AOP ID and Title:

Short Title: LXR activation leads to liver steatosis

Authors

Of the originating work: Brigitte Landesmann, Marina Goumenou, Sharon Munn and Maurice Whelan, Joint Research Centre, European Commission, Ispra, Italy

Of the content populated in the AOP-Wiki:  John R. Frisch and Travis Karschnik, General Dynamics Information Technology, Duluth, Minnesota; Daniel L. Villeneuve, US Environmental Protection Agency, Great Lakes Toxicology and Ecology Division, Duluth, MN

Status

Abstract

Liver X receptor (LXR) belongs to a class of nuclear receptors [Arhyl hydrocarbon receptor (AHR), Constitutive androstane receptor (CAR), Oestrogen receptor (ER), Farnesoid X receptor (FXR), Glucocorticoid receptor (GR), Peroxisome proliferator-activated receptor (PPAR), Pregnane X receptor (PXR), Retinoic acid receptor (RAR)] that are needed for normal liver function, but for which increased expression (i.e. activation by binding by chemical stressors) lead to liver injury, including steatosis (Mellor et al. 1996).  An increasing number of chemical stressors have been shown to increase LXR expression (Moya et al. 2020).  Activation of LXR has been linked to increased expression of a group of genes (ChREBP, SREBP-1c, FAS and SCD1) involved in increasing de novo fatty acid synthesis (Mellor et al. 1996, Schultz et al. 2000, Postic and Girard 2008).  Increases in de novo fatty acid synthesis is one of the main pathways for increases in triglycerides in livers (Angrish et al. 2016).  Increases in triglycerides can result in decreased mitochondrial biochemical function or histological changes in mitochondria structure, ultimately resulting in steatosis as a primary adverse outcome (Angrish et al. 2016; Mellor et al. 1996).

Background

This Adverse Outcome Pathway (AOP) focuses on the pathway in which activation of Liver X receptor (LXR) leads to liver steatosis through increased de novo fatty acid synthesis.  Environmental stressors result in activation of nuclear receptors linked to increases in triglyceride accumulation through several pathways.  One of the primary pathways linked to triglyceride accumulation, and focus of this AOP, is through activation of the LXR gene and coordinated molecular responses leading to increased fatty acid synthesis.  This pathway has been particular well studied in mammals (humans, lab mice, lab rats).

Summary of the AOP

Events

Molecular Initiating Events (MIE), Key Events (KE), Adverse Outcomes (AO)

Key Event Relationships

Overall Assessment of the AOP

1. Support for Biological Plausibility of Key Event Relationships: Is there a mechanistic relationship  between KEup and KEdown consistent with established biological knowledge?
Key Event Relationship (KER)	Level of Support   Strong = Extensive understanding of the KER based on extensive previous documentation and broad acceptance.
Relationship 3103: Activation, LXR leads to Increased, Expression of LXR activated genes	Moderate support.  The relationship between activation of Liver X receptor and genes linked to regulation of de novo fatty acid synthesis is broadly accepted and consistently supported across taxa.
Relationship 3104: Increased, Expression of LXR activated genes leads to Synthesis, De Novo FA	Moderate support.  The relationship between Increased, Expression of LXR activated genes and Increased de novo fatty acid synthesis is broadly accepted and consistently supported across taxa.
Relationship 110: Synthesis, De Novo FA leads to Accumulation, Triglyceride	Strong support. Increased de novo fatty acid synthesis is broadly recognized as a major pathway leading to accumulation of triglycerides, and consistently supported across taxa.
Relationship 2265: Accumulation, Triglyceride leads to Increased, Liver Steatosis	Strong support.  The relationship between accumulation of triglycerides and liver steatosis is broadly accepted and consistently supported across taxa.
Overall	Strong support.  Extensive understanding of the relationships between events from empirical studies from a variety of taxa, including frequent testing in lab mammals.

