Background: Circulating bilirubin is associated with reduced adiposity in human and animal studies. A possible explanation is provided by in vitro data that demonstrates that bilirubin inhibits mitochondrial function and decreases efficient energy production. However, it remains unclear whether hyperbilirubinemic animals have similar perturbed mitochondrial function and whether this is important for regulation of energy homeostasis. Aim: To investigate the impact of unconjugated hyperbilirubinemia on body composition, and mitochondrial function in hepatic tissue and skeletal muscle. Materials and Methods: 1) Food intake and bodyweight gain of 14-week old hyperbilirubinemic Gunn (n = 19) and normobilirubinemic littermate (control; n = 19) rats were measured over a 17-day period. 2) Body composition was determined using dual-energy X-ray absorptiometry and by measuring organ and skeletal muscle masses. 3) Mitochondrial function was assessed using high-resolution respirometry of homogenized liver and intact permeabilized extensor digitorum longus and soleus fibers. 4) Liver tissue was flash frozen for later gene (qPCR), protein (Western Blot and citrate synthase activity) and lipid analysis. Results: Female hyperbilirubinemic rats had significantly reduced fat mass (Gunn: 9.94 ± 5.35 vs. Control: 16.6 ± 6.90 g, p < 0.05) and hepatic triglyceride concentration (Gunn: 2.39 ± 0.92 vs. Control: 4.65 ± 1.67 mg g-1, p < 0.01) compared to normobilirubinemic controls. Furthermore, hyperbilirubinemic rats consumed fewer calories daily (p < 0.01) and were less energetically efficient (Gunn: 8.09 ± 5.75 vs. Control: 14.9 ± 5.10 g bodyweight kcal-1, p < 0.05). Hepatic mitochondria of hyperbilirubinemic rats demonstrated increased flux control ratio (FCR) via complex I and II (CI+II) (Gunn: 0.78 ± 0.16 vs. Control: 0.62 ± 0.09, p < 0.05). Similarly, exogenous addition of 31.3 or 62.5 μM unconjugated bilirubin to control liver homogenates significantly increased CI+II FCR (p < 0.05). Hepatic PGC-1α gene expression was significantly increased in hyperbilirubinemic females while FGF21 and ACOX1 was significantly greater in male hyperbilirubinemic rats (p < 0.05). Finally, hepatic mitochondrial complex IV subunit 1 protein expression was significantly increased in female hyperbilirubinemic rats (p < 0.01). Conclusions: This is the first study to comprehensively assess body composition, fat metabolism, and mitochondrial function in hyperbilirubinemic rats. Our findings show that hyperbilirubinemia is associated with reduced fat mass, and increased hepatic mitochondrial biogenesis, specifically in female animals, suggesting a dual role of elevated bilirubin and reduced UGT1A1 function on adiposity and body composition.

Department of Nutritional Sciences and Research Platform Active Ageing University of Vienna Vienna Austria

Institute of Biotechnology Czech Academy of Sciences Prague Czechia

School of Medical Science Griffith University Gold Coast QLD Australia

Zobrazit více v PubMed

Bailey S. A., Zidell R. H., Perry R. W. (2004). Relationships between organ weight and body/brain weight in the rat: what is the best analytical endpoint? Toxicol. Pathol. 32, 448–466. 10.1080/01926230490465874 PubMed DOI

Bakrania B., Du Toit E. F., Wagner K. H., Headrick J. P., Bulmer A. C. (2016). Pre- or post-ischemic bilirubin ditaurate treatment reduces oxidative tissue damage and improves cardiac function. Int. J. Cardiol. 202, 27–33. 10.1016/j.ijcard.2015.08.192 PubMed DOI

Boon A. C., Hawkins C. L., Bisht K., Coombes J. S., Bakrania B., Wagner K. H., et al. (2012). Reduced circulating oxidized LDL is associated with hypocholesterolemia and enhanced thiol status in Gilbert syndrome. Free Radic. Biol. Med. 52, 2120–2127. 10.1016/j.freeradbiomed.2012.03.002 PubMed DOI PMC

