Biliverdin reductase and bilirubin in hepatic disease

Abstract

The buildup of fat in the liver (hepatic steatosis) is the first step in a series of incidents that may drive hepatic disease. Obesity is the leading cause of nonalcoholic fatty liver disease (NAFLD), in which hepatic steatosis progresses to liver disease. Chronic alcohol exposure also induces fat accumulation in the liver and shares numerous similarities to obesity-induced NAFLD. Regardless of whether hepatic steatosis is due to obesity or long-term alcohol use, it still may lead to hepatic fibrosis, cirrhosis, or possibly hepatocellular carcinoma. The antioxidant bilirubin and the enzyme that generates it, biliverdin reductase A (BVRA), are components of the heme catabolic pathway that have been shown to reduce hepatic steatosis. This review discusses the roles for bilirubin and BVRA in the prevention of steatosis, their functions in the later stages of liver disease, and their potential therapeutic application.

INTRODUCTION

Obesity has grown to epidemic proportions throughout the world (36, 72). Obesity causes an increase in the release of adipokines and free fatty acids from adipose tissue, which leads to a systemic chronic inflammatory state (71). The inflammation along with hepatic lipid accumulation results in the development of nonalcoholic fatty liver disease (NAFLD), which can progress to nonalcoholic steatohepatitis (NASH; 68, 71). The incidence of NAFLD has paralleled the high rates of obesity worldwide and has become the most common cause of chronic liver disease (11, 21).

NAFLD is diagnosed by lipid accumulation within hepatocytes that accounts for >5% of the total liver weight (63). The prevalence of NAFLD in the United States is estimated at 30%, and a majority of these patients were asymptomatic (56, 81). However, 10.3% of patients with NAFLD eventually progress to a more serious hepatic disease and exhibit fibrosis (56). Hepatic steatosis is also seen in alcoholic liver disease (ALD) resulting from long-term alcohol abuse (8, 105). NAFLD and ALD have similar pathological progressions from simple hepatic steatosis to steatohepatitis, fibrosis, and then cirrhosis or possibly hepatocellular carcinoma (105). However, NAFLD usually has a greater degree of hepatic fatty degeneration whereas ALD has more pronounced inflammation and venous fibrosis (105).

Recent research has demonstrated an important function of bilirubin and the enzyme that produces it, biliverdin reductase A (BVRA), in protecting the liver against lipid accumulation and hepatic disease (54, 61, 83). Interestingly, alcoholic hepatitis, a type of ALD, is associated with higher serum conjugated bilirubin (74), whereas conversely, obese patients have lower total bilirubin serum levels (5). These findings suggest that hepatocyte damage may play a role in the development of hepatic steatosis and alterations of serum bilirubin levels in alcoholic hepatitis and obesity. This review discusses the recent findings of the protective roles of bilirubin and BVRA in hepatic diseases.

BILIRUBIN AND HEPATIC STEATOSIS

Bilirubin is generated from the breakdown of heme present in hemoproteins (e.g., hemoglobin and myoglobin) that is released from the catabolism of red blood cells. The heme ring is broken open by heme oxygenase forming biliverdin, which is reduced to bilirubin by biliverdin reductase (BVR; Fig. 1; 104). Snyder and colleagues showed that bilirubin was capable of protecting cells from the oxidative stress of a 10,000-fold increase in hydrogen peroxide (3, 91). Furthermore, they proposed a biliverdin-bilirubin redox cycle in which bilirubin, the antioxidant, reduces reactive oxygen species (ROS) and is consequently reoxidized to biliverdin. This proposed redox cycle would be capable of managing the oxidative stress perpetuated by a 10,000-fold surge in hydrogen peroxide concentration (91). However, it is possible that this redox recycling of bilirubin to biliverdin was an artifact of the cell culture conditions utilized by Sedlak and Snyder (91) as Maghzal et al. showed that the redox cycle might play an insufficient part in BVR’s role as a cellular antioxidant, at least in HeLa human cervical cancer cells (64). These authors argue that bilirubin may regenerate itself through hydrogen abstraction from the central methylene group, which would generate a resonance-stabilized tetrapyrrole radical lowering its lipophilicity (64). In cells that are more acceptable to lipid peroxidation, such as adipocytes and hepatocytes, it is plausible that the BVR-bilirubin cycle may be more pronounced. Gonzalez-Sanchez et al. showed in HepG2, Alexander, and HuH-7 human liver cell lines that BLVRA mRNA was inversely proportional to the level of ROS production (31). Indeed, a global Blvra knockout (KO) mouse had severe oxidative stress due to low plasma bilirubin levels (14). Thus, there is a need for more in vivo studies, particularly in humans, to reveal the nature of the BVR-bilirubin cycle.

