Retinoic Acid Signaling in Fatty Liver Disease

Abstract

Retinoic acid (RA) is a metabolite of vitamin A and is essential for development and growth as well as cellular metabolism. Through genomic and nongenomic actions, RA regulates a variety of physiological functions. Dysregulation of RA signaling is associated with many diseases. Targeting RA signaling has been proven valuable to human health. All-trans retinoic acid (AtRA) and anthracycline-based chemotherapy are the standard treatment of acute promyelocytic leukemia (APL). Both human and animal studies have shown a significant relationship between RA signaling and the development and progression of nonalcoholic fatty liver disease (NAFLD). In this review article, we will first summarize vitamin A metabolism and then focus on the role of RA signaling in NAFLD. AtRA inhibits the development and progression of NAFLD via regulating lipid metabolism, inflammation, thermogenesis, etc.

1. Introduction

Nonalcoholic fatty liver disease (NAFLD) is a spectrum of liver diseases ranging from simple steatosis, also known as nonalcoholic fatty liver (NAFL), to nonalcoholic steatohepatitis (NASH). The global NAFLD prevalence of NAFLD has reached 32.4% ¹, and the prevalence rate is increasing due to obesity. Many people with NASH develop liver cirrhosis and hepatocellular carcinoma (HCC) as the disease progresses.^2,3 The pathogenic mechanisms of NAFLD are complex and may involve interactions among over-nutrition, genetics, gut microbiota, etc, leading to insulin resistance, lipotoxicity, apoptosis, mitochondrial dysfunction, oxidative stress, inflammation, and fibrogenesis.^2–4 So far, no drugs have been approved for the treatment of NASH.

Retinoids are metabolites of vitamin A (retinol) that include retinaldehyde/retinal, retinyl esters, oxidized retinol, retinoic acid, and conjugates of these compounds, which are essential for cell growth and differentiation.^5,6 Abnormal retinoid levels have been linked to a wide variety of clinical issues, including cardiovascular disease, diabetes, obesity, fatty liver disease, osteoporosis, skin illnesses, and cancer.^7–10 Mammals cannot synthesize vitamin A. Vitamin A is absorbed by intestinal epithelial cells, stored in the liver, and metabolized in target cells to more biologically active metabolites, retinoic acid (RA) and 4-oxo-RA.¹¹

2. Vitamin A metabolism

Vitamin A is found in meat, dairy products, and β-carotene. In enterocytes, β-carotene is converted to retinal by β-C 15,15’ oxygenase 1 (BCO1) and then reduced to retinol by a retinal reductase. Retinyl esters are hydrolyzed to form retinol by retinyl ester hydrolase (REH) prior to absorption. Retinol is then re-esterified with long-chain fatty acids by lecithin:retinol acyltransferase (LRAT) to regenerate retinyl esters, which are secreted with chylomicrons (CM) from the intestine and up-taken by hepatocytes in the form of CM remnants (Figure 1). In hepatocytes, retinyl esters are hydrolyzed by REH to produce retinol. Retinol binds to a retinol-binding protein (RBP) for release into the circulation and is up-taken by other cell types via one of the membrane receptors, such as the signaling receptor and transporter of retinol STRA6. Within cells, cellular retinol-binding proteins (CRBP) participate in the transport and metabolism of retinol. Retinol is converted to retinal by retinol dehydrogenase (RDH) or retinyl esters by LRAT.

Figure 1. Overview of Vitamin A metabolism and RA signaling pathway.

In the intestine, retinyl esters (RE) are hydrolyzed by RE hydrolase (REH) to form retinol. Retinal dervided from β-carotene is reduced to retinol by retinaldehyde reductase. Retinol is esterifed by lecithin retinol acyltransferase (LRAT) to form REs, which are assembled into chylomicron (CM) and secreted to the circulation. REs are uptaken by hepatocytes and are hydrolyzed to form retinol by hepatic REH. Retinol is secreted into the circulation and binds to the retinol binding protein (RBP)/transthyretin (TTR) complex. The membrane protein STRA6 recognizes RBP and transports retinol into cells. In the cells, retinol is converted to retinal by retinol dehydrogenase (RDH), which is further converted to retinoic acid (RA) by retinaldehyde dehydrogenase (RALDH). All-trans RA (AtRA) activates retinoic acid receptors (RAR) whereas 9-cis RA activates retinoic X receptors (RXR). RAR and RXR form heterodimers and bind to retinoic acid elements (RARE) to regulate gene transcription and a variety of pathways, e.g. development, growth, metabolism, inflammation, tumorigenesis. RAR or RXR has three isoforms, α, β, and γ. Retinol may also be esterifed to form REs by LRAT. About 80% REs are stored in hepatic stellate cells (HSC). Loss of REs from HSC may result in HSC activation. RA may be metabolized by CYP26A1, -B1, or -C1 to form 4-ox-RA, 4-OH-RA, etc.