Bulmer A. C., Coombes J. S., Blanchfield J. T., Toth I., Fassett R. G., Taylor S. M. (2011). Bile pigment pharmacokinetics and absorption in the rat: therapeutic potential for enteral administration. Br. J. Pharmacol. 164, 1857–1870. 10.1111/j.1476-5381.2011.01413.x PubMed DOI PMC

Bulmer A. C., Verkade H. J., Wagner K. H. (2013). Bilirubin and beyond: a review of lipid status in Gilbert's syndrome and its relevance to cardiovascular disease protection. Prog. Lipid Res. 52, 193–205. 10.1016/j.plipres.2012.11.001 PubMed DOI

Bulmer A. C., Bakrania B., Du Toit E. F., Boon A.-C., Clark P. J., Powell L. W., et al. (2018). Bilirubin acts as a multipotent guardian of cardiovascular integrity: more than just a radical idea. Am. J. Physiol. Heart Circul. Physiol. 315, H429–H447. 10.1152/ajpheart.00417.2017 PubMed DOI

Busiello R. A., Savarese S., Lombardi A. (2015). Mitochondrial uncoupling proteins and energy metabolism. Front. Physiol. 6, 36. 10.3389/fphys.2015.00036 PubMed DOI PMC

Bustin S. A., Benes V., Garson J. A., Hellemans J., Huggett J., Kubista M., et al. (2009). The MIQE guidelines: Minimum information for publication of quantitative real-time PCR experiments. Clin. Chem. 55, 611–622. 10.1373/clinchem.2008.112797 PubMed DOI

Celier C., Francois D., Marsac C., Cresteil T. (1992). Impairment of mitochondrial 5-aminolevulinic acid synthase activity in Gunn rat liver. Biochem. Pharmacol. 44, 1465–1467. 10.1016/0006-2952(92)90550-3 PubMed DOI

Costford S. R., Chaudhry S. N., Salkhordeh M., Harper M. E. (2006). Effects of the presence, absence, and overexpression of uncoupling protein-3 on adiposity and fuel metabolism in congenic mice. Am. J. Physiol. Endocrinol. Metab. 290, E1304–E1312. 10.1152/ajpendo.00401.2005 PubMed DOI

Divakaruni A. S., Brand M. D. (2011). The regulation and physiology of mitochondrial proton leak. Physiology 26, 192–205. 10.1152/physiol.00046.2010 PubMed DOI

Doerrier C., Garcia-Souza L. F., Krumschnabel G., Wohlfarter Y., Mészáros A. T., Gnaiger E. (2018). High-resolution FluoRespirometry and OXPHOS protocols for human cells, permeabilized fibers from small biopsies of muscle, and isolated mitochondria. Methods Mol. Biol. 1782, 31–70. 10.1007/978-1-4939-7831-1_3 PubMed DOI

Dulloo A. G., Jacquet J., Solinas G., Montani J. P., Schutz Y. (2010). Body composition phenotypes in pathways to obesity and the metabolic syndrome. Int. J. Obes. 34 Suppl 2, S4–S17. 10.1038/ijo.2010.234 PubMed DOI

Eigentler A., Draxl A., Wiethüchter A. (2015). Laboratory protocol: citrate synthase a mitochondrial marker enzyme. Mitochondr. Physiol. Netw. 04, 1–11.

Even P. C., Rolland V., Roseau S., Bouthegourd J. C., Tomé D. (2001). Prediction of basal metabolism from organ size in the rat: relationship to strain, feeding, age, and obesity. Am. J. Physiol. Regul. Integr. Comp. Physiol. 280, R1887–R1896. 10.1152/ajpregu.2001.280.6.r1887 PubMed DOI

Fevery J. (2008). Bilirubin in clinical practice: a review. Liver Int. 28, 592–605. 10.1111/j.1478-3231.2008.01716.x PubMed DOI

Fontana-Ayoub M., Fasching M., Gnaiger E. (2016). Selected media and chemicals for respirometry with mitochondrial preparations. Mitochondr. Physiol. Netw. 02, 1–10.

