Signal Transduction and Molecular Regulation in Fatty Liver Disease

Abstract

Significance: Fatty liver disease is a major liver disorder in the modern societies. Comprehensive understanding of the pathophysiology and molecular mechanisms is essential for the prevention and treatment of the disease.

Recent Advances: Remarkable progress has been made in the recent years in basic and translational research in the field of fatty liver disease. Multiple signaling pathways have been implicated in the development of fatty liver disease, including AMP-activated protein kinase, mechanistic target of rapamycin kinase, endoplasmic reticulum stress, oxidative stress, inflammation, transforming growth factor β, and yes1-associated transcriptional regulator/transcriptional coactivator with PDZ-binding motif (YAP/TAZ). In addition, critical molecular regulations at the transcriptional and epigenetic levels have been linked to the pathogenesis of fatty liver disease.

Critical Issues: Some critical issues remain to be solved so that research findings can be translated into clinical applications. Robust and reliable biomarkers are needed for diagnosis of different stages of the fatty liver disease. Effective and safe molecular targets remain to be identified and validated. Prevention strategies require solid scientific evidence and population-wide feasibility.

Future Directions: As more data are generated with time, integrative approaches are needed to comprehensively understand the disease pathophysiology and mechanisms at multiple levels from population, organismal system, organ/tissue, to cell. The interactions between genes and environmental factors require deeper investigation for the purposes of prevention and personalized treatment of fatty liver disease. Antioxid. Redox Signal. 35, 689–717.

Introduction

Nonalcoholic fatty liver disease (NAFLD) is the most prevalent liver disease in the world, estimated to be ∼25% of the global population (29). NAFLD manifests a spectrum of hepatic disorders from simple steatosis to nonalcoholic steatohepatitis (NASH), and in some cases progresses to liver cirrhosis and cancer (29). Obesity, overnutrition, and physical inactivity are common risk factors for NAFLD (29). In addition, alcohol-related liver disease (ALD) is a common hepatic disorder in chronic alcohol drinkers (6). ALD shares similar progressive characteristics with NAFLD. In this review, we aimed to summarize recent developments in both ALD and NAFLD with a focus on signal transduction and epigenetic and transcriptional regulation.

Signal Transduction in Fatty Liver Disease

AMP-activated protein kinase signaling

AMP-activated protein kinase (AMPK) senses low energy status and plays a critical role in hepatic metabolic homeostasis (Fig. 1). AMPK is composed of three subunits: catalytic α subunit and regulatory β and γ subunits. AMPK activity is stimulated by nutrient deprivation and inhibited by overnutrition and alcohol consumption (56). Multiple factors can activate AMPK, including liver kinase B1 (LKB1), TGFβ (transforming growth factor β)-activated kinase 1 (TAK1), calcium/calmodulin-dependent protein kinase kinase β (CAMKKβ), and sestrins (SESNs) (56).

FIG. 1.

AMPK signaling and function in fatty live disease. AMPK activity is regulated by cellular energetic and nutritional status. As a response, AMPK controls multiple cellular processes, including hepatic lipid metabolism, oxidative stress, inflammation, fibrosis, and cell death. ACC1/2, acetyl-CoA carboxylases 1 and 2; AMPK, AMP-activated protein kinase; CAMKKβ, calcium/calmodulin-dependent protein kinase kinase beta; Casp6, caspase-6; CPT1, carnitine palmitoyltransferase 1; FOXO, forkhead box O; HMGCR, 3-hydroxy-3-methylglutaryl-CoA reductase; LKB1, liver kinase B1; mTORC1, mechanistic target of rapamycin kinase complex 1; NAD⁺, nicotinamide adenine dinucleotide; NF-κB, nuclear factor kappa B subunit; NIK, NF-κB-inducing kinase; p300, E1A binding protein p300; PGC1α, peroxisome proliferator-activated receptor gamma coactivator 1 alpha; Raptor, regulatory associated protein of mTOR complex 1; SIRT1, sirtuin 1; Smad3, SMAD family member 3; SREBP1/2, sterol regulatory element binding proteins 1 and 2; TAK1, transforming growth factor beta-activated kinase 1; TBK1, TANK binding kinase 1; TSC1/2, tuberous sclerosis proteins 1 and 2; ULK1, Unc-51-like autophagy activating kinase 1.

AMPK is believed to play a significant role in both ALD and NAFLD (53, 225, 238). AMPK regulates hepatic lipid homeostasis through multiple processes, including inhibition of lipogenesis and activation of fatty acid oxidation (FAO). Adenoviral overexpression of AMPKα1 in the liver reduces hepatic lipogenic gene expression and triglycerides in hyperlipidemic diabetic rats (181). Hepatic activation of AMPK also protects against diet-induced fatty liver in animal models (53). AMPK can inhibit lipogenesis by direct phosphorylation of acetyl-CoA carboxylases 1 and 2 (ACC1/2) (136). The ACC enzymatic product malonyl-CoA is not only a substrate for fatty acid synthase (FASN) but also an allosteric inhibitor for carnitine palmitoyltransferases (CPTs) that transport acyl-CoA into mitochondria for oxidation (56).

AMPK also directly phosphorylates and inhibits both sterol regulatory element binding proteins 1 and 2 (SREBP1 and SREBP2) to downregulate hepatic lipogenesis and cholesterol biosynthesis (108). In addition, AMPK phosphorylates both tuberous sclerosis complex subunit 2 (TSC2) and Raptor to inhibit mechanistic target of rapamycin kinase complex 1 (mTORC1), which activates hepatic lipogenesis by regulating SREBP maturation and activation (56, 198).

AMPK has an anti-inflammation function in the liver. Hepatic overexpression of constitutively active AMPK downregulates inflammatory genes in high-fat diet-treated mouse livers (53). AMPK also dampens hepatic inflammation through inhibition of the activity of nuclear factor kappa B subunit (NF-κB). On the one hand, AMPK can promote sirtuin 1 (SIRT1) activity by an increase of cellular NAD⁺ levels (17). As a result, SIRT1 suppresses the NF-κB transcriptional activity by deacetylation of its RelA/p65 subunit at Lys310 (222). On the other hand, AMPK can decrease the NF-κB signaling through the Unc-51 like autophagy activating kinase 1 (ULK1)–TANK binding kinase 1 (TBK1)–NF-κB-inducing kinase (NIK) axis in which AMPK phosphorylates ULK1 followed by TBK1 phosphorylation. Then TBK1 phosphorylates NIK to induce its degradation (45, 239).

AMPK can protect against hepatic apoptosis through direct phosphorylation and inhibition of procaspase 6 (238). Moreover, AMPK activation by small molecules such as A769662 reduces hepatic fibrosis in animal models (238). AMPK activation has been shown to suppress the expression of fibrogenic genes in hepatic stellate cells (HSCs). One of the potential mechanisms is that AMPK activation leads to dissociation of SMAD family member 3 (SMAD3) from its transcriptional coactivator p300 and proteasomal degradation of p300 (110). AMPK also inhibits HSC activation by control of the expression of vacuolar H⁺ adenosine triphosphatase and intracellular pH (126). Knockout of AMPKα1 in mice inhibits HSC proliferation but not fibrogenesis (33).

mTOR signaling

mTOR is a catalytic component of two protein complexes: mTORC1 and mTORC2. mTORC1 plays a critical role in metabolic homeostasis and cell growth, whereas mTORC2 activates a number of kinases, including AKT serine/threonine kinase (AKT), serum- and glucocorticoid-induced protein kinase, and protein kinase C (Fig. 2) (114).

FIG. 2.

mTOR signaling pathways in fatty liver disease. mTORC1 stimulates hepatic lipogenesis and inhibits lipid breakdown, but seems to play different roles in mild versus severe hepatic inflammation. mTORC2 also regulates lipogenesis, inflammation, and HSC activity. AKT1/2, AKT serine/threonine kinases 1 and 2; Arg, arginine; ATG13, autophagy-related gene 13; CASTOR1, cytosolic arginine sensor for mTORC1 subunit 1; EtOH, ethanol; EV, extracellular vesicle; GATOR1/2, GTPase-activating proteins 1/2 toward Rags complex; HIF1α, hypoxia inducible factor 1α; HSC, hepatic stellate cell; KC, Kupffer cell; Leu, leucine; LPS, lipopolysaccharide; Met, methionine; mTORC2, mechanistic target of rapamycin kinase complex 2; PI3K, phosphatidylinositol 3-kinase; RagA/B/C/D, Ras-related GTP binding proteins A/B/C/D; Rheb, Ras homologue enriched in brain; REDD1, protein regulated in development and DNA damage response 1; ROCK1, Rho-associated coiled-coil containing protein kinase 1; S6K1, ribosomal protein S6 kinase 1; SAM, S-adenosylmethionine; SAMTOR, S-adenosylmethionine sensor upstream of mTORC1; TFE3, transcription factor binding to IGHM enhancer 3; TFEB, transcription factor EB; UVRAG, UV radiation resistance associated.

mTORC1 promotes hepatic fatty acid and cholesterol biosynthesis by activation of SREBP1, SREBP2, and 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR) (44, 120, 221). Both ribosomal protein S6 kinase 1 (S6K1) and lipin-1 have been shown to be involved in the regulation of SREBPs (44, 221). mTORC2 also regulates hepatic lipogenesis via insulin-induced activation of AKT, glucokinase, and SREBP1c (60). SESN1/2/3 can activate AMPK and mTORC2 but inhibit mTORC1, and by doing so they promote hepatic lipid homeostasis (98, 193).

mTORC1 is a key negative regulator for autophagy (78). In the liver, autophagy helps maintain energy homeostasis and protect against cell injury or death by clearing damaged organelles and misfolded proteins. Autophagy also facilitates lipid droplet breakdown. mTORC1 inhibits the autophagy-initiating ULK complex by phosphorylation of autophagy-related gene 13 (ATG13) and ULK1/2 (78, 84). Autophagy is a double-edged sword in liver fibrosis. Most studies have demonstrated that increased autophagy activates HSCs by degrading lipid droplets (63). However, autophagy can also be used to reduce the production of fibrogenic extracellular vesicles (EVs) (52). mTORC1 overactivation in mesenchymal cells by deletion of TSC1 using Col1a2-Cre-ERT exacerbates CCl₄-induced hepatic fibrosis but not under normal conditions (182). mTOR inhibitors sirolimus and everolimus reduce bile duct ligation-induced hepatic fibrosis in rats (156). The role of mTORC2 in HSCs remains unclear. One report suggests that mTORC2 might promote HSC proliferation and activation through AKTs (166).

The role of mTORC1 in hepatic inflammation remains debatable. Chronic mTORC1 activation can promote inflammation (24). Deletion of Raptor in macrophages improves insulin sensitivity and ameliorates adipose and hepatic inflammation in mice when challenged with a high-fat diet (75). However, under some other conditions such as NASH diets or autophagy deficiency, mTORC1 is required for the resolution of inflammation (145, 196).

