Impact of Hyperbilirubinaemia on Cholesterol Metabolism and Bioenergetics

Bilirubin is a haem catabolite that is excreted through the hepatobiliary pathway and is therefore, commonly used as a biomarker of hepatic dysfunction and haemolysis in the clinical setting [1]. Although, bilirubin has been considered toxic [2], recent evidence suggests that mildly elevated circulating bilirubin concentrations may be protective against obesity, cardiovascular diseases (CVDs) and all-cause mortality [3–5]. Generally, the protective effects of bilirubin are attributed to its antioxidant potential [6–8], however, recent studies demonstrate that bilirubin modulates lipid metabolism and reduces adiposity, which could partly contribute to CVD protection [5,9–12]. However, a shortage of studies have examined the precise mechanisms of cholesterol metabolism and adiposity that could be affected by bilirubin. The main aims of this thesis were to: 1) determine whether hyperbilirubinaemia affects cholesterol synthesis, transport, and excretion; 2) explore bilirubin’s impact on body composition and bioenergetics including mitochondrial function in liver/skeletal muscle and changes in mitochondrial density and quality; 3) determine the effectiveness of oral Legalon® ingestion on circulating bilirubin concentrations, to investigate whether inducing mild hyperbilirubinaemia could impact circulating lipid concentrations in human participants. The first study measured the effect of hyperbilirubinaemia in mutant Gunn rats on circulating lipid concentrations, cholesterol synthesis, lipid excretion, and expression of hepatic genes/proteins involved in cholesterol metabolism. Female hyperbilirubinaemic (Gunn) rats had reduced serum cholesterol concentrations (0.60 ± 0.12 vs 1.56 ± 0.34 mM, P<0.001), elevated cholesterol synthesis (33.8 ± 3.77 vs 28.4 ± 5.73 % [13C]-cholesterol, P<0.05), enhanced LDL receptor (LDLr; P<0.01) expression, and increased biliary cholesterol excretion (232 ± 32.7 vs 141 ± 42.1 nmol hr-1 100g-1 bodyweight, P<0.001) compared to female normobilirubinaemic littermate (control) rats. These results indicate that female hyperbilirubinaemic Gunn rats have reduced circulating cholesterol in association with elevated LDLr expression. Increased LDLr expression and cholesterol synthesis is typical when hepatic cholesterol concentrations are decreased [13,14]. Therefore, increased cholesterol synthesis and LDLr expression observed in female Gunn rats may represent a counter-regulatory mechanism to maintain hepatic cholesterol content in the presence of elevated biliary cholesterol excretion [13,14]. The underlying mechanism explaining increased biliary lipid excretion in female Gunn rats remains unknown. However, this observation could be partly explained by greater relative biliary lipid (cholesterol+phospholipid) to bile acid excretion (0.33 ± 0.06 vs 0.24 ± 0.03 mol:mol, lipid:bile acids, P<0.01) in female Gunn rats. Previous studies have established that organic anions including bilirubin glucuronides disrupt the capacity of bile acid micelles to excrete lipids in the bile [15]. Biliary excretion of bilirubin conjugates was decreased in female (13.1 ± 2.92 vs 33.5 ± 5.09 nmol hr-1 100g-1 bodyweight, P<0.001) and male (11.0 ± 2.43 vs 43.2 ± 12.8 nmol hr-1 100g-1 bodyweight, P<0.001) Gunn rats compared to controls, due to UGT1A1 dysfunction and the inability to conjugate bilirubin. Therefore, decreased biliary excretion of bilirubin conjugates, as observed in Gunn rats, may potentially facilitate the greater coupled excretion of biliary lipids to bile acids as demonstrated in this study. It should be noted that this conclusion does not completely explain the results reported here because Gunn rats demonstrated significant sexual dimorphism in cholesterol metabolism. Male Gunn rats exhibited a non-significant reduction in circulating cholesterol concentrations (1.41 ± 0.15 vs 1.56 ± 0.23, P=0.14) and increased biliary lipid:bile acid excretion (0.31 ± 0.07 vs 0.25 ± 0.04 mol:mol, lipid:bile acid, P=0.08) compared to male normobilirubinaemic littermate (control) rats, indicating that additional mechanisms, beyond bilirubin excretion, are involved. For example, UGT1A1, which conjugates bilirubin also conjugates and facilitates the excretion of sex hormones including oestrogen. Therefore, oestrogen concentrations may be elevated in female hyperbilirubinaemic rats and synergistically impact lipid metabolism [16,17]. The second study examined the effect of hyperbilirubinaemia in vitro and in vivo on mitochondrial function and body composition. Dual X-ray absorptiometry (DEXA) analysis revealed that female Gunn rats had significantly reduced fat mass (9.94 ± 5.35 vs 16.1 ± 6.65 g, P<0.05) and lean mass (140 ± 12.1 vs 160 ± 16.0 g, P<0.05) compared to littermate controls. Female Gunn rats consumed fewer calories per day (54.1 ± 6.38 vs 63.3 ± 6.95 kcal day-1, P<0.01). However, weight gain relative to calories