Fritz-Niggli H. (1968). Inhibited oxidative phosphorylation in rat liver mitochondria of congenitally jaundiced Gunn rats and the protective action of hydroxyethylrutosides against bilirubin-induced uncoupling. Med. Exp. Int. J. Exp. Med. 18, 239–246. 10.1159/000137160 PubMed DOI

Fu Y. Y., Kang K. J., Ahn J. M., Kim H. R., Na K. Y., Chae D. W., et al. (2010). Hyperbilirubinemia reduces the streptozotocin-induced pancreatic damage through attenuating the oxidative stress in the Gunn rat. Tohoku J. Exp. Med. 222, 265–273. 10.1620/tjem.222.265 PubMed DOI

Galmés-Pascual B. M., Nadal-Casellas A., Bauza-Thorbrügge M., Sbert-Roig M., García-Palmer F. J., Proenza A. M., et al. (2017). 17β-estradiol improves hepatic mitochondrial biogenesis and function through PGC1B. J. Endocrinol. 232, 297–308. 10.1530/JOE-16-0350 PubMed DOI

Gnaiger E. (2014). Mitochondrial pathways and respiratory control an introduction to OXPHOS analysis.

Greggio C., Jha P., Kulkarni S. S., Lagarrigue S., Broskey N. T., Boutant M., et al. (2017). Enhanced respiratory chain supercomplex formation in response to exercise in human skeletal muscle. Cell Metab. 25, 301–311. 10.1016/j.cmet.2016.11.004 PubMed DOI

Hinds T. D., Hosick P. A., Chen S., Tukey R. H., Hankins M. W., Nestor-Kalinoski A., et al. (2017). Mice with hyperbilirubinemia due to Gilbert's syndrome polymorphism are resistant to hepatic steatosis by decreased serine 73 phosphorylation of PPARα. Am. J. Physiol. Endocrinol. Metab. 312, E244–E252. 10.1152/ajpendo.00396.2016 PubMed DOI PMC

Holland O. J., Cuffe J. S. M., Dekker Nitert M., Callaway L., Kwan Cheung K. A., Radenkovic F., et al. (2018). Placental mitochondrial adaptations in preeclampsia associated with progression to term delivery. Cell Death Dis 9, 1150. 10.1038/s41419-018-1190-9 PubMed DOI PMC

Holliday M. A., Potter D., Jarrah A., Bearg S. (1967). The relation of metabolic rate to body weight and organ size. Pediatr. Res. 1, 185–195. 10.1203/00006450-196705000-00005 PubMed DOI

Huang T., Peng G., Li G., Yamahara J., Roufogalis B. D., Li Y. (2006). Salacia oblonga root improves postprandial hyperlipidemia and hepatic steatosis in Zucker diabetic fatty rats: activation of PPAR-alpha. Toxicol. Appl. Pharmacol. 210, 225–235. 10.1016/j.taap.2005.05.003 PubMed DOI

Jansen P. L. M. (1999). Diagnosis and management of Crigler-Najjar syndrome. Eur. J. Pediatr. 158 Suppl 2, S89–S94. 10.1007/PL00014330 PubMed DOI

Jeong S., Yoon M. (2007). Inhibition of the actions of peroxisome proliferator-activated receptor alpha on obesity by estrogen. Obesity (Silver Spring) 15, 1430–1440. 10.1038/oby.2007.171 PubMed DOI

Jiang X., Wang X. (2004). Cytochrome C-mediated apoptosis. Annu. Rev. Biochem. 73, 87–106. 10.1146/annurev.biochem.73.011303.073706 PubMed DOI