Endoplasmic reticulum stress signaling

Endoplasmic reticulum (ER) is the major site of lipid synthesis in hepatocytes. Despite abundant ER, hepatocytes are very sensitive to the disruption of normal ER function by various ER stressors (95). ER stress is a major contributor to liver disease, including hepatic steatosis, inflammation, and fibrosis (Fig. 3) (95). In fatty liver disease, abnormal lipid accumulation often coexists with insulin resistance and proteostasis perturbation in hepatocytes. ER stress is often triggered by unfolded protein response (UPR) as misfolded or unfolded proteins are accumulated in the ER lumen (95). Worse yet, UPR can cause inflammasome activation and inflammation, and, in the case of nonresolvable ER stress, cell death (95). There are three major ER stress pathways that are mediated by the ER transmembrane proteins inositol-requiring enzyme 1 α (IRE1α), PRKR-like ER kinase (PERK), and activating transcription factor 6 α (ATF6α), respectively (95).

FIG. 3.

ER stress in fatty liver disease. There are three major ER stress signaling pathways mediated by PERK, IRE1α, and ATF6. ER stress can trigger inflammation, apoptosis, fibrogenesis, and other cellular events. ATF4/6, activating transcription factors 4 and 6; CHOP, CCAAT/enhancer-binding protein homologous protein; DNL, de novo lipogenesis; eIF2α, eukaryotic translation initiation factor 2 alpha; ER, endoplasmic reticulum; FAO, fatty acid oxidation; GADD34, growth arrest and DNA damage-inducible protein 34; HNRNPA1, heterogeneous nuclear ribonucleoprotein A1; IRE1α, inositol-requiring enzyme 1 α; JNK, JUN N-terminal kinase; miR, microRNA; NRF2, nuclear factor erythroid 2-related factor 2; PERK, PRKR-like endoplasmic reticulum kinase; PP1C, protein phosphatase 1C; PPARα, peroxisome proliferator-activated receptor α; SMAD2, SMAD family member 2; TXNIP, thioredoxin interacting protein; VLDL, very low-density lipoprotein; XBP1s, X-box binding protein 1 spliced; XBP1u, X-box binding protein 1 unspliced.

ER stress has been shown to cause hepatic steatosis (170). ER stress-induced hepatic steatosis can be regulated by multiple factors, and can happen through the regulation of lipogenesis, very low-density lipoprotein (VLDL) secretion, and FAO (170). The IRE1α–X-box binding protein 1 (XBP1) pathway plays an important role in the regulation of VLDL secretion and lipogenesis (206). Mice with hepatocyte-specific deletion of IRE1α develop hepatic steatosis and increase expression of several lipogenesis genes in the liver after the treatment with tunicamycin, an ER-stress inducer (235). In addition, XBP1 deletion decreases de novo hepatic lipogenesis, leading to reduced serum triglycerides, cholesterol, and free fatty acids through regulation of lipogenic genes, including ACC2 and stearoyl-CoA desaturase 1 (SCD1) (96). XBP1 deletion also triggers a feedback hyperactivation of IRE1α, thereby reducing plasma triglycerides and cholesterol in the XBP1 knockout mice (187). Activation of IRE1α increases the release of proinflammatory EVs from hepatocytes, whereas inhibition of IRE1α in the hepatocytes and in mice impairs hepatic inflammasome activation, liver injury, and hepatic cell apoptosis (95).

The PERK-eIF2α-ATF4 pathway also regulates hepatic steatosis. Hepatic overexpression of growth arrest and DNA damage-inducible protein 34 (GADD34), a regulatory subunit of protein phosphatase 1, which keeps eIF2α in a dephosphorylated state, protects mice from high-fat diet-induced obesity and hepatic steatosis (149). ATF4 deletion exhibits a protective effect against diet-induced hepatic steatosis in mice (104).

The ATF6α signaling pathway has also been shown to protect against hepatic steatosis. ATF6α knockout mice develop hepatic steatosis after the tunicamycin treatment probably due to the blockage of β-oxidation of fatty acids and the suppression of VLDL formation (25). ATF6α can activate peroxisome proliferator-activated receptor α (PPARα) to induce expression of FAO genes (25). ATF6α suppresses cholesterol biosynthesis by forming the ATF6-SREBP2-HDAC1 suppressive transcriptional complex (230). Furthermore, ATF6α knockout mice develop hepatic steatosis and glucose intolerance in association with increased expression of SREBP1c (197).

SESN2 can be induced by ER stress-activated CCAAT enhancer binding protein β (C/EBPβ) transcriptional factor during diet-induced obesity. Then SESN2 modulates UPR through suppression of mTORC1 and eventually suppresses the obesity-associated hepatic steatohepatitis (154). SESN2 can also be induced by ATF6 in response to ER stress, and SESN2 subsequently reduces UPR via inhibition of phosphorylation of JUN N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK) and suppresses ER stress-induced hepatocyte apoptosis and liver injury (72).

ER stress has also been implicated in hepatic inflammation and fibrosis. IRE1α increases hepatic inflammation by two distinct mechanisms. The nuclease activity of IRE1α can increase the thioredoxin interacting protein (TXNIP) mRNA levels by reducing the TXNIP-targeting microRNA 17 (miR-17). The elevation of the TXNIP promotes the NLR family pyrin domain containing 3 (NLRP3)-mediated inflammasome activation (102). The kinase activity of IRE1α, in addition to phosphorylated eIF2α and ATF6, can activate the NF-κB-mediated inflammation (76). All three major ER stress pathways have been shown to increase hepatic fibrosis. PERK can directly phosphorylate and activate heterogeneous nuclear ribonucleoprotein A1 (HNRNPA1) to upregulate SMAD2 by downregulation of miR-18 that directly targets SMAD2 during the HSC activation (92). The IRE1α-XBP1 pathway also activates HSCs by triggering autophagy (64). The role of ATF6 in hepatic fibrosis is currently unclear.

Oxidative stress signaling

Generally, cells produce reactive oxygen species (ROS) and reactive nitrogen species at certain levels according to cellular conditions. As both oxidant species are closely related, we here use ROS to represent both. ROS production is involved in different enzymatic and nonenzymatic reactions. The ROS-producing enzymes include nicotinamide adenine dinucleotide (NAD) phosphate oxidase, xanthine dehydrogenase, cytochrome P450 2 subfamily E member 1 (CYP2E1), cyclooxygenases, and lipoxygenases (57).

In fatty liver disease, accumulation of saturated free fatty acids such as palmitates, diacylglycerol, ceramides, and cholesterol in the liver can induce lipotoxicity and oxidative stress, which can lead to hepatic inflammation, mitochondrial dysfunction, cell apoptosis, and liver fibrosis (Fig. 4) (57). Alcohol metabolism also generates ROS as by-product, especially by CYP2E1 (57). In the early stage of fatty liver disease, there is an increase in mitochondrial activity as a compensatory mechanism to protect hepatic cells against the harmful lipid overflow (125). As a result, this also leads to an increase in production and accumulation of ROS.

FIG. 4.

Oxidative stress in fatty liver disease. Multiple sources including free fatty acids, cholesterol, and ethanol can lead to the production of ROS, which may cause cell damage, inflammation, and even cell death. ADH, alcohol dehydrogenase; ALDH2, aldehyde dehydrogenase 2; AP-1, activator protein 1; ASK1, apoptosis signal regulating kinase 1; CAT, catalase; CYP2E1, cytochrome P450 family 2 subfamily E member 1; DAG, diacylglycerol; IKK, inhibitor of nuclear factor kappa B kinase; p38 MAPK, p38 mitogen-activated protein kinase; PA, palmitic acid; PM, plasma membrane; ROS, reactive oxygen species.

During the development of fatty liver disease, ER stress occurs and this also increases ROS production. One of the most significant causes in the ER stress-induced ROS production is intracellular calcium flux. Excess calcium flows into the cytoplasm, which can be absorbed by mitochondria, inducing the opening of the mitochondrial permeability transition pore and causing ROS production (46). When mitochondria become dysfunctional, excessive ROS are produced due to increased electron leakage, which in turns increases oxidative stress and damage to mitochondria. ROS can cause lipid peroxidation and mitochondrial membrane damage (125). Elevated biosynthesis through mitochondrial anaplerotic/cataplerotic pathways also contributes to oxidative stress and inflammation in the liver during NAFLD (173).

ROS regulate numerous signaling molecules that may lead to oxidative stress, cell damage, or cell death. Chronic plus binge alcohol consumption increases hepatic oxidative stress that triggers activation of apoptosis signal regulating kinase 1 (ASK1) and p38 MAPK signaling pathways and subsequently hepatic steatohepatitis (122). ROS also triggers hepatic inflammation by either direct activation of the NF-κB and JNK pathways or indirectly through the ER stress pathways (57). Oxidative stress also contributes to the development of hepatic fibrosis (240).

Inflammatory signaling

Inflammation can be a physiological or pathological response to tissue injury or infection by releasing proinflammatory cytokines, chemokines, and eicosanoids during cell defense and tissue repair (179). The triggers of hepatic inflammation can originate from either extrinsic or intrinsic factors (179). Adipose tissue can produce and release numerous hormones such as leptin, resistin, and visfatin, and proinflammatory cytokines such as tumor necrosis factor α (TNFα), interleukin 1β (IL1β), and interleukin 6 (IL6), and chemokines under pathological conditions such as obesity and diabetes (179). Obesity often leads to hyperlipidemia and hyperglycemia, and both can activate inflammatory pathways via toll-like receptors (TLRs), especially TLR4 (179).

Saturated fatty acid lauric acid can activate TLR4 by promotion of the receptor dimerization and recruitment to lipid rafts (213). TLR4 expression is increased in the liver of NASH patients and can be induced by palmitate (183). Upon activation, TLR4 stimulates the signaling cascade of interleukin 1 receptor-associated kinase (IRAK)–TNF receptor-associated factor 6 (TRAF6)–TAK1. From TAK1, there are at least three branches: p38 MAPK, JNK–activator protein 1 (AP-1), and inhibitor of NF-κB kinase (IKK)–NF-κB. IKK phosphorylates the NF-κB inhibitor and triggers its degradation. As a result, NF-κB is released from the protein complex and translocates to the nucleus for transcriptional activation of numerous inflammatory chemokine and cytokine genes in hepatocytes and hepatic immune cells (27).

Lipopolysaccharides (LPS) produced by intestinal microbiota also trigger hepatic inflammation mediated by TLR4, leading to proinflammatory cytokine synthesis by activation of AP-1 and NF-κB (19). Inflammatory cytokines such as TNFα, IL1β, and IL6 can also cause hepatic insulin resistance and inflammation through their respective receptors (19). IL6 activates Janus kinases (JAKs), which in turn activate signal transducer and activator of transcription 3 (STAT3) (175). TNFα acts on the TNF receptor (TNFR) to stimulate the signaling cascade of TNFR-associated death domain protein (TRADD)–TRAF2/5–IKK–NF-κB (121). IL1β binds to IL1 receptor (IL1R) to activate both p38 MAPK and JNK signaling pathways during the fatty liver disease development (Fig. 5) (90).

FIG. 5.

