Xio Yng ,Frnk J.Gonzlez ,Min Hung ,Huichng Bi ,*

a Guangdong Provincial Key Laboratory of New Drug Design and Evaluation,School of Pharmaceutical Sciences,Sun Yat-sen University,Guangzhou,China

b Laboratory of Metabolism,Center for Cancer Research,National Cancer Institute,NIH,Bethesda,MD,USA

Keywords:

Nuclear receptors (NRs)

Non-alcoholic fatty liver disease (NAFLD)

Pregnane X receptor (PXR)

Constitutive androstane receptor (CAR)

Farnesoid X receptor (FXR)

Peroxisome proliferator activated receptor

(PPAR)

Liver X receptor (LXR)

ABSTRACT

Non-alcoholic fatty liver disease(NAFLD)has become the leading cause of chronic liver disease in adults and children worldwide.The symptoms of NAFLD range from simple steatosis and non-alcoholic steatohepatitis(NASH)to hepatic fibrosis or cirrhosis,even ultimately develops to hepatocellular carcinoma.Nuclear receptors (NRs) are a superfamily of ligand-activated transcription factors,most of which are ligand-activated that control cellular homeostasis in the liver and other tissues.A growing number of studies demonstrated the important role of NRs in NAFLD.In this review,the current findings on the role of NRs in NAFLD are summarized and future perspectives to target NRs for NAFLD are discussed.

1.Introduction

Fatty liver refers to the pathological changes of the liver with excessive accumulation of fat in liver cells,which is caused by multiple factors.Fatty liver disease (FLD) has been the second bigger liver disease following viral hepatitis that seriously threatens people’s health.1The incidence rate of FLD has a gradually increase year by year,with a disturbing trend toward younger age of onset.Fatty liver is generally divided into alcoholic fatty liver disease (AFLD) and non-alcoholic fatty liver disease (NAFLD).Alcohol is the direct cause of AFLD and alcohol abuse and addiction significantly increase the risk of alcoholic cirrhosis,most significantly in people aged 40-59 years.2NAFLD is the abnormal accumulation of lipid in the liver caused by obesity,diabetes and druginduced liver injury.The spread of sedentary lifestyle and diet westernization has obviously increased the prevalence of NAFLD worldwide.Studies have revealed that NAFLD is closely related to insulin resistance (IR) and metabolic syndrome.NAFLD causes excessive accumulation of triglycerides in the liver,which can lead to different pathological conditions,from initial hepatic steatosis to non-alcoholic steatohepatitis (NASH),and could progress to liver cirrhosis even hepatocellular carcinoma.3Therefore,NAFLD is a detrimental condition necessitating appropriate therapeutic interventions.However,there is no effective treatment for NAFLD and the underlying mechanisms of progression are poorly understood.

Nuclear receptors (NRs) are a superfamily of ligand-activated transcription factors,involved in diverse biological processes such as cell growth,differentiation,apoptosis,metabolism and gene expression during tumor formation.4-6NRs are classified into six subfamilies based on their sequence homology,and atypical NRs containing only one-conserved domain were categorized into subfamily 0 by the Nuclear Receptors Nomenclature Committee.7Therefore,NRs include 7 subfamilies (NR0-NR6).Forty-eight NRs are found in humans.8The onset and development of many diseases such as liver disease,atherosclerosis,Parkinson’s disease,and osteoarthritis,are directly or indirectly related to NR signaling and some NRs have become favored drug targets.9-11

The importance of NRs in liver diseases has been reported,suggesting NRs as key therapeutic targets in liver diseases.NRs are key modulators in the onset and progression of diverse hepatic diseases including hepatic lipid and glucose metabolism,bile acid homeostasis,drug detoxification,inflammation,regeneration,fibrosis,and tumor formation.11Increasing evidence has shown that some NRs are implicated in the development of NAFLD,which may provide novel strategies for the diagnosis and treatment of NAFLD.Moreover,the role of the NR1 subfamily in FLD has been extensively reported,including NR1C1-3,peroxisome proliferatoractivated receptor (PPAR) α/β/γ;NR1H2-3,the liver X receptor(LXR) α/β;NR1H4,the farnesoid X receptor (FXR);NR1I3,the constitutive androstane receptor(CAR);and NR1I2,the pregnane X receptor (PXR).These ligand-activated NRs form a heterodimer with NR2B1-3,the retinoid X receptor (RXR) α/β/γ,and modulate corresponding target genes in the nucleus.12,13

Knowledge about the roles of NR1 in FLD including NAFLD,and discussed future perspectives to develop promising pharmacological strategies targeting NRs for liver diseases have been the subject of a recent review.12Here we mainly focus on the new findings on the roles of NRs in NAFLD and discuss future perspectives to target NRs for NAFLD.

