BVDV infection. GW3965, an agonist for LXRs, significantly inhibited BVDV RNA and protein levels in MDBK cells. In vivo studies in a mouse model also confirmed the inhibitory role of GW3965 in BVDV replication and the ameliorating effect of GW3965 on pathological injury to the duodenum. In vitro investigations of the potential mechanisms involved showed that GW3965 significantly inhibited BVDV-induced increases in cholesterol levels and viral internalization. Furthermore, the antiviral activity of GW3965 was significantly reduced following cholesterol replenishment, thus demonstrating that cholesterol was involved in the resistance of GW3965 to BVDV replication. Further studies indicated the role of ATP-binding cassette transporter A1 (ABCA1) and cholesterol-25-hydroxylase (CH25H) in the antiviral activity of GW3965. We also demonstrated the significant antiviral effect of 25hydroxycholesterol (25HC), a product of the catalysis of cholesterol by CH25H. In addition, the anti-BVDV effects of demethoxycurcumin (DMC), cyanidin-3-O-glucoside (C3G), and saikosaponin-A (SSA), three natural agonizts of LXRs, were also confirmed in both MDBK cells and mice. However, the antiviral activities of these agents were weakened by SR9243, a synthetic inhibitor of LXRs. For the first time, our research demonstrated that the activation of LXRs can exert significant anti-BVDV effects in MDBK cells and mice.
Cholesterol-binding translocator protein TSPO regulates steatosis and bile acid synthesis in nonalcoholic fatty liver disease
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Translocator protein (TSPO, 18 kDa) levels increase in parallel with the evolution of simple steatosis (SS) to nonalcoholic steatohepatitis (NASH) in nonalcoholic fatty liver disease (NAFLD). However, TSPO function in SS and NASH is unknown. Loss of TSPO in hepatocytes in vitro downregulated acetyl-CoA acetyltransferase 2 and increased free cholesterol (FC). FC accumulation induced endoplasmic reticulum stress via IRE1A and protein kinase RNA-like ER kinase/ATF4/CCAAT-enhancer-binding protein homologous protein pathways and autophagy. TSPO deficiency activated cellular adaptive antioxidant protection; this adaptation was lost upon excessive FC accumulation. A TSPO ligand 19-Atriol blocked cholesterol binding and recapitulated many of the alterations seen in TSPO-deficient cells. These data suggest that TSPO deficiency accelerated the progression of SS. In NASH, however, loss of TSPO ameliorated liver fibrosis through downregulation of bile acid synthesis by reducing CYP7A1 and CYP27A1 levels and increasing farnesoid X receptor expression. These studies indicate a dynamic and complex role for TSPO in the evolution of NAFLD.
Regulation of intestinal LDLR by the LXR-IDOL axis
2020, Atherosclerosis
Cholesterol metabolism is tightly regulated by transcriptional and post-transcriptional mechanisms. Accordingly, dysregulation of cholesterol metabolism is a major risk factor for the development of coronary artery disease and associated complications. In recent years, it has become apparent that next to the liver, the intestine plays a key role in systemic cholesterol metabolism by governing cholesterol absorption, secretion, and incorporation into lipoprotein particles. We have previously demonstrated that the Liver X receptor (LXR)-regulated E3 ubiquitin ligase inducible degrader of LDLR (IDOL) is a regulator of cholesterol uptake owing to its ability to promote the ubiquitylation of the low-density lipoprotein receptor (LDLR). However, whether the LXR-IDOL-LDLR axis regulates the LDLR in the intestine and whether this influences intestinal cholesterol homeostasis is not known.
In this study, we evaluated the role of the LXR-IDOL-LDLR axis in enterocyte cell models and in primary enterocytes isolated from Idol^(−/−) and wild type mice. Furthermore, we studied the regulation of intestinal LDLR in Idol^(−/−) and in wild type mice treated with the LXR agonist GW3965. Finally, we assessed ezetimibe-induced trans-intestinal cholesterol efflux in Idol^(−/−) mice.
We show that in a wide range of intestinal cell lines LXR activation decreases LDLR protein abundance, cell surface occupancy, and LDL uptake in an IDOL-dependent manner. Similarly, we find that pharmacological dosing of C57BL6/N mice with the LXR agonist GW3965 increases Idol expression across the intestine with a concomitant reduction in Ldlr protein. Conversely, primary enterocytes isolated from Idol^(−/−) mice have elevated Ldlr. To test whether these changes contribute to trans-intestinal cholesterol efflux, we measured fecal cholesterol in mice following ezetimibe dosing, but found no differences between Idol^(−/−) and control mice in this setting.
In conclusion, our study establishes that the LXR-IDOL-LDLR axis is active in the intestine and is part of the molecular circuitry that maintains cholesterol homeostasis in enterocytes.
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Discovery and optimization of selective liver X receptor β (LXRβ) agonists are challenging due to the high homology of LXRα and LXRβ in the ligand-binding domain. There is only one different residue (Val versus Ile) at the ligand-binding pocket of LXRs. With machine learning methods, we identified pan LXR agonists with a novel scaffold (spiro[pyrrolidine-3,3′-oxindole]). Then, we figured out the mechanism of LXR isoform selectivity from co-crystal structures. Based on the mechanism and the new scaffold, LXRβ selective agonists were designed and synthesized. This led to the discovery of LXRβ agonists 4-7rr, 4–13 and 4-13rr with IC₅₀ values ranging from 1.78 to 6.36 μM against glioblastoma in vitro. Treatment with 50 mg/kg/day of 4-13 for 15 days significantly reduced tumor growth using an in vivo xenograft glioblastoma model.
Identify liver X receptor β modulator building blocks by developing a fluorescence polarization-based competition assay
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The liver X receptors (LXRs) of the nuclear receptor family are promising therapeutic targets of multiple diseases like lipid disorders, chronic inflammation, as well as different human cancers. To date, no LXR agonists or antagonists can be used in clinics, emphasizing the importance for discovering new LXR modulators. Fragment-based lead discovery (FBLD) is powerful for designing new scaffolds and new mechanistic drugs, but fragment screening has not been applied to LXRs yet, which might be due to the lack of a specific fragment screening method against the dynamic and hydrophobic ligand binding domain (LBD) of LXRs. Herein, a series of fluorescent tracers were designed, synthesized and tested. The tracer based on hyodeoxycholic acid exhibited a good capability for competitively detecting the ligand binding of LXRβ using a fluorescence polarization approach. Then, 1074 fragments were screened against the LBD of LXRβ (LXRβ-LBD), resulting in 27 binding hits. These fragment hits were further tested using the co-activator recruitment assay and reporter gene assay, and efforts in X-ray crystallography fortunately solved a co-crystal structure of LXRβ-LBD with the fragment F3 (tert-butyl-7-amino-3,4-dihydroisoquinoline-2(1H)-carboxylate). The fluorescence-based fragment screening tool and the newly identified LXRβ binding fragments provide the basis for developing novel LXRβ modulators.
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Nonalcoholic fatty liver disease (NAFLD) is becoming a worldwide health problem, but no approved medical treatment exists so far. Nuclear receptors are one of the drug targets for nonalcoholic steatohepatitis (NASH). Among them, liver X receptor (LXR) has been studied in recent years in tumors, metabolic diseases and inflammatory diseases, but its physiological and pharmacological effects in the treatment of NASH are controversial. Activation of LXR has the potential to modulate cholesterol homeostasis, induce anti-inflammatory effects and increase insulin sensitivity, but liver lipid deposition and hypertriglyceridemia are also increased. Inhibition of liver LXR transcriptional activity in the context of NAFLD can effectively alleviate hepatic steatosis, inflammation, and fibrosis but elevates the risk of potential cardiovascular disease. The contradictory pharmacodynamic effects of LXR in the treatment of NASH increase the difficulty of developing targeted drugs. Moreover, natural compounds play an important part in drug development, and in recent years, some natural compounds have been reported to treat NAFLD by acting on LXR or LXR pathways with fewer adverse reactions, presenting a promising therapeutic prospect. In this review, we discuss the mechanisms of LXR in NASH and summarize the natural products reported to modulate NAFLD via LXR or the LXR pathway, offering an alternative approach for LXR-related drug development in NAFLD.