Inflammatory pathways in fatty liver disease. A number of factors, including LPS, PA, ATP, IL1β, IL6, and TNFα, can trigger inflammatory signaling cascades that lead to production of cytokines and chemokines and tissue injury. ASC, apoptosis-associated speck-like protein containing a CARD; ATP, adenosine triphosphate; Casp1, caspase-1; IL1β, interleukin 1 beta; IL1R, interleukin 1 receptor; IL6, interleukin 6; IL6R, interleukin 6 receptor; IRAK, interleukin 1 receptor associated kinase; JAK, Janus kinase; K⁺, potassium ion; MAP3K1, mitogen-activated protein kinase kinase kinase 1; MEK7, MAPK/ERK kinase 7; MKK4/6/7, mitogen-activated protein kinase kinases 4/6/7; MyD88, myeloid differentiation primary response protein 88; NLRP3, NLR family pyrin domain containing 3; P2X7, purinergic receptor P2X 7; STAT3, signal transducer and activator of transcription 3; TIRAP, TIR domain containing adaptor protein; TLR4, toll-like receptor 4; TNFα, tumor necrosis factor α; TNFR, tumor necrosis factor receptor; TRADD, tumor necrosis factor receptor type 1-associated death domain protein; TRAF2/5/6, tumor necrosis factor receptor-associated factors 2/5/6.

TGFβ signaling

In the liver, TGFβ is a key regulator of liver physiology and pathology, contributing to all stages of hepatic disease progression, from initial liver injury, inflammation, fibrosis, to cirrhosis and cancer (48). Generally, the TGFβ signaling transduction network can be divided into two categories: SMAD molecule-related signaling (canonical) and non-SMAD (noncanonical) pathways (48). TGFβ acts as dimers by first binding to a pair of type II TGFβ receptors (TGFβR2) and then recruiting two type I receptors (TGFβR1) to form a stable receptor complex. Next, TGFβR1 amplifies the TGFβ signal transduction by recruiting receptor-regulated SMAD proteins (SMAD2 and SMAD3) and phosphorylating them. Activated SMAD2 and SMAD3 can form a complex with SMAD4, then together translocate into the nucleus, bind to DNA, and regulate the expression of target genes, including fibrosis-related genes. In addition, TGFβ also induces expression of the inhibitory SMAD (SMAD7) to negatively regulate the signaling pathway (48).

Those non-SMAD pathways may initiate from either TGFβR1 or TGFβR2 depending on the proteins associated with them. When TGFβ stimulates its receptors, SHC adaptor protein A can be phosphorylated and then forms a protein complex with growth factor receptor bound protein 2 and SOS Ras/Rac guanine nucleotide exchange factor to activate the MAPK cascade (99). TRAF4/6 activated by TGFβR1 facilitate recruitment of TAK1 and subsequently activate JNK and p38 MAPK and NF-κB signaling (218). Phosphatidylinositol 3-kinase (PI3K)–AKT–mTOR can be activated by the TGFβ signaling in the regulation of hepatic cell survival and function. Small GTPases, including Ras homology family member A (RHOA), cell division cycle 42, and Ras-related C3 botulinum toxin substrate (RAC), can be activated by TGFβ in hepatic fibrogenesis (93). TGFβ can also activate the JAK–STAT3 signaling pathway in liver cells, including HSCs (190).

Sesn3 is an endogenous inhibitor of the TGFβ signaling pathway and it suppresses the TGFβ-induced hepatic fibrosis by inhibiting the TGFβ receptors through recruitment of inhibitory SMAD7 and sequestration of SMAD3 to the cytoplasm (Fig. 6) (67).

FIG. 6.

TGFβ signaling pathways in fatty liver disease. TGFβ can trigger canonical Smad2/3 signaling and multiple noncanonical pathways involving several kinases. ALK1/5, anaplastic lymphoma receptor tyrosine kinases 1 and 5; MAPK, mitogen-activated protein kinase; RAF, Raf proto-oncogene serine/threonine kinase; RAS, Ras proto-oncogene GTPase; RhoA, Ras homology family member A; Sesn3, sestrin 3; Smad2/3/4/7, SMAD family members 2/3/4/7; Smurf2, SMAD specific E3 ubiquitin protein ligase 2; TGFβ, transforming growth factor β.

Hippo signaling

The Hippo signaling pathway, especially downstream mediators Yes1-associated transcriptional regulator (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ/WWTR1), has been implicated in liver metabolism, inflammation, regeneration, fibrosis, and tumorigenesis (Fig. 7) (123). Upon activation, the mammalian homologues of Hippo—mammalian STE20-like protein kinases 1 and 2 (MST1/STK4 and MST2/STK3), phosphorylate and activate large tumor suppressor kinases 1 and 2 (LATS1/2), which subsequently phosphorylate cytoplasmic YAP. Phosphorylated YAP/TAZ can be bound by 14-3-3 and sequestered in the cytoplasm and subjected to proteasomal degradation. When the Hippo signaling is off, nonphosphorylated YAP/TAZ translocate to the nucleus and coactivate the TEA domain family of transcription factors (TEADs) to induce genes involved in hepatic cell proliferation, survival, and function (123). Vestigial-like 4 (VGLL4) antagonizes nuclear YAP by interfering with the YAP-TEAD interaction (123).

FIG. 7.

Hippo signaling pathway in fatty liver disease. Multiple extracellular and intracellular factors can regulate the Hippo signaling. YAP and TAZ are two major downstream transcriptional coactivators that facilitate the TEAD transcriptional activity. AMOT, angiomotin; ARID1A, AT-rich interaction domain 1A; GPCR, G protein-coupled receptor; KIBRA, kidney and brain protein; LATS1/2, large tumor suppressor kinases 1 and 2; MAP4K, mitogen-activated protein kinase kinase kinase kinase; MOB1A/B, MOB kinase activator 1 A/B; MST1/2, mammalian STE20-like protein kinases 1 and 2; NF2, neurofibromin 2; SAV1, Salvador family WW domain containing protein 1; TAZ, transcriptional coactivator with PDZ-binding motif; TEAD, TEA domain transcription factor; VGLL4, vestigial-like family member 4; YAP, yes1-associated transcriptional regulator.

A number of upstream regulators of the Hippo pathway have been identified, including low energy, low glucose, statin, lysophosphatidic acid (LPA), glucagon, epinephrine, bile acids (BAs), and mechanical forces (41). Low glucose and low energy induce the Hippo signaling and YAP/TAZ phosphorylation through AMPK (41). In contrast, high glucose activates YAP probably through O-glycosylation (41). YAP/TAZ can also be regulated by the mevalonate pathway through posttranslational modifications of the Hippo upstream regulators with geranylgeranyl (189). LPA acts through G protein-coupled receptors (GPCRs) to inhibit LATS1/2 and thus activate YAP/TAZ (226). Glucagon and epinephrine act on their respective GPCRs to activate LATS1/2 and thus inhibit YAP/TAZ (226). BAs can induce YAP activation by either downregulation of MST1/2 and LATS1/2 expression or an increase in MST1/2 phosphorylation (4). Matrix stiffness also regulates YAP activity. Increased extracellular matrix regulates F-actin polymerization in an RHOA-dependent manner to activate YAP/TAZ nuclear translocation (43).

The Hippo signaling pathway has been implicated in hepatic metabolism. YAP can inhibit hepatic gluconeogenesis by suppression of the PPARγ coactivator 1α (PGC1α) gene expression. YAP also upregulates expression of insulin receptor substrate 2 (73). Deletion of LATS2 or MST1 gene in mice leads to fatty liver disease by induction of SREBP2 and cholesterol biosynthesis (7). YAP activation also induces glutaminolysis (42). The Hippo pathway also plays a critical role in the pathogenesis of NASH, especially through regulation of hepatocyte stress and survival and HSC activation (42, 124, 133, 210). TAZ expression is specifically induced in hepatocytes of human NASH livers but not steatotic livers (210). Knockdown of hepatocyte TAZ by siRNA improves hepatic fibrosis in a diet-induced NASH mouse model (209).

Sirtuins, microRNAs, and DNA Methylation in Fatty Liver Disease

Sirtuins

Sirtuins are NAD⁺-dependent deacetylases or deacylases. There are seven sirtuins in mammals (SIRT1–7). SIRT1 and SIRT6 are two key nuclear sirtuins implicated in fatty liver disease (Fig. 8).

FIG. 8.

SIRT1 and SIRT6 in fatty liver disease. Among seven sirtuin family members, SIRT1 and SIRT6 are significantly implicated in the regulation of the pathogenesis of fatty liver disease. CAT, catalase; ChREBP, carbohydrate response element binding protein; FGF21, fibroblast growth factor 21; HMOX1, heme oxygenase 1; MT1/2, metallothioneins 1 and 2; MTF1, metal regulatory transcription factor 1; NCOA2, nuclear receptor coactivator 2; SOD, superoxide dismutase.

Sirtuin 1

SIRT1 has a salutary role in hepatic metabolic homeostasis, antioxidative stress, anti-inflammation, and antifibrosis. SIRT1 overexpression or activation by agonists protects against diet-induced NAFLD through an increase of fatty acid β-oxidation and a decrease of inflammation in the liver (50, 202). SIRT1 also protects against hepatic steatosis through induction of fibroblast growth factor 21 (FGF21) (107). SIRT1 catalytically inactive mutant mice exhibit higher SREBP1 and SCD1 levels and lower LKB1 and AMPK phosphorylation in the liver on a high-fat diet compared with wild-type (WT) mice (26). SIRT1 can directly inhibit SREBP1c through deacetylation (161). Repopulation of human SIRT1 knockdown-induced pluripotent stem cell-derived hepatocytes in rat livers induces hepatic steatosis and inflammation (28). Sirt1 liver-specific knockout mice develop high-fat diet-induced NAFLD due to dysregulation of PPARα and PGC1α (162). SIRT1 also suppresses inflammation by deacetylation of NF-κB (177). Hepatic Sirt1 deficiency in mice exacerbates alcohol-induced hepatic steatosis and inflammation through regulation of lipin-1 and SREBP1c (223).

Sirtuin 6

SIRT6 also plays a critical role in the regulation of hepatic homeostasis and function. Hepatic Sirt6 deletion leads to age-associated hepatic steatosis through activation of glycolysis and lipogenesis and inhibition of fatty acid β-oxidation partly due to an increase in histone H3 lysine 9 (K9) acetylation in the target gene chromatin regions (83). Hepatic Sirt6 deficiency also aggravates high-fat, high-fructose, diet-induced oxidation stress and fibrosis in the mouse liver via upregulation of BTB domain and CNC homologue 1 (Bach1) and downregulation of nuclear factor, erythroid 2 like 2 (Nrf2/Nfe2l2), a key antioxidative stress transcription factor. Moreover, SIRT6 promotes NRF2 binding to the target gene promoters in response to oxidative stress (77). SIRT6 also promotes hepatic fatty acid β-oxidation through activation of PPARα by recruitment and deacetylation of nuclear receptor coactivator 2 (NCOA2), a PPARα coactivator. In addition, SIRT6 also mediates the inhibitory effect of PPARα on SREBPs for cholesterol and triglyceride biosynthesis (142). SIRT6 can directly interact with SREBP1 and SREBP2 to suppress the expression of fatty acid and cholesterol biosynthetic genes (128, 192). SIRT6 suppresses ER stress-induced hepatic steatosis through deacetylation and subsequent degradation of the deacetylated XBP1s protein via the ubiquitin/proteasome system (11).