2.NRs in NAFLD

In this section,we described the role of NRs,mainly focusing on PPAR,CAR,FXR,PXR and LXR,in liver pathophysiology and possible therapeutic strategies for the prevention and treatment of NAFLD through targeting these key receptors.

2.1.PPARs

PPAR,a ligand-activated family of transcription factors that play a significant role in the control of lipid homeostasis,is comprised of PPARα,PPARβ/δ,and PPARγ.14,15The PPAR family is expressed differently in tissues;PPARα is highly expressed in the liver,kidney,heart and intestine,PPARγ is most highly expressed in adipose tissues and PPARβ/δ is more widely expressed in different tissues,including liver,brain,kidney,heart,and adipose tissues.16,17

IR and inflammation are important steps in the development of NAFLD.PPARγ is an important regulator of inflammatory responses and lipid storage.Abnormal lipid-mediated hepatic inflammatoryimmune dysfunction and chronic low-grade inflammation are critical factors in the pathogenesis of NAFLD.18Macrophage polarization is an important mechanism in modulation of the inflammatory response.19Activation of PPARγ significantly shifted macrophage polarization from M1 towards the M2 phenotype and improved hepatic steatosis.20In diet-induced obese mice,the PPARγ agonist rosiglitazone improved fatty liver,inflammation,and IR.18PPARα in human liver is able to effectively induce the expression of genes involved in lipid transport and catabolism,notably mitochondrial and peroxisomal fatty acid β-oxidation.21PPARα was identified as a key regulator of adiponectin homologue novel osmotin protection against obesity/diabetes-induced NAFLD in ob/ob and db/db genetically-obese mouse models,which was associated with activation of the PPARα/AMP-activated protein kinase (AMPK)/sirtuins (SIRT1) axis.22Therefore,PPARα agonists are potential candidates for therapy for patients with NAFLD.It was found that GW0742,a PPARβ/δ agonist,could upregulate serum high-density lipoprotein cholesterol (HDL-C) and HDL phospholipids in NAFLD mice,which was abolished in Pparb/dnull mice.23PPARγ plays a key role in adipogenesis.Rivoglitazone can significantly reduce hyperglycemia,hyperinsulinemia,and hypertriglyceridemia by acting as a potent PPARγ agonist.24Rivoglitazone could also enhance adiponectin levels in plasma,decrease adipose mRNA levels in mice and ameliorate IR.25In clinical practice,rivoglitazone has been used for the treatment of type 2 diabetes mellitus,but further studies are needed due to its adverse effects that are similar to other thiazolidinediones PPARγ agonist.24

Interestingly,although PPARα agonists have positive functions in animal models of NAFLD,there was no obvious improvement in NAFLD patients through assessing serum biochemical index and grade of steatosis,inflammation and fibrosis.25The relatively weak potency may be attributed to species differences.PPARα expression and signaling is lower in human liver compared to mouse liver.26Some adverse effects have also been reported.Long-term thiazolidinediones treatment may cause a reduction in bone mineral density and increase fracture risks in humans.12Therefore,more studies are needed to access effects of PPAR agonists in NAFLD patients.Dual- or pan-agonists should be considered as potential therapeutic targets to enhance therapeutic effects and reduce side effects in NAFLD patients.

