The physiology of bilirubin: health and disease equilibrium

Abstract

Bilirubin has a number of physiological functions, both beneficial and harmful. In addition to having reactive oxygen species-scavenging activities, bilirubin has potent immunosuppressive effects associated with long-term pathophysiological sequelae. It has been recently recognized as a hormone with endocrine actions with interconnected effects on various cellular signaling pathways. Current studies show that bilirubin also decreases adiposity and prevents metabolic and cardiovascular diseases. All in all, the physiological importance of bilirubin is only now coming into light, and strategies for increasing plasma bilirubin levels to combat chronic diseases are starting to be considered. This review discusses the beneficial effects of increasing plasma bilirubin, incorporates emerging areas of bilirubin biology, and provides critical concepts to advance the field.

Introduction

New findings in bilirubin research suggest that it has a previously unknown function; it operates as a hormone [1,2]. Published work from the past decade reveals that bilirubin at moderately elevated levels is protective against metabolic and cardiovascular diseases (reviewed in [1,3,4]). This is contrary to the past century of thinking about bilirubin being a toxic bile substance that causes liver dysfunction and the yellowing of the skin in jaundice. This new finding has driven excitement in bilirubin research and aided in focusing on new targets for obesity, diabetes, and cardiovascular research, which we discuss further below.

Recent studies show that bilirubin activates the nuclear receptor, PPARα [5–8], which drives fat burning, lowering adiposity and blood glucose. Some have utilized this emerging concept to develop bilirubin nanoparticles as potential therapeutics for metabolic and cardiovascular diseases [1,9–12]. Others have focused on activating the heme oxygenase () system that catabolizes heme to produce bilirubin (Box 1). Newer work has also been directed at the UGT1A1 enzyme that clears bilirubin from the blood by conjugation with glucuronic acid, which after is transported to the bile and intestine [13]. Suppressing UGT1A1 to increase serum/plasma bilirubin has been considered a promising therapeutic approach [14]. This occurs naturally in with the Gilbert’s syndrome polymorphism UGT1A1*28 (TA_7/7 promoter gene variant), in whom reduced expression of the UGT1A1 enzyme serum/plasma bilirubin levels [1]. Those with the Gilbert’s syndrome polymorphism have been shown to have reduced risks of metabolic and cardiovascular diseases [1]. Humanized mice with Gilbertś syndrome gene promoter polymorphism (HuUGT1A1*28) [with knocked out from the mouse locus] were shown to have mildly elevated bilirubin levels and resistant to high-fat-diet-induced fatty liver disease and glucose intolerance [15]. These indicate that mild in plasma bilirubin levels promote health.

Box 1. Bilirubin production and catabolism.

The physiological concentrations of bilirubin levels depend on many biological factors such as genetics, sex, and age (see Figure 1 in the main text) [110]. The crucial enzyme that initiates the breakdown of heme is heme oxygenase (HMOX), which has two isoforms (HMOX1, OMIM *141250; and HMOX2, OMIM *141251) [146]. HMOX1 is the inducible isoform, believed to be the most inducible enzyme in the human body, activated by a variety of endogenous and exogenous stimuli associated with the disruption of redox homeostasis and dysregulation of immune system functions [121,147]. The primary function of the HMOX isozymes is to cleave the heme molecule into the linear tetrapyrrole biliverdin while simultaneously releasing iron and carbon monoxide (CO) as additional products. Biliverdin is then reduced to bilirubin by the enzyme biliverdin reductase A (BLVRA, OMIM *109750). In addition, biliverdin appears to be an integral component of the so-called bilirubin–biliverdin redox cycle that regenerates bilirubin scavenging activities (Figure I) [148].

The lipophilic unconjugated bilirubin is transported within the circulation bound to albumin [149]. Once dissociated from albumin and taken up by the liver cells [26,27], bilirubin is conjugated with glucuronic acid by bilirubin UDP-glucuronosyltransferase (UGT1A1, OMIM *191740). Hereditary underexpression of UGT1A1 could be due to variation in the so-called TATA box of the UGT1A1 gene promoter (mostly in Caucasians) or due to mutations in the structural UGT1A1 gene (mostly in Asians) resulting in the development of mild unconjugated hyperbilirubinemia (also known as Gilbert syndrome, OMIM *143500) [150]. This condition is very frequent, reaching a prevalence of approximately 9% in Caucasians (11.6 and 6.1% in males and females, respectively) [151], but differs geographically among various other populations [107].