SIRT6 attenuates hepatic inflammation by either suppression of the NF-κB activity in the macrophage M1 polarization or inhibition of the transcriptional activity of c-Jun for the proinflammatory genes such as IL6 and C-C motif chemokine ligand 2 (CCL2/MCP1) (101, 215). SIRT6 also plays a critical role in the control of hepatic fibrosis during NAFLD. Hepatocyte- or HSC-specific deletion of Sirt6 in mice leads to diet-induced hepatic fibrosis through the TGFβ-SMAD2/3 signaling pathway, whereas overexpression of SIRT6 completely reverses NASH (233, 241). Hepatic Sirt6 deficiency also leads to ethanol-induced liver injury due to downregulation of metallothioneins 1 and 2 (Mt1 and Mt2) and elevation of oxidative stress in mouse livers. SIRT6 overexpression dramatically improves ALD. Mechanistically, SIRT6 induces hepatic Mt1 and Mt2 gene expression by deacetylation and activation of metal regulatory transcription factor 1 (MTF1) (82).

MicroRNAs

MicroRNAs are small noncoding RNAs, ∼20–24 nucleotides in length. MicroRNAs regulate gene expression primarily at the posttranscriptional levels. A number of microRNAs have been implicated in hepatic steatosis, including miR-21, miR-22, miR-27b, miR-29, miR-33a/b, miR-34, miR-122, miR-222, miR-223, miR-375, and miR-451. Numerous microRNAs are also involved in hepatic fibrosis, including miR-16, miR-21, miR-33, miR-34, miR-122, miR-125b, miR-155, miR-192, miR-222, and miR-223 (Fig. 9) (208).

FIG. 9.

microRNAs in fatty liver disease. Numerous microRNAs are involved in the regulation of hepatic steatosis, inflammation, and fibrosis. Here only a number of well-studied microRNAs are highlighted. ABCA1, ATP-binding cassette subfamily A member 1; ABCG1, ATP-binding cassette subfamily G member 1; AGPAT1, 1-acylglycerol-3-phosphate O-acyltransferase 1; BCL2, BCL2 apoptosis regulator; CCL2, C-C motif chemokine ligand 2; CCND1, cyclin D1; CD36, CD36 molecule; CDK6, cyclin-dependent kinase 6; CEBPB, CCAAT enhancer binding protein beta; CIDEC, cell death inducing DFFA-like effector C; COL3A1, collagen type III alpha 1 chain; CTGF, connective tissue growth factor; CXCL10, C-X-C motif chemokine ligand 10; DGAT1, diacylglycerol O-acyltransferase 1; FGF11, fibroblast growth factor 11; FGFR1, fibroblast growth factor receptor 1; FXR, farnesoid X receptor; G6PC, glucose-6-phosphatase catalytic subunit; G6PD, glucose-6-phosphate dehydrogenase; GLS, glutaminase; GNA12, guanine nucleotide-binding protein subunit alpha 12; GR, glucocorticoid receptor; HGF, hepatocyte growth factor; HMGA2, high-mobility group AT-hook 2; HMGCS1/2, 3-hydroxy-3-methylglutaryl-CoA synthases 1 and 2; IGF1, insulin-like growth factor 1; IGF1R, insulin-like growth factor 1 receptor; IL1RN, interleukin 1 receptor antagonist; KLF6, Kruppel-like factor 6; LAMP1, lysosomal-associated membrane protein 1; LXRA, liver X receptor alpha; MAP3K3, mitogen-activated protein kinase kinase kinase 3; MERTK, MER proto-oncogene tyrosine kinase; NDRG3, N-Myc downstream-regulated gene 3 protein; NFYC, nuclear transcription factor Y subunit gamma; PDGFC, platelet-derived growth factor C; PIM3, Pim-3 proto-oncogene serine/threonine kinase; PKM2, pyruvate kinase M2; PPARγ, peroxisome proliferator-activated receptor γ; PTEN, phosphatase and tensin homologue; RELB, RELB proto-oncogene; RICTOR, RPTOR independent companion of mTOR complex 2; SC4MOL, sterol-C4-methyl oxidase; SCD1, stearoyl-CoA desaturase 1; SMAD2/7, SMAD family members 2 and 7; SOCS1, suppressor of cytokine signaling 1; SP3, SP3 transcription factor; SR-BI, scavenger receptor class B member 1; VHL, von Hippel–Lindau tumor suppressor; WNT3A, Wnt family member 3A; ZEB2, zinc finger E-box binding homeobox 2.

miR-16

miR-16 has been shown to inhibit hepatic fibrosis through multiple targets, including guanine nucleotide-binding protein subunit alpha 12 (GNA12), SMAD2, and Wnt family member 3A (WNT3A) (86, 152).

miR-21

miR-21 has been extensively studied in hepatic metabolism and fibrosis. Numerous miR-21 target genes have been identified, including SMAD7, forkhead box O1 (FOXO1), HMGCR, and STAT3 (20, 214). However, some data suggest that miR-21 is not required for HSC activation and liver fibrogenesis (20).

miR-29

miR-29 has been shown to regulate hepatic lipogenesis and fibrosis. Regarding regulation of hepatic lipid metabolism, the miR-29 effects are rather complicated as both positive and negative factors are targets of miR-29, including SIRT1 and CD36 (68, 111). Overexpression of miR-29a improves diet-induced hepatic steatosis and fibrosis (111). miR-29a administration improves CCl₄ and thioacetamide-induced hepatic fibrosis in mice. Collagen type I α1 (COL1A1) and platelet-derived growth factor C (PDGFC) are among the targets of miR-29a (129). Expression of miR-29 is suppressed by the TGFβ and LPS signaling pathways (168).

miR-33

miR-33 has been reported to regulate cholesterol homeostasis by targeting ATP-binding cassette transporter A1 (ABCA1), which is involved in high-density lipoprotein (HDL) biosynthesis and reverse cholesterol transport (143, 164). Short-term administration of anti-miR-33 oligonucleotides can increase HDL and decrease VLDL triglycerides in nonhuman primates. In addition, several FAO genes are also miR-33 targets (163). However, long-term silencing of miR-33 leads to elevated circulating and hepatic triglycerides in high-fat diet-treated mice due to increased lipogenesis. SREBP1 and nuclear transcription factor Y subunit gamma (NFYC) are among targets of miR-33 (55).

miR-122

miR-122 is highly abundant in the liver, which accounts for ∼70% of all hepatic microRNAs. miR-122 knockout mice develop hepatic steatohepatitis, fibrosis, and hepatocellular carcinoma (66, 195). miR-122 negatively regulates both lipogenic and FAO genes, including 1-acylglycerol-3-phosphate O-acyltransferase 1 (AGPAT1), SIRT1, and SIRT6 (47, 66, 119). Retinoic acid receptor-related orphan receptor A agonists can improve NASH by upregulating miR-122 in mice (22). miR-122 is decreased in the livers of ALD patients and mice. Overexpression of miR-122 in the liver protects against ALD partly through inhibition of hypoxia inducible factor 1 subunit alpha, a target of miR-122 (174).

miR-155

miR-155 is abundantly expressed in immune cells such as macrophages. miR-155 regulates hepatic lipid metabolism through suppression of lipogenic transcription factors such as C/EBPα and C/EBPβ, PPARγ, and liver X receptor α (LXRα) (203). miR-155 knockout mice exhibit decreased hepatic steatosis and fibrosis, but no improvement in hepatic inflammation after challenged with a methionine/choline-deficient diet (31). miR-155 can be induced by alcohol and LPS in macrophages and promotes inflammation (9, 10). In response to alcohol and LPS, miR-155 exacerbates hepatic inflammation through suppression of several negative regulators of the TLR4 signaling pathway, including IRAK3, SH2 domain-containing inositol 5′-phosphatase 1, suppressor of cytokine signaling 1 (SOCS1), and C/EBPβ in Kupffer cells (KCs) (9). miR-155 promotes alcohol-induced hepatic steatohepatitis and fibrosis through inhibition of PPARα, PPARγ, and C/EBPβ (10). miR-155 also targets lysosomal-associated membrane proteins 1 and 2 (LAMP1/2) to impair the autophagy and lysosome functions in ALD (8).

miR-192

miR-192 has been shown to mediate lipotoxicity-induced macrophage activation by targeting Rictor, a key component of mTORC2 (118). miR-192 inhibits lipogenesis in hepatocytes by targeting SCD1 and SREBP1 (112). miR-192 also aggravates oxidative stress-induced liver injury by targeting zinc finger E-box binding homeobox 2 (ZEB2) (169).

miR-223

miR-223 is highly enriched in neutrophils and it has been implicated in both ALD and NAFLD. Under the NAFLD conditions, hepatocytes can uptake miR-223-containing EVs from neutrophils via a VLDL-ApoE interaction to attenuate hepatic inflammation and fibrosis (62). In addition, macrophage-released miR-223 in the exosomes also reduces TAZ in hepatocytes to dampen hepatic fibrosis (61). Neutrophil miR-223 also attenuates hepatic inflammation and fibrosis by promoting conversion of macrophages from proinflammatory to restorative type (16). miR-223 regulates cholesterol homeostasis by targeting scavenger receptor class B member 1 and 3-hydroxy-3-methylglutaryl-CoA synthase 1 (HMGCS1) (201). In a mouse model of chronic-plus-binge ethanol feeding, miR-223 is elevated in serum and neutrophils. Deletion of the miR-223 gene leads to increased liver injury, oxidative stress, and neutrophil infiltration. Mechanistically, miR-223 directly inhibits IL6 expression and subsequently downregulates the p47 neutrophil cytosolic factor 1 (p47phox/Ncf1) gene expression in neutrophils (105).

DNA methylation

DNA methylation, especially 5-methylcytosine in the genome, is modulated by DNA methyltransferases (DNMT1, DNMT3A, and DNMT3B) and 2-oxoglutarate-dependent dioxygenases such as ten eleven translocation enzymes (TET1–3) (69). Some evidence suggests that DNA methylation is dynamically altered in the livers of ALD and NAFLD patients compared with normal controls (Fig. 10) (231). Hypermethylation of the PGC1α gene promoter has been shown to correlate with decreased PGC1α expression and mitochondrial function in the liver (188). Hypermethylation in several genes such as CPT1A and 24-dehydrocholesterol reductase (DHCR24) and hypomethylation in the dipeptidyl peptidase 4 (DPP4) gene have been associated with hepatic steatosis (13).

FIG. 10.