2.2.CAR

CAR was reported to be a key mediator in protecting against hepatic steatosis via suppressing lipogenesis and gluconeogenesis.27The relationship between CAR and lipid metabolism has been proposed.28Activation of CAR protects against fatty liver and IR.29In contrast,there was no improvement of NAFLD in Car-/-mice.30CAR activation could dramatically improve fatty liver by inhibition of lipogenesis and induction of fatty acid β-oxidation.29CAR activation could also alleviate hepatic steatosis by repressing lipogenic genes like the stearoyl-CoA desaturase-1 (Scd1),sterol regulatory element-binding protein 1c (Srebp1c) and the acetyl-CoA carboxylase (Acc) in high-fat-fed mice.28Irisin is a hormone proteolytically processed from fibronectin type III domaincontaining protein 5 (FNDC5).31In genetic-induced obese mice,CAR activation could improve hepatic steatosis and IR through inducing FNDC5/irisin expression in the liver.32Irisin transgenic mice were also protected against high-fat diet (HFD)-induced obesity and IR.32The classical CAR agonist 1,4-bis-(2-(3,5-dichloropyridyloxy)) benzene,3,3＇,5,5＇-tetrachloro-1,4-bis (pyridyloxy) benzene (TCPOBOP) was reported to markedly prevent hepatic steatosis through suppression of insulin sensitivity,lipogenic and gluconeogenic genes expression in high-fat-fed mice and obese mice.33These results support the view that CAR activation is able to alleviate hepatic steatosis.

However,there are some conflicting results demonstrating increased serum lipids and decreased liver β-oxidation with CAR activation in mice.13Inflammation and apoptosis in NASH mice were alleviated via CAR activation,but hepatic fibrosis was increased.12The number and size of lipid droplets were also increased by TCPOBOP treatment in mouse liver.28At the same time,up-regulation of the gene encoding patatin-like phospholipase domain-containing protein 3 (PNPLA3),associated with the pathogenesis of NAFLD development,was observed in mice and human hepatocytes treated with the CAR activator.34These conflicting findings can be attributed to the various models and the specific CAR activators used.

2.3.FXR

FXR is enriched in the liver,intestine,kidney,and adrenal cortex and is generally considered as a bile acid (BA) receptor.35BAs modulate lipid and glucose metabolism,and regulate inflammation in the liver and other tissues.FXR could regulate not only BA homeostasis but also lipid and glucose homeostasis.36Additionally,FXR was involved in the development of NAFLD.37FXR deficiency mice displays an increase in cholesterol and triglyceride levels,highlighting the relevance of FXR to lipid metabolism.38

BAs are products of cholesterol catabolism and play central roles in lipid metabolism.39FXR can transcriptionally drive modulation of BA synthesis,influx,efflux,and detoxification along the enterohepatic axis.40High concentration of BAs could cause NAFLD through inducing oxidative stress,inflammation,fibrosis and cirrhosis.It was shown that serum BAs were altered in NASH patients compared with those of healthy people.41In agreement with patients with NASH,the HFD-fed rats exhibited elevated expression of cholesterol 7α hydroxylase(CYP7A1),which resulted in an increase in BA production.41FXR is critical to maintain cholesterol homeostasis.FXR agonists have been investigated as candidates for the treatment of NASH.Obeticholic acid (OCA),approved for the treatment of primary biliary cholangitis,has gained significant attention due to its beneficial effects on NAFLD in animal experiments and human clinical trials.42In some animal models of NAFLD,OCA led to weight loss,improved glucose tolerance,and reduced target genes involved in fatty acid synthesis,lipogenesis and gluconeogenesis.43Improved liver histology,weight loss,decrease in serum triglyceride levels and reduction in blood pressure were observed in patients with NAFLD after OCA intervention.43FXR agonist,cilofexor,was reported to improve hepatic steatosis,liver stiffness,liver function tests and serum fibrosis markers.44A recent study showed that the FXR agonist fexaramine also mitigated ethanol-induced liver injury.45Fexaramine interventions improved BA-FXR-fibroblast growth factor(FGF)-15 signaling by modulation of hepatic CYP7A1 and lipid metabolism,and reduced steatosis in mice.45Cycloastragenol was able to improve HFD-induced liver steatosis by activating FXR signaling,which was indicated by reduction of lipid accumulation in the liver accompanied by lowered blood glucose,serum triglyceride levels and hepatic BA pool size.46These results support that FXR is a crucial player in the progression of liver diseases related to lipid metabolism especially BA homeostasis.