Conjugated bilirubin is transported by the ABCC2 protein into bile to reach the intestines [13,21]. Within the intestinal lumen, bilirubin is deconjugated by β-glucuronidase of epithelial or bacterial origin and then reduced by intestinal microbiota to a series of derivatives commonly known as urobilinoids [21]. Despite the massive production of urobilinoids (a process important for the disposal of electrons produced during fermentolytic processes in these anaerobic bacteria [152]), a bilirubin reductase has never been identified to date; thus, this enzyme is a missing puzzle in the process of bilirubin metabolism. The role of the gut microbiota in the regulation of bilirubin homeostasis is also unknown. However, a recent experimental study found that a strain of Lactobacillus can increase plasma bilirubin levels and reduce body weight [153].

Hence, UGT1A1 may influence the predisposition of fat accumulation and the development of obesity [13,16]. In fact, a recent human study showed that obese men and women had significantly higher levels of the bilirubin catabolized product, urobilin [16], which originates from the gut microbiome catabolism of conjugated bilirubin by deconjugation and then forming urobilin that is absorbed via the hepatic portal vein [13]. Individuals with Gilbert’s syndrome are known to have a lower prevalence of obesity [17], suggesting that higher bilirubin levels in the blood may mediate these actions. Indeed, plasma bilirubin levels are negatively associated with adiposity in humans [16]. The expression of UGT1A1 is under the control of hepatic nuclear receptors, such as the constitutive androstane receptor (CAR), pregnane X receptor (PXR), glucocorticoid receptor (GR), aryl hydrocarbon receptor (AHR), hepatocyte nuclear receptor 1α (HNR1α) [18], and others [14], which regulate UGT1A1 transcription via the hormone response element (HRE) in its promoter [18]. These findings open possibilities for targeting the heme-bilirubin catabolism pathway as a means of increasing bilirubin levels.

Today, bilirubin has been recognized as one of the most potent endogenous antioxidants, as a powerful immunosuppressive [19], and, quite surprisingly, as a selective cell signaling molecule [20]. Bilirubin, classically viewed as an end-product of the heme catabolic pathway, was long believed to be potentially dangerous, and it is perceived that its presence in high concentrations is an ominous sign of underlying liver disease [21]. Recent advancements support a new concept that bilirubin functions as a hormone, and several of its biological functions might be mediated his mechanism [1,2,4,13]. Here, we will discuss the new findings about bilirubin, how it functions at low (hypobilirubinemia), physiological (normobilirubinemia), mildly elevated (hyperbilirubinemia), and jaundice (severe hyperbilirubinemia) levels, and how it plays an intimate role in health and disease equilibrium.

Intracellular effects of bilirubin on different pathways

Bilirubin levels in the blood have been thought to be mostly produced by reticuloendothelial cells in the spleen [1]. However, new studies in mice lacking the biliverdin reductase-A (BVRA) enzyme [22,23], which converts bilirubin from biliverdin [24], have shown that bilirubin generation likely occurs from many tissues. Vitek first called for a clinical reclassification of bilirubin levels [25], which led Creeden et al. to propose decision limits for normobilirubinemia as ranging from 10 to 25 μmol/L depending on age, gender, and race, and hypobilirubinemia at <10 μmol/L [1]. Based on recent clinical data, the decision limits for determining the risk of pathological consequences, especially in the lower concentrations, are.

Inside the cell:

Bilirubin enters cells by multiple mechanisms [26,27]: by a still not well-defined carrier molecule(s) and another by passive diffusion. The contribution of each mechanism depends on the concentration of bilirubin [27]. Important is the intracellular binding of bilirubin to fatty acid-binding protein-1 (FABP1) [28], a fatty acid-binding transporter expressed in hepatocytes, which serves as a sink for maintaining an inward gradient. While FABP1 is almost exclusively expressed in hepatocytes, and bilirubin has actions on other tissues, bilirubin may use other FABPs or similar types of proteins, such as scavenger receptors. This is supported by bilirubin binding to other FABP proteins, such as UnaG [29,30], expressed in the Unagi freshwater eel (Anguilla japonica). UnaG interacts with unconjugated bilirubin in a lock-and-key mechanism to activate its fluorescence in the skeletal muscle [29]. There are likely other FABP proteins in humans that bilirubin can utilize for cellular entry, but these are currently unknown.

