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Bilirubin: The yellow hormone?

  • Libor Vítek
    Correspondence
    Corresponding authors. Addresses: 4th Department of Internal Medicine and Institute of Medical Biochemistry and Laboratory Diagnostics, 1st Faculty of Medicine, Charles University, Kateřinská 32, Praha 2, 12000 Czech Republic; Tel.: +420 224 964 203.
    Affiliations
    Faculty General Hospital and 1st Faculty of Medicine, Charles University in Prague, Prague, Czech Republic
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  • Claudio Tiribelli
    Correspondence
    Fondazione Italiana Fegato, Bldg Q AREA Science Park - Basovizza Campus, 34149 Trieste, Italy; Tel.: +39 040 375 7840.
    Affiliations
    Fondazione Italiana Fegato, Trieste, Italy
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      Summary

      Bilirubin is a tetrapyrrolic compound originating from heme catabolism. Although originally considered only a potentially dangerous waste product, it has become increasingly evident that this molecule represents an important modulator of various biological functions in the human body. Bilirubin appears to have versatile functions, from cell signaling (behaving almost like a “real” hormonal substance), modulation of metabolism, to immune regulation, affecting biological activities with apparent clinical and even therapeutic consequences. These activities may be the reason for the lower incidence of diseases of civilisation (cardiovascular diseases, arterial hypertension, diabetes, obesity, metabolic syndrome, certain cancers, autoimmune, and neurodegenerative diseases) observed in individuals with a chronic mild unconjugated hyperbilirubinemia, a typical sign of Gilbert’s syndrome. While higher serum concentrations of unconjugated bilirubin may serve as an important protective factor against these diseases, low levels of bilirubin are associated with the opposite effect.

      Keywords

      Introduction

      Bilirubin was long considered at best only a non-functional waste product of heme catabolism, but more often an ominous sign of liver diseases, or even a potentially neurotoxic substance. However, recent studies have shown that mildly elevated serum bilirubin concentrations, such as those typically seen in individuals with Gilbert’s syndrome (GS, benign hyperbilirubinemia), but also levels in the upper quartiles of the currently accepted physiological serum bilirubin range, may protect against diseases associated with increased oxidative stress, an exaggerated immune response and metabolic dysfunction. Since the seminal work on bilirubin metabolism performed almost a hundred years ago by Hans Fisher (the only Nobel Prize laureate in this field), our view of bilirubin has dramatically changed.

      Clinical aspects of bilirubin metabolism

      The physiological range of serum bilirubin is conventionally defined as 5–17 μmol/L (0.3–1 mg/dl). Bilirubin has a typical bimodal distribution, due to the high prevalence of GS; thus, in cohort studies, bilirubin concentrations should always be expressed as median (IQR) instead of mean (SD). Bilirubin concentrations below 10 μmol/L (0.6 mg/dl) are associated with a higher risk of various diseases, and this is even more evident for a concentration below 7 μmol/L (0.4 mg/dl) which is still within the currently accepted physiological range.
      • Novotny L.
      • Vitek L.
      Inverse relationship between serum bilirubin and atherosclerosis in men: a meta-analysis of published studies.
      ,
      • Wagner K.H.
      • Wallner M.
      • Molzer C.
      • Gazzin S.
      • Bulmer A.C.
      • Tiribelli C.
      • et al.
      Looking to the horizon: the role of bilirubin in the development and prevention of age-related chronic diseases.
      Thus, the decision limits reflecting the health risks of these lower serum bilirubin concentrations should be established, as done for HDL cholesterol in the past.
      • Vitek L.
      Bilirubin as a predictor of diseases of civilization. Is it time to establish decision limits for serum bilirubin concentrations?.
      It is also well known that the serum bilirubin concentration is lower in females than males, questioning the rationale of the current physiological value of serum bilirubin concentrations not being sex-specific. Hence, independent physiological ranges should be re-established for each sex (including a lower diagnostic cut-off value of serum bilirubin concentration for GS in females).
      • Vitek L.
      Bilirubin as a predictor of diseases of civilization. Is it time to establish decision limits for serum bilirubin concentrations?.
      ,
      • Eremiasova L.
      • Hubacek J.A.
      • Danzig V.
      • Adamkova V.
      • Mrazova L.
      • Pitha J.
      • et al.
      Serum bilirubin in the Czech population. Relationship to the risk of myocardial infarction in males.