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Trends in Endocrinology & Metabolism

ReviewAtherosclerosis: lessons from LXR and the intestine

Section snippets

Atherosclerosis, LXR, and the intestine: what is the link?

The key molecular players in the intestinal absorptive cells

Intestinal cholesterol absorption

Feasibility and challenges of developing intestine specific LXR target-therapies

Concluding remarks

Acknowledgments

J. Am. Coll. Cardiol.

J. Biol. Chem.

J. Biol. Chem.

Biochem. Biophys. Res. Commun.

Cell

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

J. Lipid Res.

J. Lipid Res.

Nutr. Metab. Cardiovasc. Dis.

J. Nutr.

J. Lipid Res.

J. Biol. Chem.

J. Lipid Res.

J. Lipid Res.

Biochem. Biophys. Res. Commun.

J. Biol. Chem.

J. Lipid Res.

J. Biol. Chem.

Cell Metab.

J. Lipid Res.

J. Lipid Res.

J. Biol. Chem.

J. Biol. Chem.

J. Lipid Res.

J. Lipid Res.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

Cell Metab.

J. Am. Coll. Cardiol.

Am. Heart J.

J. Lipid Res.

Blood cholesterol and vascular mortality by age, sex, and blood pressure: a meta-analysis of individual data from 61 prospective studies with 55,000 vascular deaths

Lancet

Major lipids, apolipoproteins, and risk of vascular disease

JAMA

Association between change in high density lipoprotein cholesterol and cardiovascular disease morbidity and mortality: systematic review and meta-regression analysis

BMJ

Cholesterol efflux capacity, high-density lipoprotein function, and atherosclerosis

N. Engl. J. Med.

Statins and stroke prevention

Expert Rev. Cardiovasc. Ther.

Control of cellular cholesterol efflux by the nuclear oxysterol receptor LXR alpha

Proc. Natl. Acad. Sci. U.S.A.

Regulation of absorption and ABC1-mediated efflux of cholesterol by RXR heterodimers

Science

LXR regulates cholesterol uptake through Idol-dependent ubiquitination of the LDL receptor

Science

Liver X receptor signaling pathways and atherosclerosis

Arterioscler. Thromb. Vasc. Biol.

Regulation of mouse sterol regulatory element-binding protein-1c gene (SREBP-1c) by oxysterol receptors, LXRalpha and LXRbeta

Genes Dev.

Role of LXRs in control of lipogenesis

Genes Dev.

Crystal structure of the heterodimeric complex of LXRalpha and RXRbeta ligand-binding domains in a fully agonistic conformation

EMBO J.

Liver LXRalpha expression is crucial for whole body cholesterol homeostasis and reverse cholesterol transport in mice

J. Clin. Invest.

Niemann–Pick C1 Like 1 protein is critical for intestinal cholesterol absorption

Science

Review
Atherosclerosis: lessons from LXR and the intestine