More than 80 % of the vitamin A in the liver is stored in hepatic stellate cells (HSC).¹² Retinal is converted by retinal dehydrogenase, also known as retinaldehyde dehydrogenase (RALDH), to all-trans RA (AtRA), 9-cis RA, 13-cis-RA, 9,13-di-cis RA, and 11-cis-RA. AtRA and 9-cis RA are the major biologically active forms of RAs. Cellular retinoic acid-binding proteins (CRABP) transport RA into the nucleus, where AtRA and 9-cis RA bind to retinoic acid receptor (RAR) and retinoic X receptor (RXR), respectively, to regulate gene transcription (Figure 1). Excess RA is metabolized by P450 family enzymes (CYP26A1, CYP26B1, and CYP26C1) into polar chemicals, including 4-hydroxy-RA and 4-oxo-RA, which are glucuronidated and then removed from the body via the kidneys or liver into bile.¹³

3. Retinoic acid receptor (RAR)/retinoic X receptor (RXR)

RAR has three isoforms, RARα (NR1B1), RARβ (NR1B2), and RARγ (NR1B3). RXR also has three isoforms, RXRα (NR2B1), RXRβ (NR2B2), and RXRγ (NR2B3). RAR heterodimerizes with RXR and the dimers bind to the retinoic acid response element (RARE) in the target genes. Ligand binding to the RAR/RXR heterodimers results in the change in associated cofactors and activation or repression of gene transcription. More than 532 genes may be regulated by RA through the traditional genomic route.¹⁴ In addition to the traditional genomic functions, RARs may also be engaged in nongenomic biological functions, such as the initiation of translation and kinase cascades,¹⁵ e.g. the p38 or ERK mitogen-activated protein kinase.^16,17 The discovery of retinoic acid has been proved valuable to human health. For instance, AtRA and anthracycline-based chemotherapy are the standard treatment of acute promyelocytic leukemia (APL),^18,19 a highly curable disease.

4. Altered retinoid metabolism in NAFLD

Hepatic stellate cell (HSC) activation is associated with reduced hepatic retinyl esters and retinol concentrations and vitamin A metabolism.²⁰ In rats, vitamin A deficiency causes HSC activation to produce extracellular matrix²¹ and potentiates carbon tetrachloride (CCl₄)-induced liver fibrosis²². In contrast, supplementation with vitamin A inhibits CCl₄-induced liver fibrosis in pigs.²³ Vitamin A is also shown to reduce mortality of animals with induced liver fibrosis by copper sulfate.²⁴ Clearly, vitamin A and its metabolites may play a key role in liver fibrogenesis.²⁵

Multiple studies on patients have shown inverse relationship with serum retinol levels and the severity of NAFLD.^26–28 Chaves et al. show that serum and hepatic retinol levels decrease in NAFLD by 35.9% and 67.9%, respectively and that a significant association exists between hepatic retinol concentrations and the severity of NAFLD.²⁹ Similarly, serum RA levels are also shown to be inversely correlated with the severity of NAFLD.³⁰ Zhong et al. show that in NAFLD patients, hepatic vitamin A metabolites, including retinyl-palmitate esters, AtRA, 13-cis-RA, and 4-oxo-atRA, are reduced while the levels of retinol (the inactive form of vitamin A) do not change.³¹ They suggest the levels of metabolites of vitamin A, rather than retinol, are more reliable for predicting the disease progression of NAFLD.³¹

In animal models of NAFLD, Trasino et al. show that hepatic retinol levels are decreased in high fat diet (HFD)-induced obese mice or genetically obese mice (db/db or ob/ob mice) accompanied by reduced RAR and RBP1 mRNA levels in HSC and elevated serum retinol levels.³² Saeed et al. report that hepatic retinyl palmitate levels are significantly increased along with upregulated hepatic mRNA levels of genes related to retinol storage and metabolism in hepatocytes of high fat/high cholesterol diet-fed mice and ob/ob mice.³³ In rats fed a methionine-choline deficient (MCD) diet, hepatic and serum retinol levels are decreased.³⁴