Domain of Applicability

Life Stage Applicability Taxonomic Applicability Sex Applicability

Life Stage: The life stage applicable to this AOP is all life stages with a liver.  Older individuals are more likely to manifest this adverse outcome pathway (adults > juveniles ) due to accumulation of triglycerides.

Sex: This AOP applies to both males and females.

Taxonomic: This AOP appears to be present broadly in vertebrates, with most representative studies in mammals (humans, lab mice, lab rats).

Essentiality of the Key Events

2. Essentiality of Key Events: Are downstream KEs and/or the AO prevented if an upstream KE is blocked?
Key Event (KE)	Level of Support Strong = Direct evidence from specifically designed experimental studies illustrating essentiality and direct relationship between key events. Moderate = Indirect evidence from experimental studies inferring essentiality of relationship between key events due to difficulty in directly measuring at least one of key events.
MIE 167 Activation, LXR	Moderate support.  Activation of Liver X receptor is a primary activator for increases in genes linked to regulation of de novo fatty acid synthesis.  However, expression of these genes can be elicited by other nuclear receptors and molecular processes.
KE 2199 Increased, Expression of LXR activated genes	Moderate support.  Increased, expression of LXR activated genes is one pathway linked to increases in de novo fatty acid synthesis.  However, a variety of molecular signals and corresponding cellular changes are required in order for de novo fatty acid synthesis to increase.
KE 89 Synthesis, De Novo FA	Moderate support.  Increase in de novo fatty acid synthesis is a primary factor in increased triglyceride levels in cells.  However, triglycerides increase in cells via a number of pathways, including increased triglyceride influx into cells.
KE 291 Accumulation, Triglyceride	Strong support. Accumulation of triglyceride is linked to liver steatosis.  Evidence is available from toxicant, gene-knockout, and high lipid diet studies.
AO 459 Increased, Liver Steatosis	Strong support. Liver steatosis occurs due to a variety of stressors and breakdown of multiple biochemical pathways and physiological changes with resulting increases in triglyceride levels.  Evidence is available from toxicant and high lipid diet studies.

Weight of Evidence Summary

3. Empirical Support for Key Event Relationship: Does empirical evidence support that a  change in KEup leads to an appropriate change in KEdown?
Key Event Relationship (KER)	Level of Support  Strong =  Experimental evidence from exposure to toxicant shows consistent change in both events across taxa and study conditions.
Relationship 3103: Activation, LXR leads to Increased, Expression of LXR activated genes	Moderate support.  Increases in Liver X receptor expression lead to increases in genes linked to regulation of de novo fatty acid synthesis, primarily from studies examining TOXCAST data, as well as changes in gene expression levels after exposure to chemical stressors.
Relationship 3104: Increased, Expression of LXR activated genes leads to Synthesis, De Novo FA	Weak support. Increases in expression of LXR activated genes lead to increases in de novo fatty acid synthesis, primarily through measured increases in gene expression and increased triglyceride levels.  Increased de novo fatty acid synthesis is inferred from increased triglyceride levels rather than directly observed.
Relationship 110: Synthesis, De Novo FA leads to Accumulation, Triglyceride	Strong support. Increases in de novo fatty acid synthesis is recognized as a primary pathway to accumulation of triglycerides.
Relationship 2265: Accumulation, Triglyceride leads to Increased, Liver Steatosis	Strong support. Increases in accumulation of triglyceride is recognized as a primary pathway to liver steatosis.
Overall	Strong support. Exposure from empirical studies shows consistent change in both events from a variety of taxa, including frequent testing in lab mammals.

References

Angrish, M.M., Kaiser, J.P., McQueen, C.A., and Chorley, B.N.  2016. Tipping the Balance: Hepatotoxicity and the 4 Apical Key Events of Hepatic Steatosis.  Toxicological Sciences 150(2): 261-268.