Bilirubin has been well-characterized as an antioxidant for over three decades. The antioxidant activity of bilirubin increases when normal atmospheric oxygen concentrations (20%) move toward levels found in tissues (2%) [31]. Many protective effects of bilirubin have been attributed to its role as an antioxidant, which is discussed in more detail by Sedlak et al. and Thomas et al. [32,33]. More recent studies have shown that bilirubin is also involved in regulating signaling pathways indicating that bilirubin has more properties than initially thought. Thus, recently bilirubin has been regarded as a hormone that binds to receptors [1] (Figure 1).

Figure 1. Bilirubin acts as a hormone.

The receptors that bilirubin has been shown to activate at physiological bilirubin (10–25 μM) and pathological levels (>100 μM).

Abbreviations: Peroxisome proliferator-activated receptor alpha (PPARα), Mas-Related G-protein coupled receptor (MRGPR), aryl hydrocarbon receptor (AHR), constitutive androstane receptor (CAR), Peroxisome proliferator-activated receptor gamma (PPARγ) Asterisks: this is an indicator that studies were performed and validated in knockout animal models.

Endocrine functions of bilirubin:

By definition, a hormone is a substance that enters the bloodstream and exerts actions on cells or tissues, which occurs by direct binding to cytoplasmic or nuclear receptors. Bilirubin has been shown to have such properties at physiological levels [1,4–8] and with potential activation of other receptors at higher pathological levels [34–37]. The earliest reports that bilirubin might activate receptors are from the late 1990s and suggested that this activation might be mediated through the AHR transcription factor [35,36]. Using Hepa1c1c7 murine hepatoma cells, Sinal et al. found that high bilirubin concentrations (100 μM) enhanced Cyp1a1 expression [35], a gene thought for many years to only be controlled by AHR. The transcriptional activity of AHR requires heterodimerization with AHRT (AhR nuclear translocator) protein on DNA recognition sequences known as DREs [38], in which the Cyp1a1 promoter contains three DRE sites [39]. It also has two PPAR response elements (PPREs) that are regulated by the PPAR nuclear receptor transcription factors, which have also been shown to induce Cyp1a1 [40,41]. As the Cyp1a1 promoter contains DREs and PPREs, a relationship exists between PPARs and AHR, as PPARα has been shown to potentiate the AHR-induced Cyp1A1 expression [42]. Whether bilirubin directly acts on AHR to induce Cyp1a1 or if this might be mediated PPARα-induction of AHR is yet to be determined. The most recent investigations show that unconjugated bilirubin directly binds to the PPARα nuclear receptor [5–8] and that this interaction occurs at physiological levels with a K_d value of 5.13 μM [5].

Bilirubin acting on PPARα at physiological levels re-defines bilirubin as a metabolic hormone essential for balancing nutrient storage (Figure 1) [1]. Bilirubin binds PPARα directly and elicits a response that reduces fat accumulation in the liver (Figure 2) and adipose tissues [1,3–8,10,13,15,43–46]. Hence, bilirubin has been proposed to be potentially useful for reducing obesity and improving metabolic dysfunction [1,3,4] (see also below). The PPARα-bilirubin interaction was validated in many different studies, which included treatment of PPARα knockout (KO) animals [8], and lentiviral knockdown of PPARα in human hepatocytes that demonstrated by RNA-sequencing analysis that bilirubin-controlled gene activity is mostly PPARα-dependent (~95%) [7]. Other studies included the standard receptor-ligand binding assays that have been classically used for nuclear receptors such as competitive ligand-binding assays [6] and pull down of PPARα using bilirubin-fused sepharose beads [8]. A competitive ligand-binding study identified the amino acids in the ligand-binding domain (LBD) of PPARα that bilirubin uses for hydrogen binding, indicating a strong affinity for the receptor [6]. These studies showed that bilirubin binds to the LBD of PPARα and competes with known ligands, such as fenofibrate, for the same binding region [6]. The interaction of bilirubin-PPARα was further supported by in silico modeling of bilirubin in the PPARα LBD, which indicated that bilirubin tightly binds into the pocket [8]. Another hypothesis that bilirubin drives PPARα activity was proposed in a study that showed that bilirubin treatment drives PPARα to the Ucp1 and Cpt1 promoters in adipocytes, demonstrated by chromatin immunoprecipitation (ChIP) assays using a PPARα-specific antibody [6]. Lastly, using state-of-the-art PamGene technology for nuclear receptor coregulator interactions, it was demonstrated that bilirubin-PPARα binding causes a molecular switch from corepressor proteins to coactivators for the PPARα interactome and, thus, induces a gene response [6]. Altogether, these investigations support the idea that bilirubin directly interacts in the LBD of PPARα and causes a change in the coregulator interactome from inhibitory to stimulatory, eliciting a gene response.