      Hepatic bilirubin biotransformation: The key regulatory step in metabolism

      Bilirubin glucuronosylation is the rate-limiting step in the disposal of unconjugated bilirubin from the human body. In humans, this process is mediated by a specific hepatic enzyme named bilirubin-UDP glucuronosyl transferase (UGT1A1, OMIM ∗191740), and specific mutations of the UGT1A1 gene account for the manifestation of mild chronic unconjugated hyperbilirubinemia (GS) (Fig. 1).
      • Bosma P.J.
      • Chowdhury J.R.
      • Bakker C.
      • Gantla S.
      • de Boer A.
      • Oostra B.A.
      • et al.
      The genetic basis of the reduced expression of bilirubin UDP-glucuronosyltransferase 1 in Gilbert's syndrome.
      The UGT1A1 gene is the major gene responsible for the systemic bilirubin concentration in the general population.
      • Lin J.P.
      • Vitek L.
      • Schwertner H.A.
      Serum bilirubin and genes controlling bilirubin concentrations as biomarkers for cardiovascular disease.
      Figure thumbnail gr1
      Fig. 1UGT1A1 gene and its variations responsible for the manifestation of mild chronic unconjugated hyperbilirubinemia (Gilbert’s syndrome).
      UGT1A1 is highly polymorphic, with about 150 allelic genotypes having been identified so far.
      • Yasar U.
      • Greenblatt D.J.
      • Guillemette C.
      • Court M.H.
      Evidence for regulation of UDP-glucuronosyltransferase (UGT) 1A1 protein expression and activity via DNA methylation in healthy human livers.
      In the majority of Caucasians, GS is associated with the UDP-glucuronosyltransferase 1A1∗28 polymorphism (rs8175347), an insertion of an additional TA-repeat into the promoter region of the UGT1A1 gene, resulting in an A(TA)7TAA promoter sequence, which reduces bilirubin glucuronosylation by 70%
      • Bosma P.J.
      • Chowdhury J.R.
      • Bakker C.
      • Gantla S.
      • de Boer A.
      • Oostra B.A.
      • et al.
      The genetic basis of the reduced expression of bilirubin UDP-glucuronosyltransferase 1 in Gilbert's syndrome.
      (Fig. 1). UGT1A1∗28 has an allelic frequency of 27–40% in the Caucasian population.
      • Barbarino J.M.
      • Haidar C.E.
      • Klein T.E.
      • Altman R.B.
      PharmGKB summary: very important pharmacogene information for UGT1A1.
      Interestingly, this allele is very rare in some populations, ranging from 0 to 5% in Melanesia and the Pacific Islands.
      • Premawardhena A.
      • Fisher C.A.
      • Liu Y.T.
      • Verma I.C.
      • de Silva S.
      • Arambepola M.
      • et al.
      The global distribution of length polymorphisms of the promoters of the glucuronosyltransferase 1 gene (UGT1A1): hematologic and evolutionary implications.
      UGT1A1∗28 homozygosity is found in 16% of European, 12% of Indian, 8% of Egyptian, and 23% of African-American individuals.
      • Strassburg C.P.
      Gilbert-Meulengracht's syndrome and pharmacogenetics: is jaundice just the tip of the iceberg?.
      The penetrance of the UGT1A1∗28 homozygous mutation is only 50%,
      • Bosma P.J.
      • Chowdhury J.R.
      • Bakker C.
      • Gantla S.
      • de Boer A.
      • Oostra B.A.
      • et al.