Mellor, C.L., Steinmetz, F.P., and Cronin, T.D.  2016.  The identification of nuclear receptors associated with hepatic steatosis to develop and extend adverse outcome pathways.  Critical Reviews in Toxicology, 46(2): 138-152.

Moya, M., Gomez-Lechon, M.J., Castell, J.V., and Jovera, R.  2010.  Enhanced steatosis by nuclear receptor ligands: A study in cultured human hepatocytes and hepatoma cells with a characterized nuclear receptor expression profile.  Chemico-Biological Interactions 184: 376–387.

Landesmann, B., Goumenou, M., Munn, S., and Whelan, M.  2012.  Description of Prototype Modes-of-Action Related to Repeated Dose Toxicity.  European Commission Report EUR 25631, 49 pages.  https://op.europa.eu/en/publication-detail/-/publication/d2b09726-8267-42de-8093-8c8981201d65/language-en

Postic, C. and Girard, J.  2008.  Contribution of de novo fatty acid synthesis to hepatic steatosis and insulin resistance: lessons from genetically engineered mice.  The Journal of Clinical Investigation 118(3): 829-838.

Schultz, J.R., Tu, H., Luk, A., Repa, J.J., Media, J.C., Li, L., Schwendner, S., Wang, S., Thoolen, M., Mangelsdorf, D.J., Lustig, K.D., and Shan, B.  2000.  Role of LXRs in control of lipogenesis.  Genes and Development 14:2831–2838.

Appendix 1

List of MIEs in this AOP

Event: 167: Activation, LXR

Short Name: Activation, LXR

Key Event Component

AOPs Including This Key Event

Biological Context

Cell term

Key Event Description

The LXR receptor

Liver X receptors are ligand-activated transcription factors of the nuclear receptor superfamily first identified in 1994 in rat liver (Apfel et al. 1994, Song 1994). There are two LXR isoforms termed a and ß (NR1H3 and NR1H2) which upon activation form heterodimers with retinoid X receptor (RXR) and bind to the LXR response element found in the promoter region of the target genes (Baranowski 2008). LXRs were shown to function as sterol sensors protecting the cells from cholesterol overload by stimulating reverse cholesterol transport and activating its conversion to bile acids in the liver (Baranowski 2008).

LXRa expression is restricted to liver, kidney, intestine, fat tissue, macrophages, lung, and spleen and is highest in liver, hence the name liver X receptor a (LXRa). LXRβ is expressed in almost all tissues and organs, hence the early name UR (ubiquitous receptor) (Ory 2004). The different pattern of expression suggests that LXRa and LXRβ have different roles in regulating physiological function. This is also supported from the observation that LXRa deficient mice do not develop hepatic steatosis when treated with LXR agonist that activates both types (Lund et al. 2006) and consequently the role of the two isoforms in relation to adverse effects could be different.

 

The molecular initiating event

Generally speaking chemicals that are able to act through NRs are usually specific ligands. These chemicals are mainly lipophilic and they mimic the action of natural hormones. However, in some cases hydrophilic chemicals (like phthalates) are also capable to act as ligands in NRs due to the molecular structure of the proteins and the pocket sites of the receptors.