Figure 2. Bilirubin responses in tissues via direct binding to receptors.

(A) Bilirubin activates the PPARα nuclear receptor transcription factor at physiological levels to regulate lipid utilization by increasing genes (ACOX1) for fat-burning β-oxidation and suppressing genes (SCD1) for de novo lipogenesis [10,15]. The PPARα-bilirubin interaction also induces lipid uptake genes such as fatty acid transporters (FATPs) and suppresses very low-density lipoprotein (VLDL) secretion from the liver and triglyceride entry to the blood to lower levels. The resultant product from PPARα-bilirubin is lipid utilization and production of the ketone β-hydroxybutyrate, which is excreted and used as an alternative energy source to glucose [146,147]. (B) The itch receptor (MRGPR) is activated by bilirubin at pathological levels (>150 μM), which increases intracellular calcium activation in MRGPR-positive neurons, activating pruritus (itching response). This figure was created by Matthew Hazzard at the University of Kentucky College of Medicine.

One report has shown that bilirubin at very high levels (>150 μM) may stimulate responses on itch receptors, and itching is known to be associated with jaundice and cholestasis. Meixiong et al. showed that at high concentrations, bilirubin interacts with the G protein-coupled receptor (GPCR) referred to as the Mas-Related G-protein coupled receptors (MRGPRs) and activated MRGPRX4 (human) and MRGPRA1 (the mouse homolog of MRGPRX4) to elicit intracellular calcium signaling response (Figure 2) [34]. Bilirubin binding to MRGPRA1 was validated by competitive binding assays and bilirubin administration in Mrgpra1 and Blvra KO mice [34]. It was found that bilirubin had a lower kinetic binding to MRGPRA1 than PPARα, with an EC₅₀ = 145.9 μM for MRGPRA1 excitation quantitated in MRGPRA1-overexpressing cells challenged with bilirubin to generate an intracellular calcium signaling response [34].

Other receptors that bilirubin may also activate are CAR, PPARγ, PPARδ, and possibly enzymes it might regulate, such as the angiotensin I-converting enzyme (ACE) (Figure 1). Huang et al. showed that bilirubin increases CAR target genes [47] though they concluded that bilirubin does not directly bind to CAR [47]. PPARα has been shown to increase CAR expression [48,49], and this mechanism likely affects CAR target genes. Bilirubin treatment increases PPARγ expression in diabetic mice [50], and bilirubin suppresses PPARγ expression in 3T3-L1 adipocytes [8]. Analysis of the interaction of bilirubin with PPARγ or PPARδ showed that bilirubin does not directly activate the transcriptional activity of either receptor [6] and has no direct interaction with PPARγ [5]. Danilov et al. showed that bilirubin at extremely high concentrations (about 250 μmol/L) might bind ACE to regulate the ectodomain shedding used for entering the circulating pool [37], but direct proof of bilirubin binding is lacking. More work is needed to reveal if bilirubin binds ACE and other targets at jaundice levels and how these might impact the cardiovascular system.

Data so far obtained indicates that there are at least two direct bilirubin receptor targets, one at physiological levels (PPARα) and another at higher pathological concentrations (MRGPR). Bilirubin likely activates AHR but this effect and the concentration (K_d or EC₅₀) at which it occurs is yet to be validated in KO animals [51] or by CRISPR KO technology in cell lines. More investigations are needed to determine whether bilirubin might bind to other receptors to control normal physiological responses.