      The genetic basis of the reduced expression of bilirubin UDP-glucuronosyltransferase 1 in Gilbert's syndrome.
      most likely due to the effects of certain modifying genes
      • Cooper D.N.
      • Krawczak M.
      • Polychronakos C.
      • Tyler-Smith C.
      • Kehrer-Sawatzki H.
      Where genotype is not predictive of phenotype: towards an understanding of the molecular basis of reduced penetrance in human inherited disease.
      as well as the presence of specific haplotypes with other UGT1A1 genotypes.
      • Ehmer U.
      • Kalthoff S.
      • Fakundiny B.
      • Pabst B.
      • Freiberg N.
      • Naumann R.
      • et al.
      Gilbert syndrome redefined: a complex genetic haplotype influences the regulation of glucuronidation.
      In addition, differential methylation of the -4 CpG site located within an upstream stimulating factor (USF) response element that regulates UGT1A1 expression may explain a proportion of the inter-individual variability in hepatic glucuronosylation by UGT1A1 (Fig. 1).
      • Yasar U.
      • Greenblatt D.J.
      • Guillemette C.
      • Court M.H.
      Evidence for regulation of UDP-glucuronosyltransferase (UGT) 1A1 protein expression and activity via DNA methylation in healthy human livers.
      All these facts highlight that GS is a phenotypic diagnosis and that UGT1A1∗28 homozygosity is only a predisposing factor.
      Although Asians have been reported to have higher serum bilirubin concentrations compared to Caucasians, the (TA)7 allele in that population is much less common.
      • Wagner K.H.
      • Shiels R.G.
      • Lang C.A.
      • Seyed Khoei N.
      • Bulmer A.C.
      Diagnostic criteria and contributors to Gilbert's syndrome.
      Instead, a heterozygous mutation in the coding exon 1 of the UGT1A1 gene (such as a presence of the UGT1A1∗6 allele) is an additional important determinant of GS in Asian populations, together with other variants in the promoter UGT1A1 region (Fig. 1),
      • Wagner K.H.
      • Shiels R.G.
      • Lang C.A.
      • Seyed Khoei N.
      • Bulmer A.C.
      Diagnostic criteria and contributors to Gilbert's syndrome.
      pointing to even more complex genetic regulation of bilirubin homeostasis evolving during phylogenesis.
      The prevalence of phenotypic GS varies between 2–12%, being much lower in females, with a male-to-female prevalence between 2–7:1 despite the lack of significant sex differences in UGT1A1∗28 genotype frequencies,
      • Liu J.Y.
      • Qu K.
      • Sferruzza A.D.
      • Bender R.A.
      Distribution of the UGT1A1∗28 polymorphism in Caucasian and Asian populations in the US: a genomic analysis of 138 healthy individuals.
      pointing to the need for normal reference values of serum bilirubin to be adjusted by sex. The prevalence markedly differs among different ethnic groups, with the lowest prevalence in African Americans and the highest in Asian populations. However, caution is needed since more accurate data are lacking, owing to the absence of homogeneity in the reported population studies.
      • Liu J.Y.
      • Qu K.
      • Sferruzza A.D.
      • Bender R.A.
      Distribution of the UGT1A1∗28 polymorphism in Caucasian and Asian populations in the US: a genomic analysis of 138 healthy individuals.