The molecular initiating event in the presented MoA is the binding to the LXR or the permissive RXR of the LXR-RXR dimer leading to activation. LXR activation can be achieved via a wide range of endogenous neutral and acidic ligands as shown by crystallographic analysis (Williams et al. 2003). There are known endogenous but also synthetic ligands that can act as agonists. Endogenous agonists for this receptor are the oxysterols (oxidized cholesterol derivatives like 22(R)-hydroxycholesterol, 24(S)-hydroxycholesterol, 27-hydroxycholesterol, and cholestenoic acid) mainly with similar affinity for the two isoforms (Baranowski 2008). Oxysterols bind directly to the typical hydrophobic pocket in the C-terminal domain (Williams et al. 2003). Other endogenous ligands are the D-glucose and D-Glucose-6-phosphate (Mitro 2007). However, the hydrophilic nature of glucose and its low affinity for LXR present a challenge to the central dogma about the nature of the NR-ligand interaction (Lazar & Wilson 2007). Unsaturated fatty acids have also been shown to bind and regulate LXRa activity in cells. However, in contrast to the role of oxysterols, the biological relevance of this observation has not been established in vivo (Pawar et al. 2003). The function of LXRs is also modulated by many currently used drugs such as statins, fibrates, and thazolidinedione derivatives (Jamroz-Wiśniewska et al. 2007). Some synthetic LXR agonists have been developed like the non-steroidal agonists T0901317 and GW3965 (Schultz et al 2000, Collins et al. 2002). LXR forms a permissive dimer with the RXR which means that chemicals that can activate this receptor can trigger the same pathway as the LXR agonists. The endogenous RXR agonist is 9-cis-retinoic acid (Heyman et al. 1992) while synthetic agonists include LGD1069 and LG100268 (Boehm et al. 1994 and 1995).

In addition to the agonist binding in the LXR there are other mechanisms for its control. LXRa gene promoter contains also functional peroxisome proliferator response element (PPRE) and peroxisome proliferator-activated receptor (PPAR) a and γ agonists were shown to stimulate LXRa expression in human and rodent (Baranowski 2008). Control of the LXRa expression is also dependent on insulin and post-translationally by protein kinase A that phosphorylates receptor protein at two sites thereby impairing its dimerization and DNA-binding (Baranowski 2008).

 

Identification of the site of action

As already mentioned above LXR isoforms are expressed in various tissues but in relation to the presented MoA we refer to LXRs that are expressed in the hepatocytes.

Nuclear receptors may be classified into two broad classes according to their sub-cellular distribution in the absence of ligand. Type I NRs (like ER and AhR) are located in the cytosol (and they are translocated into the nucleus after ligand binding) while type II NRs like LXRs (but also PXR, PPARa and PPARγ) are located in the nucleus of the cell.

The specific site of binding and the affinity of a ligand for the LXRs depend on the structure of the ligand.

 

Binding in the LXREs and target genes transcription

Upon ligand-induced activation both isoforms form obligate heterodimers with the retinoid X receptor (RXR) and regulate gene expression through binding to LXR response elements (LXREs) in the promoter regions of the target genes (Fig. 1). The LXRE consists of two idealized hexanucleotide sequences (AGGTCA) separated by four bases (DR-4 element).

Figure 1. Mechanism of transcriptional regulation mediated by LXRs. RXR - retinoid X receptor, LXRE - LXR response element (Baranowski 2008)

Target genes of LXRs are involved in cholesterol and lipid metabolism regulation (^[1], ^[2]) including:

• ABC - ATP Binding Cassette transporter isoforms A1, G1, G5, and G8
• ApoE - Apolipoprotein E
• CETP - Cholesterylester Transfer Protein
• CYP7A1 - Cytochrome P450 isoform 7A1 - cholesterol 7a-hydroxylase
• FAS - Fatty Acid Synthase
• LPL - Lipoprotein Lipase
• LXR-a - Liver X Receptor-a
• SREBP-1c - Sterol Response Element Binding Protein 1c
• ChREBP - Carbohydrate Response Element Binding Protein
• FAT/CD36 – Fatty acid uptake transporter (liver)

 

Auto-regulation of the LXRa

Human specific auto-regulated expression specifically of the LXRa has been demonstrated from several studies (Laffitte et al. 2001, Whitney et al. 2001, Li et al. 2002, Kase et al. 2007). Human LXRa gene promoter has a functional LXRE activated by both LXRa and β. In addition human liver LXRa expression is induced by both natural and synthetic LXR agonists.