Fig. 1.

Heme catabolic pathway. Heme contains a porphyrin ring and a central iron atom. In heme degradation, heme oxygenase breaks open the porphyrin ring with NADPH and oxygen (O₂) resulting in the release of iron (Fe), carbon monoxide (CO), and biliverdin. Biliverdin is reduced by biliverdin reductase to bilirubin. Reactive oxygen species (ROS) can recycle bilirubin back to biliverdin. In the liver, bilirubin is conjugated with glucuronic acid by UDP-glucuronosyltransferase 1A1 (UGT1A1) and excreted into the bile.

If bilirubin is not reoxidized to biliverdin, it is conjugated to glucuronic acid in hepatocytes by UDP-glucuronosyltransferase 1A1 (UGT1A1) to make it more soluble for biliary excretion (34, 114). The conjugated bilirubin is transported into the bile via the ATP-dependent multidrug-resistant protein transporter MRP2 (34, 114). Once in the gut, the conjugated bilirubin is reduced by the microflora to urobilinogen and further metabolized into stercobilin or urobilin, which are excreted in the feces and urine, respectively (34, 92, 102, 104). However, bilirubin is not simply a waste product of heme catabolism (109). It is also a potent antioxidant particularly when it comes to protecting lipids from oxidation (90, 109, 124). Within the last decade, bilirubin’s role has expanded as a protector against inflammation (87, 111, 121), diabetes (1, 13, 122), cardiovascular disease (47, 49, 124), metabolic syndrome (33, 55), obesity (48), and, in particular, chronic liver disease (41, 46, 53). Elevated total serum bilirubin levels have been reported to be negatively associated with NAFLD and NASH in patients undergoing routine health screening (54). In obese children, those diagnosed with NAFLD by liver ultrasonography have lower levels of total serum bilirubin than those without NAFLD (61). Puri et al. investigated children diagnosed with NAFLD and observed that total serum bilirubin levels were significantly lower in patients with NASH (83). In addition, total serum bilirubin levels were negatively correlated with the degree of steatosis and NAFLD activity score (83). These studies support an additional role of bilirubin as a protective factor against the progression and development of chronic liver disease.

Traditionally, elevated serum bilirubin levels have been considered a biomarker for liver disease and jaundice. However, it is important to differentiate between elevated serum conjugated and unconjugated bilirubin levels. Unconjugated bilirubin levels typically range from 2 to 8 µM, and for conjugated they range from 0.06 to 0.48 µM or 3–5% of total bilirubin (23, 24, 34). Patients with jaundice and hepatobiliary damage have higher concentrations of conjugated bilirubin (79 µM; 24, 34, 85), which indicates irreversible damage to hepatocytes. Meanwhile, several studies have shown a negative correlation between a patient’s serum unconjugated bilirubin levels and chronic liver disease, particularly in the severity of NAFLD and progression to NASH (41, 54, 83, 87). Salomone et al. performed a retrospective study to assess hepatic steatosis, inflammation, and fibrosis in NAFLD patients (87), in which they showed that unconjugated bilirubin levels were lower in patients with more severe inflammation and fibrosis, suggesting that unconjugated hyperbilirubinemia is protective (87). Therefore, it is imperative to report the serum conjugated and unconjugated bilirubin levels to clarify which form of bilirubin is eliciting the hepatic protection.