Additionally,BAs are critical components of the gastrointestinal tract that link the gut microbiota to hepatic and intestinal metabolism.47Small intestinal bacterial overgrowth in NAFLD and NASH patients suggest that increased exposure to intestinal bacterial products may contribute to their pathogenesis.48Animal models promoted that probiotics may reduce NASH progression.49,50The potential mechanisms of microbiota-BA-FXR in NASH,which also provide a potential strategy for NAFLD treatment targeting FXR,was recently summarized.51

Paradoxically,intestine-specific Fxr knockout mice could improve glucose homoeostasis and FXR agonist significantly aggravates disorders in lipid metabolism and IR in obesity mouse models.52Glycine-β-mucicholic acid (G-MCA),as selective highaffinity FXR inhibitor,could reduce obesity and reverse metabolic parameters in obesity mouse models.53Another report found that there were no significant differences in the severity of steatosis,inflammation,and fibrosis between wild-type (WT) and hepatocyte-specific Fxr knockout mice.54It is well known that FXR plays a different role in different organs,and therefore examining tissue specific functions of FXR in NAFLD pathogenesis is of great significance.

2.4.PXR

PXR as a membrane of NRs family is expressed in different organs,especially highly in the liver and gut.13PXR plays an important role in the regulation of genes related to expression of drug metabolizing enzymes and transporters during the disposition of xenobiotics and the homeostasis of endobiotics such as lipids,BAs,bilirubin,glucose,and retinoic acid.55It was reported that the activation of PXR affected drug-induced liver injury through increasing the expression of phase I and phase II drug metabolizing enzymes.56PXR intervenes in the onset and development of liver diseases mainly by regulating the metabolism of lipids and glucose,BA homeostasis and inflammation in the liver.57Here we mainly summarize the role of PXR in NAFLD through the regulation of lipids and/or BA metabolism.

PXR activation accelerates steatosis through increasing lipid synthesis and fatty acid uptake.56,58Several studies have shown that PXR activation can induce lipogenesis in a SREBP1cindependent manner.59Lipid accumulation and hepatic steatosis in Pxr-transgenic mice resulted in an increased expression of the fatty acid translocase (FAT/CD36) and several accessory lipogenic enzymes.60CD36 is a multi-ligand scavenger receptor,its significance in hepatic steatosis is in line with the notion that hepatic free fatty acid(FFA)uptake plays an important role in steatosis.60,61PXR activation is also associated with suppression of several genes involved in fatty acid β-oxidation,such as PPARα and thiolase.59PXR repressed fatty acid β-oxidation-related genes through interaction with the insulin-responsive forkhead box factor A2 (FoxA2)with a key regulator of β-oxidation.11PXR ablation would inhibit HFD-induced obesity,hepatic steatosis,and IR,which was associated with the inhibition of c-Jun NH2-terminal kinase (JNK) activation and down-regulation of lipin-1,encoded by a PXR target gene.62Rifampicin,as a typical hPXR agonist,was able to enhance lipid accumulation in human primary hepatocytes.62The solute carrier family 13 member 5(SLC13A5)was identified as a novel PXR target gene,and is involved in the synthesis of fatty acids.Knockdown of SLC13A5 resulted in a significant decrease of the lipid content in HepG2 cells,suggesting that PXR-mediated transcription of SLC13A5 may provide a new avenue to study PXR in NAFLD.63

However,it is noteworthy that both activation and knockout of Pxr could promote hepatic steatosis in mice.64The same results were found in human hepatoma HepG2 cells.64Further study found that activation of PXR resulted in an induction of the SREBP1-lipogenic pathway by increasing SREBP1A expression,whereas knockdown of Pxr led to an up-regulation of aldo-keto reductase(AKR)1B10,which enhanced the ACC-catalyzed reaction step of de novo lipogenesis.64These findings reveal that SREBP1A and AKR1B10 are novel PXR target genes in the lipogenic pathway.Further studies are needed to aid in the development and use of adequate PXR modulators in the progression of NAFLD.