consumed was reduced (8.09 ± 5.75 vs 14.9 ± 5.10 mg kcal-1, P<0.05) in female Gunn rats indicating that they are less energetically efficient. This led to the analysis of mitochondrial function in liver and skeletal muscle using high-resolution respirometry to ascertain the cause of reduced energetic efficiency. This analysis revealed that female Gunn rats exhibited increased oxidative phosphorylation (OXPHOS) relative to maximal noncoupled mitochondrial respiration (ETS) in hepatic mitochondria (0.78 ± 0.16 vs 0.62 ± 0.09 OXPHOS:ETS, P<0.05). The above findings were consistent with the effect of exogenous addition of unconjugated bilirubin (UCB) to control hepatic mitochondria, with 31.3 and 62.5 μM UCB increasing the OXPHOS: ETS ratio. However, exogenous UCB addition produced this effect by inhibiting ETS without affecting OXPHOS, indicating that UCB induces mitochondrial dysfunction at high concentrations. Conversely, no change in ETS (1130 ± 217 vs 1290 ± 373 pmol s-1 ng-1 citrate synthase (CS), P=0.16) or OXPHOS (901 ± 222 vs 796 ± 259 pmol s-1 ng-1 CS, P=0.36) was observed between female Gunn rats and controls. These data indicate that the greater OXPHOS:ETS ratios are a combination of increased OXPHOS and decreased ETS in female Gunn rats. Analysis of mitochondrial respiratory complexes revealed greater hepatic mitochondrial complex IV (CIV; P<0.01) expression in female Gunn rats. These findings support a conclusion that hepatic mitochondria have increased quality in female Gunn rats [18,19]. At present it remains unknown how this change in mitochondrial quality relates to reduced fat mass and energetic efficiency, however, the changes observed in female Gunn rats could represent an adaptation to bilirubin mediated inhibition of CIV as reported in vitro [20,21]. Otherwise, alterations in reproductive hormone metabolism in Gunn rats could also partially explain altered energetic states, as speculated in study one. Considering that hyperbilirubinaemia induced perturbed lipid metabolism and body composition in chapters one and two, study three sought to determine whether increasing bilirubin could alter circulating lipid profile in humans. The effect of Legalon®, containing the active ingredient silymarin, supplementation on circulating bilirubin concentrations and lipid status was investigated in a placebo controlled, single blind crossover clinical trial in healthy individuals (ACTRN12619001296123). Legalon® capsules containing 140 mg of silymarin were supplemented thrice daily (total dose of 420 mg silymarin) in a cohort of healthy males for two weeks. Two weeks of Legalon® supplementation did not change UCB concentrations compared to baseline (Legalon®: 12.5 ± 7.63 vs Baseline: 11.4 ± 4.14 μM, P=0.79). Secondary outcomes including lipid concentrations, inflammation, and total antioxidant status were also reported. Two weeks of Legalon® supplementation did not change serum cholesterol (4.80 ± 1.00 vs 4.88 ± 1.00 mM, P=0.19), triglyceride (1.07 ± 0.63 vs 1.04 ± 0.54 mM, P=0.79), C-reactive protein concentrations (1.74 ± 1.88 vs 0.92 ± 0.87 mg L-1, P=0.23) or serum antioxidant capacity (1194 ± 182 vs 1183 ± 201 mmol Fe2+ L-1, P=0.19) compared to baseline. Several clinical trials evaluating the impact of silymarin have reported changes to bilirubin concentrations following treatment [22–24]. However, these studies were conducted in patients with hepatic disease, which confounded bilirubin results, and with greater doses or different formulations of silymarin to that reported in this thesis. Although the results of this study demonstrated a negative finding, they are important because they represent the first attempt to use an orally administered, commercially approved, nutraceutical compound to increase bilirubin. These results provide important guidance to future studies that could utilise different doses or commercial preparations to induce a transient unconjugated hyperbilirubinaemia and test the impact on circulating cholesterol concentrations. In conclusion, this thesis contains three novel investigations that aimed to determine the impact of unconjugated hyperbilirubinaemia on cholesterol metabolism, synthesis and hepatic excretion; in addition to its effect on mitochondrial metabolism and body composition in Gunn rats. To determine whether these effects could be induced in humans, a nutraceutical with documented effects on circulating bilirubin was administered in a clinical trial, utilising a randomised, single-blind, crossover design. The results of this thesis suggest that bilirubin has the potential to modulate lipid and whole-body metabolism, particularly in female animals and provides the groundwork for additional studies that seek to reveal the mechanisms responsible for bilirubin’s effects. In addition, this thesis will support the discovery of more effective orally administered compounds that can modulate circulating bilirubin and lipid profile for protection against CVD.