The benefits of unconjugated bilirubin are also evident in patients with Gilbert’s syndrome (GS). These patients have unconjugated hyperbilirubinemia without hepatic damage or hemolysis due to a genetic polymorphism in the UGT1A1 gene (25). The UGT1A1 gene encodes the enzyme UDP-glucuronosyltransferase, which is responsible for the conjugation of bilirubin in the liver. UGT1A1*28 is the most common UGT1A1 polymorphism in patients with GS and contains an additional TA repeat in the UGT1A1 TATA promoter sequence. Patients with GS have a lower risk of cardiovascular disease and NAFLD (60, 89, 110). GS patients have been shown to have lower levels of serum cholesterol, triglycerides, and proinflammatory cytokines including interleukin-6 (IL-6; 112). In addition, Lin et al. found that obese children with the UGT1A1*6 variant in the coding region had slightly elevated plasma bilirubin levels and were less likely to be diagnosed with NAFLD (61). Another study determined that patients with unconjugated hyperbilirubinemia were less likely to develop NASH and had a less severe form of liver fibrosis based on the smaller degree of liver stiffness measured in these patients (53). Overall, studies continue to show that patients with UGT1A1 polymorphisms acquire health benefits from unconjugated hyperbilirubinemia. These benefits could be a result of bilirubin’s protection against lipid accumulation and chronic liver disease.

Several clinical studies on the effects of hyperbilirubinemia, particularly unconjugated hyperbilirubinemia, have demonstrated bilirubin’s protection against cardiovascular diseases (43, 97) and metabolic dysfunction (50). It is unlikely that these health benefits could be obtained solely from bilirubin’s antioxidant properties. Recently, a novel function for bilirubin has been uncovered as an activator of peroxisome proliferator-activated receptor-α (PPARα; 99). PPARα is a transcription factor that regulates genes involved in lipid metabolism, particularly in hepatic lipid homeostasis (106, 107). Bilirubin has been identified as an agonist of PPARα resulting in an increase in β-oxidation fat-burning genes resulting in a decrease in hepatic lipid accumulation (99). A humanized mouse model for GS that contains the human UGT1A1*28 polymorphism had unconjugated hyperbilirubinemia (38) and, on a high-fat diet, had decreased lipid accumulation and increased PPARα activity compared with controls (38). Mölzer et al. conducted a study with GS patients and reported similar increases in PPARα expression (73). However, the role of PPARα transcription in regard to the protective metabolic effects of bilirubin on the liver has not been directly evaluated in humans.

Bilirubin has also been shown to increase the p450 enzyme Cyp1a1 in hepatocytes, which was suggested to occur by possible activation of the aryl hydrocarbon receptor (AhR; 95). However, the Cyp1a1 promoter has two PPAR response elements that are known to be regulated by PPARα (94). The AhR is a ligand-activated transcription factor that is involved in cell differentiation and carcinogenesis, including the development of several cancers (27, 88), and inhibiting AhR levels reduces gastric tumors in mice (101, 119). Furthermore, obesity and fatty liver are prevented by inhibition of the AhR in both female and male mice (75). The fatty liver and lipid-lowering effect of bilirubin has been shown to be mediated by direct binding to PPARα (99). However, there may be other targets that bilirubin activates that also need to be investigated.

Furthermore, higher plasma bilirubin levels reduce the risks of hepatocellular carcinoma recurrence (35). In addition to PPARα, bilirubin has been shown to activate other cellular targets, but not by direct binding, which include the ERK1/2 pathway, which may mediate bilirubin’s antioncogenic effect. One study performed treated BALB/c nude mice injected with human colorectal carcinoma cells with bilirubin and found a dramatic reduction in tumor growth compared with vehicle-treated mice (78). Bilirubin inhibited tumor cell growth through the phosphorylation and activation of ERK1/2 and its upstream kinase, MEK (78). It has been well documented that hepatocellular carcinoma has an upregulation of the ERK1/2 pathway via phosphorylation of the ERK1/2 pathway components leading to cell survival and growth (59). Further studies are needed to understand the impact of bilirubin on the ERK1/2 pathway and to clarify its protective role in liver disease progression and the development of hepatocellular carcinoma.