Cardiovascular and metabolic effects of bilirubin

The role of bilirubin in human health has been characterized by population studies correlating bilirubin levels with diseases [52,53], and fully supported by the recent discovery of bilirubin endocrine activities. Higher levels of bilirubin are associated with protection from the development of cardiovascular and metabolic diseases, like obesity and diabetes, as further supported by studies in individuals with Gilbert’s syndrome [17,54]. Interestingly, Gilbert’s syndrome subjects exhibit enhanced expression of PPARα, most likely from increased plasma bilirubin levels [55]. Bilirubin-induced PPARα signaling may be the reason for the significantly lower body mass index (BMI), glucose, and insulin levels observed in these individuals [55,56]. Mice that possess the human Gilbert’s syndrome polymorphism (Hu) exhibit moderate hyperbilirubinemia and are protected from high-fat diet-induced hepatic steatosis and insulin resistance [15]. These mice also have reduced inhibitory serine-73 phosphorylation of PPARα, resulting in the enhancement of PPARα signaling in the liver [15]. Treating obese and diabetic mice with bilirubin nanoparticles improved metabolic function by lowering adiposity, blood glucose levels, and hepatic steatosis [6,10]. Several mechanisms account for these effects, including increased mitochondrial activity, altered adipose tissue remodeling, and increased ketone production [6,8,10].

Gunn rats carry a loss of function mutation in their Ugt1a1 gene, resulting in moderate-to-severe hyperbilirubinemia, and adult animals are protected against hypertension and end-stage organ damage [57–61]. Moderate hyperbilirubinemia due to targeting hepatic UGT1A1 with drugs or anti-sense morpholinos prevents angiotensin-II-induced hypertension and changes in renal vascular resistance [62,63]. The antioxidant effects of hyperbilirubinemia have long been thought to be responsible for the beneficial effects on the cardiovascular system [64–66]. However, superoxide production in angiotensin II-induced hypertension was not responsible for the antihypertensive actions of moderate hyperbilirubinemia [67], indicating that other pathways may be responsible for the blood pressure-lowering actions of moderate hyperbilirubinemia in angiotensin II-dependent hypertension. Bilirubin improves vascular function by increasing the bioavailability of nitric oxide (NO) either through direct savaging of superoxide or blockade of superoxide production by inhibition of NAD(P)H oxidase [68,69]. Additionally, bilirubin may impact NO production through actions on endothelial nitric oxide synthase (eNOS) PARα signaling. PPARα agonists stimulate AMP-activated protein kinase (AMPK) to increase eNOS serine 1177 phosphorylation and mitogen-activated protein kinase (MAPK) to enhance eNOS serine 1179 phosphorylation as well as decrease eNOS threonine 497 phosphorylation ivation of ERK and Akt [70,71]. These modifications of eNOS result in enhanced NO production, which is beneficial in conditions such as hypertension and atherosclerosis.

Modulation of bilirubin levels

Slightly higher serum bilirubin concentrations are associated with apparent health benefits, particularly decreasing the risk of developing various diseases. Mildly raising serum bilirubin levels might reduce vascular and cancer risk [72]. Importantly, even small incremental elevations in serum bilirubin concentrations may provide such benefits. Based primarily on clinical studies, each micromolar increase in serum bilirubin concentration is associated with a substantial decrease in the risks of developing atherosclerosis [73,74], arterial hypertension [75], diabetes mellitus [76], colorectal cancer [77], Crohńs disease [78], systemic lupus erythematosus [79], Fabry disease [80], or schizophrenia [81]. Hence, modulation of the bilirubin levels appears to be a reasonable potential treatment strategy (Figure 3).

Figure 3. Modulation of bilirubin level.

Several approaches can be used to mildly elevate the plasma bilirubin level above the optimal threshold. These include (i) Lifestyle approach (proper body composition, modest calorie restriction with a healthy diet rich in carbohydrates, fruit and vegetables, and sufficient aerobic activities); (ii) Nutraceutical approach, and finally; (iii) Pharmacological approach including nutraceuticals and drugs modulating HMOX/UGT1A1 activities as well as hepatic bilirubin transporting functions.

This figure was created by Matthew Hazzard at the University of Kentucky College of Medicine.