PXR also acts as a physiological sensor for toxic BAs and regulates genes involved in their detoxification.65Recent studies found that Schisandrol B or Wuzhi tablet protected against lithocholic acid(LCA)-induced cholestasis by activating PXR,and up-regulating PXR target genes involved in BA homeostasis,and regulating BA homeostasis.66,67Cytochrome P450 3A (CYP3A) enzymes encoded by PXR target genes are involved in BA metabolism.68CYP3A activity was reduced in NAFLD patients,and mRNA levels of CYP3A4 were down-regulated in liver of NASH patients compared to that of the normal liver.69Hepatic CYP3A4,major homologue of mouse CYP3A11,is a PXR target gene.70However,recent studies showed that PXR might not be the key regulator of the suppressive effect of palmitate treatment on CYP3A based on the results from loss-offunction and gain-of-function experiments.71Considering that other NRs such as CAR and FXR can also regulate CYP3A4,further studies are needed.Recent studies found that a fibroblast growth factor 21 (FGF21)-PXR pathway might be involved in decreased hepatic CYP3A4 metabolic activity in NAFLD.72

2.5.LXR

LXR is a family of NR that regulates genes involved in lipid and cholesterol homeostasis.LXRα is highly expressed in the liver and other tissues involved in lipid metabolism,whereas LXRβ is prevalently distributed at a lower level in different tissues.20

LXR is a direct regulator of genes involved in hepatic lipogenesis.73LXR controls lipid metabolism by regulating SREBP1c expression and the carbohydrate response element-binding protein (ChREBP) in NAFLD,two transcription factors involved in lipogenesis.74,75Lxra mRNA and LXRα protein were significantly increased in a rat model of NAFLD induced by HFD.76LXRα target gene mRNAs associated with lipogenesis were overexpressed in patients with NAFLD.77These results show that LXRα may be closely related to the progression of NAFLD.Hepatic inflammation is one of the critical factors in the procession of NAFLD,especially in promoting NASH.Treatment with LXR agonist could attenuate lipopolysaccharide (LPS)-induced hepatic inflammation in a HFDinduced NAFLD murine model,which was reflected by inhibiting the pro-inflammatory activity of macrophages,as evidenced by reduced tumor necrosis factor-alpha (TNF-α) and inducible nitric oxide synthase (iNOS) expression through inhibiting JNK and the phosphoinositide 3-kinase (PI3K) signaling pathway.78Activation of LXR may alleviate liver injury in part through its antiinflammatory effects and reducing hepatic cholesterol accumulation.77,78These findings indicate that LXR shows anti-inflammatory effects in steatosis.

Interestingly,SR9238,a liver-selective LXR inverse agonist,displayed the ability to reduce the expression of lipogenic genes in the liver as well as to reduce hepatic steatosis in obese mice.79In addition,SR9238 could also reduce the expression of proinflammatory cytokines and markers of hepatocellular injury in the hepatic steatosis model.In accordance with this result,Lxr-a/bdeficient mice presented lower hepatic steatosis and higher insulin sensitivity than the matched WT mice.80These findings suggest that LXR might be associated with hepatic inflammation and fibrosis in NAFLD patients.

Hepatic expression of LXRα and its related lipogenic and inflammatory genes was abnormally increased in NAFLD patients with steatosis.81Inhibition of LXRα activation might be of value for the treatment of NAFLD.Oleanolic acid (OA;3β-hydroxyolean-12-en-28-oic acid),as a pentacyclic triterpenoid,has promising pharmacological activities including hepatoprotective,antiinflammatory,and anti-cancer activities.OA has also been demonstrated to reduce hepatocellular lipid content through decreasing the promoter activity of LXR and the mRNA and protein expression of LXRα target genes.82Therefore,LXR is the main determinant in hepatic steatosis,which has been showed in both animal models and human patients with NAFLD.These contradictions highlight the need for further studies to investigate how LXRs affect NAFLD.

2.6.Cross-talk of NRs

Since all members of the NR family share common structural features,cross-talk among different NRs which function in common activation pathways have been investigated.