THE ROLE OF BILIVERDIN REDUCTASE IN HEPATIC STEATOSIS

BVRA reduces biliverdin IXα to bilirubin IXα (76), and it can also function as a signaling molecule and transcription factor (2, 65, 76). There is also a BVRB isoform that generates bilirubin IXβ, but this form of bilirubin is only produced the first few weeks after birth (7, 76, 82). A benefit of the fetal bilirubin IXβ is that it is more soluble than bilirubin IXα, which does not require conjugation for its excretion, which is required for excretion of bilirubin IXα (16). Very little is known about BVRB, and its function in adulthood has not been studied, even though it is still expressed (76). BVRA has been the most studied and is a cell surface protein that has been shown to mediate anti-inflammatory effects via the phosphatidylinositol 3-kinase (PI3K) and Akt pathway (116). The protective actions of BVRA were recently demonstrated by a global BVRA-null mouse (14). BVRA has also been shown to prevent hepatic lipid accumulation and disease progression (37). Hinds et al. developed a liver-specific BVRA KO mouse and found significantly higher liver weight, hepatic triglycerides, and Oil Red O staining in the liver after high-fat diet feeding in the KO compared with the wild-type mice (37). In addition, the liver-specific BVRA KO mice had upregulated de novo lipogenesis enzymes including fatty acid synthase, an enzyme involved in synthesizing fatty acids (37). Moreover, liver-specific BVRA KO mice had a decrease in phospho-AMPK, a downstream inhibitor of enzymes involved in fatty acid synthesis, and an increase in the active form of acetyl-CoA carboxylase, the rate-limiting enzyme in fatty acid synthesis (37). These results imply that BVRA or bilirubin in the liver is an essential factor that mediates fat accumulation.

Glycogen synthase kinase-3β (GSK3β) breaks down glycogen and promotes lipid synthesis in the liver through its inhibition of lipid metabolic enzyme transcription (79). GSK3β activity is regulated by phosphorylation at serine 9 (pSer9), which inactivates the enzyme (98). BVRA has been shown to control the Ser9 phosphorylation of GSK3β (29). GSK3β is a target of Akt, and its activity is most likely regulated by the BVR-PI3K-Akt pathway [reviewed in more detail by O’Brien et al. (76) and Rochette et al. (86)], as was shown in liver-specific BVRA KO mice (37) and by others (69). Phosphatase and tensin homolog (PTEN) negatively regulates the PI3K-Akt pathway through its phosphatase activity (67, 100, 116), but the impact of BVRA on PTEN has yet to be investigated. In liver-specific BVRA KO mice, there was an increase in GSK3β activity as demonstrated by a significant decrease in the levels of pSer9 GSK3β in the liver (37). PPARα is also a phosphoprotein that is regulated by phosphorylation at several different residues (9). One such residue is serine 73 (pSer73), which marks the protein for degradation via the ubiquitin pathway (Fig. 2; 37). Phosphorylation of PPARα at pSer73 is mediated by GSK3β, and BVRA KO mice exhibited an increase in pSer73 PPARα in the liver as well as a decrease in the total levels of PPARα (37). In addition, liver-specific BVRA KO mice exhibited significantly decreased levels of known PPARα target genes such as fibroblast growth factor 21 (FGF21; 37). Through PPARα activation, FGF21 has been shown to regulate hepatic lipid metabolism and attenuate hepatic steatosis (10, 18, 37, 39, 42, 66). PPARα KO mice exhibit an enhanced fatty liver and lower serum FGF21 levels (15). Most likely, BVRA plays a pivotal role in preventing the inhibition of PPARα by phosphorylating and inhibiting GSK3β, which allows for the upregulation of β-oxidation and the downregulation of de novo lipogenesis genes to promote lipid metabolism and glycogen storage in the liver (37, 107).

Fig. 2.