Lifestyle modifications approach

Ideal body composition

In 2009, a clinical investigation of more than 10,000 subjects by Andersson et al. described a significant negative relationship between body composition and serum bilirubin levels [82]. In this work, they observed that bilirubin levels increased linearly with each 1% decrease in weight loss and were associated with a significantly higher bilirubin level in both men and women [82]. A negative correlation has been observed between bilirubin concentrations and BMI [17,83–85], with the lowest bilirubin concentrations observed in obese patients with visceral obesity and additional metabolic complications [84,86]. Consistent with these data, body fat percentage was the primary determinant of bilirubin concentrations in young obese subjects [87], the most likely explanation being bilirubin overconsumption due to obesity-induced oxidative stress [88], although this remains to be determined. In addition, bilirubin levels are positively associated with muscle mass, as demonstrated in patients with sarcopenia [89] and elite athletes [90]. Further supporting this is that diet-induced obese mice treated with bilirubin nanoparticles had significantly higher muscle mass and reduced adiposity [6].

Dietary factors

Fasting and calorie restriction are associated with increased serum bilirubin concentrations [91], and long-term caloric restriction is positively associated with increased lifespan [92,93]. Whether coincidental or causal, mildly elevated serum bilirubin concentrations have been associated with increased lifespan [94,95]. This has been supported by studies in people with Gilbert’s syndrome, which were reported to have only half the mortality rates compared with normobilirubinemic individuals [96]. A greater telomere length was also found in individuals with the Gilbert’s syndrome polymorphism [97].

In addition, the composition of the diet has a modulating effect on serum bilirubin concentrations. For instance, increased consumption of fruits and vegetables was positively associated with bilirubin levels in the US NHANES study [98]. Increased serum bilirubin concentrations in response to a higher carbohydrate diet were also observed in pregnant women [99], which validates previous reports [100,101]. It is noteworthy to mention that a fast-food diet was associated with significantly lower bilirubin levels [102]. Similarly, a high-fat diet that increased visceral fat content had the same bilirubin-reducing effect [103]. It is unknown whether these observations are the results of a feedback mechanism of the human body to cope with increased metabolic and oxidative stress; however, considering current knowledge, this could be a possible explanation.

Aerobic activities

In a recent study, serum bilirubin concentrations were higher in elite athletes [90], and interestingly, also the prevalence of Gilbert’s syndrome was significantly higher in athletes (9.6 vs. 22%) [90]. From these observations, one might speculate that a mild elevation of serum bilirubin predisposes one to better sports performance, as supported by the observation that regular physical activity results in an increase in serum bilirubin concentrations [104,105] (reviewed in [33,106]). This aspect was confirmed in rats where high aerobic exercise elevated plasma bilirubin concentrations [44].

bilirubin improving exercise performance (reviewed in [106]) possible and affected byoglobin levels as the greatest contributor to aerobic performance, and individuals with Gilbert’s syndrome possess increased total hemoglobin and decreased red blood cell survival [107]). Hence, it remains to be determined whether increases in circulating bilirubin predispose to or maybe increase sports performance. In addition, the antioxidant activity of bilirubin has the potential to modulate training-induced adaptation [33], similar to other antioxidant regimens [108]. However, these questions remain to be elucidated, and future studies are necessary to understand better bilirubin’s function in exercise.

Nutraceutical supplementation

Many natural compounds are often used as nutraceutical supplements that can affect bilirubin metabolism by inducing HMOX1 [109], inhibiting bilirubin uptake, suppressing UGT1A1 activity, improving the antioxidant capacity of the human body, or saving bilirubin from consumption or clearance [110]. These compounds include various plant polyphenols such as curcuminoids, flavonoids, or some flavonolignans from the silymarin complex (for a review, see [110]). Elevated serum bilirubin levels have been reported in clinical studies with patients treated with milk thistle extract (Silybum marianum (L.) Gaertn.) via the inhibition of the UGT1A1 activity by flavonolignans from the silymarin complex [111]. These data align with the results from studies in which silymarin flavonolignans dehydrosilybin A and B increased serum bilirubin concentrations [112]. On the other hand, it should be noted that some clinical trials have shown no effect of milk thistle capsules containing 140 mg silymarin on bilirubin levels when taken three times daily in healthy participants [113]. UGT1A1 has been shown to be increased in the livers of obese mice [114,115]. The differential findings in the clinical studies might be that silymarin is more effective in obese people with higher UGT1A1 and lower bilirubin levels [1]. The other explanation could be that clinically used silymarin drugs/supplements do not contain sufficient amounts of standardized silymarin. Thus, the effects of silymarin need further studies and require assessing optimal dosing and specific metabolites responsible via pharmacokinetic studies.