      Mild hyperbilirubinemia is associated with health benefits

      The beneficial associations of mildly elevated systemic bilirubin concentrations with diseases of civilization (in particular those affecting the cardiovascular system) have been reported in numerous studies.
      • Wagner K.H.
      • Wallner M.
      • Molzer C.
      • Gazzin S.
      • Bulmer A.C.
      • Tiribelli C.
      • et al.
      Looking to the horizon: the role of bilirubin in the development and prevention of age-related chronic diseases.
      ,
      • Vitek L.
      Bilirubin as a predictor of diseases of civilization. Is it time to establish decision limits for serum bilirubin concentrations?.
      ,
      • Vitek L.
      The role of bilirubin in diabetes, metabolic syndrome, and cardiovascular diseases.
      • Vitek L.
      Bilirubin and atherosclerotic diseases.
      • Gazzin S.
      • Vitek L.
      • Watchko J.
      • Shapiro S.M.
      • Tiribelli C.
      A novel perspective on the biology of bilirubin in health and disease.
      Besides, mild unconjugated hyperbilirubinemia is also associated with lower adiposity, a decreased risk of metabolic syndrome, non-alcoholic fatty liver disease (NAFLD),
      • Weaver L.
      • Hamoud A.R.
      • Stec D.E.
      • Hinds Jr., T.D.
      Biliverdin reductase and bilirubin in hepatic disease.
      and diabetes,
      • Wagner K.H.
      • Wallner M.
      • Molzer C.
      • Gazzin S.
      • Bulmer A.C.
      • Tiribelli C.
      • et al.
      Looking to the horizon: the role of bilirubin in the development and prevention of age-related chronic diseases.
      ,
      • Vitek L.
      The role of bilirubin in diabetes, metabolic syndrome, and cardiovascular diseases.
      as well as overall cancer risk.
      • Wagner K.H.
      • Wallner M.
      • Molzer C.
      • Gazzin S.
      • Bulmer A.C.
      • Tiribelli C.
      • et al.
      Looking to the horizon: the role of bilirubin in the development and prevention of age-related chronic diseases.
      Bilirubin downregulates almost all functions of the immune system,
      • Jangi S.
      • Otterbein L.
      • Robson S.
      The molecular basis for the immunomodulatory activities of unconjugated bilirubin.
      a phenomenon certainly implicated in the low frequency of various inflammatory, autoimmune, and degenerative diseases in individuals with GS.
      • Wagner K.H.
      • Wallner M.
      • Molzer C.
      • Gazzin S.
      • Bulmer A.C.
      • Tiribelli C.
      • et al.
      Looking to the horizon: the role of bilirubin in the development and prevention of age-related chronic diseases.
      All these observations may account for the reduced mortality rates recently reported
      • Wagner K.H.
      • Wallner M.
      • Molzer C.
      • Gazzin S.
      • Bulmer A.C.
      • Tiribelli C.
      • et al.
      Looking to the horizon: the role of bilirubin in the development and prevention of age-related chronic diseases.
      in those with GS compared to the normobilirubinemic population. Although these associations were originally ascribed to the powerful antioxidant activities of bilirubin,
      • Stocker R.
      • Yamamoto Y.
      • McDonagh A.F.
      • Glazer A.N.
      • Ames B.N.
      Bilirubin is an antioxidant of possible physiological importance.
      several additional and more biologically potent activities that could account for these beneficial effects have recently been described. These activities, which can be collectively covered by the term “bilirubinomics”,
      • Gazzin S.
      • Vitek L.
      • Watchko J.
      • Shapiro S.M.
      • Tiribelli C.
      A novel perspective on the biology of bilirubin in health and disease.
      include the modulation of cell signaling; protein phosphorylation
      • Hansen T.W.
      • Mathiesen S.B.
      • Walaas S.I.
      Bilirubin has widespread inhibitory effects on protein phosphorylation.
      ; or the activation of cytoplasmic and nuclear receptors, as well as binding to other molecules within the vascular bed and also the cell interior (Table 1), which upon complexing with bilirubin exert further biological activities (Fig. 2) (for review see
      • Vitek L.
      Bilirubin as a signaling molecule.
      ). Bilirubin thus acts as a ‘real’ endocrine molecule, with effects similar to those recently proposed for bile acids.
      • Vitek L.
      • Haluzik M.
      The role of bile acids in metabolic regulation.
      These receptors involve those implemented in energy homeostasis (such as peroxisome proliferator-activated receptors [PPARs], aryl hydrocarbon receptor [AhR], or constitutive androstane receptor [CAR]), biotransformation processes (such as CAR, pregnane X receptor [PXR)), sensitive perception (via MRGPRX4 (Mas-related G protein-coupled receptor X4]).
      • Vitek L.
      Bilirubin as a signaling molecule.
      Also, high-affinity binding molecules, belonging mostly to a lipocalin superfamily of proteins (such as fatty acid-binding protein [FABP1] or apolipoprotein D [apoD]) (Table 1), activate, upon complexing with bilirubin, various additional cell-signaling pathways (Fig. 2).
      • Vitek L.
      Bilirubin as a signaling molecule.
      Table 1Bilirubin as a ligand of biological targets in the human body.
      CompartmentBinding toFunction
      Known as well as putative functions.
      Cell nucleusAhR
      • Phelan D.
      • Winter G.M.
      • Rogers W.J.
      • Lam J.C.
      • Denison M.S.
      Activation of the Ah receptor signal transduction pathway by bilirubin and biliverdin.
      Energy homeostasis