Keipert S., Ost M., Chadt A., Voigt A., Ayala V., Portero-Otin M., et al. (2013). Skeletal muscle uncoupling-induced longevity in mice is linked to increased substrate metabolism and induction of the endogenous antioxidant defense system. Am. J. Physiol. Endocrinol. Metab. 304, E495–E506. 10.1152/ajpendo.00518.2012 PubMed DOI

Kummitha C. M., Kalhan S. C., Saidel G. M., Lai N. (2014). Relating tissue/organ energy expenditure to metabolic fluxes in mouse and human: experimental data integrated with mathematical modeling. Physiol. Rep. 2, 1–20. 10.14814/phy2.12159 PubMed DOI PMC

Larsen S., Kraunsøe R., Gram M., Gnaiger E., Helge J. W., Dela F. (2014). The best approach: homogenization or manual permeabilization of human skeletal muscle fibers for respirometry? Anal. Biochem. 446, 64–68. 10.1016/j.ab.2013.10.023 PubMed DOI

Lemieux H., Semsroth S., Antretter H., Höfer D., Gnaiger E. (2011). Mitochondrial respiratory control and early defects of oxidative phosphorylation in the failing human heart. Int. J. Biochem. Cell Biol. 43, 1729–1738. 10.1016/j.biocel.2011.08.008 PubMed DOI

Liu J., Dong H., Zhang Y., Cao M., Song L., Pan Q., et al. (2015). Bilirubin increases insulin sensitivity by regulating cholesterol metabolism, adipokines and PPARγ levels. Sci. Rep. 5, 9886. 10.1038/srep09886 PubMed DOI PMC

Malik S. G., Irwanto K. A., Ostrow J. D., Tiribelli C. (2010). Effect of bilirubin on cytochrome c oxidase activity of mitochondria from mouse brain and liver. BMC Res. Notes 3, 162. 10.1186/1756-0500-3-162 PubMed DOI PMC

Mustafa M. G., Cowger M. L., King T. E. (1969). Effects of bilirubin on mitochondrial reactions. J. Biol. Chem. 244, 6403–6414. 10.1016/s0021-9258(18)63479-9 PubMed DOI

Mustafa M. G., King T. E. (1970). Binding of bilirubin with lipid. A possible mechanism of its toxic reactions in mitochondria. J. Biol. Chem. 245, 1084–1089. 10.1016/s0021-9258(18)63292-2 PubMed DOI

Naveenkumar S. K., Thushara R. M., Sundaram M. S., Hemshekhar M., Paul M., Thirunavukkarasu C., et al. (2015). Unconjugated bilirubin exerts pro-apoptotic effect on platelets via p38-MAPK activation. Sci. Rep. 5, 1–16. 10.1038/srep15045 PubMed DOI PMC

Neuzil J., Stocker R. (1994). Free and albumin-bound bilirubin are efficient co-antioxidants for alpha-tocopherol, inhibiting plasma and low density lipoprotein lipid peroxidation. J. Biol. Chem. 269, 16712–16719. 10.1016/s0021-9258(19)89449-8 PubMed DOI

Nguyen P., Leray V., Diez M., Serisier S., Le Bloch J. B., Dumon H. (2008). Liver lipid metabolism. J. Anim. Physiol. Anim. Nutr. (Berl) 92, 272–283. 10.1111/j.1439-0396.2007.00752.x PubMed DOI

Odell G. B. (1966). The distribution of bilirubin between albumin and mitochondria. J. Pediatr. 68, 164–180. 10.1016/S0022-3476(66)80147-6 DOI

Ost M., Werner F., Dokas J., Klaus S., Voigt A. (2014). Activation of AMPKα2 is not crucial for mitochondrial uncoupling-induced metabolic effects but required to maintain skeletal muscle integrity. PLoS One 9, e94689. 10.1371/journal.pone.0094689 PubMed DOI PMC