Inhibitory effects toward UGT1A1 were also described for epigallocatechin gallate and, to a lesser extent, for echinacea and saw palmetto [116]. It is highly likely that many other natural compounds used, especially in traditional Asian medicine, could have similar effects, although clinical data supporting these are lacking [117–119].

In contrast, the intake of certain fruits and vegetables, and coffee, is associated with a decrease in serum bilirubin concentrations due to the opposite modulation of the enzymatic and transporting mechanisms of bilirubin metabolism (for a review, see [110]).

Pharmacological applications

Many drugs used in routine clinical practice interfere with bilirubin metabolism by induction of HMOX1 that induces bilirubin production [120] or decreasing bilirubin clearance by inhibiting its uptake or biotransformation [110].

HMOX1 induction is considered one of the most inducible genes in the human body, being upregulated by numerous endogenous and exogenous factors [109,120,121]. The induction of HMOX1 has been reported for many clinically used drugs, including commonly prescribed NSAIDs, hypolipidemic, anti-aggregation drugs, H2 antihistamines, and some antihypertensive medications, as well as natural substances used as nutraceuticals [109,110]. It is important to note that the induction of HMOX1 may not significantly affect bilirubin concentrations in circulation, as implied from clinical studies on the HMOX1 gene polymorphism [122,123]. Likely, bilirubin concentrations increase intracellularly, as demonstrated in previous experimental studies [124,125]. Because of the biological importance of HMOX1 induction, several clinical trials with therapeutic drugs and nutraceuticals are currently ongoing to assess the clinical impact of this approach (www.clinicaltrials.gov, accessed January 20, 2023). The induction of HMOX1 is a promising approach to increasing bilirubin production and improving conditions affected by lower bilirubin levels (hypobilirubinemia, described in [1]).

Indeed, some drugs have clinical data showing that their use is associated with a mild increase in serum bilirubin concentrations. A typical example is atazanavir, an HIV replication inhibitor, which suppresses UGT1A1 activity and significantly increases serum bilirubin, decreases the risk of cardiovascular disease [126], and slows the progression of carotid intima-media thickness progression [127,128]. Similar UGT1A1-suppressing activities resulting in mild systemic hyperbilirubinemia have been reported for several other anticancer and antiviral pharmaceuticals [110]. Another typical example of bilirubin-inducing drugs are statins, inhibitors of HMG-CoA reductase commonly used to lower serum cholesterol levels, which interfere with bilirubin metabolism by competing for its basolateral transporting proteins on the liver cell membrane. A significant elevation of serum bilirubin has been reported in patients with hypercholesterolemia treated with rosuvastatin, simvastatin, and atorvastatin [129–131]. Similar bilirubin-increasing effects were observed in human subjects treated with niacin [132] or ursodeoxycholic acid [133].

The need for comprehensive pharmacological research with well-controlled clinical studies to test candidate drugs for their bilirubin-modulating activities is apparent.

Future applications

The potential therapeutic use of bilirubin

In the past, a direct parenteral administration of bilirubin in humans has been used frequently as a therapeutic approach. Up to 40 studies covering almost 1,500 human subjects have been reported since the 1920s in the literature using intravenous infusions of bilirubin with a relatively good safety profile (for a review of these studies, see [134]).

Recent advancements using nanotheranostics to deliver bilirubin to pathologically altered tissues and organs are currently a hot pharmacological topic in experimental medicine. Bilirubin nanoparticles bound to various carrier molecules such as polyethylene glycol, chitosan, or gold, often further modified and conjugated with other active substances such as cytostatics (for a review, see [1,135]), were constructed to treat metabolic, inflammatory, and cancer diseases [1,12,136–141]. Although important risks and hazards are associated with using these delivery particles, particularly from the long-term perspective [142], bilirubin nanoparticles have indisputable therapeutic potential, especially with the development of new self-generated and self-enhanced biodegradable materials in this rapidly evolving field [143].

Concluding remarks and future directions

As common in research, the more problems that are solved, the more questions arise. We are still not close to completely understanding the interrelated intracellular events elicited by bilirubin. The studies on experimental systems such as cell culture or organoids provide some potential answers and clues into its mechanisms. Identification of these molecular events will also allow us to understand better the interplay between bilirubin and several metabolic and immune pathways (see Outstanding questions). Defining whether modulation of bilirubin levels by dieting and exercise and possibly dietary supplementation may be useful for improving health, as suggested by population studies. Bilirubin and induction of HMOX1 to produce bilirubin may serve as new therapeutics for diseases with metabolic and inflammatory dysfunction. Newer studies to determine the impact of hypobilirubinemia [1] may offer advantages to better understanding diseases such as cardiovascular and fatty liver diseases, where low levels might be a factor.