      Biotransformation

      Immune system cell differentiation

      Anti-microbial effects infectious

      Anti-inflammatory effects

      Anti-atherogenic effects

      Inhibition of protein phosphorylation

      Anti-cancer effects

      Role in circadian rhytmicity
      CAR
      • Huang W.
      • Zhang J.
      • Chua S.S.
      • Qatanani M.
      • Han Y.
      • Granata R.
      • et al.
      Induction of bilirubin clearance by the constitutive androstane receptor (CAR).
      Energy homeostasis

      Biotransformation
      PXRBiotransformation
      PPARα
      • Stec D.E.
      • John K.
      • Trabbic C.J.
      • Luniwal A.
      • Hankins M.W.
      • Baum J.
      • et al.
      Bilirubin binding to PPARalpha inhibits lipid accumulation.
      Energy homeostasis
      PPARγ
      • Liu J.
      • Dong H.
      • Zhang Y.
      • Cao M.
      • Song L.
      • Pan Q.
      • et al.
      Bilirubin increases insulin sensitivity by regulating cholesterol metabolism, adipokines and PPARgamma levels.
      Energy homeostasis
      apoD
      • Goessling W.
      • Zucker S.D.
      Role of apolipoprotein D in the transport of bilirubin in plasma.
      See below
      CytoplasmFABP1
      • Levi A.J.
      • Gatmaitan Z.
      • Arias I.M.
      Two hepatic cytoplasmic protein fractions, Y and Z, and their possible role in the hepatic uptake of bilirubin, sulfobromophthalein, and other anions.
      Metabolic and immune system signaling

      PPARs transactivation
      apoD
      • Goessling W.
      • Zucker S.D.
      Role of apolipoprotein D in the transport of bilirubin in plasma.
      See below
      NADPH oxidaseSuppression of superoxide-mediated cell signaling with multiple biological effects
      Cell membraneMRGPRX4
      • Meixiong J.
      • Vasavda C.
      • Green D.
      • Zheng Q.
      • Qi L.
      • Kwatra S.G.
      • et al.
      Identification of a bilirubin receptor that may mediate a component of cholestatic itch.
      Sensitive perception

      Anti-inflammatory effects

      Energy homeostasis

      Anti-cancer effects

      Antihypertensive effects

      Cardioprotective activities
      apoD
      • Goessling W.
      • Zucker S.D.
      Role of apolipoprotein D in the transport of bilirubin in plasma.
      Anti-cancer effects

      Angiogenesis

      Immune system modulation

      Cholesterol metabolism

      Sex hormone signaling
      AFP (via its cell receptor)Cell growth, differentiation, regeneration, apoptosis and transformation during onto- as well as oncogenesis
      IntravascularapoD
      • Goessling W.
      • Zucker S.D.
      Role of apolipoprotein D in the transport of bilirubin in plasma.
      Lipid metabolism

      Oxidative stress defense
      AFPSee above
      AlbuminOxidative stress defense
      PGDS
      • Beuckmann C.T.
      • Aoyagi M.
      • Okazaki I.
      • Hiroike T.
      • Toh H.
      • Hayaishi O.
      • et al.
      Binding of biliverdin, bilirubin, and thyroid hormones to lipocalin-type prostaglandin D synthase.
      Energy homeostasis