Ostrow J. D., Pascolo L., Brites D., Tiribelli C. (2004). Molecular basis of bilirubin-induced neurotoxicity. Trends Mol. Med. 10, 65–70. 10.1016/j.molmed.2003.12.003 PubMed DOI

Pennell E. N., Shiels R., Vidimce J., Wagner K.-H., Shibeeb S., Bulmer A. C. (2019). The impact of bilirubin ditaurate on platelet quality during storage. Platelets 31, 884–813. 10.1080/09537104.2019.1693038 PubMed DOI

Rakhshandehroo M., Knoch B., Müller M., Kersten S. (2010). Peroxisome proliferator-activated receptor alpha target genes. PPAR Res. 2010, 1–20. 10.1155/2010/612089 PubMed DOI PMC

Rodrigues C. M., Solá S., Brites D. (2002a). Bilirubin induces apoptosis via the mitochondrial pathway in developing rat brain neurons. Hepatology 35, 1186–1195. 10.1053/jhep.2002.32967 PubMed DOI

Rodrigues C. M., Solá S., Brito M. A., Brites D., Moura J. J. (2002b). Bilirubin directly disrupts membrane lipid polarity and fluidity, protein order, and redox status in rat mitochondria. J. Hepatol. 36, 335–341. 10.1016/S0168-8278(01)00279-3 PubMed DOI

Rodrigues C. M., Solá S., Silva R., Brites D. (2000). Bilirubin and amyloid-beta peptide induce cytochrome c release through mitochondrial membrane permeabilization. Mol. Med. 6, 936–946. 10.1007/bf03401828 PubMed DOI PMC

Rolf B., Stern L. (1980). Introduction: bilirubin encephalopathy - the preventive role of bilirubin binding to albumin. Crit. Rev. Clin. Lab. Sci. 11, 307–399.

Rossi F., Francese M., Iodice R. M., Falcone E., Vetrella S., Punzo F., et al. (2005). [Inherited disorders of bilirubin metabolism]. Minerva Pediatr. 57, 53–63. 10.1016/S0168-8278(02)00359-8 PubMed DOI

Sambasivarao S. V. (2013). Glucuronidation of the steroid enantiomers ent-17β-estradiol ent-androsterone and ent-etiocholanolone by the human UDP-glucuronosyltransferases. 18, 1199–1216. 10.1016/j.micinf.2011.07.011.Innate PubMed DOI PMC

Scarpulla R. C. (2011). Metabolic control of mitochondrial biogenesis through the PGC-1 family regulatory network. Biochim. Biophys. Acta 1813, 1269–1278. 10.1016/j.bbamcr.2010.09.019 PubMed DOI PMC

Seyed Khoei N., Grindel A., Wallner M., Mölzer C., Doberer D., Marculescu R., et al. (2018). Mild hyperbilirubinaemia as an endogenous mitigator of overweight and obesity: implications for improved metabolic health. Atherosclerosis 269, 306–311. 10.1016/j.atherosclerosis.2017.12.021 PubMed DOI

Shiels R. G., Vidimce J., Pearson A. G., Matthews B., Wagner K. H., Battle A. R., et al. (2019). Unprecedented microbial conversion of biliverdin into bilirubin-10-sulfonate. Sci. Rep. 9, 1–10. 10.1038/s41598-019-39548-w PubMed DOI PMC

Siliart B. A., Boveris A., Garaza Pereira A. M., Stoppani A. O. (1972). Bilirubin: a multi-site inhibitor of mitochondrial respiration. FEBS Lett. 27, 270. 10.1016/0014-5793(72)80638-0 PubMed DOI

Stanford J. A., Shuler J. M., Fowler S. C., Stanford K. G., Ma D., Bittel D. C., et al. (2015). Hyperactivity in the Gunn rat model of neonatal jaundice: age-related attenuation and emergence of gait deficits. Pediatr. Res. 77, 434–439. 10.1038/pr.2014.199 PubMed DOI PMC