Biliverdin reductase signaling reduces hepatic fat accumulation. Biliverdin reductase A (BVRA) generation of unconjugated bilirubin from biliverdin activates peroxisome proliferator-activated receptor-α (PPARα; 99), which is a transcription factor that interacts with the retinoid X receptor (RXR) to increase genes in the β-oxidation pathway that burn fatty acids in the mitochondria. BVRA activates Akt, which inhibits glycogen synthase kinase-3β (GSK3β) through increased phosphorylation at serine 9 (S9) to prevent hepatic steatosis (37). BVRA can also directly interact with GSK3β to regulate activity (69). Inhibition of GSK3β results in increased activity of PPARα via decreased phosphorylation (P) at serine 73 (37). Z, zinc finger DNA binding domains. The signaling functions of BVRA and bilirubin have been reviewed further by Hamoud et al. (34), O’Brien et al. (76), and Rochette et al. (86).

BILIRUBIN, BILIVERDIN REDUCTASE, AND INFLAMMATION

Inflammation is a key component in the progression of simple hepatic steatosis to steatohepatitis. Inflammation is regulated by a multitude of factors including the toll-like receptor 4 (TLR4) signaling pathway. Experiments performed by Seki et al. on TLR4 mutant mice demonstrated that 3 wk post-bile duct ligation, the TLR4 mutant mice had reduced hepatic fibrosis, decreased expression of fibrotic marker α-smooth muscle actin (α-SMA), and decreased Sirius red staining for collagen (93). In comparison, the wild-type mice showed overt hepatic fibrosis in this same time frame (93). Five days post-bile duct ligation the TLR4 mutant mice showed an active suppression of hepatic fibrogenesis markers including collagen-α₁(I), α-SMA, TGF-β1, and tissue inhibitor of metalloproteinases 1 (TIMP-1), which are known to be upregulated during hepatic stellate cell (HSC) activation and fibrosis (26, 93). Recently, TLR5 knockout mice were shown to develop hepatic lipogenesis and the metabolic syndrome (96), but whether BVRA or bilirubin impacts this receptor is unknown. Thus, these experiments suggest that the TLR4, and possibly TLR5, signaling pathway is a key player in HSC activation and fibrogenesis (93)

Lipopolysaccharide (LPS) is secreted by the gut microbiota and is a ligand for TLR4 (80). When mice were treated with antibiotics to reduce LPS secretion by the gut microbiota, TLR4 activation was reduced resulting in a downregulation of HSC activation as measured by a significant reduction in collagen and α-SMA expression (93). This study also found that quiescent HSCs are the cells most influenced by TLR4 activation in the liver (93). In addition, LPS, the TLR4 ligand, also increases HSC sensitivity to TGF-β1, a profibrogenic cytokine and proposed activator of HSCs (93). Also, BVRA may regulate the TLR4 signaling pathway in HSCs. Wegiel et al. identified BVRA as an inhibitor of TLR4 in liver macrophages (117). Biliverdin induces the nitrosylation of BVRA by nitric oxide, which results in transport to the nucleus, where it binds to the TLR4 promoter to inhibit its expression (117). Thus, BVRA may prevent fibrogenesis by antagonizing the TLR4 signaling pathway not only in macrophages but also in HSCs (Fig. 3). However, more work is needed to determine the role of BVRA and bilirubin in TLR4- or TLR5-mediated hepatic steatosis and fibrosis.

Fig. 3.

Proposed inhibitory role of biliverdin reductase A (BVRA) in fibrogenesis. Hepatic stellate cells (HSCs) are activated by lipopolysaccharide (LPS) binding to toll-like receptor 4 (TLR4) or stimulation by transforming growth factor-β (TGF-β). Activated HSCs increase collagen production as well as several other factors to promote liver fibrogenesis. BVRA can attenuate fibrogenesis through antagonism of TLR4. α-SMA, α-smooth muscle actin; ROS, reactive oxygen species; TIMP-1, tissue inhibitor of metalloproteinases 1.

Macrophages are an important component of the inflammatory cascade in the liver and can be divided into two distinct classes: M1, proinflammatory, and M2, anti-inflammatory macrophages (66). M1 macrophages release inflammatory cytokines such as interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α; 66), and IL-12 and promote the inflammatory process. M2 macrophages, however, are reparative in nature and produce high levels of IL-10 and TGF-β and low levels of IL-12. Recently, Hu et al. reported the critical role of BVRA in the polarization of M2 macrophages in vitro and in response to renal ischemia-reperfusion injury in vivo (44). In a series of elegant experiments, the authors established that overexpression of BVRA in cultured macrophages promoted the M2 phenotype as indexed by expression of IL-10 levels whereas knockdown of BVRA promoted an M1-like phenotype (44). There is little known about the role of BVRA in the liver, especially on inflammation and hepatic immune cells. Bilirubin, a marker for liver disease, may have another protective role as an antioxidant to reduce inflammation.