      Carcinogenesis

      Immune system modulation
      Cerebrospinal fluidPGDS
      • Beuckmann C.T.
      • Aoyagi M.
      • Okazaki I.
      • Hiroike T.
      • Toh H.
      • Hayaishi O.
      • et al.
      Binding of biliverdin, bilirubin, and thyroid hormones to lipocalin-type prostaglandin D synthase.
      Immune system modulation
      AFP, α1-fetoprotein; AhR, aryl hydrocarbon receptor; apoD, apolipoprotein D; CAR, constitutive androstane receptor; FABP, fatty acid binding protein; MRGPRX4, Mas-related G protein-coupled receptor X4; PGDS, (lipocalin-type) prostaglandin D synthase; PPAR, peroxisome proliferator-activated receptor; PXR, pregnane X receptor. Adapted according to.
      • Vitek L.
      Bilirubin as a signaling molecule.
      Known as well as putative functions.
      Figure thumbnail gr2
      Fig. 2Involvement of bilirubin in cell signaling pathways.
      This scheme represents possible involvement of bilirubin in cell signaling based on either clinically proved, experimentally proposed, or theoretically justified data reported in the recent literature. Only the key signaling pathways are illustrated. Adapted according to.
      • Vitek L.
      Bilirubin as a signaling molecule.
      AA, arachidonic acid; AhR, aryl hydrocarbon receptor; AMPK, AMP-activated protein kinase; apoD, apolipoprotein D; CAR, constitutive androstane receptor; FGF21, fibroblast growth factor 21, Glut 1, glucose transporter-1; MAPK, mitogen-activated protein kinases; MRGPRX4, Mas-related G protein-coupled receptor X4; mTOR, mammalian target of rapamycin; PGC-1α, proliferator-activated receptor g coactivator 1α; PPAR, peroxisome proliferator-activated receptor; PXR, pregnane X receptor; ROS, reactive oxygen species; S1P, sphingosine-1-phosphate; SRB1, scavenger receptor B1; SREBP-1c, sterol regulatory element-binding protein 1.
      Therefore, it is not surprising that mild elevation of serum bilirubin, binding to and activating a wide array of cell targets involved in cell signaling and metabolic homeostasis, is associated with lower adiposity, a lowered risk of metabolic syndrome, NAFLD,
      • Weaver L.
      • Hamoud A.R.
      • Stec D.E.
      • Hinds Jr., T.D.
      Biliverdin reductase and bilirubin in hepatic disease.
      diabetes,
      • Vitek L.
      The role of bilirubin in diabetes, metabolic syndrome, and cardiovascular diseases.
      and an overall better metabolic status.
      • Seyed Khoei N.
      • Grindel A.
      • Wallner M.
      • Molzer C.
      • Doberer D.
      • Marculescu R.
      • et al.
      Mild hyperbilirubinaemia as an endogenous mitigator of overweight and obesity: implications for improved metabolic health.
      A hormone (from the Greek participle ὁρμῶν, "setting in motion") is any member of a class of signaling molecules in multicellular organisms that are transported to distant organs to regulate physiology and/or behavior. Hence, as we proposed previously
      • Vitek L.
      Bilirubin and atherosclerotic diseases.
      and now in detail here, bilirubin acts as a ‘real’ hormonal substance fulfilling most of the required criteria – being a ‘chemical substance secreted into the bloodstream and acting on distant tissues, usually in a regulatory fashion upon binding to specific receptors’.
      • Kronenberg H.M.
      • Melmed S.
      • Larsen P.R.
      • Polonsky K.S.
      Principles of endocrinology.
      Our proposal is in line with the change of the classical endocrinology paradigm which has occurred during recent years with discoveries of novel hormonal substances originated from musculoskeletal, adipose, enteric, cardiac, or hepatic tissues.
      • Bouillon R.
      • Drucker D.J.
      • Ferrannini E.
      • Grinspoon S.
      • Rosen C.J.
      • Zimmet P.
      The past 10 years-new hormones, new functions, new endocrine organs.
      • Ogawa T.
      • de Bold A.J.
      The heart as an endocrine organ.
      • Watt M.J.
      • Miotto P.M.
      • De Nardo W.
      • Montgomery M.K.
      The liver as an endocrine organ-linking NAFLD and insulin resistance.