The changes in hepatic vitamin A and their metabolite levels are likely due to the change in genes involved in retinol metabolism. Another gene associated with NAFLD is aldo-keto reductase family 1 member B10 (AKR1B10), which is a key enzyme of retinol metabolism with a very efficient and high all-trans-retinaldehyde reductase activity in converting all-trans-retinaldehyde to retinol, is significantly overexpressed in human NASH liver and HCC tumors.³⁵ Pettinelli et al. show that NASH patients have highly induced AKR1B10 expression and reduced ALDH1A2 and ALDH1A3 expression in the liver as well as elevated plasma retinol levels.³⁶ 17-beta hydroxysteroid dehydrogenase 13 (HSD17B13) has RDH activity and a loss-of-function mutation in HSD17B13 reduces the progression of NAFLD.³⁷ Patatin-like phospholipase domain-containing 3 (PNPLA3), is reported to have retinyl-palmitate lipase activity, releasing retinol from lipid droplets in hepatic stellate cells.³⁸ The genetic association studies show that the genetic variant in I148M (rs738409), is a the risk factor for NAFLD as it reduces the lipase activity and decreases circulating serum retinol levels in NAFLD patients.^38–40 Thus, it is evident that disruption in the retinoid metabolism is often associated with NAFLD.

5. Mechanisms underlying the regulation of NAFLD by RA signaling

Hepatic lipid accumulation occurs from an imbalance between lipid absorption/uptake, synthesis, and secretion/disposal, which are regulated by several pathways, including uptake of circulating free fatty acids (FFAs), de novo lipogenesis (DNL), lipolysis, fatty acid oxidation (FAO), and secretion of lipids in very low-density lipoproteins (VLDL) or cholesterol to bile. Obesity is also associated with the development of NAFLD. Next, we will discuss how the RA signaling affects hepatic lipid metabolism, inflammation, fibrogenesis, and obesity.

5.1. RA signaling in hepatic lipid metabolism

Accumulation of FFAs may cause lipotoxicity. Amengual et al. show that AtRA treatment induces hepatic expression of peroxisome proliferator-activated receptor alpha (PPARα), retinoid X receptor alpha (RXRα), uncoupling protein 2 (UCP2), liver-type carnitine palmitoyltransferase 1 (CPT1), and carnitine/acylcarnitine carrier (CAC), and a reduction in the mRNA expression levels of sterol regulatory element binding protein 1c (SREBP1c) and fatty acid synthase (FASN),⁴¹ and reduces hepatic triglyceride (TG) levels and VLDL secretion and increases circulating 3-hydroxybutyrate levels.⁴¹ AtRA is also shown to induce FAO in HepG2 cells⁴² and mouse primary hepatocytes.⁴³ We show that AtRA induces FAO independent of activation of RARα.⁴³ PPARα is a key regulator of FAO; activating PPARα protects from trans-fatty acid induced steatohepatitis while PPARα inhibition increases the susceptibility to steatohepatitis.⁴⁴ PPARα binds to DNA as a heterodimer with RXR. Both AtRA and 9-cis-RA can induce the expression of RXR which in turn activates PPAR:RXR heterodimers leading to the transcription of PPARα target genes.⁴⁵ In addition to reducing hepatic TG accumulation, activation of PPARα:RXR also decreases the production of TG-rich VLDL and plasma TG levels.⁴⁶ Peroxisome proliferator-activated receptor beta/delta (PPARβ/δ) is another transcription factor that is known to stimulate FAO. PPARβ/δ may prevent dyslipidemia, insulin resistance, obesity, and NAFLD by regulating hepatic glucose catabolism and FAO and by inhibiting DNL via AMPK signaling.⁴⁴ Apart from its canonical RARs, AtRA binds to PPARβ/δ with high affinity depending on the expression levels of CRABPII and FABP5 which delivers AtRA to RAR and PPARβ/δ respectively.⁴⁷

Circulating FFA uptake is a major source of the FA pool in the liver. During fasting and insulin resistance, hepatocytes extract FFAs which increase lipogenesis and lipotoxicity. Fatty acid translocase (CD36/FAT) is a transmembrane glycoprotein that acts as scavenger receptor capable of binding several ligands, including long-chain fatty acids, lipoproteins, and oxidized lipids. Even though CD36 expression is considered low in the normal liver, its expression is increased in the liver of NAFLD patients.⁴⁸ It is well known that CD36 increases FFA uptake and drives hepatosteatosis onset and to its progression to NASH.⁴⁹ CD36 is a well-characterized PPARγ target.^50,51 It is reported recently that Alisol B, a natural compound isolated from a plant called Alisma orientalis, attenuates HFD and carbon tetrachloride (CCL4)-induced liver steatosis by inhibiting CD36 by regulating the RARα-HNF4α-PPARγ transcriptional cascade.⁵² Tang et al. show that activating RARβ2 inhibits PPARγ and CD36 levels in HFD-fed mice.⁵³ We show that AtRA inhibits hepatocyte fatty acid uptake and CD36 expression and that the inhibition of CD36 expression is dependent on activation of RARα.⁴³