Bilirubin has a complex effect on the inflammatory response in the periphery versus the central nervous system. Both induction of heme oxygenase as well as bilirubin treatment have been demonstrated to have anti-inflammatory actions in many inflammatory conditions such as colitis and organ transplantation (113, 118, 120, 123). Bilirubin treatment in these conditions has been shown to decrease the levels of proinflammatory markers including myeloperoxidase, TNF-α, and IL-1β (120). Bilirubin treatment can also lower the levels of proinflammatory and proapoptotic genes including monocyte chemoattractant protein-1, caspase-3, and caspase-8 to improve allograft function and survival (113). Despite these reported anti-inflammatory effects, bilirubin has also been reported to promote inflammation in microglia and nerve cells (22, 32, 62), albeit at extremely high levels. This is consistent with the toxic effects of bilirubin on the central nervous system in infants with hyperbilirubinemia (77) and highlights the dichotomous relationship between bilirubin and inflammation. Further investigations are needed to determine whether the hyperbilirubinemia consists of unconjugated or conjugated bilirubin and to investigate whether high levels of bilirubin IXβ cause similar complications for infants.

BILIRUBIN AND BILIVERDIN REDUCTASE IN FIBROSIS AND LIVER DISEASE

The HSC has been identified as the primary contributor to the development of hepatic fibrosis in liver disease (26). Upon liver injury, the HSCs become activated causing them to transform into contractile myofibroblast-like cells that have an increase in α-SMA expression, a protein involved in fibroblast contractility (Fig. 3; 4, 26, 40). Washington et al. analyzed liver biopsies of NAFLD and NASH patients and found that 74/76 of the NAFLD and NASH liver biopsies had an increase in α-SMA, the strongest marker for HSC activation, compared with controls (115). Reeves et al. examined liver biopsies from ALD patients and observed that the patients’ biopsies contained more activated HSCs than controls, demonstrated by a higher amount of α-SMA (84). The HSC activation was also positively correlated with the degree of hepatic steatosis (84). HSCs are spindle-shaped cells located in the space of Disse between the basolateral side of the hepatocytes and the sinusoidal endothelial cells (26). Quiescent HSCs have characteristic vitamin A droplets. The activated HSCs are characterized by a loss of vitamin A droplets, proliferation, and increased extracellular matrix (ECM) production causing a dramatic alteration in the liver parenchyma (26).

Another important pathological feature of both NAFLD and ALD is the oxidative stress mediated by the increase in ROS production (6). In ALD, oxidative stress results from the CYP2E1-catalyzed breakdown of ethanol to acetaldehyde to acetate (12). This reaction generates potent amounts of ROS leading to lipid peroxidation and oxidative liver damage through the formation of DNA adducts (12). NAFLD has also been shown to have increased levels of ROS and damaging lipid peroxidation products (6). Elevated serum bilirubin is a biomarker for liver disease and is often associated with jaundice (74). However, Tang et al. showed that bilirubin is most likely preventive in HSC activation (103), which may occur as an antioxidant or signaling through PPARα. However, this function still remains unknown.