      Can mild hyperbilirubinemia improve longevity? Lessons from the evolution

      As mentioned above, a positive association between serum bilirubin concentrations and longevity has been reported in large human epidemiological studies (for review see
      • Wagner K.H.
      • Wallner M.
      • Molzer C.
      • Gazzin S.
      • Bulmer A.C.
      • Tiribelli C.
      • et al.
      Looking to the horizon: the role of bilirubin in the development and prevention of age-related chronic diseases.
      ). In this respect, it is interesting to look at the possible evolutionary aspects of this association. The UGT1A1 promoter in chimpanzees contains only 4 TA repeats, suggesting a much higher transcription of UGT1A1. This results in substantially lower systemic bilirubin levels in the great apes, where the median serum bilirubin level is lower than 1.7 μmol/L.
      • Ronke C.
      • Dannemann M.
      • Halbwax M.
      • Fischer A.
      • Helmschrodt C.
      • Brugel M.
      • et al.
      Lineage-specific changes in biomarkers in great apes and humans.
      An additional interesting observation is that chimpanzees have a higher level of oxidative stress and a greater risk of cardiovascular diseases compared with humans of equivalent age,
      • Videan E.N.
      • Heward C.B.
      • Chowdhury K.
      • Plummer J.
      • Su Y.
      • Cutler R.G.
      Comparison of biomarkers of oxidative stress and cardiovascular disease in humans and chimpanzees (Pan troglodytes).
      and live half as long as humans, which might be due to the evolutionary acquisition of a TATA box genetic mutation in humans.
      • Zhao G.
      • Guo S.
      • Somel M.
      • Khaitovich P.
      Evolution of human longevity uncoupled from caloric restriction mechanisms.
      As mentioned above, the lowest UGT1A1∗28 allele frequency is found in Melanesia and the Pacific islands,
      • Premawardhena A.
      • Fisher C.A.
      • Liu Y.T.
      • Verma I.C.
      • de Silva S.
      • Arambepola M.
      • et al.
      The global distribution of length polymorphisms of the promoters of the glucuronosyltransferase 1 gene (UGT1A1): hematologic and evolutionary implications.
      and this is associated with significantly lower serum bilirubin levels as reported in Melanesian children
      • Manning L.
      • Laman M.
      • Townsend M.A.
      • Chubb S.P.
      • Siba P.M.
      • Mueller I.
      • et al.
      Reference intervals for common laboratory tests in Melanesian children.
      and adult Tongans.
      • Brown A.C.
      • Onopa J.
      • Holck P.
      • Kaufusi P.
      • Kabasawa D.
      • Craig W.J.
      • et al.
      Traditional kava beverage consumption and liver function tests in a predominantly Tongan population in Hawaii.
      It is tempting to speculate that the shorter lifespan in Oceania
      • Taylor R.
      • Bampton D.
      • Lopez A.D.
      Contemporary patterns of Pacific Island mortality.
      might at least in part be affected by the low serum bilirubin levels due to the negative association reported between serum bilirubin concentrations and all-cause mortality (for review see,
      • Wagner K.H.
      • Wallner M.
      • Molzer C.
      • Gazzin S.
      • Bulmer A.C.
      • Tiribelli C.
      • et al.
      Looking to the horizon: the role of bilirubin in the development and prevention of age-related chronic diseases.
      and above).

      Therapeutic implications of bilirubin and bilirubin-related pigments

      Bilirubin behaves as a typical yin-yang substance, with deleterious effects at high concentrations, yet providing substantial benefits for human health when only mildly elevated, such as in those with GS (the double-faced Janus effect). This is the reason for the attempts to iatrogenically mimic GS to enhance protection against diseases of civilisation.
      • Vitek L.
      • Bellarosa C.
      • Tiribelli C.
      Induction of mild hyperbilirubinemia: hype or real therapeutic opportunity?.
      Even a tiny increase in serum bilirubin concentration seems biologically relevant since each micromolar increase (even still within the current physiological range) is associated with a significant decrease in the risk of various oxidative stress-mediated diseases.
      • Vitek L.
      Bilirubin as a predictor of diseases of civilization. Is it time to establish decision limits for serum bilirubin concentrations?.
      ,
      • Vitek L.
      Bilirubin and atherosclerotic diseases.
      There are several ways to increase systemic levels of bilirubin. A decrease in the efficacy of the liver cell to conjugate bilirubin with glucuronic acid by partial inhibition of UGT1A1 was reported for numerous other xenobiotics routinely used in clinical medicine (for review see
      • Vitek L.
      • Bellarosa C.
      • Tiribelli C.
      Induction of mild hyperbilirubinemia: hype or real therapeutic opportunity?.
      ), but also for naturally occurring compounds such as plant flavonolignanes.
      • Suk J.
      • Jasprova J.
      • Biedermann D.
      • Petraskova L.
      • Valentova K.
      • Kren V.
      • et al.
      Isolated silymarin flavonoids increase systemic and hepatic bilirubin concentrations and lower lipoperoxidation in mice.
      A more elegant approach to enhance the bile pigment pool seems to be dietary supplementation with bilirubin-like structures that commonly occur in Nature. One example is the blue-green algae, which contain substantial amounts of phycobilins (tetrapyrolic compounds resembling the structures of biliverdin or bilirubin
      • McCarty M.F.
      Clinical potential of Spirulina as a source of phycocyanobilin.
      ).
      Interestingly, in the last 5 years novel therapeutic approaches for delivering bilirubin to pathologically altered tissues and organs have been investigated. Bilirubin has been incorporated into various forms of nanoparticles, and the biological efficacy of these particles has been experimentally verified in recent studies focused on the treatment of inflammatory diseases, conditions associated with increased oxidative stress or cancer.
      • Vitek L.
      • Bellarosa C.
      • Tiribelli C.
      Induction of mild hyperbilirubinemia: hype or real therapeutic opportunity?.
      ,
      • Vitek L.
      • Tiribelli C.T.
      Bilirubin, intestinal integrity, the microbiome, and inflammation.
      As an example, bilirubin encapsulated into nanoparticles was reported in preclinical studies to inhibit inflammatory processes in the pancreas as well as intestine (on a mouse model of pancreatitis and inflammatory bowel disease, respectively) via modulation of a wide array of effectors of both innate as well as adaptive immunity. Bilirubin was also shown to protect gut barrier integrity as well as to maintain microbiome richness and diversity, further expanding the pathophysiological implications and therapeutic potential of bilirubin.
      • Vitek L.
      • Tiribelli C.T.
      Bilirubin, intestinal integrity, the microbiome, and inflammation.