Outstanding questions.

• What are the receptor targets of bilirubin?

• How do high levels of plasma/serum unconjugated bilirubin protect against cardiovascular and metabolic diseases?

• How can plasma/serum levels of bilirubin be safely elevated to protect against cardiovascular and metabolic diseases?

• What diseases are affected by hypobilirubinemia?

• Could we reassess the current reference values of bilirubin concentrations in plasma/serum?

Figure I. Heme catabolism to bilirubin.

Heme is catabolized by heme oxygenase to biliverdin while releasing oxygen (O²), iron (Fe²⁺), and carbon monoxide (CO). Then, biliverdin reductase reduces biliverdin to bilirubin IXalpha by an NADPH to NADP mechanism. Bilirubin is cleared by hepatocytes (liver cells) via the UGT1A1 UDP-glucuronosyltransferase (UGT1A1, OMIM *191740) in microsomes by conjugation with glucuronide giving rise to bilirubin diglucuronide (also named bilirubin diglucuronoside according to IUBMB Enzyme Nomenclature), which is then excreted into the bile.

Clinician’s corner.

• The serum/plasma concentration of bilirubin has traditionally been considered a marker of hepatic disease. However, emerging data show that increased levels of plasma bilirubin are protective and that low levels (0.58 mg/dl) can predispose patients to the development of cardiovascular and metabolic diseases. This raises several important questions that need to be addressed clinically. First, should bilirubin concentration be followed as a potential indicator of risk for cardiovascular and metabolic disease? Second, what decision limits should be used to define “normal” plasma bilirubin concentrations? Third, how can we effectively increase low levels of bilirubin in still-healthy subjects as well as in patients?

• The results of several large-scale population studies have consistently demonstrated that plasma bilirubin concentrations correlate with protection against the development of cardiovascular and metabolic disease. These results suggest that physicians should routinely monitor the levels of bilirubin, especially in those subjects with risk factors for cardiovascular and metabolic diseases such as obesity, hypertension, type II diabetes, metabolic syndrome, metabolic dysfunction-associated liver disease (MAFLD), or those with a family history of cardiovascular and/or metabolic diseases.

• The median serum/plasma bilirubin concentrations in the general population is around 10 μmol/l [1] μ [1] but the real range of “normal” bilirubin level still needs to be defined. However, the bilirubin levels can vary widely depending on the conditions in which plasma is collected. For example, prolonged fasting results in slightly increased levels of plasma bilirubin, and conditions like obesity are associated with decreased levels. Establishment of decision limits of bilirubin concentrations needs to be established so that individuals who exhibit levels below the established lower decision limit can be identified for treatments to increase plasma bilirubin levels.

• There are currently no specific therapies approved for the treatment of low levels of plasma bilirubin. Potential therapies include: (i) formulations of bilirubin solubilized in a water-based formula which could be administered to patients orally or via injection; (ii) drugs that interfere with the natural conjugation of bilirubin in the liver; (iii) natural supplements that also target endogenous bilirubin metabolism, and (iv) a new concept that certain probiotics might alter the metabolism of bilirubin in the gut.

• In light of the emerging data on the important role of bilirubin levels in health and disease, clinicians should recognize the need to measure bilirubin concentrations in patients at risk for cardiovascular and/or metabolic diseases. There needs to be an effort to specifically define “normal” bilirubin levels and develop a specific treatment for individuals who exhibit “low” levels of bilirubin (hypobilirubinemia).

Highlights.

• Recent studies indicate that mildly elevated bilirubin levels in the blood have many health benefits.

• The physiological mechanisms of bilirubin action in the human body are discussed with a focus on translational importance.

• Bilirubin can be considered a hormone, in particular through its interaction with PPARα.

• Increased concentrations of blood bilirubin levels above 10 μM are associated with protection from cardiovascular and metabolic diseases.

• Bilirubin levels can be regulated by diet, aerobic activity, natural compounds, and some medications.