DNL is the process of the synthesis of endogenous fatty acids from acetyl-CoA produced by other metabolic pathways such as glycolysis. About 26% of TG in the livers of NAFLD patients come from DNL suggesting that impairment in DNL contributes to the pathogenesis of NAFLD.⁵⁴ Two major pathways downstream of the insulin receptor activates sterol regulatory element-binding protein 1C (SREBP1C), both involving the phosphoinositide-3 kinase (PI3K)/protein kinase B (PKB) pathway, one resulting in the phosphorylation of the nascent SREBP1c itself and the other in the activation of the liver X receptor (LXR).⁵⁵ Insulin resistance leads to hypertriglyceridemia and hepatic steatosis which is associated with increased SREBP1c activity. Therefore, inhibiting SREBP1C activation has the potential for treatment of hypertriglyceridemia and NAFLD.⁵⁶ Treatment of HFD-fed mice by RA reduces hepatosteatosis and this effect is suggested through sirtuin 1 (SIRT1)-mediated inhibition of SREBP1C.⁵⁷ Although AtRA inhibits lipogenic genes in the liver , DNL is not affected when mice are injected with heavy water followed by analysis of newly synthesized fatty acids or triglycerides,⁴³ suggesting that AtRA lowers hepatic TG levels likely independent of DNL.

Hepatic TG and cholesterol esters are secreted to the circulation in the form of VLDL. AtRA is shown to lower lipid contents in VLDL.⁴¹ Nonetheless, controversial data have been reported on the role of AtRA in hepatic lipogenesis and VLDL secretion.⁴⁵ Retinoids have been reported to induce hypertriglyceridemia due to enhanced hepatic lipogenesis and VLDL production and secretion as well as VLDL clearance.⁴⁵

5.2. RA signaling in hepatic inflammation

Retinoids have been known to possess anti-inflammatory effects for 40 years,^58,59 which may be mediated through downregulation of Th1 cytokines, such as tumor necrosis factor α (TNFα),^60,61 interferon gamma (IFNγ),⁶² and IL-12.⁶³ In the THP-1 monocyte/macrophage cell line, AtRA reduces liposaccharide (LPS)-induced production of the proinflammatory cytokines TNFα and IL-12 and enhances IL-10 production.⁶⁴ Mechanistically, retinoids inhibit the phosphorylation of IκB kinase α/β (IKKα/β),⁶⁵ the nuclear factor kappa B (NFκB)-DNA interaction or its translocation to the nucleus.^63,66 Consistent with these observations, deletion of RARα in macrophages⁶⁷ or hepatocytes⁴³ aggravates inflammatory response whereas RARβ2 activation inhibits inflammatory cytokine secretion,⁶⁸ suggesting a critical role of RARα and RARβ2 in mediating retinoid’s effects on inflammation.

5.3. RA in hepatic fibrosis and HSC activation

Fibrosis is a wound healing process characterized by extracellular matrix (ECM) accumulation that causes scarring and impaired liver function. The effects of RA on ECM accumulation and fibrosis are controversial. It is shown that loss of retinyl esters in hepatic stellate cells (HSC) is often a characteristic for HSC activation during liver injury.⁶⁹ Earlier studies show that 9-cis-RA enhanced plasminogen activator (PA)/plasmin levels and thereby induced proteolytic activation of TGF-β1, a strong fibrogenic cytokine, resulting in enhanced ECM production.⁷⁰ However, 9-cis-RA activates RXR, which can form heterodimers with a variety of nuclear receptors to exert its functions. Later reports show a protective effect of RA signaling on liver fibrosis. Hisamori et al. show that AtRA is shown to attenuate CCl4-induced liver fibrosis by reducing the production of TGFβ, IL-6 and collagen from HSCs in mice.⁷¹ They further show that AtRA inhibits TGFβ-dependent transdifferentiation of the cells and the activities of NFκB p65 and p38 mitogen-activated protein kinase ⁷¹. Wang et al. also show that AtRA reduces liver fibrosis induced by common bile duct ligation via inhibition of TGFβ and connective tissue growth factor (CTGF) in rats.⁷² In vitro studies show that AtRA inhibits HSC proliferation and collagen production via suppressing active protein-1, c-Jun N-terminal kinase signal, and expression of profibrogenic genes (TGF-β(1), CTGF, MMP-2, TIMP-1, TIMP-2, PAI-1), and inducing MMP-3 and MMP-13.⁷³ RA may also synergize with PPARγ to reverse fibrosis via modulating senescence of HSC.⁷⁴ In terms of specific RARs, expression of dominant negative form RARα is shown to induce fibrosis.⁷⁵ However, genetic ablation of RARα in the liver does not affect fibrogenesis.⁴³