DNA methylation in fatty liver disease. In both human and animal NAFLD livers, both hypermethylation and hypomethylation sites in numerous genes have been associated with different disease conditions. CPT1A, carnitine palmitoyltransferase 1A; CYP27A1, cytochrome P450 family 27 subfamily A member 1; DHCR24, 24-dehydrocholesterol reductase; DNMT3A, DNA methyltransferase 3 alpha; DPP4, dipeptidyl peptidase 4; FASN, fatty acid synthase; FGFR2, fibroblast growth factor receptor 2; MAT1A, methionine adenosyltransferase 1A; NAFLD, nonalcoholic fatty liver disease; NPC1L1, NPC1-like intracellular cholesterol transporter 1; PRC1, protein regulator of cytokinesis 1; SLC9A3R1, SLC9A3 regulator 1; SLC27A5, solute carrier family 27 member 5; SLC43A1, solute carrier family 43 member A1; SLC51A, solute carrier family 51 subunit alpha; TG, triglyceride; TRIM4, tripartite motif containing 4; TUBA1B, tubulin alpha 1b.

In the NASH livers, DNA methylation in the ATP citrate lyase (ACLY) gene CpG sites is decreased, whereas it is increased in the insulin-like growth factor 1 (IGF1) and IGF binding protein 2 (IGFBP2) genes (2). Hypermethylation in the genes of cytochrome P450 family 27 subfamily A member 1 (CYP27A1), NPC1 like intracellular cholesterol transporter 1 (NPC1L1), and solute carrier family 51 subunit α (SLC51A) has been associated with NASH (139). Hypomethylation in the genes of protein regulator of cytokinesis 1 (PRC1) and tripartite motif containing 4 (TRIM4) has been associated with NASH (194). When severe and mild NAFLD liver samples are compared, several CpG sites in the promoters of PPARα and PPARδ genes are hypermethylated, whereas the TGFB1 and PDGFA gene promoters are hypomethylated (231).

When liver samples of normal control and ALD patients are analyzed for DNA methylation profiles, both PPARα and PPARδ gene promoters are hypermethylated in the ALD livers, whereas the COL1A1 gene promoter is hypomethylated (231). Hepatic TET2 protein levels are decreased and DNMT3B protein levels are increased in the ALD patients compared with normal controls (151).

In rat HSCs, DNMT3A and DNMT3B are induced during transdifferentiation to myofibroblasts (151). Hypermethylation in the genes of methionine adenosyltransferase 1A (MAT1A), PPARα, and PPARγ has been associated with hepatic fibrosis (137). Hypomethylation of the genes of caspase-1 (CASP1) and fibroblast growth factor receptor 2 (FGFR2) has been associated with hepatic fibrosis (137). Interestingly, a dual G9a/DNMT1 inhibitor—CM272, suppresses the TGFβ-induced fibrogenesis in human LX-2 HSCs and improves hepatic fibrosis in CCl₄ and bile duct ligation mouse models (12). Although its protein levels are not altered in human fibrotic livers, the chromatin occupancies of DNMT3A at the SREBP1 and FASN gene promoters are decreased in the livers of human steatosis and NASH patients (87).

Transcriptional Regulation of Hepatic Steatosis, Inflammation, and Fibrosis

Carbohydrate response element binding protein

Carbohydrate response element binding protein (ChREBP) is a basic/helix–loop–helix/leucine zipper transcription factor that is responsive to carbohydrate load, especially glucose. There are two isoforms – ChREBPα and ChREBPβ. ChREBPα has a low-glucose inhibitory domain (LID) and a glucose-response activation conserved element in the N-terminal of the protein (106); however, ChREBPβ does not have the LID motif (106). Glucose metabolites facilitate the nuclear translocation and transcriptional action of ChREBP through binding to a carbohydrate response element (ChoRE) in collaboration with Max-like protein X. Numerous ChREBP target genes have at least one ChoRE in their promoter region, for example, pyruvate kinase L/R (PKLR), FASN, ACC1, and SCD1 (148). ChREBP can be activated by O-GlcNAcylation and lysine acetylation (220).

Deletion of the ChREBP gene in mice leads to reduced glycolysis and lipogenesis (70). In an ob/ob mouse model, liver-specific knockdown of ChREBP using adenovirus-mediated shRNAs improves hepatic steatosis and insulin sensitivity due to reduced hepatic lipogenesis (37). Hepatic ChREBP overexpression in mice leads to increased hepatic steatosis but improved insulin signaling on a high-fat diet (14). ChREBP also modulates an adaptive response to high-fructose diet by induction of FGF21 to control fructose metabolism and lipogenesis in the liver (51). In response to a high-fructose diet, ChREBP can protect liver from overproduction of cholesterol and CCAAT/enhancer-binding protein homologous protein (CHOP)-mediated hepatic injury through an interaction with SREBP2 and promotion of its degradation (Fig. 11) (232).

FIG. 11.

Key transcription factors in the regulation of hepatic lipogenesis. A number of key transcription factors that have been implicated in the regulation of hepatic lipogenesis are highlighted here: ChREBP, LXR, SREBP1c, and FOXO1. AAs, amino acids; Ac, acetyl; ATG14, autophagy-related gene 14; ATGL, adipose triglyceride lipase; BAs, bile acids; CDKN1B, cyclin-dependent kinase inhibitor 1B; CFLAR, CASP8 and FADD apoptosis regulator; CYP7A1, cytochrome P450 family 7 subfamily A member 1; ERK, extracellular signal-regulated kinase; GCK, glucokinase; GlcNAc, N-acetyl-d-glucosamine; HSL, hormone-sensitive lipase; IKBE, NFKB inhibitor epsilon; LPL, lipoprotein lipase; OGT, O-linked N-acetylglucosamine transferase; PKA, protein kinase A; PP2A, protein phosphatase 2A; RAs, retinoic acids; RXR, retinoid X receptor.

Liver X receptor

The LXR family has two members—LXRα and LXRβ. Whereas LXRβ is ubiquitously expressed, LXRα is abundant in metabolically active tissues, including adipose tissue and liver (204). LXRs function as heterodimers with retinoid X receptor α (RXRα). Transcriptional activity of LXRs is induced by elevated levels of cholesterol or other endogenous ligands in the cell. LXRs regulate multiple lipid metabolic processes, including lipogenesis, cholesterol absorption, transport, and conversion to BAs (204). Regarding lipogenesis, LXRs activate the transcription of the SREBP1, ChREBP, FASN, and SCD1 genes (21, 165, 204).

LXRs play an important role in cholesterol metabolism. Cytochrome P450 family 7 subfamily A member 1 (CYP7A1), the rate-limiting enzyme in the biosynthesis of BAs, is a direct transcriptional target of LXRs (158). LXRs also induce expression of biliary cholesterol excretion genes such as ATP-binding cassette subfamily G members 5 and 8 (ABCG5/8) (228). Two reverse cholesterol transport genes ABCA1 and ABCG1 are also regulated by LXRs (80, 200). LXRs have been implicated in the NAFLD pathogenesis. LXR exerts anti-inflammation function through regulation of the activity of KCs and infiltrated macrophages and cholesterol homeostasis in the liver (158). However, data on targeting LXR for NAFLD are not very consistent as both salutary and harmful effects have been reported (Fig. 11) (88).

Sterol regulatory element binding proteins

SREBPs are synthesized as membrane proteins that are situated in the ER. In the absence of sterols, SREBP interacts with the SREBP-cleavage activating protein (SCAP) and then translocates to the Golgi complex where the SREBP precursor is processed by proteases S1P and S2P to generate a mature form of SREBP transcription factor. There are three members of SREBP—SREBP1a, SREBP1c, and SREBP2. Proteolysis of SREBP1 is not strongly regulated by sterols, but the maturation process is inhibited by polyunsaturated fatty acids and promoted by insulin and high levels of glucose (185).

SREBP1c activates lipogenic genes such as ACC1, FASN, and SCD1 (184). Deletion of the SREBP1 gene in ob/ob mice partially improves hepatic steatosis, whereas deletion of the SCAP gene in ob/ob mice or high-fat-induced obese mice prevents hepatic steatosis (132, 217). SREBP1c levels are elevated in the liver of human hepatic steatosis patients (141). SREBP2 mRNA and free cholesterol levels are increased in the livers of human NASH patients compared with normal controls (131). The SREBP2 upregulation may be attributed to hyperinsulinemia, inflammatory cytokines, or miR-122 downregulation (Fig. 11) (138).

Forkhead box O

FOXO transcription factors are members of the O subgroup of the Forkhead family that contain a conserved DNA binding domain. There are four FOXO genes—FOXO1/3/4/6 in mammals. FOXO transcriptional activities are tightly regulated by posttranslational modifications that control either cytosolic or nuclear localization of FOXOs. Phosphorylation at several conserved sites (FOXO1-T24/S256/S319, FOXO3-T32/S253/S315, FOXO4-T32/S197/S262), primarily catalyzed by Akt, induces the nuclear exclusion of FOXOs (39).

FOXOs regulate glucose homeostasis through promotion of hepatic gluconeogenesis and inhibition of glycolysis (40, 59). FOXOs also regulate hepatic lipid metabolism by activating lipolysis and lipophagy through upregulation of adipose triglyceride lipase (ATGL), hormone-sensitive lipase (HSL), lipoprotein lipase (LPL), CPT1A, ATG5, and ATG14 (39, 216, 236). FOXO1 also suppresses de novo lipogenesis by downregulation of SREBP1 and other lipogenic genes (36, 237). Deletion of hepatic FOXO1/3 or FOXO1/3/4 in mice leads to hepatic steatosis (191, 234). FOXO1/3/4 liver-specific knockout mice develop severe NASH on a high-fat, high-cholesterol diet (Fig. 11) (153).

FOXO1 plays a significant role in the regulation of macrophage activity. FOXO1 promotes apoptosis of insulin-resistant macrophages by inhibition of p65 NF-κB via induction of NF-κB inhibitor ɛ (IKBE) (180). Under obesity and insulin resistance conditions, FOXO1 and TLR4 form a feedback circuitry to modulate an inflammatory response in which FOXO1 transcriptionally activates TLR4, and subsequently, the TLR4 pathway inhibits the FOXO1 transcriptional activity in macrophages (Fig. 11) (49).

FOXO1 can inhibit the proliferation and transdifferentiation of HSCs through induction of p27Kip1 encoded by cyclin-dependent kinase inhibitor 1B (CDKN1B) and manganese-containing superoxide dismutase encoded by superoxide dismutase 2 (SOD2). FoxO1^+/− mice are more susceptible to hepatic fibrosis induced by bile-duct ligation compared with WT mice (1). FOXOs are negatively regulated by the PI3K-AKT signaling pathway. In human HSC line LX-2 cells, TNF-related apoptosis inducing ligand inhibits the PI3K/AKT signaling and thus activates FOXOs. As a result, expression of an apoptosis suppressor CASP8 and FADD like apoptosis regulator (CFLAR) is downregulated and this leads to HSC apoptosis through several apoptosis-activating molecules such as caspase-3, caspase-8, and BH3 interacting domain death agonist (155). Liver-specific deletion of FOXO1/3/4 in mice leads to severe hepatic steatosis and fibrosis with elevated expression of Col1a1, Pdgfrb, Tgfb1, and TIMP metallopeptidase inhibitor 1 (Timp1) in the liver on a high-fat diet compared with that in WT controls (Fig. 11) (153).