Studies have shown that bilirubin attenuates the activation of HSCs and reduces their harmful effects on the liver. As a known antioxidant, bilirubin reduces ROS production by HSCs, which is an important feature of HSC activation. Increased ROS production reacts with lipids of the cell membrane, DNA bases, and mitochondria resulting in cellular oxidative stress and damage (45, 103). Tang et al. showed that when bilirubin levels are increased, there was a decrease in α-SMA expression and therefore a decrease in HSC activation (103). In a clinical study of pediatric biliary atresia, 19 patients demonstrated a negative correlation between α-SMA expression and serum bilirubin (20). These studies suggest that bilirubin could play a role in preventing HSC activation and subsequent hepatic fibrosis. Bilirubin also attenuates the ECM accumulation and HSC proliferation upon hepatic injury. TIMPs inhibit matrix metalloproteinases, or MMPs, preventing ECM degradation and increasing net accumulation. An increase in ECM accumulation is an important sign of liver fibrosis (103). Bilirubin decreases the TIMP-1-to-MMP-2 ratio in the liver signifying higher ECM degradation and lower accumulation (103). In addition, bilirubin has been shown to play a role in inhibiting HSC proliferation and increased apoptosis. Proliferation and survival of activated HSCs are observed in liver disease (Fig. 3; 103). Since HSCs are the main contributors to fibrosis, it is likely that bilirubin is exerting its effects on HSCs by preventing their activation and profibrotic activity.

POTENTIAL THERAPEUTIC APPLICATIONS OF BILIRUBIN AND BILIVERDIN REDUCTASE

Although several preclinical studies have demonstrated protective effects of bilirubin on the liver, one challenge is translating these findings to patients suffering from liver diseases. One issue with bilirubin is its insolubility in aqueous solutions making it difficult to deliver by traditional mechanisms. Recently, the solubility of bilirubin in aqueous solutions was markedly improved by covalent attachment of polyethylene glycol (PEG) resulting in PEGylated bilirubin nanoparticles (58). These PEGylated bilirubin nanoparticles were found to be effective against hepatocellular injury by reducing oxidative stress, proinflammatory cytokine production, and recruitment of neutrophils in a mouse model of ischemia-reperfusion injury (52). Bilirubin levels can also be increased in patients via antagonism of hepatic UGT1A1 to create a moderate hyperbilirubinemia. Antagonism of UGT1A1 can be achieved by several methods, including protease inhibitor drugs such as indinavir and atazanavir, antisense morpholinos, or a natural antagonist such as silymarin (17, 19, 108). All of these approaches have their limitations, and none have been specifically demonstrated to be protective against NAFLD.

Delivery of BVRA is another potential therapeutic avenue against NAFLD. Proteins such as BVRA when fused with a protein transduction domain, such as Tat, possess the ability to traverse the lipid bilayer membrane of mammalian cells. Recent studies have utilized this strategy to deliver BVRA both in vitro and in vivo to protect against inflammation (51, 57). Another encouraging area is the delivery of peptide sequences of BVRA. Several studies have demonstrated that cell-signaling activities of BVRA can be replicated via delivery of specific peptide sequences of the protein (28, 70). BVR peptides when combined with nanospheres were found to attenuate the development of hyperglycemia in obese, diabetic ob/ob mice (30). Although very promising in the potential regulation of blood glucose levels, the effects of BVR peptides on NAFLD have yet to be reported.

CONCLUSIONS

As NAFLD continues to be problematic worldwide, it is essential to uncover the mechanism involved and prevent the progression of hepatic disease. Some studies have shown a link between bilirubin and BVRA in the prevention of hepatic steatosis. Bilirubin possibly inhibits hepatocellular carcinomas, but more studies are needed to be conclusive. Suppression of UGT1A1 to increase unconjugated bilirubin or delivery of BVRA nanoparticles may serve as a useful therapeutic target for liver diseases such as NAFLD and NASH. Furthermore, bilirubin and BVRA may have potential treatment value in preventing the development and progression of chronic liver diseases and cancer. More studies are needed to elucidate their therapeutic potential and roles in the different stages of liver disease.

GRANTS

This work was supported by National Institutes of Health Grants L32-MD-009154 (T. D. Hinds, Jr.), K01-HL-125445 (T. D. Hinds, Jr.), P01-HL-051971 (D. E. Stec), P01-HL-088421 (D. E. Stec), and P20-GM-104357 (D. E. Stec).

DISCLAIMERS

The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

L.W. and T.D.H. conceived and designed research; L.W. and T.D.H. prepared figures; L.W., A.-R.H., D.E.S., and T.D.H. drafted manuscript; L.W., A.-R.H., D.E.S., and T.D.H. edited and revised manuscript; L.W., A.-R.H., D.E.S., and T.D.H. approved final version of manuscript.