5.4. RA signaling in obesity and insulin resistance

Obesity and insulin resistance are among the most common risk factors for NAFLD as the majority of obese and diabetic patients have NAFLD.^76,77 Thus, treating insulin resistance may help to fight NAFLD. Circulating RA concentrations are lower in subjects with NAFLD and are associated with hepatic lipid metabolism and insulin resistance.³⁰ AtRA treatment is known to attenuate obesity and insulin resistance.⁷⁸ The effect of AtRA on obesity is likely through inhibition of adipogenesis and induction of energy expenditure.^79,80 At molecular levels, AtRA is suggested to inhibit obesity via activation of both PPARβ/δ and RAR.⁸¹ We show that hepatic RARα plays an important role in mediating AtRA’s effect on diet-induced obesity.⁴³ Tsuchiya et al. show that AtRA improves insulin sensitivity likely via induction of leptin receptor-mediated phosphorylation of signal transducer and activator of transcription 3 (STAT3) and insulin receptor substrate 1 (IRS1) and RARα activation is important for these effects.⁸² Thus, the inhibition of obesity may play a role in AtRA-mediated amelioration of NAFLD.

6. Therapeutic potential of RA in NAFLD

Some studies have been aimed to identify the therapeutic potential of vitamin A metabolites in the treatment of NAFLD. Matsumoto et al. show that feeding obese Zucker (fa/fa) rats, an animal model of NAFLD, with brown rice increases RA synthesis which in turn, protects against NAFLD by increasing fatty acid oxidation (FAO) and VLDL secretion.⁸³ Zarei et al. report that AtRA significantly reduces liver steatosis in high fat diet (HFD)-fed rabbits.⁸⁴ We show that AtRA prevents and reverses Western diet-induced liver steatosis in mice.⁴³ Zhu et al.⁸⁵ and Kim et al.⁸⁶ also show that AtRA prevents HFD-induced liver steatosis in mice. Liu et al. report that RA levels are significantly reduced in NAFLD patients and correlated with hepatic TG levels.³⁰ It is also reported that the intake of β-carotene is inversely associated with liver steatosis in humans.⁸⁷ However, it remains to be investigated whether AtRA or other retinoids attenuates liver steatosis in humans.

7. Closing remarks and future directions

In this review, we primarily discuss the role of RA signaling in liver and to some extent in adipose tissue. However, RA signaling in other cells and tissues affects the progression of NAFLD. The gut-liver axis plays a key role in the pathogenesis of liver diseases, including NAFLD.^88,89 Dysregulation of gut microbiota, barrier and permeability contributes to the development of NAFLD.^90–92 There is a rich array of literature describing the role of RA signaling in reshaping gut microbiomes, inflammation, immunity, and barrier functions.^93,94 Serum retinol levels and gut permeability display an inverse relationship.^95,96 RA protects against a leaky gut likely through direct modulation of intestinal permeability and autoimmunity as well as regulating gut microbiota (e.g. Lactobacillus spp.) (see review ⁹³). RA inhibits gut microbiota dysbiosis,⁹⁴ which may in turn regulate nutrient absorption, gut permeability, and hepatic metabolism thus protecting against NAFLD.

Over the past decades, many studies from different groups investigated the role of retinoids, particularly AtRA, in metabolic homeostasis and cancer development. In addition to APL, AtRA is being used to treat a range of human cancers in clinical trials. ^97,98 In rodents, AtRA attenuates Western diet-induced liver steatosis, inflammation, and fibrosis likely via inducing FAO and energy expenditure and inhibiting fatty acid uptake, NF-κB, and TGFβ (Figure 2). AtRA activates RARα, RARβ, and RARγ. The relative role of these RARs in the liver and other cell types/organs (e.g. adipocytes, intestine) in the development of NAFLD should be elucidated. Understanding the cell-specific effects of RA signaling and the functions of other less-studied retinoids may offer new therapeutic approaches to treatment of NAFLD.

Figure 2. Regulation of the development of NAFLD by the RA/RAR signaling.

Activation of RAR by RA inhibits the development of NAFLD likely via several pathways. Activation of RAR induces fatty acid oxidation (FAO) and thermogenesis and inhibits fatty acid (FA) uptake, NF-κB, and TGFβ, leading to a reduction in hepatic lipid accumulation, inflammation and fibrogenesis. The role of RAR activation in inhibiton of de novo lipogenesis (DNL) remains to be further clarified.