References

1. ↑ Peet 1998 - Peet D.J., Cholesterol and Bile Acid Metabolism Are Impaired in Mice Lacking the
   Nuclear Oxysterol Receptor LXRa in mammals, Cell, 93, 693–704, 1998
2. ↑ Edwardsa et al. 2002 - Edwardsa P.A., et al, LXRs; Oxysterol-activated nuclear receptors that regulate genes
   controlling lipid homeostasis, (Oxidized Lipids as Potential Mediators of
   Atherosclerosis), Vascular Pharmacology, 38 (No 4), 249–256, 2002

List of Key Events in the AOP

Event: 2199: Increased, Expression of LXR activated genes

Short Name: Increased, Expression of LXR activated genes

AOPs Including This Key Event

Biological Context

Event: 89: Synthesis, De Novo FA

Short Name: Synthesis, De Novo FA

Key Event Component

AOPs Including This Key Event

Biological Context

Cell term

Key Event Description

A number of pathways and a great number of enzymes like GK, L-PK, ACC, FAS and SCD-1 are involved in the de novo FA synthesis ^[1]. As it is already discussed above these enzymes are induced by LXR agonists (FAS, SCD1), the SREBP-1c (GK, ACC, FAS) and the ChREBP (L-PK, ACC, FAS) leading to enhancement of the de novo FA synthesis.

Figure 1. Metabolic pathway for de novo FA synthesis and TG formation ^[1]

As proposed from Diraison et al 1997 the de novo FA synthesis contributes maximum 5% to the synthesis of FA and TG under normal conditions. Conditions associated with high rates of lipogenesis, such as low fat - high carbohydrate (LF/HC) diet, hyperglycemia, and hyperinsulinemia are associated with a shift in cellular metabolism from lipid oxidation to TG esterification, thereby increasing the availability of TGs derived from VLDL synthesis and secretion.

References

1. ↑ ^{1.0 1.1} Postic & Girard 2008 - Postic C., Girard J., Contribution of de novo fatty acid synthesis to hepatic steatosis and
   insulin resistance: lessons from genetically engineered mice, J. Clin. Invest. 118 (No 3),
   829–838, 2008

Event: 291: Accumulation, Triglyceride

Short Name: Accumulation, Triglyceride

Key Event Component

AOPs Including This Key Event

Biological Context

Cell term

Key Event Description

Leads to Fatty Liver Cells.

List of Adverse Outcomes in this AOP

Event: 459: Increased, Liver Steatosis

Short Name: Increased, Liver Steatosis

AOPs Including This Key Event

Biological Context

Organ term

Domain of Applicability

Taxonomic Applicability Life Stage Applicability Sex Applicability

Steatosis is the result of perturbations in well-known metabolic pathways that are well-studied and well-known in many taxa.

Key Event Description

Biological state: liver steatosis is the inappropriate storage of fat in hepatocytes.

Biological compartment: steatosis is generally an organ-level diagnosis; however, the pathology occurs within the hepatocytes.

Role in biology: steatosis is an adverse endpoint. 

 

Description from EU-ToxRisk:

Activation of stellate cells results in collagen accumulation and change in extracellular matrix composition in the liver causing fibrosis. (Landesmann, 2016)(Koo et al 2016)

How it is Measured or Detected

Steatosis is measured by lipidomics approaches that measure lipid levels, or by histology.

Regulatory Significance of the AO

Steatosis is a regulatory endpoint and has been used as an endpoint in many US EPA assessments, including IRIS assessments.

References

Landesmann, B. (2016). Adverse Outcome Pathway on Protein Alkylation Leading to Liver Fibrosis, (2).

https://doi.org/10.1016/j.molcel.2005.08.010

Koo, J. H., Lee, H. J., Kim, W., & Kim, S. G. (2016). Endoplasmic Reticulum Stress in Hepatic Stellate Cells Promotes Liver Fibrosis via PERK-Mediated Degradation of HNRNPA1 and Up-regulation of SMAD2. Gastroenterology, 150(1), 181–193.e8. https://doi.org/10.1053/j.gastro.2015.09.039

Appendix 2

List of Key Event Relationships in the AOP