      Perspectives and conclusion

      From being considered a waste substance associated with liver disease, our understanding of bilirubin has come a very long way. Bilirubin is now regarded as a molecule with many intricate biological functions, while our view on mildly hyperbilirubinemic individuals with GS has changed dramatically. While in the past these individuals were regarded as predisposed to chronic fatigue and various gastrointestinal problems, often being advised against physical exercise while being kept on strict diets, it is now clear that there is no reason to label individuals with GS as “patients”. Instead, GS is likely to represent a selective advantage due to the potent beneficial biological effects of bilirubin which protect against various diseases of civilisation.
      Indeed, as one of the most abundant intrinsic antioxidants in the body, bilirubin may protect us from cardiovascular disease and cancer etc. As always in Nature, nothing is fully good or bad, but the dark side of the coin (specifically represented by an impaired capacity to biotransform certain xenobiotics in individuals with GS, and by neurotoxicity in severe neonatal hyperbilirubinemia) is outweighed by the benefits associated with a mild elevation of bilirubin. The modulation of bilirubin concentration may be a suitable way to prevent/treat different diseases in the future; however, only time will tell whether this option is feasible and realistic.
      Although the current experimental and clinical evidence strongly suggest a causative relationship between bilirubin and several clinical conditions which may be explained by its “endocrine” activities, future research is certainly needed to assess the possible contribution of reverse causality or other potential confounding factors. These may include the effects of other bioactive molecules (such as carbon monoxide formed during heme catabolism) or decreased clearance of such biomolecules due to impaired hepatic biotransformation.
      The future of the “yellow research” will require the active participation of chemists, physiologists, immunologists, cell biologists, epidemiologists, and clinicians resulting in real translational research. In any case, based on the evidence presented herein and remembering the true meaning of the Greek word hormone (ὁρμῶν, "setting in motion"), we believe we can safely remove the question mark from the title, as bilirubin can certainly be viewed as “the yellow hormone”.

      Abbreviations

      GS, Gilbert’s syndrome; NAFLD, non-alcoholic fatty liver disease; UGT1A1, bilirubin-UDP glucuronosyl transferase.

      Financial support

      This work was supported by grants RVO-VFN64165/2021 from the Czech Ministry of Health , NV18-07-00342 from the Czech Health Research Council and 21-01799S from the Czech Science Foundation (LV), and by an intramural grant from Fondazione Italiana Fegato (CT). The funding bodies had no role in the collection, management, analysis, and interpretation of the data; preparation, review, or approval of the manuscript; and decision to submit the manuscript for publication.

      Authors’ contributions

      Both authors contributed equally.

      Conflict of interest

      The authors declare no conflicts of interest that pertain to this work.
      Please refer to the accompanying ICMJE disclosure forms for further details.

      Supplementary data

      The following is the supplementary data to this article:

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