Peroxisome proliferator-activated receptor α

Among three PPAR nuclear receptors, expression of PPARα is the most abundant in the liver. It is responsible for the clearance of lipids in the liver through regulation of numerous genes involved in FAO, including CPT1A and acyl-CoA oxidase 1 (ACOX1) (130). PPARα also controls plasma triglyceride levels through upregulation of LPL (176). Moreover, PPARα affects lipogenesis through regulation of expression of malonyl-CoA decarboxylase, an enzyme that degrades fatty acid synthesis precursor malonyl-CoA (97). PPARα whole-body or liver-specific knockout mice exhibit severe steatohepatitis after either fasting or feeding with a high-fat diet (Fig. 12) (81). PPARα has anti-inflammation function in the liver. One of the main mechanisms is to transrepress inflammatory transcription factors such as NF-κB, AP-1, and STATs (157). PPARα also controls the duration of inflammation by regulating the breakdown of its ligand leukotriene B4, an inflammatory inducer (38). PPARα-null macrophages have increased NF-κB, JNK, and p38 MAPK activities and inflammatory mediators compared with their WT counterparts (Fig. 12) (30).

FIG. 12.

PPARs and FXR in fatty liver disease. Main functions of PPARα, PPARδ, PPARγ, and FXR in the liver are summarized here. 9-HODE, 9-hydroxyoctadecadienoic acid; 15-HETE, 15-hydroxyeicosatetraenoic acid; ACOX1, acyl-CoA oxidase 1; ACTA2, actin alpha 2, smooth muscle; APOA4, apolipoprotein A4; CD68, CD68 molecule; CEBPD, CCAAT enhancer binding protein delta; CLEC4F, C-type lectin domain family 4 member F; COL1A1, collagen type I alpha 1 chain; CYP8B1, cytochrome P450 family 8 subfamily B member 1; ETS1, ETS proto-oncogene 1; FABP1, fatty acid binding protein 1; FAs, fatty acids; FBP1, fructose-bisphosphatase 1; HDAC3, histone deacetylase 3; IKBKE, inhibitor of nuclear factor kappa B kinase subunit epsilon; LIPG, lipase G; LTB4, leukotriene B4; M2 φ, M2 macrophage; NCOR, nuclear receptor corepressor; PKLR, pyruvate kinase L/R; PPARδ, peroxisome proliferator-activated receptor δ; SHP, small heterodimer partner; SUMO, sumoylation; VLDLR, very low-density lipoprotein receptor.

Peroxisome proliferator-activated receptor δ

PPARδ, structurally very similar to PPARα, is ubiquitously expressed in most tissues. Like the other members of the PPAR family, it forms a heterodimer with RXR and regulates expression of its target genes by binding to specific PPAR response elements. It is expressed in all cell types in the liver (229). In contrast to PPARα-null mice, PPARδ knockout mice have no significant alterations in plasma free fatty acids and β-hydroxybutyrate and hepatic triglycerides and glycogen. Transcriptional profiling reveals that PPARδ positively regulates PKLR and fructose-bisphosphatase 1 (FBP1) genes in glucose metabolism and apolipoprotein A4 (APOA4), LIPG, and VLDLR for lipoprotein metabolism, and negatively regulates inflammatory genes such as STAT1, IKBKE, and CD68 (Fig. 12) (172). Hepatic overexpression of PPARδ in mice decreases fasting blood glucose levels but increases hepatic glycogen and triglyceride deposition. However, this does not lead to hepatic injury possibly due to an increase in monounsaturated fatty acids and a decrease in saturated fatty acids (116). PPARδ exerts protective effects against hepatic inflammation. PPARδ promotes alternative M2 activation of hepatic KCs and ameliorates obesity-induced inflammation (147). Activation of PPARδ by specific agonists reduces expression of inflammatory and ER stress genes in the liver (Fig. 12) (100).

Peroxisome proliferator-activated receptor γ

PPARγ expression is low in the normal liver, but it is elevated in the liver of NAFLD patients (159). PPARγ promotes steatosis probably due to its role in lipogenesis. PPARγ knockout mice are protected from hepatic steatosis but develop severe hyperglycemia and insulin resistance (Fig. 12) (54, 134).

PPARγ has anti-inflammatory function, especially through control of macrophage activation. In macrophages, activation of PPARγ downregulates inflammatory genes partly by antagonizing transcription factors such as AP-1, NF-κB, and STAT (167). PPARγ also induces monocyte differentiation toward anti-inflammatory M2 macrophages. PPARγ activation has been shown to protect from hepatic inflammation through inhibition of M1 macrophage polarization and promotion of alternative M2 activation under high-fat diet conditions (Fig. 12) (146).

PPARγ plays a critical role in the maintenance of HSC quiescence. Deletion of PPARγ in HSCs using an aP2-Cre leads to exacerbated hepatic fibrosis in mice when challenged with CCl₄ (135). PPARγ is downregulated during the HSC activation and re-expressed during the fibrosis resolution. Knockout of PPARγ specifically in mouse HSCs leads to downregulation of C/EBPδ, SREBP1, and ETS proto-oncogene 1 (ETS1). After the CCl₄ treatment, HSC-specific PPARγ-deficient mice have 40% more hepatic fibrosis and are 30% slower in resolution of hepatic fibrosis than that in WT mice. Administration of rosiglitazone, a PPARγ agonist, improves the hepatic fibrosis regression in WT mice but not in PPARγ HSC-specific knockout mice (Fig. 12) (117).

Farnesoid X receptor

Farnesoid X receptor (FXR) is a receptor for BAs, with chenodeoxycholic acid being the most potent followed by deoxycholic acid, cholic acid, and lithocholic acid. FXR plays a major role in the feedback regulation of BA biosynthesis by suppression of the CYP7A1 and cytochrome P450 family 8 subfamily B member 1 (CYP8B1) genes via small heterodimer partner and FGF19 (91, 186). In addition, activation of FXR also regulates glucose and lipid homeostasis (85). FXR knockout mice have elevated levels of hepatic triglycerides and serum triglycerides and cholesterol (186). FXR inhibits hepatic lipogenesis by suppression of the transcriptional activity of LXR and consequently the expression of SREBP1c and ChREBP (18, 211). FXR activation also induces the PPARα gene expression for promotion of FAO (Fig. 12) (160).

Activator protein-1

AP-1 is a transcription factor that is composed of a homodimer or a heterodimer with at least one protein from the Jun family (c-Jun, JunB, and JunD) and another from the Fos family (c-Fos, FosB, Fra1, Fra2, and ΔFosB) or ATFs (65). Expression of c-Jun is increased in both hepatocytes and nonparenchymal liver cells (NPLCs) in the livers of NASH patients compared with hepatic steatotic patients (178). Conditional knockout of c-Jun in mouse livers using Alfp-Cre or Mx1-Cre reveals that c-Jun in the hepatocytes promotes cell survival and restrains biliary ductal reaction, whereas c-Jun in NPLCs promotes ductal reaction and hepatic fibrogenesis via regulation of osteopontin (OPN) and CD44 (Fig. 13) (178).

FIG. 13.

Key transcription factors in the regulation of hepatic inflammation. Numerous transcription factors are involved in the regulation of hepatic inflammation. AP-1, NF-κB, PU.1, STAT1, and KLF4 are highlighted here. ARG1, arginase 1; CCL5, C-C motif chemokine ligand 5; CD44, CD44 molecule; C/EBPβ, CCAAT enhancer binding protein β; FIZZ1, found in inflammatory zone 1; IFNγ, interferon gamma; KLF4, Kruppel-like factor 4; MCPIP, monocyte chemotactic protein-induced protein; MRC1, mannose receptor C-type 1; NASH, nonalcoholic steatohepatitis; NPLCs, nonparenchymal liver cells; OPN, osteopontin; PU.1, transcription factor PU.1.

Nuclear factor kappa B subunit

NF-κB is composed of a heterodimer or homodimer of the Rel family members encoded by the NFKB1, NFKB2, REL, RELA, and RELB genes. It is often activated in various chronic liver diseases, including ALD and NAFLD, and a number of proinflammatory cytokine and chemokine genes such as IL1β, IL6, TNFα, CCL2, and CCL5 are often upregulated by NF-κB (121). NF-κB plays a significant role in the development of methionine/choline-deficient (MCD) diet-induced NASH in mice (34). Pharmacological inhibition of IKK2, an upstream activator of NF-κB, in a high-sucrose diet-induced NASH model, remarkably reduces hepatic steatosis and inflammation (Fig. 13) (15). The proinflammatory activity of NF-κB in macrophages can be suppressed by SIRT1 via deacetylation (177).

Transcription factor PU.1

PU.1 transcription factor, a member of the ETS family, plays an important role in the differentiation of various immune cells, including macrophages, neutrophils, dendritic cells, T lymphoid cells, and B lymphoid cells (103). Knockdown or inhibition of PU.1 significantly suppresses hepatic inflammatory genes, including IL1β, IL6, and TNFα, especially in macrophages, in a high-fat diet-induced NAFLD or db/db mouse model (Fig. 13) (115).

Signal transducer and activator of transcription 1

STAT1 plays a different role than STAT3 in diet-induced NASH. Hepatic Stat1 and Stat3 are both activated in the liver of C57BL/6 mice after feeding with high-fat diets. Reduction of Stat1, but not Stat3, levels in the liver impedes T cell infiltration and NASH development in mice, and several inflammatory and fibrotic genes such as smooth muscle actin α2 (Acta2), Tgfb1, Il1b, Il6, and Tnf are downregulated (Fig. 13) (58).

Kruppel-like factor 4

Kruppel-like factor 4 (KLF4) is a zinc finger-containing transcription factor and it regulates macrophage polarization (109). After exposure to IL4, KLF4 is induced as well as STAT6. Through upregulation of MCP1-induced protein (MCPIP), KLF4 promotes M2 polarization via PPARγ and C/EBPβ and suppresses M1 polarization via NF-κB (79). In an ALD mouse model, knockdown of KLF4 by siRNA increases M1 macrophage markers, while KLF4 overexpression decreases M1 macrophage markers and increases M2 macrophage markers such as arginase 1 (Arg1) and Il10 (171). Sumoylation of KLF4 induced by IL4 promotes macrophage M2 polarization (Fig. 13) (205).

ETS proto-oncogene 1

Both in vitro and in vivo data suggest that ETS1 plays a critical role in the maintenance of HSC quiescence (117). ETS1 gene expression is high in fresh quiescent rat HSCs but diminishes after 7 days of culture (activation) (89). Knockdown of Ets1 in mouse primary HSCs by shRNA leads to upregulation of proliferation genes such as cyclin-dependent kinases 2 and 4 (Cdk2/4) and cyclin D1 (Ccnd1), fibrosis genes such as Col1a1, Timp1, and lysyl oxidase like 2 (Loxl2), and inflammation genes such as Il1b, Il6, C-X-C motif chemokine ligand 1 (Cxcl1), and Cxcl5, and downregulation of quiescence-associated genes such as Pparγ and insulin-induced gene 1 (Insig1). Knockout of one allele of the Ets1 gene in mouse HSCs using Lrat-Cre leads to worse hepatic fibrosis than WT mice after the CCl₄ treatment (117). In addition to the control of the HSC quiescent state, ETS1 also functions against fibrosis through other mechanisms. For example, ETS1 increases matrix metallopeptidase 1 (MMP1) gene expression in response to hepatocyte growth factor (150). In fibroblasts, ETS1 plays an antagonistic role against the TGFβ-induced fibrotic activity potentially by sequestering SMAD transcription factors from their target genes (Fig. 14) (32).