PPARα,PXR,CAR and LXR are activated in the progression of HFD-induced NAFLD in mice,which was characteristic of hepatic steatosis,lobular inflammation,and progressive fibrosis.Further studies demonstrated that the gene expression of hepatic NRs such as PXR,PPARα/γ/δ,CAR,LXRα,FXR and their target genes such as Cyp3a11,Cyp2b10,ATP-binding cassette sub-family G member 5/8(Abcg5/8) were changed in a time-dependent manner during the progression of NAFLD induced by a HFD in mice.83CD36,a free fatty acid uptake transporter,is a PPARγ target gene.84PXR-induced lipid accumulation was reported to be associated with the increased expression of Cd36 and Cd36 is a direct transcriptional target of PXR in mice.60,61LXR may also regulate Cd36 expression in the liver and hepatic steatosis induced by LXR agonists was largely reversed in the Cd36-null mice.60These studies support the notion that Cd36 is a shared target of LXR,PXR,and PPARγ,indicating that these three NRs may cooperate to increase hepatic steatosis by increasing the expression of fatty acid transporter CD36.Inflammation is an important step in the development of NAFLD,especially NASH.PPARα induces the expression of inhibitor of kappa B(IkB)to inhibit the nuclear factor kappa-B(NF-kB)signaling.85Activation of PPARγ reduces the expression of inflammatory factors,such as TNF-α.LXR activation also attenuates hepatic inflammation and reduces hepatic cholesterol accumulation.86LXRs and PPARγ may control immunity by mediating pro-inflammatory gene transrepression through parallel SUMOylation-dependent pathways.87These data provide the basis of cross-talk between PPARγ and LXRs in hepatic inflammation during steatosis.The nuclear factor (erythroidderived 2)-like 2 (NRF2)/FXR/LXRα pathways also synergistically regulate both endogenous and exogenous metabolism in NAFLD mice.88A recent animal study showed that acanthoic acid (AA),with anti-inflammatory and hepatic-protective effects,may target FXR/LXRs-mediated AMPK-SIRT1 signaling to eliminate lipid accumulation in NAFLD.89Moreover,AA attenuated lipid accumulation via FXR and LXRs activation in steatotic alpha mouse liver(AML)-12 cells.These data suggest that the FXR-LXRs axis may play crucial roles together in NAFLD.

Since all members of the NR family share common structural features,the study of cross-talk among different NRs may also provoke novel ideas for molecular mechanisms of NAFLD induced by multiple factors.In order to increase the beneficial effects of NRs in NAFLD,combination therapies of different NRs agonist/antagonist should also be examined when targeting NRs as potential therapeutic intervention strategies for NAFLD.12

3.Summary and future perspectives

NAFLD is the most common chronic liver disease worldwide and is characterized by excessive deposition of lipid in the liver.More studies suggest that NRs play essential roles in the development of NAFLD by mediating target genes linked to lipid,BA and glucose metabolism and genes involved in inflammation response,IR and obesity.The possible pathways modulated by NRs in NAFLD are summarized in

Fig.1.Targeting NRs may be beneficial for the prevention and treatment of NAFLD.However,the underlying mechanisms of NR signaling during the onset and development of NAFLD remain to be clarified.Considering that all members of the NR family sharing common structural features,cross-talk among different NRs which function in common activation pathways should continue to be investigated.

Fig.1.A simplified summary for NRs in NAFLD.

On the other hand,activation of NR1 is expected to attenuate hepatic steatosis,inflammation,fibrosis,IR and dyslipidemia.However,some NR agonists exerted insufficient or paradoxical effects,which may be due to low potency and/or low specificity for the target organ/tissue.Thus,more attention should be paid to these controversial findings and the development of more potent and more stable single agonist is urgently needed.Novel pharmacological interventions including those mentioned such as combination of agonist/antagonist,dual/triple agonist,organ-specific agonist/antagonist,and NR modulator,would be critical to obtain the most benefits of NR activation and to minimize those adverse effects when targeting NRs as the promising therapeutic intervention strategies for NAFLD in the future.

Authors’contributions

X.Yang wrote the draft manuscript.H.Bi designed the structure and framework of the manuscript and H.Bi,F.J.Gonzalez and M.Huang revised the manuscript.All authors read and approved the final manuscript.

Declaration of competing interest

The authors declare that they have no conflict of interest.

Acknowledgements

The work was supported by the Natural Science Foundation of China (81973392,81320108027),the National Key Research and Development Program of China (2017YFE0109900),the Natural Science Foundation of Guangdong Province (2017A030310330,2017A030311018),China Postdoctoral Science Foundation(2019TQ0398)and the Fundamental Research Funds for the Central Universities (19ykyjs34).