Stec D. E., John K., Trabbic C. J., Luniwal A., Hankins M. W., Baum J., et al. (2016). Bilirubin binding to PPARα inhibits lipid accumulation. PLoS One 11, e0153427. 10.1371/journal.pone.0153427 PubMed DOI PMC

Stocker R., Yamamoto Y., McDonagh A. F., Glazer A. N., Ames B. N. (1987). Bilirubin is an antioxidant of possible physiological importance. Science 235, 1043–1046. 10.1126/science.3029864 PubMed DOI

Vaz A. R., Delgado-Esteban M., Brito M. A., Bolaños J. P., Brites D., Almeida A. (2010). Bilirubin selectively inhibits cytochrome c oxidase activity and induces apoptosis in immature cortical neurons: assessment of the protective effects of glycoursodeoxycholic acid. J. Neurochem. 112, 56–65. 10.1111/j.1471-4159.2009.06429.x PubMed DOI

Vítek L., Ostrow J. (2009). Bilirubin chemistry and metabolism; harmful and protective aspects. Curr. Pharm. Des. 15, 2869–2883. 10.2174/138161209789058237 PubMed DOI

Vítek L. (2012). The role of bilirubin in diabetes, metabolic syndrome, and cardiovascular diseases. Front. Pharmacol. 3, 1–7. 10.3389/fphar.2012.00055 PubMed DOI PMC

Wallner M., Marculescu R., Doberer D., Wolzt M., Wagner O., Vitek L., et al. (2013). Protection from age-related increase in lipid biomarkers and inflammation contributes to cardiovascular protection in Gilbert's syndrome. Clin. Sci. 125, 257–264. 10.1042/Cs20120661 PubMed DOI

Watchko J. F., Tiribelli C. (2013). Bilirubin-induced neurologic damage--mechanisms and management approaches. N. Engl. J. Med. 369, 2021–2030. 10.1056/NEJMra1308124 PubMed DOI

Wu Z., Puigserver P., Andersson U., Zhang C., Adelmant G., Mootha V., et al. (1999). Mechanisms controlling mitochondrial biogenesis and respiration through the thermogenic coactivator PGC-1. Cell 98, 115–124. 10.1016/S0092-8674(00)80611-X PubMed DOI

Yoon M., Jeong S., Nicol C. J., Lee H., Han M., Kim J. J., et al. (2002). Fenofibrate regulates obesity and lipid metabolism with sexual dimorphism. Exp. Mol. Med. 34, 481–488. 10.1038/emm.2002.67 PubMed DOI

Zelenka J., Dvořák A., Alán L., Zadinová M., Haluzík M., Vítek L. (2016). Hyperbilirubinemia protects against aging-associated inflammation and metabolic deterioration. Oxid. Med. Cell. Longev. 26, 6190609. 10.1155/2016/6190609 PubMed DOI PMC

Zeng Q., Dong S. Y., Sun X. N., Xie J., Cui Y. (2012). Percent body fat is a better predictor of cardiovascular risk factors than body mass index. Braz. J. Med. Biol. Res. 45, 591–600. 10.1590/S0100-879X2012007500059 PubMed DOI PMC

Zhou J., Tracy T. S., Remmel R. P. (2011). Correlation between bilirubin glucuronidation and estradiol-3-gluronidation in the presence of model UDP-glucuronosyltransferase 1A1 substrates/inhibitors. Drug Metab. Dispos. 39, 322–329. 10.1124/dmd.110.035030 PubMed DOI PMC

Zucker S. D., Storch J., Zeidel M. L., Gollan J. L. (1992). Mechanism of the spontaneous transfer of unconjugated bilirubin between small unilamellar phosphatidylcholine vesicles. Biochemistry 31, 3184–3192. 10.1021/bi00127a020 PubMed DOI