FIG. 14.

Key transcription factors in the regulation of hepatic fibrosis. Numerous transcription factors have been implicated in the regulation of hepatic fibrosis. ETS1, GATA4, TCF21, β-catenin, SMAD2/3, YAP, and TAZ are highlighted here. BIRC5, baculoviral IAP repeat containing 5; CXCL5, C-X-C motif chemokine ligand 5; CYR61, cysteine-rich angiogenic inducer 61; FN1, fibronectin 1; FZD, frizzled class receptor; GATA4/6, GATA binding proteins 4/6; GLI2/3, GLI family zinc finger 2/3; IHH, Indian hedgehog signaling molecule; INSIG1, insulin-induced gene 1; LOXL2, lysyl oxidase-like 2; LRP5/6, LDL receptor-related proteins 5/6; MMP7/14, matrix metallopeptidases 7/14; PAI-1, plasminogen activator inhibitor 1; PDGFB, platelet-derived growth factor B; PDGFRB, platelet-derived growth factor receptor B; SMAD2/3, SMAD family members 2/3; TCF21, transcription factor 21; TGFBR1, transforming growth factor beta receptor 1; TIMP1, TIMP metallopeptidase inhibitor 1.

GATA binding protein 4

GATA binding protein 4 (GATA4) plays a protective role in hepatic fibrogenesis (35). In conditional heterozygous Gata4 flox/+:G2-Cre mice, CCl₄ treatment leads to severe fibrogenesis compared with CCl₄-treated WT mice. Transfection of GATA4 in LX-2 cells downregulates expression of fibrosis marker genes such as ACTA2 and collagen (35). Knockdown of Gata4 in mouse HSCs decreases Pparγ and increases Col1a1, Timp1, Loxl2, Il6, Ccl5, and Cxcl5 (117). Interestingly, endothelial Gata4 also regulates hepatic fibrosis through an angiocrine signaling manner, specifically by suppressing the Pdgfb gene expression in liver sinusoidal endothelial cells (Fig. 14) (212).

Transcription factor 21

Transcription factor 21 (TCF21) is a basic helix-loop-helix transcription factor and plays a role in the regulation of cell fate and differentiation during development (5). Through a cell-based transcription factor screening, TCF21 has been identified as a suppressor of the HSC activation. Tcf21 expression is downregulated during fibrogenesis and re-expressed during fibrosis regression in mice treated with CCl₄. AAV6-mediated TCF21 overexpression protects against CCl₄-induced hepatic injury and fibrosis. Mechanistically, TCF21 suppresses the TGFβ-induced fibrogenic gene program, including ACTA2 and COL1A1 (Fig. 14) (144).

β-Catenin

β-Catenin has been implicated in HSC activation and hepatic fibrosis. Gene expression profiling suggests that Wnt signaling genes such as frizzled class receptor 2 (FZD2), WNT4, and WNT5 are upregulated in the activated rat HSCs compared with control quiescent cells (74). WNT3A promotes HSC activation and survival, whereas secreted frizzled-related protein 1 (sFRP1) inhibits both (140). sFRP5 also inhibits HSC proliferation and migration in vitro and attenuates CCl₄-induced hepatic fibrosis in mice (23). LDL receptor-related protein 6 (LRP6), a coreceptor for WNT, is elevated in livers of fibrotic patients. LRP6 gene knockout in cultured rat HSCs and in CCl₄-treated rats reduces HSC activation and hepatic fibrosis (Fig. 14) (227).

SMAD3

SMAD3 is a key transcription factor mediating the TGFβ profibrogenic activity. Numerous fibrotic genes, including COL1A1 and TIMP1, are direct targets of SMAD3 (199). In vitro data suggest that TGFβ activates SMAD2 in early cultured HSCs and SMAD3 in activated myofibroblasts (113). Ablation of the Smad3 gene in mice leads to resistance to dimethylnitrosamine-induced liver fibrosis (94). After acute liver injury, both SMAD2 and SMAD3 induce the collagen gene expression stimulated by TGFβ and PDGF. During chronic liver disease, SMAD2 and SMAD3 may be regulated differently in response to TGFβ and PDGF (224). SMAD3 transcriptional activity is modulated by lysine acetylation primarily by p300 and deacetylation by SIRT1 and SIRT6 (71, 241). HSC-specific knockout of Sirt6 leads to hyperacetylated Smad2/3 and exacerbated hepatic fibrosis (233, 241). Aryl hydrocarbon receptor also suppresses HSC activation and hepatic fibrosis through disruption of the interaction between SMAD3 and β-catenin (Fig. 14) (219).

YAP and TAZ

As key downstream mediators of the Hippo signaling pathway, both YAP and TAZ/WWTR1 have been implicated in hepatic fibrosis. YAP is activated in HSCs in CCl₄-treated mice and fibrotic human patients (124). Expression of YAP target genes such as connective tissue growth factor (CTGF) and ankyrin repeat domain 1 (ANKRD1) is increased during the initial stage of HSC activation in vitro. Knockdown of YAP or pharmacological inhibition of YAP blocks HSC activation in vitro. Administration of verteporfin, a chemical compound that disrupts the YAP/TEAD complex, to CCl₄-treated mice remarkably improves hepatic fibrosis (124). Overexpression of YAP in hepatocytes induces hepatic inflammation and fibrosis as early as 2 weeks. Deletion of Yap alone or together with Taz from hepatocytes improves CCl₄-induced inflammation and fibrosis. Cysteine-rich angiogenic inducer 61 (CYR61/CCN1), a transcriptional target of YAP and TAZ, is shown to mediate, at least partially, the YAP/TAZ effects on hepatic inflammation and fibrosis (133). YAP has been shown to mediate the integrin b1-triggered fibrogenesis (127). YAP activation also drives HSC transdifferentiation by induction of glutaminolysis (Fig. 14) (42).

TAZ is highly expressed in NASH livers (210). TAZ undergoes similar regulation to YAP by LATS1/2 with regard to its phosphorylation and cellular localization. Unlike YAP, the role of TAZ in HSCs remains elusive. Increasing evidence suggests that hepatocyte TAZ can indirectly promote HSC activation by induction and secretion of Indian hedgehog, an inducer of HSC activation. TAZ expression is elevated in hepatocytes in human and mouse livers during the transition from steatosis to NASH (210). Further study suggests that cholesterol is a trigger for the upregulation of TAZ in the NASH hepatocytes through a soluble adenylyl cyclase—protein kinase A—Ca²⁺—RHOA pathway that prevents the proteasomal degradation of TAZ (207). Administration of siRNA against hepatocyte Taz attenuates hepatic injury, inflammation, and fibrosis in a diet-induced NASH mouse model and also partially reverses the established NASH (209). Ceramide has also been shown to inhibit the YAP/TAZ signaling and hepatic fibrosis by promoting phosphorylation and proteasomal degradation of YAP/TAZ (Fig. 14) (3).

Concluding Remarks

As fatty liver disease is a complex metabolic disease, a variety of factors ranging from environmental (diet, alcohol, life style, and so forth), genetic (common and rare gene mutations), and epigenetic (DNA methylation, histone modifications, noncoding RNAs, and others) should be considered for both research and clinical purposes. New technologies such as next-generation sequencing, proteomics, and metabolomics are expected to further enhance our understanding of the pathogenesis and potential therapeutic targets for the fatty liver disease. It is foreseeable that effective therapeutics for this very common liver disease will become available in the near future.

Abbreviations Used

9-HODE

9-hydroxyoctadecadienoic acid

15-HETE

15-hydroxyeicosatetraenoic acid

AA

amino acid

ABCA1

ATP-binding cassette subfamily A member 1

ABCG1/5/8

ATP-binding cassette subfamily G members 1, 5, and 8

Ac

acetyl

ACC1/2

acetyl-CoA carboxylases 1 and 2

ACOX1

acyl-CoA oxidase 1

ACTA2

actin alpha 2, smooth muscle

ADH

alcohol dehydrogenase

AGPAT1

1-acylglycerol-3-phosphate O-acyltransferase 1

AKT1/2

AKT serine/threonine kinases 1 and 2

ALD

alcoholic liver disease

ALDH2

aldehyde dehydrogenase 2

ALK1/5

anaplastic lymphoma receptor tyrosine kinases 1 and 5

AMOT

angiomotin

AMPK

AMP-activated protein kinase

AP-1

activator protein 1

APOA4

apolipoprotein A4

ARG1

arginase 1

ARID1A

AT-rich interaction domain 1A

ASC

apoptosis-associated speck-like protein containing a CARD

ASK1

apoptosis signal regulating kinase 1

ATF

activating transcription factor

ATG13

autophagy-related gene 13

ATG14

autophagy-related gene 14

ATGL

adipose triglyceride lipase

ATP

adenosine triphosphate

BA

bile acid

Bach1

BTB domain and CNC homologue 1

BCL2

BCL2 apoptosis regulator

BIRC5

baculoviral IAP repeat containing 5

CAMKKβ

calcium/calmodulin-dependent protein kinase kinase β

CASP

caspase

CASTOR1

cytosolic arginine sensor for mTORC1 subunit 1

CAT

catalase

CEBPB

CCAAT enhancer binding protein beta

CEBPD

CCAAT enhancer binding protein delta

CCL2/5

C-C motif chemokine ligands 2 and 5

CCND1

cyclin D1

CD36

CD36 molecule

CD44

CD44 molecule

CD68

CD68 molecule

CDK6

cyclin-dependent kinase 6

CDKN1B

cyclin-dependent kinase inhibitor 1B

C/EBP

CCAAT enhancer binding protein

CFLAR

CASP8 and FADD apoptosis regulator

CHOP

CCAAT/enhancer-binding protein homologous protein

ChoRE

carbohydrate response element

ChREBP

carbohydrate response element binding protein

CIDEC

cell death inducing DFFA-like effector C

CLEC4F

C-type lectin domain family 4 member F

COL1A1

collagen type I alpha 1 chain

COL3A1

collagen type III alpha 1 chain

CPT1

carnitine palmitoyltransferase 1

CPT1A

carnitine palmitoyltransferase 1A

CTGF

connective tissue growth factor

CXCL1/5/10

C-X-C motif chemokine ligands 1, 5, and 10

CYP2E1

cytochrome P450 family 2 subfamily E member 1

CYP7A1

cytochrome P450 family 7 subfamily A member 1

CYP8B1

cytochrome P450 family 8 subfamily B member 1

CYP27A1

cytochrome P450 family 27 subfamily A member 1

CYR61

cysteine-rich angiogenic inducer 61

DAG

diacylglycerol

DGAT1

diacylglycerol O-acyltransferase 1

DHCR24

24-dehydrocholesterol reductase

DNL

de novo lipogenesis

DNMT3A

DNA methyltransferase 3 alpha

DPP4

dipeptidyl peptidase 4

eIF2

eukaryotic translation initiation factor 2

ER

endoplasmic reticulum

ERK

extracellular signal-regulated kinase

EtOH

ethanol

ETS1

ETS proto-oncogene 1

EV

extracellular vesicle

FABP1

fatty acid binding protein 1

FAO

fatty acid oxidation

FASN

fatty acid synthase

FBP1

fructose-bisphosphatase 1

FGF11

fibroblast growth factor 11

FGF21

fibroblast growth factor 21

FGFR1/2

fibroblast growth factor receptors 1 and 2

FIZZ1

found in inflammatory zone 1

FN1

fibronectin 1

Fos

Fos proto-oncogene

FOXO1/3

forkhead box O 1/3

FXR

farnesoid X receptor

FZD

frizzled class receptor

G6PC

glucose-6-phosphatase catalytic subunit

G6PD

glucose-6-phosphate dehydrogenase

GADD34

growth arrest and DNA damage-inducible protein 34

GATA4/6

GATA binding proteins 4 and 6

GATOR1/2

GTPase-activating proteins 1/2 toward Rags complex

GCK

glucokinase

GlcNAc

N-acetyl-d-glucosamine

GLI2/3

GLI family zinc finger 2/3

GLS

glutaminase

GNA12

guanine nucleotide-binding protein subunit alpha 12

GPCR

G protein-coupled receptor

GR

glucocorticoid receptor

HDAC3

histone deacetylase 3

HDL

high-density lipoprotein

HGF

hepatocyte growth factor

HIF1α

hypoxia inducible factor 1α

HMGA2

high-mobility group AT-hook 2

HMGCR

3-hydroxy-3-methylglutaryl-CoA reductase

HMGCS1/2

3-hydroxy-3-methylglutaryl-CoA synthases 1 and 2

HMOX1

heme oxygenase 1

HNRNPA1

heterogeneous nuclear ribonucleoprotein A1

HSC

hepatic stellate cell

HSL

hormone-sensitive lipase

IFNγ

interferon gamma

IGF1

insulin-like growth factor 1

IGF1R

insulin-like growth factor 1 receptor

IGFBP2

IGF binding protein 2

IHH

Indian hedgehog signaling molecule

IKBE

NFKB inhibitor epsilon

IKBKE

inhibitor of nuclear factor kappa B kinase subunit epsilon

IKK

inhibitor of nuclear factor kappa B kinase

IL1β

interleukin 1β

IL6

interleukin 6

IL1R

interleukin 1 receptor

IL1RN

interleukin 1 receptor antagonist

IL6R

interleukin 6 receptor

INSIG1

insulin-induced gene 1

IRAK

interleukin 1 receptor-associated kinase

IRE1α

inositol-requiring enzyme 1 α

JAK

Janus kinase

JNK

JUN N-terminal kinase

JUN

JUN proto-oncogene

K⁺

potassium ion

KC

Kupffer cell

KIBRA

kidney and brain protein

KLF4/6

Kruppel-like factors 4 and 6

LAMP1

lysosomal-associated membrane protein 1

LATS1/2

large tumor suppressor kinases 1 and 2

Leu

leucine

LID

low-glucose inhibitory domain

LIPG

lipase G

LKB1

liver kinase B1

LOXL2

lysyl oxidase-like 2

LPA

lysophosphatidic acid

LPL

lipoprotein lipase

LPS

lipopolysaccharide

LRP5/6

LDL receptor-related proteins 5 and 6

LTB4

leukotriene B4

LXR

liver X receptor

M2 φ

M2 macrophage

MAP3K1

mitogen-activated protein kinase kinase kinase 1

MAP3K3

mitogen-activated protein kinase kinase kinase 3

MAP4K

mitogen-activated protein kinase kinase kinase kinase

MAPK

mitogen-activated protein kinase

MAT1A

methionine adenosyltransferase 1A

MCPIP

monocyte chemotactic protein-induced protein

MEK7

MAPK/ERK kinase 7

MERTK

MER proto-oncogene tyrosine kinase

Met

methionine

miR

microRNA

MKK

mitogen-activated protein kinase kinase

MMP1/7/14

matrix metallopeptidases 1, 7, and 14

MOB1A/B

MOB kinase activator 1 A/B

MRC1

mannose receptor C-type 1

MST1/2

mammalian STE20-like protein kinases 1 and 2

MT

metallothionein

MTF1

metal regulatory transcription factor 1

mTOR

mechanistic target of rapamycin kinase

mTORC1/2

mechanistic target of rapamycin kinase complexes 1 and 2

MYC

MYC proto-oncogene

MyD88

myeloid differentiation primary response protein 88

NAD

nicotinamide adenine dinucleotide

NAFLD

nonalcoholic fatty liver disease

NASH

nonalcoholic steatohepatitis

NCOA2

nuclear receptor coactivator 2

NCOR

nuclear receptor corepressor

NDRG3

N-Myc downstream-regulated gene 3 protein

NF2

neurofibromin 2

NF-κB

nuclear factor kappa B subunit

NFYC

nuclear transcription factor Y subunit gamma

NIK

NF-κB-inducing kinase

NLRP3

NLR family pyrin domain containing 3

NPC1L1

NPC1-like intracellular cholesterol transporter 1

NPLCs

nonparenchymal liver cells

NRF2

nuclear factor erythroid 2-related factor 2

OGT

O-linked N-acetylglucosamine transferase

OPN

osteopontin

P2X7

purinergic receptor P2X 7

p38 MAPK

p38 mitogen-activated protein kinase

p300

E1A binding protein p300

PA

palmitic acid

PAI-1

plasminogen activator inhibitor 1

PDGF

platelet-derived growth factor

PDGFB

platelet-derived growth factor B

PDGFC

platelet-derived growth factor C

PDGFRB

platelet-derived growth factor receptor B

PERK

PRKR-like endoplasmic reticulum kinase

PGC1α

peroxisome proliferator-activated receptor gamma coactivator 1 alpha

PI3K

phosphatidylinositol 3-kinase

PIM3

Pim-3 proto-oncogene serine/threonine kinase

PKA

protein kinase A

PKLR

pyruvate kinase L/R

PKM2

pyruvate kinase M2

PM

plasma membrane

PP1C

protein phosphatase 1C

PP2A

protein phosphatase 2A

PPAR

peroxisome proliferator-activated receptor

PRC1

protein regulator of cytokinesis 1

PTEN

phosphatase and tensin homologue

PU.1

transcription factor PU.1

RA

retinoic acid

RAF

Raf proto-oncogene serine/threonine kinase

RAG

Ras-related GTP binding protein

RAS

Ras proto-oncogene GTPase

Raptor

regulatory associated protein of mTOR complex 1

REDD1

protein regulated in development and DNA damage response 1

RELB

RELB proto-oncogene

RHEB

Ras homologue enriched in brain

RHOA

Ras homology family member A

RICTOR

RPTOR independent companion of MTOR complex 2

RIDD

regulated IRE1-dependent decay

ROCK1

Rho-associated coiled-coil containing protein kinase 1

ROS

reactive oxygen species

RXR

retinoid X receptor

S6K1

ribosomal protein S6 kinase 1

SAM

S-adenosylmethionine

SAMTOR

S-adenosylmethionine sensor upstream of mTORC1

SAV1

Salvador family WW domain containing protein 1

SC4MOL

sterol-C4-methyl oxidase

SCAP

SREBP-cleavage activating protein

SCD1

stearoyl-CoA desaturase 1

SESN

sestrin

sFRP

secreted frizzled related protein

SHP

small heterodimer partner

SIRT

sirtuin

SLC9A3R1

SLC9A3 regulator 1

SLC27A5

solute carrier family 27 member 5

SLC43A1

solute carrier family 43 member A1

SLC51A

solute carrier family 51 subunit alpha

SMAD

SMAD family member

SMURF

SMAD-specific E3 ubiquitin protein ligase

SOCS1

suppressor of cytokine signaling 1

SOD

superoxide dismutase

SP3

SP3 transcription factor

SR-BI

scavenger receptor class B member 1

SREBP

sterol regulatory element binding protein

STAT

signal transducer and activator of transcription

SUMO

sumoylation

TAK1

transforming growth factor beta-activated kinase 1

TAZ

transcriptional coactivator with PDZ-binding motif

TBK1

TANK binding kinase 1

TCF21

transcription factor 21

TEAD

TEA domain transcription factor

TET

ten eleven translocation

TFE3

transcription factor binding to IGHM enhancer 3

TFEB

transcription factor EB

TG

triglyceride

TGFβ

transforming growth factor β

TGFBR1

transforming growth factor beta receptor 1

TIMP1

TIMP metallopeptidase inhibitor 1

TIRAP

TIR domain containing adaptor protein

TLR4

toll-like receptor 4

TNF

tumor necrosis factor

TNFR

tumor necrosis factor receptor

TRADD

tumor necrosis factor receptor type 1-associated death domain protein

TRAF

tumor necrosis factor receptor-associated factor

TRIM4

tripartite motif containing 4

TSC

tuberous sclerosis complex subunit

TUBA1B

tubulin alpha 1b

TXNIP

thioredoxin interacting protein

ULK1

Unc-51-like autophagy activating kinase 1

UPR

unfolded protein response

UVRAG

UV radiation resistance associated

VGLL4

vestigial-like family member 4

VHL

von Hippel–Lindau tumor suppressor

VLDL

very low-density lipoprotein

VLDLR

very low-density lipoprotein receptor

WNT

Wnt family member

WT

wild type

WWTR1

WW domain containing transcription regulator 1

XBP1

X-box binding protein 1

XBP1s

X-box binding protein 1 spliced

XBP1u

X-box binding protein 1 unspliced

YAP

yes1-associated transcriptional regulator

ZEB2

zinc finger E-box binding homeobox 2

Authors' Contributions

X.C.D. contributed to the concept development, literature searches, illustration preparation, and article writing and revision. M.H. contributed to the article draft preparation, especially the signal transduction section. H.G.K. contributed to the article draft preparation, especially the section of sirtuins, microRNAs, and DNA methylation. K.C. contributed to the article draft preparation, especially the section of Transcriptional Regulation of Hepatic Steatosis, Inflammation, and Fibrosis.

Author Disclosure Statement

No competing financial interests exist.

Funding Information

This work was supported by the U.S. National Institute of Diabetes and Digestive and Kidney Diseases of the National Institutes of Health under award numbers R01DK120689 (Xiaocheng Charlie Dong) and R01DK121925 (Xiaocheng Charlie Dong) and the Indiana Clinical and Translational Sciences Institute funded, in part, by award number UL1TR002529 from the National Center for Advancing Translational Sciences of the National Institutes of Health. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.